
==== Front
World J Diabetes
WJD
World Journal of Diabetes
1948-9358
Baishideng Publishing Group Inc

jWJD.v15.i9.pg1962
10.4239/wjd.v15.i9.1962
97164
Basic Study
Dexmedetomidine ameliorates diabetic intestinal injury by promoting the polarization of M2 macrophages through the MMP23B pathway
Lu M et al. DEX and MMP23B in diabetic gut injury
Lu Man Department of Anesthesiology, The First Affiliated Hospital of Zhejiang Chinese Medical University (Zhejiang Provincial Hospital of Traditional Chinese Medicine), Hangzhou 310006, Zhejiang Province, China

Guo Xiao-Wen Department of Anesthesiology, The First Affiliated Hospital of Zhejiang Chinese Medical University (Zhejiang Provincial Hospital of Traditional Chinese Medicine), Hangzhou 310006, Zhejiang Province, China

Zhang Fang-Fang Department of Anesthesiology, The First Affiliated Hospital of Zhejiang Chinese Medical University (Zhejiang Provincial Hospital of Traditional Chinese Medicine), Hangzhou 310006, Zhejiang Province, China

Wu Dan-Hong Department of Anesthesiology, The First Affiliated Hospital of Zhejiang Chinese Medical University (Zhejiang Provincial Hospital of Traditional Chinese Medicine), Hangzhou 310006, Zhejiang Province, China

Xie Di Department of Emergency, Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China

Luo Feng-Qin Department of Anesthesiology, The First Affiliated Hospital of Zhejiang Chinese Medical University (Zhejiang Provincial Hospital of Traditional Chinese Medicine), Hangzhou 310006, Zhejiang Province, China. 3021702500@qq.com

Author contributions: Luo FQ designed the research study; Lu M, Guo XW, Zhang FF, and Wu DH performed the research; Xie D contributed new reagents and analytic tools; Lu M, Guo XW, Zhang FF, Wu DH, and Luo FQ analyzed the data and wrote the manuscript. All authors have read and approved the final manuscript.

Corresponding author: Feng-Qin Luo, MM, Attending Doctor, Department of Anesthesiology, The First Affiliated Hospital of Zhejiang Chinese Medical University (Zhejiang Provincial Hospital of Traditional Chinese Medicine), No. 54 Youdian Lane, Shangcheng District, Hangzhou 310006, Zhejiang Province, China. 3021702500@qq.com

15 9 2024
15 9 2024
15 9 19621978
5 6 2024
17 7 2024
12 8 2024
©The Author(s) 2024. Published by Baishideng Publishing Group Inc. All rights reserved.
2024
https://creativecommons.org/licenses/by-nc/4.0/ This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial.
BACKGROUND

Diabetes is often associated with gastrointestinal dysfunctions, which can lead to hypoglycemia. Dexmedetomidine (DEX) is a commonly used sedative in perioperative diabetic patients and may affect gastrointestinal function.

AIM

To investigate whether sedative doses of DEX alleviate diabetes-caused intestinal dysfunction.

METHODS

Sedation/anesthesia scores and vital signs of streptozotocin (STZ)-induced diabetic mice under DEX sedation were observed. Diabetic mice were divided into saline and DEX groups. After injecting sedatives intraperitoneally, tight junctions (TJs) and apoptotic levels were evaluated 24 hours later to assess the intestinal barrier function. The role of DEX was validated using Villin-MMP23B flox/flox mice with intestinal epithelial deletion. In vitro, high glucose and hyperosmolarity were used to culture Caco-2 monolayer cells with STZ inter-vention. Immunofluorescence techniques were used to monitor the barrier and mitochondrial functions.

RESULTS

MMP23B protein levels in the intestinal tissue of STZ-induced diabetic mice were significantly higher than those in the intestinal tissue of control mice, with the DEX group displaying decreased MMP23B levels. Diabetes-mediated TJ dis-ruption, increased intestinal mucosal permeability, and systemic inflammation in wild-type mice might be reversed by DEX. In Caco-2 cells, MMP23B was associated with increased reactive oxygen species accumulation, mitochondrial membrane potential depolarization, and TJ disruption.

CONCLUSION

DEX reduces MMP23B, which may potentially contribute to STZ-induced intestinal barrier dysfunction, affecting TJ modification through mitochondrial dysfunction.

Diabetes
Dexmedetomidine
Intestinal barrier
Piezo1
Tight junctions
==== Body
pmc Core Tip: This study investigates the protective role of dexmedetomidine (DEX) in diabetic intestinal injury through the MMP23B pathway. The findings reveal that DEX acts as a sedative and enhances intestinal barrier function by promoting M2 macrophage polarization and reducing mitochondrial dysfunction. Streptozotocin-induced diabetic mice and Caco-2 cell models provide robust evidence of the potential therapeutic benefits of DEX, offering insights into its dual functionality in managing sedation and intestinal healing under diabetic conditions. This research can pave the way for the development of new treatment strategies targeting intestinal complications in diabetic patients.

