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10.1371/journal.pone.0309903
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Maggot extract accelerates skin wound healing of diabetic rats via enhancing STAT3 signaling
Maggot extract accelerated diabetic rat skin wound healing
Wu Mo-Li Conceptualization Data curation Formal analysis Investigation Methodology Writing – original draft Writing – review & editing 1
Yang Zhe-Ming Data curation Formal analysis Investigation Methodology Writing – original draft Writing – review & editing 1
Dong Hai-Chao Formal analysis 2
Zhang Hong Data curation 2
Zheng Xu Investigation 1
Yuan Bo Investigation 1 2
Yang Yang Investigation 1
https://orcid.org/0009-0004-9275-6506
Liu Jia Conceptualization Funding acquisition Supervision Writing – review & editing 1 *
Li Pei-Nan Conceptualization Data curation Funding acquisition Supervision 2 *
1 Department of Cell Biology, College of Basic Medical Sciences, Dalian Medical University, Dalian, China
2 Department of Orthopedic Surgery, Second Affiliated Hospital, Dalian Medical University, Dalian, China
Hafeez Kamran Sairah Editor
Lahore College for Women University, PAKISTAN
Competing Interests: The authors have declared that no competing interests exist.

* E-mail: jialiudl2022@163.com (JL); lipndl@163.com (PNL)
6 9 2024
2024
19 9 e03099037 4 2024
21 8 2024
© 2024 Wu et al
2024
Wu et al
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Background

Diabetic skin wound is a complex problem due to the disruption of normal repairing program and lack of effective remedy. Lucilia sericata larvae (maggot) is a folk method to treat chronic skin wound, while its therapeutic effects on that caused by diabetic remains unknown.

Objective

This study aims to investigate the therapeutic effects of maggot extract (M.E.) on diabetic skin wound and its molecular mechanism by establishing the skin wound model of diabetic Sprague Dawley (SD) rats.

Methods

Diabetic model was established by injecting intraperitoneally streptozotocin in SD rats under specific pathogen-free (SPF) conditions. The rat fasting blood glucose values were ≧16.7 mmol/L 72 hours after intraperitoneal streptozotocin (60mg/kg body weight) injection. The rats were divided into five groups (n = 10/group): normal group: normal SD rats without any treatment, diabetic blank group: the diabetic rats without any treatment, Vaseline group: the diabetic rats dressed with Vaseline, recombinant human epidermal-growth-factor (rhEGF) group: the diabetic rats dressed with a mixture of Vaseline and 200 μg/g rhEGF, M.E. group: the diabetic rats dressed with a mixture of Vaseline and 150 μg/ml maggot extract. The round open wounds (1.0 cm in diameter) down to the muscle fascia were made on both sides of rat dorsa by removing the skin layer (epidermis and dermis) and were daily photographed for calculating their healing rates. Hematoxylin-eosin (HE) and Masson’s trichrome staining were performed on skin wound sections to analyze re-epithelialization and granulation tissue formation. Immunohistochemical (IHC), immunofluorescent (IF) stainings and Western blotting were conducted to analyze the statuses of STAT3 signaling.

Results

The average wound healing rates on the 14th day were 91.7% in the normal, 79.6% in M.E., 71% in rhEGF, 55.1% in vaseline and 43.3% in the diabetes blank group. Morphological staining showed more active granulation tissue formation, re-epithelialization and neovascularization in M.E.-group than those in the blank and the vaseline-treated groups. Decreased p-STAT3 nuclear tranlocation and down-regulated Bcl-2, CyclinD1 and vascular endothelial growth factor (VEGF) expression were evidenced in the diabetic rats, which could be improved by rhEGF and especially M.E.

Conclusion

Maggot extract would be an alternative and/or adjuvant candidate for the better management of diabetic skin wounds because of its activity in enhancing STAT3 activation.

http://dx.doi.org/10.13039/501100001809 National Natural Science Foundation of China 81450016 https://orcid.org/0009-0004-9275-6506
Liu Jia http://dx.doi.org/10.13039/501100001809 National Natural Science Foundation of China 81272786 https://orcid.org/0009-0004-9275-6506
Liu Jia http://dx.doi.org/10.13039/501100005047 Natural Science Foundation of Liaoning Province 2019-ZD-0650 Li Pei-Nan This work was supported by the Grants from National Natural Science Foundation of China [Nos. 81450016 and 81272786 to J.L.] and Natural Science Foundation of Liaoning Province, China [No. 2019-ZD-0650 to P.N. L.]. The funders have roles in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data AvailabilityAll relevant data are within the manuscript and its Supporting Information files.
Data Availability

All relevant data are within the manuscript and its Supporting Information files.
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pmcIntroduction

Diabetes is a chronic metabolic disease, which has become a growing international health concern [1,2]. Diabetes (type 1 and 2) involves many complications, and the most common one is the delayed healing and even nonunion of acute and chronic skin wounds [3]. Multiple factors or molecular machinaries responsible for wound healing are altered in diabetic individuals, leading to the reduced regenerative activity of skin wounds. It has been proposed that the local peripheral nerve and vascular alterations are the main reasons for the difficulty of skin wound healing [4]. Current options for wound management of diabetic patients are: 1) strict control of blood glucose; 2) application of vasodilators to improve microvascular circulation; 3) antibacterial and anti-inflammatory treatments; 4) surgical debridement and 5) local nursing and health care [5]. Although the above measures include both the whole and local treatments, the incidence of diabetic wounds and the disability rate caused by them remain almost unchanged, indicating the unsatisfactory efficacy of the above approaches [6]. Therefore, it would be necessary to alleviate the influence of pathogenic factors and meanwhile to accelerate the wound healing. Because of the complexity of the pathogenic factors of diabetic wounds, the new agent (drug) containing beneficial components for neovascularization and cell proliferation would be helpful.

Lucilia sericata (Meigen 1826, Diptera: Calliphoridae) is a widespread blow fly species. The medicinal value of Lucilia sericata larvae (maggot) has long been recognized [7]. The maggots are traditionally used to remove the necrotic tissue in the deep wound where the conventional surgery fails to reach [8]. In addition, the maggot body has a strong self-repairing ability after injury, because of its richness in regeneration promoting elements [9]. Even though, the traditional dry baking method significantly reduced the bio-activity of the maggot product [10]. For this reason, we had prepared fresh maggot extract and shown its beneficial effects on normal rat skin wound healing [11]. Moreover, the enhanced signal transducer and activator of transcription 3 (STAT3) activation and upregulation of the anti-apoptotic gene B-cell lymphoma-2 (Bcl-2) were found in maggot extract treated skin wounds in normal rats [11]. Because activated STAT3 signaling controls proliferation and differentiation during wound healing and skin remodeling [12], the above results provide scientific evidence for the effectiveness of maggot extract in improving wound healing [13].

