
==== Front
Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

39294294
72593
10.1038/s41598-024-72593-8
Article
Cell-free adipose tissue extracts as a novel treatment for rosacea by downregulating TRPV1
Zhou Liuyi 1
Chen Lulu 12
Li Ting 1
Wang Lu 1
Lin Shiqi 12
Zhao Ye 1
Wu Sufan sufanwu@163.com

1
Jin Tingting tingtingjin@bjmu.edu.cn

1
1 Center for Plastic & Reconstructive Surgery, Department of Plastic & Reconstructive Surgery, Zhejiang Provincial People’s Hospital, Affiliated People’s Hospital, Hangzhou Medical College, Hangzhou, Zhejiang, China
2 https://ror.org/04epb4p87 grid.268505.c 0000 0000 8744 8924 The Second Clinical Medical College, Zhejiang Chinese Medical University, Hangzhou, China
18 9 2024
18 9 2024
2024
14 2175926 4 2024
9 9 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Rosacea is a chronic inflammatory skin disease that typically affects the central facial area. Its main clinical symptoms include paroxysmal flushing, telangiectasia, and non-temporary erythema. Cell-free adipose tissue extracts (ATEs) are liquid components extracted from human adipose tissue that contain large amounts of growth factors. Despite the scar-reducing, anti-aging, and wound-healing effects of ATEs, the efficacy of ATEs in rosacea remains unknown. Therefore, the anti-rosacea effects of ATEs were investigated in human cathelicidin peptide (LL-37) induced rosacea mice and capsaicin (CAP)-stimulated HaCaT keratinocytes. In vitro, ATEs significantly reduced TRPV1 expression, intracellular calcium ions influx and the release of inflammatory factors (such as KLK5, IL-6, IL-8 and TNF-α) after intervening in CAP-stimulated cells. The in vivo results revealed that ATEs alleviated rosacea symptoms, such as erythema score, erythema area, transepidermal water loss, abnormal epidermal thickness, mast cell infiltration and telangiectasia upon downregulating TRPV1 and CD31 expression. Moreover, the up-regulated TRPV1 protein expression was also recovered by ATEs administration in vivo and in vitro. Meanwhile, ATEs demonstrated good biocompatibility. In summary, ATEs could be a potential therapeutic agent for rosacea by regulating inflammation and alleviating telangiectasia.

Keywords

Rosacea
Cell-free adipose tissue extracts
TRPV1
Inflammation
Subject terms

Cell biology
Skin diseases
Natural Science Foundation of China82305040 Li Ting Zhejiang Provincial Health Science and Technology Program202466176 2024660434 Li Ting Jin Tingting Hangzhou Medical College basic research fund basic research programKYYB2023020 Li Ting issue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

Rosacea is a chronic inflammatory facial skin disorder characterized by erythema, inflammatory papules, rash, telangiectasia, pustules, and ocular manifestations, which seriously affects the quality of people’s lives1. The etiology of rosacea appears to involve a complex interplay of factors, including neurogenic neurovascular dysregulation and dysregulation of immune (innate, adaptive, inflammasome), etc2,3. Existing treatment options primarily focus on symptom management via metronidazole, azelaic acid, and ivermectin, with limited success in achieving long-term disease control4. Although these medications effectively reduce erythema and inflammatory lesions, they have little effect on vascular dilation. They may cause side effects such as local skin dryness, itching, burning, and stinging, and excessive use may even worsen facial skin barrier damage2,3.

Recent studies have reported the potential role of transient receptor potential (TRP) in the pathogenesis of rosacea5,6. TRP is a family of non-selective cation channels permeable to Ca2+, which is expressed in sensory neurons throughout the body, including the skin7. TRP plays a crucial role in various physiological processes such as thermoregulation, nociception, and inflammation8. Interestingly, research has shown that activation of transient receptor potential vanilloid 1 (TRPV1), a subfamily of TRP, contributes to the characteristic symptoms of rosacea5,9. Recently, several studies have shown increased expression of TRPV1 in the facial skin of patients with rosacea compared to healthy controls10. Additionally, TRPV1 can be activated by various stimuli, such as heat, capsaicin (CAP), mustard oil, and acidic environments, thereby leading to the release of mediators, inducing inflammation and vasodilation9,11. Growing evidence indicates that TRPV1 may be a viable therapeutic target for rosacea. However, current TRPV1 antagonists are limited by their poor skin permeability, systemic side effects, and insufficient efficacy12,13. Therefore, innovative therapeutic strategies should be developed to down-regulate TRPV1 expression to treat rosacea.

Cell-based therapy represents a promising approach for the treatment of inflammatory conditions. Among these, adipose-derived stem cells (ASCs) have been shown to possess immunomodulatory and regenerative properties14. ASCs have been shown to significantly improve scleroderma, wound healing, and chronic pressure ulcers15,16. However, isolating, culturing, and expanding ASCs is a complex and time-consuming process. The adipose tissue is an abundant source of ASCs and secretes large amounts of basic fibroblast growth factor (b-FGF), vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF), etc., which promote angiogenesis and adipogenesis17,18. The adipose tissue is inherently enriched with a variety of bioactive factors that can be directly isolated for clinical application, circumventing the need for cell isolation or cultivation. Cell-free adipose tissue extracts (ATEs) refer to the purified liquid fraction and are derived from adipose tissue using a mechanical approach to remove cellular components and lipid remnants19. This new kind of autologous extract was first developed in 2018. Previous studies have confirmed that ATEs contain a variety of growth factors, including brain-derived neurotrophic factor (BDNF), transforming growth factor-β (TGF-β), hepatocyte growth factor (HGF), b-FGF, PDGF, epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1) and neurotrophic factor (NT-3), contributing positive therapeutic effects of ATEs on skin repair19–21. ATEs have been reported to possess anti-inflammatory, antioxidant, and anti-bacterial properties, as evidenced by multiple pre-clinical studies on tissue regeneration, inflammatory conditions, and wound healing22–24. Additionally, ATEs possess advantageous characteristics in clinical applications, as follows: (1) they are cell-free, have good biocompatibility, and are non-immunogenic; (2) they are derived from the body with controllable quality; (3) they are simple and safe to prepare, with the availability of long-term storage at low temperatures for consecutive applications; (4) they are rich in growth factors similar to ASCs and have the potential to replace ASCs. Thus, ATEs represent a promising avenue for future clinical applications in skin diseases25–27.

Considering the aforementioned advantages of ATEs, this study investigated the potential of ATEs as a therapeutic strategy for rosacea. ATEs were hypothesized to alleviate the inflammatory and vasodilatory processes associated with rosacea. Therefore, the anti-rosacea effects of ATEs were evaluated in vitro and in vivo, and the underlying mechanisms were explored. This study aimed to provide a promising therapeutic modality for the treatment of rosacea by elucidating the mechanisms underlying the regulatory effects of ATEs on TRPV1.

Materials and methods

ATEs preparation

This study performed in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Zhejiang Provincial People’s Hospital (Affiliated People’s Hospital, Hangzhou Medical College). Written informed consent was obtained from each donor. All experiments were performed in accordance with relevant guidelines and regulations.

ATEs were extracted from human fresh fat tissue as described in a previous paper19. With the informed consent of two healthy female patients who underwent liposuction between March 2023 and June 2023 at Zhejiang Provincial People’s Hospital, Hangzhou, China, the fresh fat tissue was obtained after liposuction. Briefly, the lipoaspirate was washed with normal saline and centrifuged at 1200 g for 3 min to remove oil and blood cells. After centrifugation, the upper oil layer and bottom fluid layer were removed, and the median fat layer was collected and mechanically emulsified with high-speed blender for 2 min. The emulsified fat was centrifuged at 1200 g for 5 min and filtered through a 0.22 μm filter (Corning Glass Works, Corning, NY). The final ATEs were obtained and stored at −20 ℃ for further analysis. To determine the protein concentration of ATEs, the BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China) was employed.