INTRODUCTION

Type 2 diabetes mellitus (T2DM) is a multifactorial disease characterized by tissue-specific dysregulation, and a complex interplay of genetic and environmental factors contribute to its pathogenesis[1-3]. Although the endocrine aspects of T2DM have been extensively studied, its intestinal component remains poorly explored. The gut, often overlooked, plays a pivotal role in glucose homeostasis and may be a key intervention area.

Intestinal macrophages are key players involved in the homeostasis of the gastrointestinal tract, performing several functions including supporting intestinal neurons, facilitating gastrointestinal motility, participating in oral tolerance to food antigens, and contributing to pathogen clearance. However, intestinal macrophages are also implicated in T2DM pathogenesis. Emerging evidence suggests that alterations in the functional profile of intestinal macrophages can incite metabolic inflammation, thereby accelerating obesity and T2DM progression[4-6].

T2DM can lead to gastrointestinal motility disorders, such as decreased gastric motility. However, the specific mechanisms linking T2DM to these gastrointestinal changes are not fully elucidated. Anesthetics, including dex-medetomidine (DEX), influence various physiological processes, but their impact on intestinal macrophages in diabetic contexts remains poorly understood.

DEX, a selective alpha-2 adrenergic receptor agonist, is widely used for its sedative, analgesic, and anxiolytic properties. In the context of infection and other pathological conditions, DEX enhances intestinal microcirculation, alleviates postoperative pain, and decreases opioid requirements[7-12]. Given its potential influence on inflammatory and immune responses, DEX may modulate the activity of intestinal macrophages, affecting T2DM-associated gastrointestinal dysfunctions.

In the present study, we aimed to delineate the intricate relationship between macrophages and DEX in the context of T2DM. By establishing a streptozotocin (STZ)-induced diabetic mouse model, we investigated the role of DEX in diabetes-induced gastrointestinal injury through comprehensive in vivo and in vitro experiments. Understanding the interaction between DEX and intestinal macrophages can unveil novel mechanisms underlying diabetes-related gastrointestinal complications and identify targets for therapeutic intervention. Our findings provide a fresh perspective on the intricate dynamics of diabetes-related intestinal dysfunction and may pave the way for the development of innovative treatment strategies that leverage the pharmacological potential of DEX to modulate macrophage activity and ameliorate gastrointestinal disturbances in diabetic patients.

MATERIALS AND METHODS

Analysis of the GSE63992 dataset

Differential gene analysis was performed on data sourced from the Gene Expression Omnibus database GSE63992. Volcano plots and heatmaps were generated, and R language packages were used for data processing[7,8].

Gene set enrichment analysis

A Gene Ontology/Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was performed on the data sourced from the Gene Expression Omnibus database GSE63992. R language packages were applied for data processing, and the corresponding bubble, circular, and bar charts, which also facilitated the analysis of gene overlaps within the identified Gene Ontology terms, were obtained. Furthermore, the involvement of differentially expressed genes (DEGs) in specific pathways was analyzed using DAVID[9,10].

Identification of hub genes

Gene networks were visualized using Cytoscape 3.6.1, and further analyzed within the STRING database to investigate biological processes.

Mice

Villin-CreERT, MMP23B flox/flox, and wild-type (WT) C57BL/6 male mice aged 6-8 weeks were provided by the Xinhua Hospital affiliated with the Shanghai Jiao Tong University School of Medicine. All animals were handled according to the 3R principle and in compliance with animal welfare standards.

Diabetic mouse model

The mice were divided into the following groups: Control, diabetes (DB), DB + MMP23B knockout (KO), DEX + DB, and DEX. The DB and DEX + DB mice were injected with STZ (40 mg/kg, intraperitoneal) daily for 5 days[11,12].

Macrophage depletion

We used clodronate liposomes (Cls), which are taken up by macrophages through endocytosis; the release of clodronate by lysosomes induces apoptosis and depletes macrophages. Mice were randomly assigned to one of the following four groups: Sham + phosphate buffered saline (PBS) (Sham; n = 4), DB (n = 4), MMP23B KO + DB + Cls (MMDC; n = 4), and MMP23B KO + PBS (MMP; n = 4). Cls or control (PBS) were intraperitoneally administered at a 24-hour interval before the DB procedure[13,14].

Caco-2 cell culture and treatment

We obtained Caco-2 cells from the Cell Bank of the Chinese Academy of Sciences, and four groups were randomly assigned: PBS (n = 4), MMP23B (n = 4), MMP23B + GM6001 (n = 4).