It has to be pointed out that the results so far obtained from our previous study are all from the healthy experimental animals and the skin wounds of those animals are repairable as a matter of course. Alternatively, the maggot extract merely shortens the healing time to a certain extent. Therefore, the key point of the medication value of the maggot extract should be its therapeutic efficacy for the refractory wounds such as diabetic skin wounds. It has been evidenced that the remarkable reduction of phospho-STAT3 (p-STAT3) immunopositive keratinocytes and down-regulation of some growth factors such as interleukin-22 (IL-22) and forkhead box M1 (FOXM1) are responsible for the delayed wound healing of diabetic wounds [14,15]. For these reasons, we consider that maggot extract might also exert beneficial effects on diabetic rat skin wound healing via improving the activity of STAT3 signaling. The current study aims to address the therapeutic effects of maggot extract on diabetic skin wound and its molecular mechanism.

Materials and methods

Ethics approval

This research project was approved by Animal Care and Use Committee of Dalian Medical University (DMU) in the ethics approval number AEE18055 to guarantee that the number of experimental animals should be restrictively controlled and all works involving experimental animals were performed in full compliance with NIH (National Institutes of Health, USA) Guidelines for the Care and Use of Laboratory Animals.

Establishment of type 1 diabetic rat model

50 male Sprague Dawley (SD) rats (aged 5–6 weeks, 200–220 g), were provided by SPF (specific pathogen free) experimental animal center of Dalian Medical University. 4 mg/ml streptozotocin (Sigma-Aldrich, St. Louis, MO, USA) in citric acid/sodium citrate buffer solution was prepared for injection by the method described elsewhere [16,17]. All rats were fed with standard diet under 20–26°C. After two weeks of adaptive feeding, 40 rats were randomly selected for inducing diabetic model. Briefly, the rats were fasted for 12 hours, followed by one-time intraperitoneal injection of 60 mg/kg streptozotocin. Rats were fasted for 12 hours after injection. 72 hours after streptozotocin injection, the fasting tail venous blood of the treated rats was sampled and checked by blood glucose meter (Accu-chek active; Roche). The rats were diagnosed with diabetes when their fasting blood glucose levels were over 16.7 mmol/L [18].

Maggot extract preparation

Lucilia sericata blowflies were obtained from BIOWIM Technology Development Co., Ltd (Dalian, China) and maintained in a closed container with a net at a constant temperature of about 22°C. Their larvae were collected when they were reared to later-second or early-third stages. The collected maggots were rinsed three times with autoclaved pure water, placed in 3.5% formaldehyde normal saline solution for 5 minutes, in 2% H2O2 for 3 minutes, in 1% hydrochloride acid for 5 minutes and finally subjected to three washes with autoclaved pure water. The collected maggots were directly snap frozen in liquid nitrogen and stored at −80°C until extract preparation. The extracts of maggots were prepared by series sectioning the frozen maggot tissues into pieces (5 μm in thickness) and then putting the frozen sections into the test tubes containing pH7.5 phosphate buffered saline (PBS). By this way, the high-quality maggot extracts were prepared in high yields [11]. As a comparison, maggot extract was treated via the traditional method. Briefly, 400 mg of the frozen maggots were weighed. One half (200mg) of them was sectioned into pieces (5 μm in thickness) and then put into ice-cold lysis buffer (Beyotime P0013B), and the other half was dried at 35°C for 12 hours by the method described elsewhere [19], smashed them into powder and put the powder into the same volume (200 μl) ice-cold lysis buffer. The same volume of the protein samples exacted by the two methods were separated by 10% sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE).

Skin wound model and treatments

10 normal and 40 diabetic rats were anesthetized with ketamine (75mg/kg body weight) and xylazine (10mg/kg body weight) intraperitonealy. The hair in the dorsal skin of rats was shaved and the round open wounds (1.0 cm in diameter) down to the muscle fascia were made on both sides of rat dorsa by removing the skin layer (epidermis and dermis). All of the operated rats were separately fed (one rat/cage). 50 SD rats were divided into 5 experimental groups (10 rats/group): Group 1, normal SD rats without any treatment (normal group); Group 2, the diabetic rats without any treatment (diabetic blank group); Group 3, diabetic rats dressed with Vaseline (Rhawn, Shanghai, China, Vaseline group) [11]; Group 4, diabetic rats dressed with a mixture of Vaseline [11] and 200 μg/g recombinant epidermal growth factor (Pavay Gene Pharmaceutical, Guilin, China, rhEGF group); Group 5, the diabetic rats dressed with a mixture of Vaseline [11] and 150 μg/ml maggot extract (M.E. group). The experimental treatments lasted for 14 days by daily dressing the reagents mentioned above. Maggot extract was quantified by Ultraviolet spectrophotometer-based calculation of protein concentration.

Wound examination

The wound areas were measured and the wound healing rates were calculated to evaluate the wound healing in different experimental groups. The status of wound healing and the contraction of wound regions were observed and photographed. The electronic images were saved to computer and the wound areas recorded in them were measured by Image J software (National Institutes of Health, USA) with the method described elsewhere [20]. The average wound healing rate in each of the experimental groups was calculated with the formula: wound healing rate = (initial wound area—uninformed area) / initial wound area × 100%.

Wound edge biopsy

The rats were anesthetized and sacrificed through cervical dislocation performed by appropriately trained and competent personnel at the end of the experiment. The skin tissues in the size of 0.3 × 0.3 × 0.2 cm were biopsied from the wound margins of the five experimental groups. Half of the tissues was snap-frozen in liquid nitrogen, and then moved to the refrigerator at—80°C for later molecular analyses. The remaining part of the tissues was fixed in 10% neutral formalin buffer to prepare paraffin-embedded tissue block for histological, immunohistochemical and immunofluorescent stainings.

Histological, immunohistochemical and immunofluorescent analyses

Hematoxylin-eosin (HE) and Masson’s trichrome staining (Solarbio Science & Technology Co. Ltd, Beijing, China) were performed on 4 mm-thick central wound sections to analyze re-epithelialization and granulation tissue formation, respectively. Immunohistochemical (IHC) and immunofluorescent (IF) staining were conducted on wound tissue sections by the method described elsewhere [21]. The antibodies used were: Bcl-2 (bs-0032R, Bioss Inc., Beijing, China; 1:250); STAT3 (sc-8019, Santa Cruz Inc., USA; 1:200); VEGF (bs-1313R, Bioss Inc., Beijing, China; 1:200); CyclinD1 (bs-20596R, Bioss Inc., Beijing, China; 1:250); p-STAT3 (sc-8059, Santa Cruz Inc., USA; 1:200). The color reaction was performed by using 3, 3’-diaminobenzidine tetrahydrochloride (DAB) after the binding of the primary antibody (Vector Laboratories, Burlingame, CA, USA). For double IF staining, the tissue sections were washed with pH 7.4 PBS, incubated in 3% H2O2 for 10 min and then with the first antibody at 4°C overnight in a humid chamber. Finally, the tissues were co-incubated with FITC-conjugated goat anti-mouse IgG and PE-conjugated goat anti-rabbit IgG (both 1:100; Santa Cruz Biotech, Santa Cruz, CA, USA) at 37°C for 60 min in the dark, sealed with fluorescence mounting medium, and observed and imaged under a fluorescence microscope (BX53F, Olympus, Tokyo, Japan).