Cell culture and cell viability assay

HaCaT were purchased from Shanghai Cell Bank of Chinese Academy of Sciences (SCSP-5091). HaCaT cells were cultured in high-DMEM supplemented with 10% fetal bovine serum (FBS, Dalian Meilun Biotechnology Co., Ltd., China), penicillin (100 U/ml), and streptomycin (100 U/ml) and maintained at 37 ℃ with 5% CO2.

Biocompatibility of ATEs

The biocompatibility of ATEs was evaluated by Live/Dead staining at 24 h. HaCaT cells were treated with different protein concentration of ATEs (50 μg/ml, 200 μg/ml, 350 μg/ml), and the concentration of capsaicin (10 μM, 20 μM, 30 μM) and capsazepine (10 μM, 20 μM, 30 μM). Cells were seeded in the plate and cultured at 37 ℃ for 24 h. After the supernatant was aspirated, 1 ml Calcein AM/PI reagent (Calcein AM: PI: PBS = 1 μl: 1 μl: 1 ml, Beyotime Biotechnology, Shanghai, China) was added to each well of the plate. After incubation for 30 min, the images of cell staining were captured by fluorescence microscopy (EVOS M7000, Thermo Fisher, USA) and analyzed by image J software (Media Cybernetics, Bethesda, MD, USA).

Western blot analysis

Total proteins were harvested from the dorsal skin samples or HaCaT cells using radioimmunoprecipitation assay (RIPA) buffer with proteinase inhibitor cocktail for 30 min on ice, and the protein was quantified by BCA protein assay. The targeted protein was separated by 8–12% SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was blocked with 5% nonfat milk for 2 h, which was followed by overnight incubation at 4 ℃ with the primary antibodies (anti-β-actin or anti-TRPV1, 1:1000 dilution). Following incubation with peroxidase-conjugated goat antirabbit/mouse IgG at 4 ℃ for 2 h, each protein was visualized using Western Lightning Plus ECL, densitometry was performed using Bio-Rad Quantity One software (V6.1.0, Bio-Rad Laboratories, Inc., USA).

Measurement of the intracellular Ca2+ concentration

Cells were plated on 6-well plates at a density of 105 cells/well in 2 ml medium. HaCaT cells were treated with CPZ (10 μM), or CPZ (10 μM) plus CAP (20 μM), or ATEs (50 μg/ml), or ATEs (50 μg/ml) plus CAP (20 μM). and cultured at 37℃ for 24 h. Intracellular Ca2+ concentration was measured using a Ca2+ probe, Fluo-4AM (S1060, Beyotime Biotechnology, Shanghai, China). After the supernatant was aspirated and cells were washed with Hank’s Balanced Salt Solution (HBSS, H1046, Solarbio Science & Technology Co., Ltd., Beijing, China) three times, cells were incubated with Ca2+ probe (Fluo-4AM, 3 μM) for 30 min, followed by HBSS washing three times. Finally, cells with fluorescent probes were visualized under a fluorescence microscope, and the immunoreactivity was quantified by Image J software.

Flow cytometry

Cells with fluorescent probes were suspended in HBSS and washed three times, then they were filtered through a 70 μm filter (Corning Glass Works, Corning, NY), and run through a BD Accuri™ C6 Plus Flow Cytometer (BD Biosciences, USA). Data were analyzed with the FlowJo software (V10).

Enzyme-linked immunosorbent assay (ELISA)

The concentrations of serum KLK5, IL-6, IL-8 and TNF-α were measured by human ELISA kit (U96-1917E, U96-1510E, U96-1513E, U96-3716E, Yobibio, Shanghai, China). All the procedures were performed following the manufacturer’s instructions.

Animal experiments

This study was carried out in compliance with the ARRIVE (Animal Research: Reporting of In vivo Animal Experiments) guidelines. Six-week-old female BALB/c mice were purchased from Shanghai Slack Company (animal production license number: SCXK: 2022–0004, Shanghai, China). All experiments on the mice were approved by the Laboratory Animal Management and Ethics Committee of Zhejiang Provincial People’s Hospital (Approval number: SYXK: 2019‐0013, Zhejiang, China). All experiments were performed in accordance with relevant guidelines and regulations.

After acclimation for 1 week, their dorsal hairs were removed using an electric shaver, and they were randomly assigned to one of five groups (n = 3 per group, total 15): (1) a baseline-applied normal group (NC), (2) normal saline control group, (3) CAP, (positive control), (4) CPZ group, and (5) ATEs group. To induce rosacea-like skin lesions, the shaved dorsal area was treated with 200 μL LL-37 (Shanghai Aladdin Biochemical Tech Co., Ltd, China) by local intradermal injection once a day for 3 days. On the first day of rosacea induction, the mice were injected intradermally with normal saline, CAP, or CPZ, or ATEs in accordance with the conditions of each group, 30 min after LL-37 treatment. The experimental scheme is summarized in Fig. 1A. Animals are allowed to move, eat and drink freely during modeling without anesthesia or other treatment. At the end of study (Day 8), all the mice were sacrificed with an overdose of sodium pentobarbital (200 mg/kg; i.p.) and the pieces of skin from the back were taken for histological and biochemical examination.Fig.1 Biocompatibility of CAP, CPZ, and ATEs and regulation of their transient receptor potential vanilloid 1 (TRPV 1) expression on HaCaT keratinocytes. (A) Live and dead staining for CAP, CPZ and ATEs. Scale bar = 275 μm. (B) Quantitative analysis for live and dead stains and cell viability is represented as the total ratio of live cells to live and dead cells. (C, D) Total proteins were prepared and analyzed for TRPV1 by Western blot. β-Actin was used as internal control. The protein levels were quantified by band density in proportion to the expression of the total form. All the above data are expressed as mean ± SD. *p < 0.05 or **p < 0.01 or ***p < 0.001 vs. NC group by one-way ANOVA followed by LSD multiple comparison. The experiments were repeated three times to ensure the accuracy.

Measurement of erythema score

The erythema score was utilized to assess the severity of LL-37 induced rosacea in mice, incorporating symptoms such as scarring, dryness, erythema and hemorrhage as primary indicators28. And it was scored as 0 (none), 1 (mild; being faint erythema), 2 (moderate; light erythema), 3 (severe; clear and distinct erythema) and 4 (extremely severe; dark erythema with clear borders). This evaluation was conducted once a day until the end of experiment.

Trans‑epidermal water loss (TEWL)

To investigate the effect on dorsal skin dryness, the mice were assessed the value of TEWL using gpskin barrier light (GPower, Seoul, Republic of Korea). TEWL was measured at the center of the shaved dorsum area of mouse under specific conditions at approximately 21 ± 2 ℃ and 50–55% humidity. The data were measured once the TEWL readings reached a state of equilibrium, approximately 30 s post the probe’s placement on the skin. The statistical value was shown in terms of fold change by comparison with the control group and subsequently represented in units of g/m2/hours. The TEWL was assessed every day along with clinical symptoms.