Immunofluorescence

Immunofluorescence staining was performed on frozen and paraffin tissue sections. Cells and tissues were fixed on glass slides or centrifuge tubes using 4% paraformaldehyde to preserve their morphological structure and protein distribution. Samples were treated with 0.1% Triton X-100 to increase antibody entry and binding. Nonspecific binding sites were blocked by adding an appropriate concentration of bovine serum albumin. Specific antibodies targeting the proteins of interest [CD 86 (AiFang, AF16377, 1:1000, rabbit polyclonal IgG), ZO-1 (Santa Cruz, sc-33725, 1:100, rat monoclonal IgG), and occludin (Santa Cruz, sc-133256, 1:100, mouse monoclonal IgG)] were added, and samples were incubated overnight at 4 °C. Secondary antibodies labeled with specific dyes [FITC-labeled donkey anti-rabbit IgG (Servicebio, GB22403, 1:200) and Cy3-labeled goat anti-mouse IgG (Servicebio, GB21303, 1:200)] were used for fluorescent signal detection. Samples were washed several times with a buffer solution to remove unbound secondary antibodies and other nonspecifically binding molecules. Slides were placed under a fluorescence microscope, and fluorescent signals were observed, detected, and recorded after illuminating with a specific wavelength. All images were captured using an Olympus fluorescence microscope (Olympus, BX53)[15,16].

Enzyme-linked immunosorbent assay

Samples (tissue extracts) were collected and diluted or processed as needed. The target molecule was dissolved in a buffer solution and added to the wells of an enzyme-linked immunosorbent assay plate. The solid phase was allowed to interact with the target molecule, and the plate was incubated at room temperature or under refrigerated conditions. This step allows the target molecule to adsorb onto the solid phase[17].

Mitochondrial membrane potential measurement

The mitochondrial membrane potential (ΔΨm) was determined according to the manufacturer’s instructions. After washing the cells, a JC-1 working solution was labeled for 20 minutes (37 °C). The dual fluorescence imaging of JC-1 was completed using a RuoChuang camera[18]. Using the JC-1 monomer level to assess mitochondrial health is of great significance as it can reflect changes in ΔΨm. ΔΨm is an important indicator of mitochondrial function, and its decrease is usually associated with early apoptosis events. As a fluorescent probe, JC-1 can exhibit different fluorescent properties under different ΔΨm states. When ΔΨm is high, JC-1 is in a polymeric form inside the mitochondria, emitting red fluorescence; however, when ΔΨm decreases, JC-1 exists in a monomeric form in the cytoplasm, emitting green fluorescence. This change in fluorescence color provides an intuitive and sensitive method for detecting ΔΨm.

In research on diabetic intestinal injury, a specific detection method using JC-1 can help reveal changes in mito-chondrial function under diabetic conditions. Diabetes may lead to mitochondrial dysfunction, including decreased ΔΨm, thereby affecting the energy metabolism of intestinal cells and cell apoptosis. Through JC-1 detection, researchers can observe changes in ΔΨm of intestinal cells in a diabetic mouse model and further explore the impact of diabetes on mitochondrial function in intestinal cells.

Furthermore, the versatility of the JC-1 staining method makes it suitable for various cell types, including muscle cells and neurons, and as an indicator of ΔΨm in intact tissues and isolated mitochondria. The high affinity of JC-1 staining reagents for mitochondria and its application in flow cytometry analysis makes it an indispensable tool for studying mitochondrial health and apoptosis.

Statistical analysis

Statistical analysis was conducted using GraphPad Prism 9.0. The normality of the data was assessed using the Kolmogorov-Smirnov test. Subsequently, Tukey’s post hoc test was used for multiple group comparisons conducted through a one-way analysis of variance. Quantitative data are presented as mean ± SD. P value of < 0.05 were considered statistically significant.

RESULTS

GSE63992 of DEGs

The heatmap of gene differences and volcano plots are presented in the figures. In total, 34687 genes were identified based on the following criteria: |LogFC| > 2 and P value < 0.05 (3 genes); |LogFC| > 1 and P value < 0.05 (75 genes); and |LogFC| > 0.58 and P value < 0.05 (514 genes). Among identified genes, FAM83D, FCN3, PLAC9, MMP23B, FAM107A, SELM, ISG15, AAAS, RASL10A, and KLC2 were upregulated in samples from diabetic individuals (Figure 1).

Figure 1 Data processing and differentially expressed gene identification. A: Heatmap of sample-to-sample correlation. The diabetes group displayed a stronger intragroup correlation than the control group; B: Volcano plot generated using the limma R tool, showcasing all differentially expressed genes in the control and diabetes groups.