Protein extraction and Western blotting

Western blotting was conducted using antibodies against Bcl-2 (bs-0032R, Bioss Inc., Beijing, China; 1:500); STAT3 (sc-8019, Santa Cruz Inc., USA; 1:600); VEGF (bs-1313R, Bioss Inc., Beijing, China; 1:500); CyclinD1 antibody (bs-20596R, Bioss Inc., Beijing, China; 1:500); p-STAT3 (sc-8059, Santa Cruz Inc., USA; 1:500), respectively. The experiment was performed by the method described elsewhere [11]. Briefly, the sample proteins (20μg/well) were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membrane (Amersham, Buckin ghamshire, UK). The membrane was blocked in 5% skimmed milk (Sigma-Aldrich) Tris-buffered saline (TBS-T) (10 mmol/L Tris–HCl, pH 8.0, 0.5% Tween 20 and 150 mmol/L NaCl) at 4°C. After three washes with TBS-T, the membrane was incubated for 3h with the primary antibody at room temperature. Appropriate secondary HRP-conjugated antibodies were used for detection with chemiluminescent ECL reagents (Roche GmbH, Mannheim,Germany). In addition, an antibody against β-actin (ProteinTech 66009–1) was used as a loading control. The labeling signal was removed with a stripping buffer (62.5 mmol/L Tris–HCl, pH 6.7, 100 mmol/L β-mercaptoethanol, 2% sodium dodecyl sulfate (SDS), and the membrane was re-incubated with another antibody until all the parameters were examined.

Statistical analysis

Statistical analysis was performed using SPSS 21.0 (IBM Corp., Armonk, NY, USA). Data are presented as mean ± standard deviation. Data comparisons among experimental groups were performed using Student’s t test and analysis of variance (ANOVA), with a value of P < 0.05 being considered as statistically significant.

Results

Better quality of fresh maggot extract

Ultraviolet spectrophotometer-based calculation of protein concentration revealed that the protein yield per unit weight of fresh maggots (15.695 μg/mg) is 1.4 folds higher than that (11.545 μg/mg) prepared from the dried maggot powder. The SDS-PAGE result further demonstrated that the protein content and composition of fresh maggot extract (M.E.) is better than that of the maggot powder (Fig 1). The extract prepared from fresh maggots (M.E.) was selected for further experiments.

10.1371/journal.pone.0309903.g001 Fig 1 Comparison of the quality of maggot extracts prepared from the baked maggots and fresh maggots by SDS-PAGE electrophoresis.

Establishment of diabetic rat model

The typical symptoms of diabetes include polydipsia, polyphagia and polyuria, which was observed in rats on the third day after streptozotocin injection. As shown in Fig 2, the diabetic rats but not the normal ones showed weight loss, whereas the fasting blood glucose levels of normal rats were less than 7.0 mmol/L, and in streptozotocin-treated rats were distinctly increased (ranging from 25.6 to 27.3 mmol/L). The difference of fasting blood glucose levels between the normal and streptozotocin-treated rats was statistically significant (P < 0.01; Table 1).

10.1371/journal.pone.0309903.g002 Fig 2 Establishment of diabetic rat model via streptozotocin injection.

A, The appearance and blood glucose measurement of normal rats and streptozotocin-treated rats. B, The average fasting blood glucose levels (BGL) of SD rats in the 5 groups. C, The average body weights of SD rats in the experimental groups at different time points. The data are presented as mean ± SD. n = 10. *, P< 0.01, compared with normal rats.

10.1371/journal.pone.0309903.t001 Table 1 Average fasting blood glucose levels and body weights of the rats in different groups during the treatments.

Model group	Average fasting blood glucose levels (mmol/L) /Average body weight (g)	
Before streptozotocin injection	After streptozotocin injection	
Day 3	Day 7	Day 14	
Untreated diabetic rats	5.5±0.4/205±6	25.8±1.7/208±5	26.1±1.3/212±6	26.3±1.5/225±8	
Vaseline-treated	5.1±0.8/204±4	27.3±1.3/208±5	26.9±1.8/215±8	27.0±1.5/219±8	
Maggot extract-treated	5.6±0.3/203±5	25.6±1.7/210±7	26.0±1.8/219±6	25.8±1.5/228±5	
rhEGF-treated	5.5±0.4/208±6	26.3±1.4/211±9	26.5±1.3/219±7	26.7±1.3/225±8	
Normal group	Average fasting blood glucose levels (mmol/L) /Average body weight (g)
measured at the same time as the model groups	
Normal rats	5.3±0.5/202±3	5.1±0.6*/212±5	5.3±0.6*/236±9#	5.2±0.5*/269±12#	
*, Significant difference (P < 0.01) between normal rats and diabetic rats without and with the treatments

#, Significant difference (P < 0.01) between normal rats and diabetic rats without and with the treatments; Model group, rats with streptozotocin injection; Normal group, rats without streptozotocin injection.

Different wound healing rates of experimental groups

Two weeks after the establishment of diabetic model, the skin circular wounds in 1.0 cm diameter were made on both sides of rat dorsa (Fig 3A). The reduction of wound diameter of all groups appeared 3 days after wounding in different speeds. The wound contraction, tissue granulation and epidermal growth of the normal group were faster than those of the diabetic groups. Of the diabetic groups, wound healing efficiency of M.E. group was quicker than rhEGF-treated, vaseline-treated and diabetic blank group (Fig 3B–3D). As shown in Table 2 and Fig 3C, the wound healing rates of normal group (7th day: 47.6% ± 2.8%, 14th day: 91.7% ± 3.7%) were much higher than those of diabetic blank group (7th day: 21.6% ± 2.1%, 14th day: 43.3% ± 2.3%, P < 0.05). On the 7th and the 14th day, the wound healing rates of ME group were 38.2% ± 2.5% and 79.6% ± 4.2%, rhEGF group were 33.6% ± 3.1% and 71.0%±4.0%, and vaseline group were 22.8% ± 3.1% and 55.1%±5.0%, respectively. Statistical analyses showed that wound healing rate of M.E. group and rhEGF group were significantly higher (P < 0.05) than that of the vaseline group on the 7th and 14th day after wound modeling.

10.1371/journal.pone.0309903.g003 Fig 3 The effects of different treatments on wound healing at different time points.

A, A round skin wound was made on rat dorsa. B, The wound model rats were divided into 5 experimental groups and their wounds were locally treated by Vaseline, rhEGF gel (rhEGF) and maggot extract (M.E.), respectively. The wounds of normal rats (N) and the diabetic rats (N/D) without treatment were cited as blank controls. C, The wound healing rates of five experimental groups. D, Analogic diagram reflecting the progress of wound healing. The data are shown as mean ± SD, n = 10. *, P < 0.05.

10.1371/journal.pone.0309903.t002 Table 2 Average wound healing rates of five experimental groups at three time points.