Histological analysis

At the dissection day, all mice skin was fixed with 10% formalin for 24 h at room temperature. Then each sample was embedded in paraffin and sectioned into 4 μm, followed by H&E staining and Toluidine Blue Staining. The images of stained sections were captured under topical microscopy and analyzed by Image J software. In immunohistochemical (IHC) staining, the specimens were incubated in rabbit monoclonal anti-CD31 antibody (ab182981; Abcam, Cambridge, UK) overnight at 4 ℃. HRP-labeled polymer anti-rabbit antibody (Gene Tech, Shanghai, China) was then added for 30 min at 37 ℃. A 3,3’-diaminobenzidine substrate kit (Gene Tech, Shanghai, China) was used to visualize the reaction. Unstained replicates of the sections were incubated overnight at 4 ℃ with PBS-diluted primary antibody TRPV1 (ab305299; Abcam, Cambridge, UK) for Immunofluorescence. After PBS wash, the sections were incubated with Horseradish peroxidase (HRP) conducted secondary antibodies for 1 h at room temperature, followed by DAPI Staining Solution for 5 min. The sections were visualized under a fluorescence microscope. The immunoreactivity of TRPV1 was quantified by Image J software.

Statistical analysis

All data were analyzed using IBM SPSS Statistics software (V22, IBM Corporation, USA) and expressed as mean values ± standard deviation of triplicate experiments. Data from different groups were compared using one-way ANOVA or non-parametric tests. A P-value < 0.05 was considered as a significant difference, and a P-value < 0.01 was considered to indicate a very significant difference.

Results

Effect of CAP, CPZ, and ATEs on HaCaT keratinocytes

To evaluate the biocompatibility and determine the optimal dosage for further experiments, the effects of capsaicin (CAP), capsazepine (CPZ), and ATEs were assessed on HaCaT keratinocytes using the Live/Dead staining. As shown in Fig. 1A,B, 30 μM of CAP, 20 μM of CPZ, and 30 μM of CPZ significantly reduced the cell viability (all p < 0.001). In contrast, ATEs exerted no cytotoxic effects at the treated concentrations. Based on the results, the concentrations of 10 μM CAP, 20 μM CAP, 10 μM CPZ, and 50 μg/ml ATEs, 200 μg/ml ATEs, and 350 μg/ml ATEs were selected for further experiments. Capsaicin is the active component in chili peppers and is a well-known activator of the transient receptor potential vanilloid 1 (TRPV1) receptor11. In contrast to capsaicin, capsazepine acts as a TRPV1 antagonist, binding to the same receptor site as capsaicin but preventing the activation of the channel29. Notably, the effects of capsaicin and capsazepine may vary depending on the cell type and the specific experimental conditions. Currently, the role of ATEs with respect to TRPV1 remains unclear. To verify the effects on TRPV1, Western blot analysis was performed using the selected drug concentrations. Compared with the NC group, the protein expression of TRPV1 was most significantly upregulated at 20 μM of CAP, whereas 10 μM CPZ inhibited the upregulation of TRPV1 induced by CAP (all p < 0.001) (Fig. 1C,D). The efficacy of the drugs was not affected by the time interval (p > 0.05, simultaneous addition of CAP and CPZ vs. CPZ added 1 h after CAP administration). Regarding ATEs, TRPV1 protein expression was significantly down-regulated following treatment with 50 μg/ml of ATEs (all p < 0.001), indicating that ATEs may act as antagonists. Hence, 20 µM of CAP, 10 µM of CPZ, and 50 μg/ml of ATEs were used in all subsequent studies, unless otherwise specified. These concentrations were selected as they exhibited good cell viability while ensuring sufficient potency for targeted signaling pathway analysis.

ATEs regulate the expression of TRPV1 and proinflammatory mediators in HaCaT keratinocytes

Upregulation of TRPV1 represents a pivotal aspect of rosacea pathogenesis, and CAP serves as an agonist of TRPV130,31. Therefore, CAP was employed to induce up-regulation of the TRPV1 pathway to simulate rosacea pathology. The effects of ATEs on the protein expression of HaCaT cells and cellular calcium inward flow under CAP stimulation were examined. As shown in Fig. 2A,B, the protein expression of TRPV1 was significantly downregulated in the CPZ group and ATEs group compared with the NC group (all p < 0.001). Moreover, treatment of HaCaT cells with CPZ or ATEs also significantly reduced the protein levels of TRPV1 induced by CAP (all p < 0.001 vs. CAP). No statistically significant difference was observed between the CPZ and ATEs in altering the effects of CAP induction (p > 0.05, CPZ + CAP vs. ATEs + CAP). TRPV1 is a non-selective cation channel that is highly permeable to calcium ions9. Activation of TRPV1 can lead to calcium influx, which in turn induces the release of inflammatory mediators32. Calcium fluorescent probe loading and flow cytometry were performed on the treated HaCaT keratinocytes, revealing a significant increase in fluorescence intensity compared to the NC group, whereas CPZ or ATEs showed a significant decrease (all p < 0.001) (Fig. 2C,E). Additionally, the flow cytometry assay confirmed that both ATEs and CPZ reduced CAP-induced intracellular calcium influx, showing no significant difference between the two, which was consistent with the WB and immunofluorescence results (all p < 0.001, ATEs + CAP vs. CAP, CPZ + CAP vs. CAP) (p > 0.05, CPZ + CAP vs. ATEs + CAP). The ELISA to examine whether ATEs could suppress the level of KLK5, IL-6, IL-8 and TNF-α proinflammatory mediators of rosacea. As a result, the treatment of ATEs inhibited KLK5, IL-6, IL-8 and TNF-α in vitro (Fig. 2F,G,H and I). Moreover, after CAP stimulation, the secretion of KLK5, IL-6, IL-8 and TNF-α increased, and ATEs could inhibit the increase of these proinflammatory mediators (all p < 0.001, CAP vs. ATEs + CAP). Based on the above results, the down-regulating effects of ATEs (as an antagonist) on proinflammatory mediators and TRPV1 pathways may be presented as a rosacea-therapeutic strategy.Fig.2 Effect of ATEs on the expression of TRPV1 and intracellular calcium flow in a vitro model. (A, B) Total proteins were prepared and immunoblotted for TRPV1 by using specific antibodies. β-Actin was used as internal control. The protein levels were quantified by band density in proportion to the expression of total form. (C) Fluorescence images of untreated (NC), or HaCaT cells treated with CPZ, CPZ plus CAP, CAP, ATEs, ATEs plus CAP for 24 h, loaded with Fluo-4 AM for thirty minutes. Scale bar = 275 μm. (D) Quantitative calcium fluorescence expression of HaCaT keratinocytes via flow cytometry. (E) The mean fluorescence intensity of intracellular calcium was quantified. (F, G, H, I) ELISA evaluated concentration of KLK5, IL-6, IL-8 and TNF-α in cell serum with untreated (NC), or HaCaT cells serum treated with CPZ, CPZ plus CAP, CAP, ATEs, ATEs plus CAP. All the above data are expressed as mean ± SD. *p < 0.05 or **p < 0.01 or ***p < 0.001 vs. NC group by one-way ANOVA followed by LSD multiple comparison. The experiments were repeated three times to ensure the accuracy.