GSE63992 was performed on the Gene Set Enrichment Analysis result

The enriched genes demonstrated significant involvement in diverse biological processes, including the KEGG metabolism of xenobiotics by cytochrome P450, reactome biological oxidations, KEGG drug metabolism cytochrome P450, NABA ECM glycoproteins, KEGG retinol metabolism, NABA core matrisome, reactome drug ADME, KEGG steroid hormone biosynthesis, reactome phase II conjugation of compounds, and WP metapathway biotransformation phase I and II (Figure 2).

Figure 2 Functional and pathway enrichment analyses performed on the Gene Set Enrichment Analysis results. Functional and pathway enrichment analyses were conducted using DAVID to gain a comprehensive understanding of the role of the 10 differentially regulated pathways identified in the intestinal tissue dataset. A-D: Genes displaying significant enrichment in various biological processes: Reactome metabolism of fat-soluble vitamins, Kyoto Encyclopedia of Genes and Genomes complement and coagulation cascades, wp network map of the sarscov2 signaling pathway, reactome olfactory signaling pathway, reactome metabolism of vitamins and cofactors, reactome biological oxidations, reactome neutrophil degranulation, reactome metabolism of amino acids and derivatives, reactome metabolism of lipids, and reactome platelet activation signaling and aggregation. KEGG: Kyoto Encyclopedia of Genes and Genomes.

Functional and pathway enrichment

Figure 3A displays the expression of multiple inflammatory factors in the control and experimental groups. These factors are primarily linked to cellular hormone metabolic processes, hormone metabolism, digestion, responses to toxic substances, retinol metabolism, collagen-containing extracellular matrix, brush border, and cluster of actin-based cell projections space. They are also enriched in various KEGG pathways (Figure 3).

Figure 3 Functional and pathway enrichment. Functional and pathway enrichment analyses were conducted using DAVID to gain a comprehensive understanding of the role played by the 31 differentially expressed genes identified in the brain tissue dataset. A-C: Main identified pathways: Cellular hormone metabolic process, hormone metabolic process, digestion, response to toxic substance, retinol metabolic process, collagen-containing extracellular matrix, brush border, and cluster of actin-based cell projections. KEGG: Kyoto Encyclopedia of Genes and Genomes; BP: Biological process; CC: Cell composition; MF: Molecular function; GO:0034754: Cellular hormone metabolic process; GO:0042445: Hormone metabolic process; GO:0007586: Digestion; GO:0062023: Collagen-containing extracellular matrix; GO:0005903: Brush border; GO:0098862: Cluster of actin-based cell projections; GO:0016616: Oxidoreductase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor; GO:0016614: Oxidoreductase activity, acting on CH-OH group of donors; GO:0004032: Alditol:NADP+ 1-oxidoreductase activity; hsa00982: Drug metabolism-cytochrome P450; hsa00980: Metabolism of xenobiotics by cytochrome P450; hsa00830: Retinol metabolism.

Protein–protein interaction network analysis and inflammatory factor detection

The integrated analysis of LogFC and KEGG pathways highlighted the consistent involvement of MMP23B in these biological processes. Subsequently, a protein-protein interaction network comprising 11 DEGs was established using the STRING database and illustrated through the utilization of the Cytoscape tool (Figure 4A and B). In the diabetic nephropathy (DN) group, MMP23B, MMP19, and HPX were upregulated. Interestingly, DEX treatment reduced the expression levels of inflammatory markers in contrast to the DB group (Figure 4C-E).

Figure 4 Protein-protein interaction network analysis and detected inflammatory factors. The protein-protein interaction network comprised five nodes representing molecular pathways and processes associated with the five core differentially expressed genes. A and B: Working modules were developed using the MCODE plugin for Cytoscape; C-E: The results revealed a module of nine genes. The expressions of HPX, MMP19, and MMP23 were elevated in the diabetes group. The expression levels of inflammatory factors decreased upon dexmedetomidine compared with those in the control group. dP < 0.0001. DB: Diabetes group; DEX: Dexmedetomidine.

The extent of STZ-induced intestinal injury is positively correlated with MMP23B in mice

We used MMP23B KO mice to understand the role of MMP23B in intestinal injury. Hematoxylin and eosin staining indicated less severe injury in MMP23B KO mice than in WT ones (Figure 5A-C). Immunofluorescence analysis indicated that the intestinal injury marker was markedly reduced after 24 hours of treatment (Figure 5D-I). Furthermore, protein expression levels of occludin and ZO-1 were markedly increased in the MMP23B KO group compared with the WT group, as observed by immunofluorescence (Figure 5J and K).

Figure 5 The severity of diabetes-induced intestinal injury was positively correlated with MMP23 expression in mice. MMP23 KO mice were used to investigate the role of MMP23 in intestinal injury. A-C: Hematoxylin and eosin staining revealed a less severe injury in the intestines of MMP23 knockout mice; D-I: Immunofluorescence analysis demonstrated a significant reduction in the intestinal injury marker after 24 hours of treatment; J and K: The protein expression of occludin and ZO-1 was significantly upregulated following MMP23 knockout, as revealed by immunofluorescence analysis. Scale bar = 50 μm. bP < 0.01, cP < 0.001, dP < 0.0001. DB: Diabetes group.