Group	Wound healing rates (%, n = 10)	
Day 3	Day 7	Day 14	
Normal rats	25.7±1.9 *	47.6±2.8 *	91.7±3.7 *	
Untreated diabetic rats	9.9±2.4	21.6±2.1	43.3±2.3	
Vaseline-treated	10.2±3.6	22.8±3.1	55.1±5.0	
Maggot extract-treated	14.7±2.1#&	38.2±2.5#&	79.6±4.2#&	
rhEGF-treated	14.8±2.5#	33.6±3.1#	71.0±4.0#	
*, P < 0.05 in comparison with untreated diabetic group at the same time

#, P < 0.05 in comparison with Vaseline-treated group at the same time

&, P < 0.05 in comparison with normal group at the same time.

Active regeneration of maggot extract- and rhEGF-treated wounds

To investigate wound regeneration, re-epithelialization and granulation tissue formation were checked by HE and Masson’s trichrome staining performed on the whole layer skin wound tissues biopsied from each experimental group on the 14th day. As shown in Fig 4, a large number of infiltrating inflammatory cells, disordered tissue architecture, lack of granulation tissue structure, less neovascularization and incomplete re-epithelialization were found in the diabetic blank group and vaseline-dressed group. In contrast with the control groups, lesser inflammatory cell infiltration, dense granulation tissue structure, abundant collagen fibers as well as capillary formation were observed in the wound tissues of maggot extract-treated and rhEGF-treated groups.

10.1371/journal.pone.0309903.g004 Fig 4 The morphology analysis of wound healing in different experimental groups on the 14th day of the experiment.

A, Composite images of hematoxilyn/eosin-stained central wound sections of five experimental groups to show re-epithelialization (×50). Epithelial tongues are highlighted by dash line. Insets: the regions in higher magnification (×200). B, Masson’s trichrome staining for evaluating granulation tissue (gt) composition (×50). Red color: cytoplasm, muscle fibers; blue color: collagen fibers. Insets: the regions in higher magnification (×200). C and D, Quantitative analysis of re-epithelialization (percentage of distance covered by epidermis) and granulation tissue (percentage of collagen fibers in granulation tissue). *, P < 0.01; #, P > 0.05.

Differential STAT3 and its downstream gene expression in normal and diabetic rats

Immunohistochemical (Fig 5A) and immunofluorescent (Fig 5B) staining revealed that STAT3 protein as well as Bcl-2, Cyclin D1 and VEGF were widely distributed in regenerative wound tissues of normal rats, accompanied with frequent p-STAT3 nuclear translocation. In contrast, the expression intensities of the above parameters in untreated or vaseline-dressed wounds of diabetic rats were distinctly weak. The results of Western blotting were in accordance with that of IHC in terms of increased p-STAT3 level and Bcl-2, Cyclin D1 and VEGF expression in the wound tissues of normal rather than the diabetic rats (Fig 5C; P < 0.05).

10.1371/journal.pone.0309903.g005 Fig 5 Distinct STAT3 activation and its downstream gene expression patterns between normal and diabetic rat wound tissues.

Immunohistochemical staining (A) and immunofluorescent labeling (B) were performed on the wound tissues. Western blotting (C) was conducted on the sample proteins prepared from the wound tissues of normal (N) and diabetic rats without any treatments (N/D). The data are shown as mean ± SD. n = 10. *, P < 0.05.

Enhanced STAT3 activation in maggot extract- and rhEGF-treated tissues

Because recombinant epidermal growth factor (rhEGF) was commonly used in the clinical treatment of skin wound healing, the statuses of STAT3 signaling and Bcl-2, Cyclin D1 and VEGF expressions in the wounds treated with rhEGF were checked and compared with the results from maggot extract-treated wound tissues. It was found that the immunolabelings of p-STAT3, Bcl-2, Cyclin D1 and VEGF were strongly positive in the wound tissues of diabetic rats treated with maggot extract and rhEGF (Fig 6). Western blotting further demonstrated that Bcl-2, Cyclin D1 and VEGF levels in maggot extract-treated group were higher than those in the vaseline-treated and untreated group while in coherence with those of rhEGF-treated and the normal control group (Fig 6B; P < 0.05).

10.1371/journal.pone.0309903.g006 Fig 6 Enhanced STAT3 activation and upregulated VEGF, Cyclin D1 and Bcl-2 in maggot extract and rhEGF treated wound tissues.

Immunohistochemical stainings (A), Western blottings (B) and immunofluorescent labelings (C) were performed on the samples of the vaseline, maggot extract (M.E.) and rhEGF treated wounds of the diabetic rats. The data are shown as mean ± SD. n = 10. *, P < 0.05.

Discussion

Diabetes is a metabolic disease characterized by elevated blood glucose and its incidence keeps increasing [22]. Instances of chronic, hard-healing, or non-healing diabetic wounds and ulcers are predicted to increase, which seriously affects patients’ quality of life. Several standard treatments of diabetic wounds have been proposed, while the effects are limited in terms of accelerating the course of wound healing. For instance, becaplermin (platelet derived factor, PDGF) is the only drug approved by Food and Drug Administration (FDA), USA for the treatment of diabetic foot ulcer, but its efficacy is poor and usually increases the risk of death [23]. This therapeutic dilemma is largely due to the complexity of pathogenesis of diabetic wounds [24]. It would be of clinical significance to explore multipotent novel agent(s) that promotes the diabetic skin wound healing without causing side effects. To fulfill this purpose, we established rat diabetic model by streptozocin injection and made skin wounds on rat dorsa. The results of wound area calculation showed that the skin wound contraction of the untreated diabetic rats was significantly delayed compared with that of the untreated normal ones. The wounds of normal rats were almost healed (healing rates: 91.7% ± 3.7%), while the wounds of the corresponding diabetic group healed partly (healing rates: 43.3% ± 2.3%) on the 14th day. The significant delay of wound healing makes the diabetic rats a suitable model for experimental therapy and investigation of potential agent for better treatment of diabetic skin wounds.

Normally, wound healing involves hemostasis, proliferation and remodeling, while this stepwise process becomes disordered due to the lack of wound repair promoters in the diabetic skin [25]. Therefore, the biological effects of new agent should be multifaceted and maggot extract, as a mixture of natural products, is a potential candidate because of its richness in bioactive substances [26]. The roles of maggots in promoting wound healing have been recognized [27,28] and multiple bioactive substances were identified in its extract [9]. However, the traditional dry baking method reduces the components and especially the biological activity of maggot extract. We had therefore developed an unique method by which the maggot extract with high quality and high yield was prepared and its beneficial effects on the skin wound healing of normal adult rats were evidenced [11]. Even though, we proposed that the real clinical medicinal value of the extract is its effectiveness on refractory wounds such as skin injuries in diabetic patients. To address this issue, the influence of maggot extract in diabetic skin wound healing was elucidated by citing Vaseline as a conventional dressing control, rhEGF as the clinical treatment control and the untreated wounds of the normal and the diabetic rats as blank controls. The results showed that on the 14th day of treatments, the wound healing rates of maggot extract-treated (79.6%±4.2%) and rhEGF-treated group (71.0%±4.0%) were similar and faster than those of other groups. Because rhEGF has been commonly used to treat skin wounds, our results indicated that maggot extract may equivalently promote diabetic wound healing. This notion is supported by reduced local inflammation, active neovascularization and accelerated re-epithelization observed in maggot extract-treated wound regions. Further investigation of the underlying mechanism of maggot extract promoted wound healing can provide scientific basis for the practical use of this natural product to treat diabetic wounds.