ATEs attenuate rosacea-like clinical symptoms of rosacea skin

To validate the therapeutic efficacy of ATEs on rosacea, an LL-37 induced mouse model of rosacea-like phenotype was established. The most used method is intradermal injection of LL-37. Erythema and inflammation appeared on the skin of the injection area in mice approximately 12 h after the third injection, resulting in skin lesions resembling rosacea28. Following model establishment, normal saline (CON), CAP, CPZ, and ATEs were administered to the mice. The results of the experiment were displayed in Fig. 3A. the control group given only normal saline showed worsened visual symptoms and erythema score of rosacea, including erythema, drying, edema, and scarring. From the general photographs and the simulated map of erythema area changes, CAP effectively delayed the healing of rosacea, while CPZ and ATEs accelerated the healing process (Fig. 3B ,C). Specifically, the erythema area and score of rosacea were remarkably reduced by CPZ and ATEs (p < 0.001). The area of erythema was evaluated on the last day of assessment, as illustrated in Fig. 3F. ATEs administration mitigated the erythema area, as opposed to the increased expression of the control group (p < 0.001). More importantly, there was no significant difference in the improvement of erythema area between the ATEs group and the inhibitor CPZ group (p > 0.05, ATEs vs. CPZ). Furthermore, the TEWL value, representing skin hydration and barrier state, was evaluated on the dorsal side of the mice on the last day. A similar degree of reduction was found in the CPZ group and the ATEs group, whereas the CAP group exhibited an increase in TEWL (Fig. 3G). The specific average and standard deviation (SD) values of TEWL (g/m2/h) for each group were: NC 2.14 ± 0.7257, CON 7 17.98 ± 0.9060, CAP 32.84 ± 1.816, CPZ 6.441 ± 0.1762, ATEs 6.83 ± 0.6210 (all p < 0.01).Fig.3 ATEs ameliorate the symptom of LL-37 induced rosacea-like mice. (A) Scheme of the experimental schedule. (B) Representative photographs of rosacea area in mice after treatment with different formulations. The black square is 1.8 cm in size. (C) Simulation diagram and (D) quantitative analysis of the change in erythema area. (E) Erythema scores were measured once a day for 8 days. The erythema score was the average value of the sum of scores for each mouse. (F) The final erythema area of each group after treatments on day 8. (G) Measurement of Trans-epidermal water loss (TEWL) in skin on day 8. All the above data are expressed as mean ± SD. *p < 0.05 or **p < 0.01 or ***p < 0.001 vs. CON group by one-way ANOVA followed by LSD multiple comparison. The experiments were repeated three times to ensure the accuracy.

ATEs inhibit abnormal epidermal thickening, mast cell infiltration and vascular hyperplasia of rosacea skin in mice

To investigate the anti-rosacea effects of ATEs in cutaneous tissue, the epidermal thickness, the mast cell infiltration, and telangiectasia were assessed by histological examination. Through the H&E staining (Fig. 4A,B and S1), epidermal hyperplasia and hyperkeratosis were shown in the control group (LL-37 model group). The CAP group demonstrated massive thickening of the epidermal layer, as well as abundant inflammatory cell infiltration. In contrast, ATEs treatment attenuated the abnormal thickened epidermis, and its reduction effect was similar to that of the CPZ group. The specific average and SD values of the epidermal thickness (μm) for each group were as followed: CON 165.4 ± 26.24, CAP 264.2 ± 27.95, CPZ 86.43 ± 10.69, and ATEs 80.63 ± 21.26 (all p < 0.001). Additionally, H&E staining revealed that ATEs and CPZ treatment ameliorated the infiltration of inflammatory cells into the dermis. Regarding telangiectasia, the immuno-histochemistry results showed a significant increase in the expression of CD31 (a marker of blood vessels) in the control group at the skin lesions, and there was more severe abnormal vascular expression in the CAP group (Fig. 4C,D). Conversely, the expression of CD31 in the ATEs group and the CPZ group was significantly decreased. No statistically significant difference was found between the ATEs group and the CPZ group (p > 0.05, ATEs vs. CPZ). As shown in Fig. 4E,F, Toluidine blue staining analysis showed that the mast cell infiltration in the dermis of mice skin lesions in the CAP group increased significantly (p < 0.001 vs. CON). While, compared with the control group, the mast cell infiltration in the CPZ group and ATEs group was significantly reduced (both p < 0.001). Collectively, these results suggested that the ATEs administration methods showed great potential for alleviating skin inflammation.Fig.4 Effect of ATEs on epidermal thickness and vascular hyperplasia of mice with LL-37 induced rosacea. (A, B) Epidermal thickness was examined by H&E staining of skin lesions. Scale bar = 275 μm. (C) Immunohistochemistry analysis of CD31 of mice skin lesions. Scale bar = 275 μm. (D) Quantitative analysis of CD31 expression at skin lesions. (E, F) Mast cell was examined by Toluidine blue staining of skin lesions. Scale bar = 275 μm. All the above data are expressed as mean ± SD. *p < 0.05 or ** p < 0.01 or ***p < 0.001 vs. CON group by one-way ANOVA followed by LSD multiple comparison. The experiments were repeated three times to ensure the accuracy.

ATEs suppress TRPV1 activation in rosacea mouse skin

Our above findings indeed confirmed that the regulatory effect of ATEs on TRPV1 in vitro. Immunofluorescence and Western blot analysis were performed to figure out whether ATEs inhibit TRPV1 activation and expression in vivo. As demonstrated in Fig. 5A,B, the immunofluorescence staining of TRPV1 was significantly increased in the rosacea-like lesion, indicating the activation of TRPV1. Compared to the control group, the TRPV1 expression of the CAP group was significantly increased (p < 0.001). In contrast, the ATEs group and the CPZ group revealed significantly reduced TRPV1 expression (all p < 0.001), and there was no statistical difference between these two groups (p > 0.05). Moreover, control group increased the expression of TRPV1 protein, while the ATEs treatment demonstrated a significant decrease (Fig. 5C,D). In conclusion, these results indicated that ATEs could suppress the activation of TRPV1 in rosacea mice skin.Fig.5 ATEs suppresses TRPV1 activation in the lesional skin. (A) Immunofluorescence staining of TRPV1 protein (labelled with FITC, green) in skin biopsies from rosacea mice. (B) Quantitative analysis of TRPV1 expression was performed by the ratio of fluorescent area of TRPV1 (green) in relation to total area of DAPI (blue) from randomly selected 3 sections in each group. (C) Western blot analysis of TRPV1 in skin biopsies. (D) The protein levels of TRPV1 were quantified by band density in proportion to the expression of total form. β-actin was used as internal control. All the above data are expressed as mean ± SD. *p < 0.05 or ** p < 0.01 or *** p < 0.001 vs. CON group by one-way ANOVA followed by LSD multiple comparison. The experiments were repeated three times to ensure the accuracy.

Discussion

Rosacea is a relapsing, chronic inflammatory disease, which has obscured causes and treatments1. Due to the financial burden and side effects of existing treatments on rosacea patients, research has been conducted to develop new treatments. In this study, the anti-rosacea efficacy of ATEs was verified in HaCaT keratinocytes and LL-37 induced rosacea mouse model. The HaCaT cell line is a representative epithelial keratinocyte used in rosacea experiments33,34. In rosacea patients, keratinocytes are activated by pervading antigens in the impaired skin barrier and then, stimulating upregulation of TRPV1 expression, thereby triggering the release of inflammatory mediators10,30. Furthermore, LL-37 is the most widely used method to establish animal models of rosacea. Previous studies demonstrated the link between LL-37 and rosacea in a mouse model, which resulted in telangiectasia, erythema, and inflammation, and the skin lesion expressed higher levels of TLR-2, KLK-5, TNF-α, IL-1β, and IL-635,36. LL-37 is known to cause various allergic inflammatory diseases by triggering proinflammatory mediators via MAPKs, TLR-2, or TRPV1 pathways37. Therefore, a mouse disease model of rosacea was induced by intradermal injection of LL-37 to explore the molecular mechanisms and histopathological characteristics of rosacea treated with ATEs.