In vivo experiments demonstrated that macrophage clearance can improve intestinal function and the epithelium

To elucidate the role of macrophages in intestinal injury, we depleted macrophages using Cls in mice. Hematoxylin and eosin staining revealed mild injury in mice treated with Cls (Figure 6A-D). Immunofluorescence analysis demonstrated a significant induction in ZO-1 and reduction in CD86 levels after 24 hours of Cl treatment (Figure 6E-L). In addition, immunofluorescence analysis revealed a significant increase in the protein expression of CD86 and ZO-1 in MMP23B KO mice (Figure 6M and N).

Figure 6 In vivo experiments demonstrated that macrophage clearance can improve intestinal function and epithelium integrity. We depleted macrophages using clodronate liposomes in mice to elucidate the role of macrophages in intestinal injury. A-D: Hematoxylin and eosin staining revealed less severe injury in mice treated with clodronate liposomes; E-L: Immunofluorescence analysis demonstrated a significant induction in ZO-1 and reduction in CD86 levels 24 hours after clodronate liposome treatment; M and N: Immunofluorescence analysis revealed a significant increase in the protein expression of CD86 and ZO-1 in MMP23 knockout mice. Scale bar = 50 μm. aP < 0.05, bP < 0.01, cP < 0.001, dP < 0.0001. DB: Diabetes group.

Macrophage clearance can improve intestinal function and the epithelium in diabetic mice

Immunofluorescence analysis demonstrated significantly induced ZO-1 and occludin levels after 24 hours of Cl treatment (Figure 7A-J). Immunofluorescence analysis also revealed a significant decrease in the protein expression of occludin and ZO-1 in MMP23B KO mice (Figure 7K and L).

Figure 7 Macrophage clearance enhances intestinal function and improves the epithelium in diabetes mice. A-J: After 24 hours of treatment with clodronate liposomes, immunofluorescence analysis revealed a substantial decrease in ZO-1 and occludin levels; K and L: The immunofluorescence analysis demonstrated a notable elevation in the protein expression of occludin and ZO-1 following DB. Scale bar = 50 μm. aP < 0.05, cP < 0.001, dP < 0.0001. DB: Diabetes group; Cls: Clodronate liposomes.

In vivo studies indicated that MMP23B KO induces the expression of arginase-1 and interleukin-10

In vivo experiments demonstrated that MMP23B KO resulted in M2 polarization. Following DB induction with STZ, the fluorescence intensity of arginase-1 (Arg-1) (Figure 8A-I) and interleukin (IL)-10 (Figure 8J-R) significantly decreased. Compared with the DB pretreatment levels, the fluorescence intensity levels were induced in MMP23B KO mice (Figure 8S and T).

Figure 8 In vivo studies demonstrated that MMP23 knockout upregulates arginase-1 and interleukin-10. A-R: MMP23 knockout led to M2 polarization. After inducing diabetes with the diabetes treatment, the fluorescence intensity of arginase-1 (A-I) and interleukin-10 (J-R) significantly decreased compared with that before treatment; S and T: Indicating fluorescence intensity levels after MMP23 knockout treatment. Scale bar = 50 μm. cP < 0.001, dP < 0.0001. DB: Diabetes group; Arg-1: Arginase-1; IL-10: Interleukin-10.

In vitro models demonstrated that GM6001 inhibits intestinal epithelium injury

Fluorescence intensity levels of occludin (Figure 9A-C) and ZO-1 (Figure 9D-F) significantly decreased after MMP23B treatment compared with the baseline levels. Treating the MMP23B group with GM6001 improved the fluorescence intensity levels of occludin (Figure 9G) and ZO-1 (Figure 9H).

Figure 9 In vitro models demonstrated that GM6001 inhibits intestinal epithelium damage. A-F: Following lipopolysaccharide treatment, a substantial decrease was observed in the fluorescence intensity levels of occludin (A-C) and ZO-1 (D-F) compared with the baseline levels; G and H: Administering GM6001 to the MMP23B group improved the fluorescence intensity levels of occludin (G) and ZO-1 (H). Scale bar = 50 μm. cP < 0.001, dP < 0.0001. HG: High glucose.

DEX induces mitochondrial impairment in Caco-2 cells in vitro

We investigated the effects of DEX on MMP23B-induced mitochondrial dysfunction in Caco-2 cells. Our results revealed that compared with a high glucose environment alone, treating cells with DEX decreases JC-1 monomer levels and increases ΔΨm, indicating that DEX can reverse the mitochondrial damage induced by high glucose (Figure 10).