It is known that trauma as a stimulator can trigger chain reactions of tissue remodeling [29]. In the skin, the re-epithelialization process includes the migration, proliferation and differentiation of keratinocytes, elimination of damaged tissues, and production of extracellular matrix. These events are mainly mediated by extracellular signal transduction pathways in which STAT3 signaling plays active roles [30] because some of its downstream target genes such as VEGF, CyclinD1 and Bcl-2 are highly expressed in the wound healing area and act as key factors to accelerate wound healing [13]. Our previous works demonstrated that the main mechanism of maggot extract-promoted wound healing of normal rat skin is to increase STAT3 signaling activity and then to upregulate the expression of the above-mentioned damage repair promoting genes [11]. Given this evidence, we speculate that maggot extract may also promote the healing of diabetic skin injury through similar cellular and molecular mechanisms. To address this issue, immunohistochemical staining and Western blot analyses were conducted to check the statuses of STAT3, p-STAT3, VEGF, CyclinD1 and Bcl-2 in the wound tissues of the experimental groups. The results showed that the nuclear translocation of p-STAT3 and VEGF, CyclinD1 and Bcl-2 production in skin wounds of normal rats were significantly higher than that of the diabetic blank and vaseline-treated tissues and this situation can be reversed, at least in large, by rhEGF as well as maggot extract. These results thus confirm the lack of wound repair promoting factors in diabetic skin tissue and suggest that both maggot extract and rhEGF promote diabetic wound healing via similar molecular route, i.e. the enhancement of STAT3 signaling transduction, because the interplay of EGF and STAT3 signaling has been well known [31].

The application of maggot and its extract in wound care keeps increasing and has been proved effective [32]. To the best of our knowledge, the comparison of experimental therapeutic efficacy of maggot extract with that of conventional wound care agent(s) is still lesser reported. Because EGF-containing ointment has been commonly used in clinical management of skin wounds and the data concerning its molecular effects on the wound tissues have been well documented, it was selected as the clinically relevant control. The results show that the promoting effects of maggot extract on wound healing of diabetic rats (79.6% ± 4.2%) is relatively better than that of rhEGF-treated group (71.0%±4.0%), indicating the presence of additional biological effects of maggot extract on the skin wounds beyond stimulating STAT3 activation. Elucidation of the influence(s) of pathway inhibitors such as STAT selective inhibitor AG490 in maggot extract-promoted wound healing of diabetic rat skin would further strengthen this notion.

Conclusions

The results of current study demonstrate the effectiveness of maggot extract in improving skin wound healing of diabetic rats via promoting the activity of STAT3 signaling and up-regulating the expression of STAT3 downstream target genes Bcl-2, Cyclin D1 and VEGF. In this context, maggot extract would be an ideal agent to resolve the complexity of skin wound healing of diabetic patients.

Supporting information

S1 Raw images (PDF)

S1 Raw data (PDF)

S2 Raw data (PDF)

S3 Raw data (PDF)

10.1371/journal.pone.0309903.r001
Decision Letter 0
Hafeez Kamran Sairah Academic Editor
© 2024 Sairah Hafeez Kamran
2024
Sairah Hafeez Kamran
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version0
21 May 2024

PONE-D-24-13961Maggot extract accelerates skin wound healing of diabetic rats via enhancing STAT3 signalingPLOS ONE

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Reviewer #1: Yes

Reviewer #2: Partly

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #2: No

**********

5. Review Comments to the Author

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Reviewer #1: Review Report:

The manuscript titled "Maggot extract accelerates skin wound healing of diabetic rats via enhancing STAT3 signaling" suggests an intriguing investigation into the potential therapeutic effects of maggot extract on wound healing, particularly in diabetic rats. The study likely delves into the mechanisms by which maggot extract may expedite the healing process, with a specific focus on its impact on STAT3 signaling pathways. The use of maggot extract for wound healing purposes represents a novel and potentially innovative approach, especially if the study provides evidence supporting its efficacy. Overall, the manuscript appears to present a promising study with implications for both basic research and clinical practice in the field of wound healing and diabetes management.

Note: I reviewed the file named (PONE-D-24-13961) downloaded from journal online portal and all my comments are according to the line numberings of the pdf file “PONE-D-24-13961”

Detailed Comments/Recommendations:

Title:

� The title of the study, "Maggot extract accelerates skin wound healing of diabetic rats via enhancing STAT3 signaling," appears to be apt and engaging. It succinctly captures the main focus of the research while also hinting at the potential mechanisms involved. This descriptive and informative title is likely to attract readers' attention and convey the significance of the study's findings.

Abstract:

� Background: Rewrite the starting line of the paragraph in a meaningful and in scientific representative way. The scientific name of Lucilia sericata must be italicized in the abstract and in everywhere of the manuscript.

� Methods: The dose of the streptozotocin must be mentioned. Similarly, it is not mentioned that how the wounds were made in rats. Rats groupings is confusing. Please elaborate the groups according to their treatment protocols. Kindly briefly describe the information about wound formation.

� Results: Rewrite the first line of this section and 14th, must be written in this way.

� Conclusion: In objective the authors claimed to explore the molecular mechanism of healing but the conclusion of the study in the last of abstract is not exactly supporting the aims and objective.

� Key Words: Authors used 4 out of 5 same words as mentioned in title. The key words should be different from the words used in title so that the reader may explore the study with different more focused words.

Note: The authors are requested to resolve the above mentioned points and rewrite the abstract in an appropriate comprehensive scientific language and give focused key words.

Introduction:

� There is no coordination in starting both paragraphs of the introduction. It will be more suitable if authors write the introduction in sequence in term of following points; introduction of diabetes, its types, complications, wounds pathogenesis, treatment regimens available, and treatment failures.

� Starting in 3rd paragraph of introduction, there is lacking about the introduction (family, genus, distribution, habitat etc) of Lucilia sericata.

� The last paragraph of Introduction is well written.

� Kindly add on the aims and objectives of the current study.

Materials and Methods:

Maggot Extract Preparation:

� Lucilia cuprina should be written in italic as Lucilia cuprina. In the Material and Method, subsection Maggot Extract Preparation, Lucilia cuprina is mentioned while in abstract and introduction Lucilia sericata is discussed.

� For extract preparation of Maggots, from where the larvae and its organism were identified? Kindly mention the details from where the organism was collected or obtained and give identification details as well.

� Kindly describe the standard conditions at which the blowflies were maintained.