Firstly, in vitro experiments indicated that ATEs could block CAP-induced activation of TRPV1. Previous studies reported TRPV1 expression was increased in all rosacea subtypes compared with normal skin. To investigate the probable effect of ATEs, CAP was selected as a TRPV1 agonist, and CPZ was selected as a TRPV1 antagonist. Experiments on TRPV1 protein expression revealed that CAP boosted TRPV1 protein expression, whereas CPZ and ATEs decreased TRPV1 protein expression (Fig. 1). Notably, ATEs also showed excellent biocompatibility. To further explore the therapeutic effects of ATEs, CAP was selected to model rosacea in vitro. ATEs were found to effectively counteract the upregulation of TRPV1 protein expression induced by CAP, which was similar to the CPZ antagonist (Fig. 2). In addition to TRPV1 activation in human epidermal keratinocytes, CAP evoked an increase in the Ca2+ concentration. Calcium ion influx is a conduction pathway closely related to the release of inflammatory mediators, and TRPV1 is a non-selective cation channel with high permeability to calcium ions32. As described in a previous study, CAP activated TRPV1 and stimulated intracellular calcium signaling. We proved that CAP-induced Ca2+ influx was also blocked by ATEs or CPZ (Fig. 2). In fact, several studies have shown that exposure of keratinocytes to CAP leads to TRPV1-mediated Ca2+ influx, resulting in the release of KLK5 and other inflammatory factors (such as IL-6, IL-8, TNF-a), which promote the inflammatory response and cause erythema, telangiectasia and pain5,30,38. In this study, ATEs significantly reduced the release of inflammatory factors such as KLK5, IL-6, IL-8 and TNF-α after intervening in CAP-stimulated cells (Fig. 2). These results further support the idea that ATEs downregulate the TRPV1 channel, inhibit Ca2+ influx and the release of proinflammatory mediators, thereby relieving rosacea-like symptoms. However, further research is required.

In vivo studies using LL-37 induced rosacea skin models were conducted to further demonstrate the efficacy of ATEs as a therapeutic strategy. ATEs showed anti-inflammatory effects by downregulating TRPV1 and alleviated rosacea symptoms by inhibiting epidermal thickening and telangiectasia (CD31 expression), promoting skin barrier repair. In the present experiment, ATEs attenuated the visible phenotype and histological phenomena of rosacea, including erythema, drying, bleeding scab, epidermal hyperplasia, and increased mast cell infiltration (Fig. 3 and Fig. 4). In addition, the erythema area and score of the ATEs group was significantly smaller than that of other groups, accompanied by increased skin hydration. Rosacea is characterized by a damaged cutaneous barrier that leads to increased TEWL1,3. Therefore, skin hydration plays an essential role in creating a favorable environment that promotes the repair of the skin barrier. As an important pathogenesis for rosacea, vascular dysfunction is characterized by telangiectasia and angiogenesis1. Such vascular dysfunction could be related to the elevated levels of VEGF and TRP channels in rosacea patients4. Nonetheless, several studies showed that ATEs could exert therapeutic effects on wound healing by promoting angiogenesis17,19,24. In this study, ATEs significantly reduced telangiectasia in a rosacea-like mouse model. Most previous studies used a concentration of 500 μg/ml total protein for ATEs, whereas our study used only 1/10 of that concentration. This difference may be attributed to the VEGF released from ATEs promoting the growth of abnormally proliferated capillaries. However, our research is limited to the visual symptoms of animals, and further research is needed to confirm the results. Moreover, the immunofluorescence experiments confirmed the in vitro findings and underscored the potential of ATEs to target TRPV1. The activation of TRPV1 stimulates the C fibers of sensory neurons to release vasoactive peptides, such as CGRP and SP, leading to vasomotor dysfunction and high neurovascular reactivity manifesting as paroxysmal flushing, persistent erythema, and telangiectasia5,9. Moreover, TRPV1 activation plays a crucial role in heat heat-sensitive regulation of inflammation12. By suppressing TRPV1 activation, ATEs may disrupt the inflammatory cascade that contributes to the development and progression of rosacea. Thus, ATEs are a potential option for rosacea treatment (Fig. 6).Fig.6 A graphical abstract for illustrating the role of ATEs on rosacea mice and HaCaT keratinocytes. In vivo and in vitro models, ATEs alleviated rosacea symptoms by regulating TRPV1. TRPV1 contributes to the increase of calcium influx, while ATEs could play anti-inflammatory and anti-telangiectasia effects by inhibiting TRPV1. This suggested that ATEs have the potential to treat rosacea.

Previous studies have shown that ATEs contain a variety of effective active growth factors, and that their combination may synergistically result in the best outcome19,20. For example, IGF-I is capable of maintaining skin surface lipids and thickness39; EGF can stimulate keratinocyte and fibroblast proliferation and increase fibronectin production40; PDGF, TGF-β and b-FGF contribute to ATEs’ ability to promote fibroblast proliferation and collagen synthesis in the dermis41; HGF promotes monocyte migration, regulates collagen fiber synthesis and inflammatory response25,41; and other substances involved in promoting tissue regeneration and repair41. To date, the concrete roles and contributions of these grown factors in ATEs remain to be elucidated. Whether some other growth factors or ingredients in ATEs also contribute to ATEs, warrants further investigation. Although ATEs have been shown to have anti-aging, anti-inflammatory, and wound-healing effects, the rosacea-mitigating effects of ATEs have not been investigated. In this study, the anti-rosacea efficacy of ATEs was demonstrated in HaCaT keratinocytes and LL-37 induced mouse model. ATEs may have outstanding effects in repairing skin inflammation.

Interestingly, besides the beneficial effects on rosacea, this study also demonstrates the importance of subcutaneous adipose tissue for overall skin health. Subcutaneous adipose layer physically shapes, and supports facial skin through its vasculature and secretion of adipokines42. Notably, adipose tissue itself influences cell behavior in the tissue microenvironment by releasing and secreting various growth factors, as well as plays a role in tissue repair and inflammation in the skin, heart, skeletal muscle, and breast26,43–45. For example, Pallua et al.18 found that fresh lipoaspirate contained a certain amount of growth factors (e.g. b-FGF, PDGF, etc.). These results indicated that adipose tissue is inherently enriched with a variety of bioactive factors that might be directly isolated for clinical application without the cell isolation or cultivation process. ATEs are rich in many active growth factors and cytokines derived from adipose tissue, including IGF-I, HGF, PDGF, b-FGF, adiponectin, etc., which directly or indirectly promote tissue regeneration, remodeling and repair19–21,26. Through in vivo and in vitro experiments, this study found that administration of ATEs alleviated the expression of pro-inflammatory cytokines and mast cell infiltration, and restored the level of intracellular Ca2+, epidermal hyperplasia, and TEWL in terms of skin barrier function. Other studies have demonstrated that ATEs improve the proliferation and multipotency of adipose stem cell, thereby providing support and elasticity to the skin25,46,47.