Figure 10 Dexmedetomidine induces mitochondrial impairment in Caco-2 cells in vitro. A-M: During inflammation, mitochondrial oxidative energy metabolism plays a pivotal role in maintaining the integrity of the gut epithelial barrier. We investigated the potential effects of dexmedetomidine on high glucose-induced mitochondrial dysfunction in Caco-2 cells. Comparative analysis revealed that dexmedetomidine treatment, as opposed to high glucose treatment alone, substantially reduced mitochondrial dysfunction, manifested through reduced levels of JC-1 monomers and enhanced ΔΨm. aP < 0.05, cP < 0.001. HG: High glucose; DEX: Dexmedetomidine; PBS: Phosphate buffer saline; GM: GM6001.

DISCUSSION

Our study reveals a novel role of DEX in modulating intestinal function, particularly in the context of diabetes. DEX significantly influences intestinal motility and immune responses by interacting with the MMP23B pathway[19,20]. This finding is pivotal as it suggests that DEX counteracts diabetes-related intestinal tissue damage by regulating the activity of M1 macrophages and expression of MMP23B, a key inflammatory factor.

In the diabetic state, the intestinal microenvironment is altered, leading to inflammation and subsequent damage. This state is characterized by the release of cytokines, which can cause tissue damage when dysregulated[21,22]. Our bioinformatics analysis revealed that in addition to MMP19 and MMP8, MMP23B was upregulated in the diabetic group, indicating its potential role in the pathogenesis of diabetes-related intestinal complications.

Furthermore, our mechanistic experiments revealed that DEX decreased the expression levels of these inflammatory factors, suggesting its protective effect on intestinal tissues. MMP23B, with its capacity to degrade extracellular matrix proteins, is implicated in various biological processes, including central nervous system development and inflammatory responses. Its modulation by DEX may be central to mitigating diabetes-induced intestinal disruptions. Elucidating the effect of DEX on MMP23B can provide a new perspective on the management of diabetes-related gastrointestinal complications and open avenues for future research into targeted therapies that leverage this pathway for intestinal protection and repair.

These cytokines are crucial in regulating immune responses and the inflammatory process, but their excessive or improper release may damage intestinal tissues. Through bioinformatics analysis, we identified three genes with Log|FC| > 2 and P value < 0.05 in the DB group, among which the expression of the inflammatory factor MMP23B was elevated. Further gene enrichment analysis revealed cellular metabolism and extracellular matrix glycoprotein secretion. In the DN group, the expression of MMP23B, MMP19, and MMP8 was elevated. DEX decreased the expression levels of inflammatory factors compared with those in the DB group.

MMP23B is involved in central nervous system development, inflammatory responses, and invasion and metastasis. It has a typical metalloproteinase structure, including a signal peptide sequence, a protease active site domain, and a cysteine/cysteine repeat sequence. The zinc ions in the MMP23B active domain can participate in the degradation of extracellular matrix proteins, playing a role in reshaping the extracellular matrix and regulating cellular behavior[23,24]. However, the impact of MMP23B on intestinal macrophages remains unclear.

M1 macrophages, also known as classically activated macrophages, promote inflammation and host defense in response to infection or injury[25,26]. In diabetes-related intestinal tissue, M1 macrophages can contribute to the immune response and tissue damage. During diabetes, the intestinal tissue can be exposed to bacterial pathogens or their components due to increased intestinal permeability. This exposure triggers an inflammatory response, leading to M1 macrophage activation in the intestinal tissue. These cytokines contribute to recruiting and activating other immune cells, enhancing the inflammatory response in diabetes-related intestinal tissues[27,28]. M1 macrophages have enhanced phagocytic capacity and microbial killing abilities. They can engulf and eliminate pathogens, thereby helping in controlling infection in individuals with diabetes. However, excessive M1 macrophage activation can lead to tissue damage due to the release of cytotoxic molecules. Suppression of IL-10 promotes inflammatory reactions and impairs M1 macrophage polarization by targeting MMP23B, which is critical in macrophage polarization[29-31].

Our study demonstrated that MMP23B is responsible for macrophage polarization, highlighting its unique role in M1 macrophage polarization and potential contribution to the development of diabetes-related intestinal damage in mice. Hematoxylin and eosin staining indicated less severe injury in MMP23B KO mice. Immunofluorescence analysis indicated that the intestinal injury marker was markedly reduced after 24 hours of treatment. Furthermore, the protein expression of occludin and ZO-1 was markedly increased after MMP23B KO, as observed via immunofluorescence analysis.