� Kindly write full form of PBS.

� Kindly remove the space between µ m. it should be written as µm.

� rhEGF details are missing. From where it was obtained? Give specification and supplier details.

� What are the dressing reagents? Give elaboration at the end of subsection Skin Wound Model and Treatment.

� In subsection Wound Examination, the critera for wound healing and its markers etc are not mentioned.

� Wound edge biopsy: the procedure of taking biopsy is not mentioned.

Results:

� Establishment of diabetic rat model: “The typical symptoms of diabetes include the increased drinking, eating and urinating, which appeared to the rats on the third day after streptozotocin injection. As shown in Fig 2, the model rats but not the normal ones showed lost weight, and the fasting blood glucose levels of normal SD rats were less than 7.0 mmol/L, and in streptozotocin-treated rats were distinctly increased in the range from 25.6 to 27.3 mmol/L and 26.5 ± 0.8 mmol/L in average” This paragraph should be rewritten in correct meaningful way.

� Active regeneration of maggot extract- and rhEGF-treated wounds: Rewrite this sentence: “HE and Masson’s trichrome stainings were performed on the whole layer skin wound tissues biopsied from each experimental group on the 14th day to analyze re-epithelialization and granulation tissue formation”.

Discussion:

� Over all the discussion is well written and quite satisfactory.

� On the 14th day, should be written as; On the 14th day.

� It is written in discussion that; “The beneficial effects of maggots on wound healing have long been recognized[27,28] and its extract contains alkaloids, oils, polypeptides and proteins that promote local angiogenesis and cell proliferation [9]”. Please mention the name of any oil, alkaloid, polypeptide etc which has been isolated so from maggot and is used for wound healing.

� The authors are requested to check out all the in text citation that there must be space between text and citation. At most of places it is written as; recognized[27,28], It should be like that; recognized [27,28].

� There is lacking any scientific evidence about the key constituent of maggot responsible for the wound healing.

Conclusion:

� It should be rewritten by keeping in view the aims and objective of study and proceeding towards their achievement.

References:

� Reference no 31: Mol Cell Endocrinol. Why the authors write journal name in italic. Kindly remove the italics.

� The authors are advised to review all the references for the strict adherence of the uniformity of Journal’s references style.

Concluding Remarks:

The paper necessitates the aforementioned corrections, inclusive of rectifying any errors highlighted, coupled with a meticulous proofreading by a proficient language expert to refine its English language usage. Upon addressing these amendments, the paper will meet the required standards for acceptance.

Reviewer #2: The study is good contribution to the diabetic wound healing research and will help others once published.

I would suggest:

Enhance the overall quality of manuscript.

Better phrases can be used.

Abbreviations need to be explained.

Ensure soundness of data, results and their presentation.

A lot is needed to be done before making it to final publishing.

**********

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Reviewer #1: Yes: Dr. Aamir Mushtaq

Reviewer #2: No

**********

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Attachment Submitted filename: Review Comments May 24.docx

Attachment Submitted filename: PONE-D-24-13961.docx

10.1371/journal.pone.0309903.r002
Author response to Decision Letter 0
Submission Version1
29 Jun 2024

Rebuttal Letter

1. To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (1) methods of sacrifice, (2) efforts to alleviate suffering.

Response: Thanks. To alleviate suffering, the rats were first anesthetized with ketamine and xylazine intraperitonealy and then sacrificed through cervical dislocation performed by appropriately trained and competent personnel at the end of the experiment, which was described in Methods section and highlighted in red as “The rats were anesthetized and sacrificed through cervical dislocation performed by appropriately trained and competent personnel at the end of the experiment. The skin tissues in the size of 0.3 × 0.3 × 0.2 cm .……”

2. Please change the highlighted text in your manuscript with black text.

Response: Thanks. The highlighted text has been changed to black in the manuscript.

3. "We note that your Data Availability Statement is currently as follows: [All relevant data are within the manuscript and its Supporting Information files.]

Please confirm at this time whether or not your submission contains all raw data required to replicate the results of your study. Authors must share the “minimal data set” for their submission. PLOS defines the minimal data set to consist of the data required to replicate all study findings reported in the article, as well as related metadata and methods (https://journals.plos.org/plosone/s/data-availability#loc-minimal-data-set-definition).

For example, authors should submit the following data:

- The values behind the means, standard deviations and other measures reported;

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Response: According to the requirements above, we have uploaded the raw data named as S2 Raw data, S3 Raw data and S4 Raw data in Supporting Information files.

Reviewer #1: Review Report:

The manuscript titled "Maggot extract accelerates skin wound healing of diabetic rats via enhancing STAT3 signaling" suggests an intriguing investigation into the potential therapeutic effects of maggot extract on wound healing, particularly in diabetic rats. The study likely delves into the mechanisms by which maggot extract may expedite the healing process, with a specific focus on its impact on STAT3 signaling pathways. The use of maggot extract for wound healing purposes represents a novel and potentially innovative approach, especially if the study provides evidence supporting its efficacy. Overall, the manuscript appears to present a promising study with implications for both basic research and clinical practice in the field of wound healing and diabetes management.

Note: I reviewed the file named (PONE-D-24-13961) downloaded from journal online portal and all my comments are according to the line numberings of the pdf file “PONE-D-24-13961”

Detailed Comments/Recommendations:

Title: The title of the study, "Maggot extract accelerates skin wound healing of diabetic rats via enhancing STAT3 signaling," appears to be apt and engaging. It succinctly captures the main focus of the research while also hinting at the potential mechanisms involved. This descriptive and informative title is likely to attract readers' attention and convey the significance of the study's findings.

Abstract: Background: Rewrite the starting line of the paragraph in a meaningful and in scientific representative way. The scientific name of Lucilia sericata must be italicized in the abstract and in everywhere of the manuscript.

Response: Thank you for your suggestion. We have rewritten the starting line of the “Background” paragraph and highlighted in red in ABSTRACT section as: Diabetic skin wound is a complex problem due to the disruption of normal repairing program and lack of effective remedy. The scientific name of Lucilia sericata have been corrected as italicized in the abstract and in everywhere of the manuscript.

Methods: The dose of the streptozotocin must be mentioned. Similarly, it is not mentioned that how the wounds were made in rats. Rats groupings is confusing. Please elaborate the groups according to their treatment protocols. Kindly briefly describe the information about wound formation.

Response:

1) According to your suggestion, the dose of the streptozotocin has been added in the “Methods” paragraph and highlighted in red as: The rat fasting blood glucose values were ≧16.7 mmol/L 72 hours after intraperitoneal streptozotocin (60 mg/kg body weight) injection.

2) The groups in the “Methods” paragraph and highlighted in red as: The rats were divided into five groups (n = 10/group): normal group (normal SD rats without any treatment), diabetic blank group (the diabetic rats without any treatment), Vaseline group (the diabetic rats dressed with Vaseline), rhEGF (recombinant human epidermal-growth-factor) group (the diabetic rats dressed with a mixture of Vaseline and 200 μg/g rhEGF), M.E. group (the diabetic rats dressed with a mixture of Vaseline and 150 μg/ml maggot extract) .