As previously stated, ATEs possess advantageous characteristics in clinical applications. ATEs are autologous extracts and represent a safe and reliable alternative to current commercial growth factors, as they do not pose any risk of immune rejection or ethical concerns. Additionally, compared to a similar product, Platelet-rich plasma (PRP), which comes from blood, ATEs has the potential to break the limitations of autologous production for own use. That is standardized preparation of ATEs from medical waste emulsified fat and commercial availability for other people. However, if applying ATEs commercially, more strict consideration must be given48. Rosacea always goes with skin barrier damage, resulting in erythema, inflammatory papules and telangiectasia. In terms of therapeutic delivery, the development of an injection or microneedle patch enriched with appropriate concentration of ATEs holds immense promise as a daily caring treatment for rosacea patients, offering a soothing and anti-inflammatory protective layer to alleviate their symptoms.(Supplementary file)

Conclusion

In vivo and in vitro experiments revealed that intradermal injection of ATEs alleviated the expression of TRPV1, intracellular calcium ions influx and the release of inflammatory mediators in terms of anti-inflammatory properties, as well as restored the level of epidermal hyperplasia, telangiectasia, and TEWL in terms of skin barrier function. This study provides strong evidence for the therapeutic potential of ATEs as a novel approach to managing rosacea. By down-regulating TRPV1, ATEs offer a promising strategy for the treatment of rosacea. Further research is warranted to fully elucidate the mechanisms of action and translate these findings into effective clinical applications, ultimately leading to improved rosacea patient outcomes.

Supplementary Information

Supplementary Information.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-72593-8.

Acknowledgements

This work was supported by Natural Science Foundation of China (Grant number 82305040), Zhejiang Provincial Health Science and Technology Program (Grant number 202466176, 2024660434), and Hangzhou Medical College basic research fund basic research program (KYYB2023020).

Author contributions

LZ and LC conducted the main work of this study; LZ contributed to the experimental methodology and writing; LC and SL contributed to the animal experiment; LZ and YZ contributed to the molecular experiment of ATEs; TJ improved the experimental design and funded this study; LW participated to the experimental design; TL and SW contributed to the experimental design; SW funded this study and contributed the revision; TJ designed, drafted, and funded this study.

Data availability

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare no competing interests.

Ethics statement

The animal study was reviewed and approved by the Laboratory Animal Management and Ethics Committee of Zhejiang Provincial People’s Hospital (Affiliated People’s Hospital, Hangzhou Medical College) (Approval number: SYXK: 2019‐0013, Zhejiang, China). This study was carried out in compliance with the ARRIVE (Animal Research: Reporting of In vivo Animal Experiments) guidelines. The human tissue sample study was approved by the Ethics Committee of Zhejiang Provincial People’s Hospital (Affiliated People’s Hospital, Hangzhou Medical College), and performed in accordance with the Declaration of Helsinki. All experiments were performed in accordance with relevant guidelines and regulations.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Liuyi Zhou and Lulu Chen.
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References