While the role of MMP23B in the intestine remains unclear, our study revealed that knocking out MMP23B downregulates the intestinal tight junction (TJ) proteins ZO-1 and occludin and upregulates proinflammatory factors. These results provide a deeper understanding of MMP23B. Previous studies have reported that IL-1β stimulates periodontal inflammation during RR, inducing IL-10 and tumor necrosis factor-α synthesis and secretion. However, the initial source of IL-10 and the M2 subtype that possesses anti-inflammatory properties remain unclear. ZO-1, the earliest discovered epithelial TJ protein, is crucial in cell function and permeability[32-36]. Accordingly, blocking or removing ZO-1 in cells did not prevent the assembly of TJs or hinder the development of barrier functions.

We depleted macrophages using Cls in mice to elucidate the role of macrophages in intestinal injury[37,38]. Hematoxylin and eosin staining revealed mild injury in mice treated with Cls. Immunofluorescence analysis demonstrated a significant reduction in ZO-1 and CD86 levels after 24 hours of Cl treatment and a significant increase in the protein expression of CD86 and ZO-1 after MMP23 knockout. In vivo experiments revealed that knocking out MMP23B led to M2 polarization.

Upon STZ induction through DB treatment, fluorescence intensity levels of Arg-1 and IL-10 were notably reduced. Compared with the pretreatment levels, the fluorescence intensity levels displayed a decrease after MMP23B KO. Mitochondria have various critical functions in cells and are closely related to cell damage. During energy production, mitochondria produce some byproducts, such as reactive oxygen species. When reactive oxygen species are produced excessively, they may lead to oxidative stress, thereby damaging mitochondria and other organelles and further exacerbating cell damage[39]. The findings of the present study indicated that treatment with DEX, as opposed to treatment with MMP23B alone, significantly reduced mitochondrial dysfunction, as evidenced by decreased levels of JC-1 monomers and an improved ΔΨm.

Although animal models have provided valuable insights, the complexity of the human diabetic intestinal damage has not been fully understood. Careful consideration and rigorous validation are required to translate our findings from mouse experiments into clinical applications. Moreover, further investigation into the exact role of MMP23B in diabetes-related inflammation is needed.

CONCLUSION

In conclusion, our study indicated that DEX reduces MMP23B, which is a potential contributor to intestinal barrier dysfunction that may induce diabetes, affecting TJ changes through mitochondrial dysfunction.

Data sharing statement

Raw data can be obtained by contacting the corresponding author.

Institutional animal care and use committee statement: This study was reviewed and approved by the Medical Ethics Committee of Xinhua Hospital, Shanghai Jiao Tong University School of Medicine (Approval No. XHEC-STCSM-2021-028).

Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article.

ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.

Provenance and peer review: Unsolicited article; Externally peer reviewed.