3) The information about wound formation has been described and highlighted in red in the “Methods” paragraph as: The round open wounds (1.0 cm in diameter) down to the muscle fascia were made on both sides of rat dorsa by removing the skin layer (epidermis and dermis) and were daily photographed for calculating their healing rates.

Results: Rewrite the first line of this section and 14th, must be written in this way.

Response: The first line of this section was rewritten as “The average wound healing rates on the 14th day were 91.7 % in the normal, 79.6% in M.E., 71% in rhEGF, 55.1% in vaseline and 43.3% in the diabetes blank group”, which was highlighted in red.

Conclusion: In objective the authors claimed to explore the molecular mechanism of healing but the conclusion of the study in the last of abstract is not exactly supporting the aims and objective.

Response: Thank you for this valuable suggestion. The “Conclusion” has been rewritten as “Maggot extract would be an alternative and/or adjuvant candidate for the better management of diabetic skin wounds because of its activity in enhancing STAT3 activation.”

Key Words: Authors used 4 out of 5 same words as mentioned in title. The key words should be different from the words used in title so that the reader may explore the study with different more focused words.

Response: According to your advice, Key Words have been changed to “Lucilia sericata larvae extract, diabetic skin wound, streptozotocin, signal transducer and activator of transcription 3, wound healing related genes”.

Note: The authors are requested to resolve the above mentioned points and rewrite the abstract in an appropriate comprehensive scientific language and give focused key words.

Introduction:

� There is no coordination in starting both paragraphs of the introduction. It will be more suitable if authors write the introduction in sequence in term of following points; introduction of diabetes, its types, complications, wounds pathogenesis, treatment regimens available, and treatment failures.

� Response: According to your comment. we have integrated starting both paragraphs of the introduction into one paragraph in the order of introduction of diabetes, its types, complications, wounds pathogenesis, treatment regimens available, and treatment failures, which is shown in Introduction part of text in red: “Diabetes is a chronic metabolic disease, which has become a growing international health concern [1, 2]. Diabetes (type 1 and 2) involves many complications, and the most common one is the delayed healing and even nonunion of acute and chronic skin wounds [3]. Multiple factors or molecular machinaries responsible for wound healing are altered in diabetic individuals, leading to the reduced regenerative activity of skin wounds. It has been proposed that the local peripheral nerve and vascular alterations are the main reasons for the difficulty of skin wound healing [4]. Current options for wound management of diabetic patients are: 1) strict control of blood glucose; 2) application of vasodilators to improve microvascular circulation; 3) antibacterial and anti-inflammatory treatments; 4) surgical debridement and 5) local nursing and health care [5]. Although the above measures include both the whole and local treatments, the incidence of diabetic wounds and the disability rate caused by them remain almost unchanged, indicating the unsatisfactory efficacy of the above approaches [6]. Therefore, it would be necessary to alleviate the influence of pathogenic factors and meanwhile to accelerate the wound healing. Because of the complexity of the pathogenic factors of diabetic wounds, the new agent (drug) containing beneficial components for neovascularization and cell proliferation would be helpful.”

Starting in 3rd paragraph of introduction, there is lacking about the introduction (family, genus, distribution, habitat etc) of Lucilia sericata.

� Response: According to your suggestion, the introduction of Lucilia sericata have been added in the “Introduction” part and highlighted in red as: “Lucilia sericata (Meigen 1826, Diptera: Calliphoridae) is a widespread blow fly species.........”.

The last paragraph of Introduction is well written.

Kindly add on the aims and objectives of the current study.

Response: Thank you for your comments. the aims and objectives of the current study have been added at the end of the “Introduction” part and highlighted in red as: “The current study aims to address the therapeutic effects of maggot extract on diabetic skin wound and its molecular mechanism.”

Materials and Methods:

Maggot Extract Preparation:

� Lucilia cuprina should be written in italic as Lucilia cuprina. In the Material and Method, subsection Maggot Extract Preparation, Lucilia cuprina is mentioned while in abstract and introduction Lucilia sericata is discussed.

� Response: Thank you for your comments. Lucilia cuprina have been corrected to Lucilia sericata.

For extract preparation of Maggots, from where the larvae and its organism were identified? Kindly mention the details from where the organism was collected or obtained and give identification details as well.

Kindly describe the standard conditions at which the blowflies were maintained.

Response: Thank you for your comments. The related information have been added in the text as “Lucilia sericata blowflies were obtained from BIOWIM Technology Development Co., Ltd (Dalian, China) and maintained in a closed container with a net at a constant temperature of about 22℃. Their larvae were collected when they were reared to later-second or early-third stages. The collected maggots were rinsed three times with autoclaved pure water, placed in 3.5% formaldehyde normal saline solution for 5 minutes, in 2% H2O2 for 3 minutes, in 1% hydrochloride acid for 5 minutes and finally subjected to three washes with autoclaved pure water. ”

�

Kindly write full form of PBS.

Response: Thank you for your comments. The full form of PBS is phosphate buffered saline, which has been added in the text as “ ……putting the frozen sections into the test tubes containing pH7.5 phosphate buffered saline (PBS). ”

Kindly remove the space between µ m. it should be written as µm.

Response: Thank you for your careful reading. The typo has been corrected.

rhEGF details are missing. From where it was obtained? Give specification and supplier details.

What are the dressing reagents? Give elaboration at the end of subsection Skin Wound Model and Treatment.

Response: Thanks for your comments. rhEGF was purchased from Guilin Pavay Gene Pharmaceutical Co., Ltd (Guilin, China). rhEGF details and the dressing reagents have been added to the text as “ ……Group 4, diabetic rats dressed with a mixture of Vaseline and 200 μg/g recombinant epidermal growth factor (Pavay Gene Pharmaceutical, Guilin, China, rhEGF group); Group 5, the diabetic rats dressed with a mixture of Vaseline and 150 μg/ml maggot extract (M.E. group). The experimental treatments lasted for 14 days by daily dressing the reagents mentioned above. ”

� In subsection Wound Examination, the critera for wound healing and its markers etc are not mentioned.

Response: Thanks for your suggestion. The way to evaluate wound healing has been described in the text as “The wound areas were measured and the wound healing rates were calculated to evaluate the wound healing in differen experimental groups.”

Wound edge biopsy: the procedure of taking biopsy is not mentioned.

Response: Thanks for your suggestions. The procedure of taking biopsy has been described in “Wound edge biopsy” subsection as “The rats were anesthetized and sacrificed through cervical dislocation performed by appropriately trained and competent personnel at the end of the experiment. The skin tissues in the size of 0.3 × 0.3 × 0.2 cm were biopsied from the wound margins of the five experimental groups. Half of the tissues was snap-frozen in liquid nitrogen……”

Results:

� Establishment of diabetic rat model: “The typical symptoms of diabetes include the increased drinking, eating and urinating, which appeared to the rats on the third day after streptozotocin injection. As shown in Fig 2, the model rats but not the normal ones showed lost weight, and the fasting blood glucose levels of normal SD rats were less than 7.0 mmol/L, and in streptozotocin-treated rats were distinctly increased in the range from 25.6 to 27.3 mmol/L and 26.5 ± 0.8 mmol/L in average” This paragraph should be rewritten in correct meaningful way.