1. van Zuuren EJ Rosacea N. Engl. J. Med. 2017 377 1754 1764 10.1056/NEJMcp1506630 29091565
van Zuuren, E. J. Rosacea. N. Engl. J. Med. 377, 1754–1764 (2017).29091565
2. Paiva-Santos AC Rosacea topical treatment and care: From traditional to new drug delivery systems Mol. Pharm. 2023 20 3804 3828 10.1021/acs.molpharmaceut.3c00324 37478169
Paiva-Santos, A. C. et al. Rosacea topical treatment and care: From traditional to new drug delivery systems. Mol. Pharm. 20, 3804–3828 (2023).37478169
3. van Zuuren EJ Rosacea: New concepts in classification and treatment Am. J. Clin. Dermatol. 2021 22 457 465 10.1007/s40257-021-00595-7 33759078
van Zuuren, E. J. et al. Rosacea: New concepts in classification and treatment. Am. J. Clin. Dermatol. 22, 457–465 (2021).33759078
4. Thiboutot D Standard management options for rosacea: The 2019 update by the national rosacea society expert committee J. Am. Acad. Dermatol. 2020 82 1501 1510 10.1016/j.jaad.2020.01.077 32035944
Thiboutot, D. et al. Standard management options for rosacea: The 2019 update by the national rosacea society expert committee. J. Am. Acad. Dermatol. 82, 1501–1510 (2020).32035944
5. Xiao T Sun M Zhao C Kang J TRPV1: A promising therapeutic target for skin aging and inflammatory skin diseases Front. Pharmacol. 2023 14 1037925 10.3389/fphar.2023.1037925 36874007
Xiao, T., Sun, M., Zhao, C. & Kang, J. TRPV1: A promising therapeutic target for skin aging and inflammatory skin diseases. Front. Pharmacol. 14, 1037925 (2023).36874007
6. Steinhoff M Schauber J Leyden JJ New insights into rosacea pathophysiology: A review of recent findings J. Am. Acad. Dermatol. 2013 69 S15 26 10.1016/j.jaad.2013.04.045 24229632
Steinhoff, M., Schauber, J. & Leyden, J. J. New insights into rosacea pathophysiology: A review of recent findings. J. Am. Acad. Dermatol. 69, S15-26 (2013).24229632
7. Clapham D E. snapshot: Mammalian TRP channels Cell 2007 129 220 10.1016/j.cell.2007.03.034 17418797
Clapham, D. E. snapshot: Mammalian TRP channels. Cell 129, 220 (2007).17418797
8. Gao N Li M Wang W Liu Z Guo Y A bibliometrics analysis and visualization study of TRPV1 channel Front. Pharmacol. 2023 14 1076921 10.3389/fphar.2023.1076921 37025492
Gao, N., Li, M., Wang, W., Liu, Z. & Guo, Y. A bibliometrics analysis and visualization study of TRPV1 channel. Front. Pharmacol. 14, 1076921 (2023).37025492
9. Gouin O TRPV1 and TRPA1 in cutaneous neurogenic and chronic inflammation: Pro-inflammatory response induced by their activation and their sensitization Protein Cell 2017 8 644 661 10.1007/s13238-017-0395-5 28364279
Gouin, O. et al. TRPV1 and TRPA1 in cutaneous neurogenic and chronic inflammation: Pro-inflammatory response induced by their activation and their sensitization. Protein Cell 8, 644–661 (2017).28364279
10. Oh S Son M Park J Kang D Byun K Radiofrequency irradiation modulates TRPV1-related burning sensation in rosacea Molecules 2021 26 1424 10.3390/molecules26051424 33800730
Oh, S., Son, M., Park, J., Kang, D. & Byun, K. Radiofrequency irradiation modulates TRPV1-related burning sensation in rosacea. Molecules 26, 1424 (2021).33800730
11. Yang F Zheng J Understand spiciness: mechanism of TRPV1 channel activation by capsaicin Protein Cell 2017 8 169 177 10.1007/s13238-016-0353-7 28044278
Yang, F. & Zheng, J. Understand spiciness: mechanism of TRPV1 channel activation by capsaicin. Protein Cell 8, 169–177 (2017).28044278
12. Garami A Hyperthermia induced by transient receptor potential vanilloid-1 (TRPV1) antagonists in human clinical trials: Insights from mathematical modeling and meta-analysis Pharmacology & Therapeutics 2020 208 107474 10.1016/j.pharmthera.2020.107474 31926897
Garami, A. et al. Hyperthermia induced by transient receptor potential vanilloid-1 (TRPV1) antagonists in human clinical trials: Insights from mathematical modeling and meta-analysis. Pharmacology & Therapeutics 208, 107474 (2020).31926897
13. Chizh BA The effects of the TRPV1 antagonist SB-705498 on TRPV1 receptor-mediated activity and inflammatory hyperalgesia in humans Pain 2007 132 132 141 10.1016/j.pain.2007.06.006 17659837
Chizh, B. A. et al. The effects of the TRPV1 antagonist SB-705498 on TRPV1 receptor-mediated activity and inflammatory hyperalgesia in humans. Pain 132, 132–141 (2007).17659837
14. Qin Y An update on adipose-derived stem cells for regenerative medicine: Where challenge meets opportunity Adv. Sci. (Weinh) 2023 10 e2207334 10.1002/advs.202207334 37162248
Qin, Y. et al. An update on adipose-derived stem cells for regenerative medicine: Where challenge meets opportunity. Adv. Sci. (Weinh) 10, e2207334 (2023).37162248
15. Xue Y LNP-RNA-engineered adipose stem cells for accelerated diabetic wound healing Nat. Commun. 2024 15 739 10.1038/s41467-024-45094-5 38272900
Xue, Y. et al. LNP-RNA-engineered adipose stem cells for accelerated diabetic wound healing. Nat. Commun. 15, 739 (2024).38272900
16. Quan Y Zhang Y Li J Lu F Cai J Transplantation of in vitro prefabricated adipose organoids attenuates skin fibrosis by restoring subcutaneous fat and inducing dermal adipogenesis FASEB J. 2023 37 e23076 10.1096/fj.202202117R 37432650
Quan, Y., Zhang, Y., Li, J., Lu, F. & Cai, J. Transplantation of in vitro prefabricated adipose organoids attenuates skin fibrosis by restoring subcutaneous fat and inducing dermal adipogenesis. FASEB J. 37, e23076 (2023).37432650
17. Sarkanen J-R Human adipose tissue extract induces angiogenesis and adipogenesis in vitro Tissue Eng. Part A 2012 18 17 25 10.1089/ten.tea.2010.0712 21902602
Sarkanen, J.-R. et al. Human adipose tissue extract induces angiogenesis and adipogenesis in vitro. Tissue Eng. Part A 18, 17–25 (2012).21902602
18. Pallua N Pulsfort AK Suschek C Wolter TP Content of the growth factors bFGF, IGF-1, VEGF, and PDGF-BB in freshly harvested lipoaspirate after centrifugation and incubation Plast. Reconstr. Surg. 2009 123 826 833 10.1097/PRS.0b013e318199ef31 19319045
Pallua, N., Pulsfort, A. K., Suschek, C. & Wolter, T. P. Content of the growth factors bFGF, IGF-1, VEGF, and PDGF-BB in freshly harvested lipoaspirate after centrifugation and incubation. Plast. Reconstr. Surg. 123, 826–833 (2009).19319045
19. Yu Z Fat extract promotes angiogenesis in a murine model of limb ischemia: A novel cell-free therapeutic strategy Stem Cell Res. Ther. 2018 9 294 10.1186/s13287-018-1014-y 30409190
Yu, Z. et al. Fat extract promotes angiogenesis in a murine model of limb ischemia: A novel cell-free therapeutic strategy. Stem Cell Res. Ther. 9, 294 (2018).30409190
20. Zheng H Fat extract improves fat graft survival via proangiogenic, anti-apoptotic and pro-proliferative activities Stem Cell Res. Ther. 2019 10 174 10.1186/s13287-019-1290-1 31196213
Zheng, H. et al. Fat extract improves fat graft survival via proangiogenic, anti-apoptotic and pro-proliferative activities. Stem Cell Res. Ther. 10, 174 (2019).31196213
21. López JF Adipose tissue extract shows potential for wound healing: In vitro proliferation and migration of cell types contributing to wound healing in the presence of adipose tissue preparation and platelet rich plasma Cytotechnology 2018 70 1193 1204 10.1007/s10616-018-0211-y 29511946
López, J. F. et al. Adipose tissue extract shows potential for wound healing: In vitro proliferation and migration of cell types contributing to wound healing in the presence of adipose tissue preparation and platelet rich plasma. Cytotechnology 70, 1193–1204 (2018).29511946
22. Xu G-Y Cell-free extracts from human fat tissue with a hyaluronan-based hydrogel attenuate inflammation in a spinal cord injury model through m2 microglia/microphage polarization Small 2022 18 e2107838 10.1002/smll.202107838 35333441
Xu, G.-Y. et al. Cell-free extracts from human fat tissue with a hyaluronan-based hydrogel attenuate inflammation in a spinal cord injury model through m2 microglia/microphage polarization. Small 18, e2107838 (2022).35333441
23. Kan T Cell-free fat extract-loaded microneedles attenuate inflammation-induced apoptosis and mitochondrial damage in tendinopathy Mater. Today Bio. 2023 22 100738 10.1016/j.mtbio.2023.100738 37600349
Kan, T. et al. Cell-free fat extract-loaded microneedles attenuate inflammation-induced apoptosis and mitochondrial damage in tendinopathy. Mater. Today Bio. 22, 100738 (2023).37600349
24. Yin M γ-PGA hydrogel loaded with cell-free fat extract promotes the healing of diabetic wounds J. Mater. Chem. B 2020 8 8395 8404 10.1039/D0TB01190H 32966542
Yin, M. et al. γ-PGA hydrogel loaded with cell-free fat extract promotes the healing of diabetic wounds. J. Mater. Chem. B 8, 8395–8404 (2020).32966542
25. Cai Y Li J Jia C He Y Deng C Therapeutic applications of adipose cell-free derivatives: A review Stem Cell Res. Ther. 2020 11 312 10.1186/s13287-020-01831-3 32698868
Cai, Y., Li, J., Jia, C., He, Y. & Deng, C. Therapeutic applications of adipose cell-free derivatives: A review. Stem Cell Res. Ther. 11, 312 (2020).32698868
26. Wang J A multilayered nanofibrous patch functionalized with adipose tissue extract for the treatment of bladder regeneration Mater. Design. 2022 220 110821 10.1016/j.matdes.2022.110821
Wang, J. et al. A multilayered nanofibrous patch functionalized with adipose tissue extract for the treatment of bladder regeneration. Mater. Design. 220, 110821 (2022).
27. Deng M Protective effect of fat extract on UVB-induced photoaging in vitro and in vivo Oxid. Med. Cell. Longev. 2019 2019 6146942 10.1155/2019/6146942 31531185
Deng, M. et al. Protective effect of fat extract on UVB-induced photoaging in vitro and in vivo. Oxid. Med. Cell. Longev. 2019, 6146942 (2019).31531185
28. Rodrigues-Braz D Cutaneous and ocular rosacea: Common and specific physiopathogenic mechanisms and study models Mol. Vis. 2021 27 323 353 34035646
Rodrigues-Braz, D. et al. Cutaneous and ocular rosacea: Common and specific physiopathogenic mechanisms and study models. Mol. Vis. 27, 323–353 (2021).34035646
29. Sung B Prasad S Ravindran J Yadav VR Aggarwal BB Capsazepine, a TRPV1 antagonist, sensitizes colorectal cancer cells to apoptosis by TRAIL through ROSg–JNK–CHOP-mediated upregulation of death receptors Free Radic. Biol. Med. 2012 53 1977 1987 10.1016/j.freeradbiomed.2012.08.012 22922338
Sung, B., Prasad, S., Ravindran, J., Yadav, V. R. & Aggarwal, B. B. Capsazepine, a TRPV1 antagonist, sensitizes colorectal cancer cells to apoptosis by TRAIL through ROSg–JNK–CHOP-mediated upregulation of death receptors. Free Radic. Biol. Med. 53, 1977–1987 (2012).22922338
30. Sg L Cutaneous neurogenic inflammation mediated by TRPV1-NGF-TRKA pathway activation in rosacea is exacerbated by the presence of Demodex mites J. Eur. Acad. Dermatol. Venereol JEADV 2023 37 2589 10.1111/jdv.19449 37606610
Sg, L. et al. Cutaneous neurogenic inflammation mediated by TRPV1-NGF-TRKA pathway activation in rosacea is exacerbated by the presence of Demodex mites. J. Eur. Acad. Dermatol. Venereol JEADV 37, 2589 (2023).37606610
31. Ho KW Ward NJ Calkins DJ TRPV1: A stress response protein in the central nervous system Am. J. Neurodegener. Dis. 2012 1 1 14 22737633
Ho, K. W., Ward, N. J. & Calkins, D. J. TRPV1: A stress response protein in the central nervous system. Am. J. Neurodegener. Dis. 1, 1–14 (2012).22737633
32. Zhai K Liskova A Kubatka P Büsselberg D Calcium entry through TRPV1: A potential target for the regulation of proliferation and apoptosis in cancerous and healthy cells Int. J. Mol. Sci. 2020 21 4177 10.3390/ijms21114177 32545311
Zhai, K., Liskova, A., Kubatka, P. & Büsselberg, D. Calcium entry through TRPV1: A potential target for the regulation of proliferation and apoptosis in cancerous and healthy cells. Int. J. Mol. Sci. 21, 4177 (2020).32545311
33. Deng Z A positive feedback loop between mTORC1 and cathelicidin promotes skin inflammation in rosacea EMBO Mol. Med. 2021 13 e13560 10.15252/emmm.202013560 33734592
Deng, Z. et al. A positive feedback loop between mTORC1 and cathelicidin promotes skin inflammation in rosacea. EMBO Mol. Med. 13, e13560 (2021).33734592
34. Zhang Y Nav1 8 in keratinocytes contributes to ROS-mediated inflammation in inflammatory skin diseases Redox Biol. 2022 55 102427 10.1016/j.redox.2022.102427 35952475
Zhang, Y. et al. Nav1 8 in keratinocytes contributes to ROS-mediated inflammation in inflammatory skin diseases. Redox Biol. 55, 102427 (2022).35952475
35. Yamasaki K Increased serine protease activity and cathelicidin promotes skin inflammation in rosacea Nat. Med. 2007 13 975 980 10.1038/nm1616 17676051
Yamasaki, K. et al. Increased serine protease activity and cathelicidin promotes skin inflammation in rosacea. Nat. Med. 13, 975–980 (2007).17676051
36. Yuan X Artemisinin, a potential option to inhibit inflammation and angiogenesis in rosacea Biomed. Pharmacother. 2019 117 109181 10.1016/j.biopha.2019.109181 31387196
Yuan, X. et al. Artemisinin, a potential option to inhibit inflammation and angiogenesis in rosacea. Biomed. Pharmacother. 117, 109181 (2019).31387196
37. Miura S Cathelicidin antimicrobial peptide ll37 induces toll-like receptor 8 and amplifies IL-36γ and IL-17C in human keratinocytes J. Invest Dermatol. 2023 143 832 841.e4 10.1016/j.jid.2022.10.017 36496195
Miura, S. et al. Cathelicidin antimicrobial peptide ll37 induces toll-like receptor 8 and amplifies IL-36γ and IL-17C in human keratinocytes. J. Invest Dermatol. 143, 832-841.e4 (2023).36496195
38. Lee YM A novel role for the TRPV1 channel in UV-induced matrix metalloproteinase (MMP)-1 expression in HaCaT cells J. Cell Physiol. 2009 219 766 775 10.1002/jcp.21729 19206161
Lee, Y. M. et al. A novel role for the TRPV1 channel in UV-induced matrix metalloproteinase (MMP)-1 expression in HaCaT cells. J. Cell Physiol. 219, 766–775 (2009).19206161
39. Makrantonaki E Interplay of IGF-I and 17beta-estradiol at age-specific levels in human sebocytes and fibroblasts in vitro Exp. Gerontol. 2008 43 939 946 10.1016/j.exger.2008.07.005 18755261
Makrantonaki, E. et al. Interplay of IGF-I and 17beta-estradiol at age-specific levels in human sebocytes and fibroblasts in vitro. Exp. Gerontol. 43, 939–946 (2008).18755261
40. Gainza G Villullas S Pedraz JL Hernandez RM Igartua M Advances in drug delivery systems (DDSs) to release growth factors for wound healing and skin regeneration Nanomedicine 2015 11 1551 1573 10.1016/j.nano.2015.03.002 25804415
Gainza, G., Villullas, S., Pedraz, J. L., Hernandez, R. M. & Igartua, M. Advances in drug delivery systems (DDSs) to release growth factors for wound healing and skin regeneration. Nanomedicine 11, 1551–1573 (2015).25804415
41. Xu Y Cell-free fat extract increases dermal thickness by enhancing angiogenesis and extracellular matrix production in nude mice Aesthet. Surg. J. 2020 40 904 913 10.1093/asj/sjz306 31679030
Xu, Y. et al. Cell-free fat extract increases dermal thickness by enhancing angiogenesis and extracellular matrix production in nude mice. Aesthet. Surg. J. 40, 904–913 (2020).31679030
42. Bojanowski K Hypodermal delivery of cosmetic actives for improved facial skin morphology and functionality Int. J. Cosmetic Sci. 2013 35 562 567 10.1111/ics.12077
Bojanowski, K. Hypodermal delivery of cosmetic actives for improved facial skin morphology and functionality. Int. J. Cosmetic Sci. 35, 562–567 (2013).
43. Liu M Lu F Feng J Aging and homeostasis of the hypodermis in the age-related deterioration of skin function Cell Death Dis. 2024 15 443 10.1038/s41419-024-06818-z 38914551
Liu, M., Lu, F. & Feng, J. Aging and homeostasis of the hypodermis in the age-related deterioration of skin function. Cell Death Dis. 15, 443 (2024).38914551
44. Sugita K Adipose tissue remodeling via TSLP-mediated IL-4/IL-13 signaling: Implications for atopic dermatitis and skin barrier J. Allergy Clin. Immunol. 2024 154 282 284 10.1016/j.jaci.2024.06.002 38871185
Sugita, K. Adipose tissue remodeling via TSLP-mediated IL-4/IL-13 signaling: Implications for atopic dermatitis and skin barrier. J. Allergy Clin. Immunol. 154, 282–284 (2024).38871185
45. Ibrahim MM Subcutaneous and visceral adipose tissue: Structural and functional differences Obes. Rev. 2010 11 11 18 10.1111/j.1467-789X.2009.00623.x 19656312
Ibrahim, M. M. Subcutaneous and visceral adipose tissue: Structural and functional differences. Obes. Rev. 11, 11–18 (2010).19656312
46. Gaur M Dobke M Lunyak VV Mesenchymal stem cells from adipose tissue in clinical applications for dermatological indications and skin aging Int. J. Mol. Sci. 2017 18 208 10.3390/ijms18010208 28117680
Gaur, M., Dobke, M. & Lunyak, V. V. Mesenchymal stem cells from adipose tissue in clinical applications for dermatological indications and skin aging. Int. J. Mol. Sci. 18, 208 (2017).28117680
47. Yuan C Adipose-derived stem cell-based optimization strategies for musculoskeletal regeneration: Recent advances and perspectives Stem Cell Res. Ther. 2024 15 91 10.1186/s13287-024-03703-6 38539224
Yuan, C. et al. Adipose-derived stem cell-based optimization strategies for musculoskeletal regeneration: Recent advances and perspectives. Stem Cell Res. Ther. 15, 91 (2024).38539224
48. Alam M Effect of platelet-rich plasma injection for rejuvenation of photoaged facial skin JAMA Dermatol. 2018 154 1447 1452 10.1001/jamadermatol.2018.3977 30419125
Alam, M. et al. Effect of platelet-rich plasma injection for rejuvenation of photoaged facial skin. JAMA Dermatol. 154, 1447–1452 (2018).30419125