Response: According to your suggestion, this paragraph has been rewritten in the text as “The typical symptoms of diabetes include polydipsia, polyphagia and polyuria, which was observed in rats on the third day after streptozotocin injection. As shown in Fig 2, the diabetic rats but not the normal ones showed weight loss, whereas the fasting blood glucose levels of normal rats were less than 7.0 mmol/L, and in streptozotocin-treated rats were distinctly increased (ranging from 25.6 to 27.3 mmol/L). The difference of fasting blood glucose levels between the normal and streptozotocin-treated rats was statistically significant (P < 0.01; Table 1).”

Active regeneration of maggot extract- and rhEGF-treated wounds: Rewrite this sentence: “HE and Masson’s trichrome stainings were performed on the whole layer skin wound tissues biopsied from each experimental group on the 14th day to analyze re-epithelialization and granulation tissue formation”.

Response: Thanks for your suggestion. This sentence has been rewritten in the text as “To investigate wound regeneration, re-epithelialization and granulation tissue formation were checked by HE and Masson’s trichrome staining performed on the whole layer skin wound tissues biopsied from each experimental group on the 14th day. ”

Discussion:

Over all the discussion is well written and quite satisfactory.

On the 14th day, should be written as; On the 14th day.

Response: Thanks for your comments. The sentence has been corrected in the text as “The wounds of normal rats were almost healed (healing rates: 91.7% ± 3.7%), while the wounds of the corresponding diabetic group healed partly (healing rates: 43.3% ± 2.3

Attachment Submitted filename: Response to reviewers.docx

10.1371/journal.pone.0309903.r003
Decision Letter 1
Hafeez Kamran Sairah Academic Editor
© 2024 Sairah Hafeez Kamran
2024
Sairah Hafeez Kamran
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version1
31 Jul 2024

PONE-D-24-13961R1Maggot extract accelerates skin wound healing of diabetic rats via enhancing STAT3 signalingPLOS ONE

Dear Dr. Liu,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Kind regards,

Sairah Hafeez Kamran, PhD

Academic Editor

PLOS ONE

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Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

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Comments to the Author

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Reviewer #1: All comments have been addressed

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #3: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: (No Response)

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #3: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #3: (No Response)

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The revised version of paper entitled "The manuscript titled "Maggot extract accelerates skin wound healing of diabetic rats via enhancing STAT3 signaling" has been reviewed by me and it has been found that the authors have resolved all the mentioned technical issues. However, at certain points there are few English language related errors. For the well balanced presentation of the paper i would like to advise the authors to get this paper proof read by a proficient language expert to refine its English language usage. From me side it is accepted for the publication as the authors have successfully addressed all of my comments.

Reviewer #3: I greatly appreciate considering me as a reviewer for the manuscript PONE-D-24-13961R1.

1) In the section of Skin wound model and treatments, author should add reference that illustrate using vaseline as dressing for diabetic wound.

2) All figures resolution should be improved.

3) Quantity of Maggot extract should be mentioned

**********

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Reviewer #1: Yes: Dr. Aamir Mushtaq

Reviewer #3: No

**********

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10.1371/journal.pone.0309903.r004
Author response to Decision Letter 1
Submission Version2
13 Aug 2024

Rebuttal Letter

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Response: According to the requirements above, we have checked carefully all the references one by one to ensure that each is complete and correct and have not cited any papers that have been retracted.

Reviewer #1: The revised version of paper entitled "The manuscript titled "Maggot extract accelerates skin wound healing of diabetic rats via enhancing STAT3 signaling" has been reviewed by me and it has been found that the authors have resolved all the mentioned technical issues. However, at certain points there are few English language related errors. For the well balanced presentation of the paper i would like to advise the authors to get this paper proof read by a proficient language expert to refine its English language usage. From me side it is accepted for the publication as the authors have successfully addressed all of my comments.

Response: Thanks for your comments. We have got this paper read carefully and corrected the grammatical errors and typos found in the secondly revised manuscript.

Reviewer #3: I greatly appreciate considering me as a reviewer for the manuscript PONE-D-24-13961R1.

1) In the section of Skin wound model and treatments, author should add reference that illustrate using vaseline as dressing for diabetic wound.

Response: Thanks for your suggestion. We have added the reference that illustrate using vaseline as dressing for diabetic wound in the section of Skin wound model and treatments and highlighted it in red as “……Group 3, diabetic rats dressed with Vaseline (Rhawn, Shanghai, China, Vaseline group) [11]; Group 4, diabetic rats dressed with a mixture of Vaseline [11] and 200 μg/g recombinant epidermal growth factor (Pavay Gene Pharmaceutical, Guilin, China, rhEGF group); Group 5, the diabetic rats dressed with a mixture of Vaseline [11] and 150 μg/ml maggot extract (M.E. group)” .

2) All figures resolution should be improved.

Response: Yes. We have improved the resolution of all figures.

3) Quantity of Maggot extract should be mentioned.

Response: Thank you for your suggestion. The quantity of maggot extract and the quantitative method have been added in the section of Skin wound model and treatments and highlighted in red as “……Group 5, the diabetic rats dressed with a mixture of Vaseline [11] and 150 μg/ml maggot extract (M.E. group). The experimental treatments lasted for 14 days by daily dressing the reagents mentioned above. Maggot extract was quantified by Ultraviolet spectrophotometer-based calculation of protein concentration. ”

Attachment Submitted filename: Response to reviewers.docx

10.1371/journal.pone.0309903.r005
Decision Letter 2
Hafeez Kamran Sairah Academic Editor
© 2024 Sairah Hafeez Kamran
2024
Sairah Hafeez Kamran
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version2
21 Aug 2024

Maggot extract accelerates skin wound healing of diabetic rats via enhancing STAT3 signaling

PONE-D-24-13961R2

Dear Dr. Liu,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Sairah Hafeez Kamran, PhD

Academic Editor

PLOS ONE

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #3: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: (No Response)

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #3: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #3: (No Response)

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #3: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #3: No

**********

10.1371/journal.pone.0309903.r006
Acceptance letter
Hafeez Kamran Sairah Academic Editor
© 2024 Sairah Hafeez Kamran
2024
Sairah Hafeez Kamran
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
27 Aug 2024

PONE-D-24-13961R2

PLOS ONE

Dear Dr. Liu,

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team.

At this stage, our production department will prepare your paper for publication. This includes ensuring the following:

* All references, tables, and figures are properly cited

* All relevant supporting information is included in the manuscript submission,

* There are no issues that prevent the paper from being properly typeset

If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps.

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Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Sairah Hafeez Kamran

Academic Editor

PLOS ONE
==== Refs
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