Peer-review model: Single blind

Specialty type: Endocrinology and metabolism

Country of origin: China

Peer-review report’s classification

Scientific Quality: Grade B, Grade B, Grade C, Grade C

Novelty: Grade B, Grade B

Creativity or Innovation: Grade B, Grade C

Scientific Significance: Grade B, Grade B

P-Reviewer: Barbalho SM; Dąbrowski M; Horowitz M; Sahu SK S-Editor: Wang JJ L-Editor: A P-Editor: Zheng XM
==== Refs
1 Elashi AA Toor SM Umlai UI Al-Sarraj YA Taheri S Suhre K Abou-Samra AB Albagha OME Correction: Genome-wide association study and trans-ethnic meta-analysis identify novel susceptibility loci for type 2 diabetes mellitus BMC Med Genomics 2024 17 131 38755684
2 Merlo EM Tutino R Myles LAM Lia MC Minasi D Alexithymia, intolerance to uncertainty and mental health difficulties in adolescents with Type 1 diabetes mellitus Ital J Pediatr 2024 50 99 38755698
3 Zeng X Huang S Ye X Song S He J Hu L Deng S Liu F Impact of HbA1c control and type 2 diabetes mellitus exposure on the oral microbiome profile in the elderly population J Oral Microbiol 2024 16 2345942 38756148
4 Ahn JH Lopez L Tibbs T Yu YR Tighe R Denson L Arthur J Zinc restriction by intestinal E. coli - produced Yersiniabactin activates HIF-1α in macrophages to promote CD-associated fibrosis Inflamm Bowel Dis 2024 30 S62 S62
5 Zhang S Zhang C Yan H Yang L Shi N Liu C Chen Y Sacral Nerve Stimulation Alleviates Intestinal Inflammation Through Regulating the Autophagy of Macrophages and Activating the Inflammasome Mediated by a Cholinergic Antiinflammatory Pathway in Colitis Rats Neuromodulation 2024 27 302 311 36740464
6 Zhu L Dou Z Wu W Hou Q Wang S Yuan Z Li B Liu J Ghrelin/GHSR Axis Induced M2 Macrophage and Alleviated Intestinal Barrier Dysfunction in a Sepsis Rat Model by Inactivating E2F1/NF-κB Signaling Can J Gastroenterol Hepatol 2023 2023 1629777 38187112
7 Deng S Li W Li Z Wang P Ma Q Bright luminescent Zn(2)GeO(4):Mn NP/MXene hydrogel-based ECL biosensor for glioblastoma diagnosis Talanta 2024 276 126214 38718647
8 Liu C Xu R Dexmedetomidine protects H9C2 rat cardiomyocytes against hypoxia/reoxygenation injury by regulating the long non-coding RNA colon cancer-associated transcript 1/microRNA-8063/Wnt/β-catenin axis Bioengineered 2022 13 13300 13311 35635079
9 Pluskiewicz W Werner A Bach M Adamczyk P Drozdzowska B Fracture risk prediction in postmenopausal women from GO Study: the comparison between FRAX, Garvan, and POL-RISK algorithms Arch Osteoporos 2024 19 39 38755326
10 Rocca MA Valsasina P Romanò F Tedone N Amato MP Brichetto G Boccia VD Chataway J Chiaravalloti ND Cutter G Dalgas U DeLuca J Farrell RA Feys P Freeman J Inglese M Meza C Motl RW Salter A Sandroff BM Feinstein A Filippi M Cogex Research Team Cognitive rehabilitation effects on grey matter volume and Go-NoGo activity in progressive multiple sclerosis: results from the CogEx trial J Neurol Neurosurg Psychiatry 2024
11 Chen S Li Y Song W Cheng Y Gao Y Xie L Huang M Yan X Insulin eye drops improve corneal wound healing in STZ-induced diabetic mice by regulating corneal inflammation and neuropeptide release BMC Ophthalmol 2024 24 155 38594682
12 Tabaa MME Tabaa MME Rashad E Elballal MS Elazazy O Harmine alleviated STZ-induced rat diabetic nephropathy: A potential role via regulating AMPK/Nrf2 pathway and deactivating ataxia-telangiectasia mutated (ATM) signaling Int Immunopharmacol 2024 132 111954 38554444
13 Culemann S Knab K Euler M Wegner A Garibagaoglu H Ackermann J Fischer K Kienhöfer D Crainiciuc G Hahn J Grüneboom A Nimmerjahn F Uderhardt S Hidalgo A Schett G Hoffmann MH Krönke G Addendum: Stunning of neutrophils accounts for the anti-inflammatory effects of clodronate liposomes J Exp Med 2023 220
14 Mochalova EN Egorova EA Komarova KS Shipunova VO Khabibullina NF Nikitin PI Nikitin MP Comparative Study of Nanoparticle Blood Circulation after Forced Clearance of Own Erythrocytes (Mononuclear Phagocyte System-Cytoblockade) or Administration of Cytotoxic Doxorubicin- or Clodronate-Loaded Liposomes Int J Mol Sci 2023 24
15 Fang M Yin W Qiu C Song T Lin B Wang Y Xiong H Wu S Stromal B Lymphocytes Affecting Prognosis in Triple-Negative Breast Cancer by Opal/TSA Multiplexed Immunofluorescence Int J Womens Health 2024 16 755 767 38706691
16 Tomimatsu K Fujii T Bise R Hosoda K Taniguchi Y Ochiai H Ohishi H Ando K Minami R Tanaka K Tachibana T Mori S Harada A Maehara K Nagasaki M Uchida S Kimura H Narita M Ohkawa Y Precise immunofluorescence canceling for highly multiplexed imaging to capture specific cell states Nat Commun 2024 15 3657 38719795
17 Roelofs L Frössling J Rosander A Bjerketorp J Belaghi RA Hansson I Frosth S Digital dermatitis in Swedish dairy herds assessed by ELISA targeting Treponema phagedenis in bulk tank milk BMC Vet Res 2024 20 168 38698418
18 Małota K Student S Świątek P Low mitochondrial activity within developing earthworm male germ-line cysts revealed by JC-1 Mitochondrion 2019 44 111 121 29398303
19 Ekkapat G Kampitak W Theerasuwipakorn N Kittipongpattana J Engsusophon P Phannajit J Chokengarmwong N A Comparison of Efficacy between Low-dose Dexmedetomidine and Propofol for Prophylaxis of Postoperative Delirium in Elderly Patients Undergoing Hip Fracture Surgery: A Randomized Controlled Trial Indian J Crit Care Med 2024 28 467 474 38738208
20 Sun Y Darmani NA A Comparative Study of the Antiemetic Effects of α(2)-Adrenergic Receptor Agonists Clonidine and Dexmedetomidine against Diverse Emetogens in the Least Shrew (Cryptotis parva) Model of Emesis Int J Mol Sci 2024 25
21 Frühbeck G Gómez-Ambrosi J Ramírez B Becerril S Rodríguez A Mentxaka A Valentí V Moncada R Reina G Baixauli J Casado M Silva C Escalada J Catalán V Decreased expression of the NLRP6 inflammasome is associated with increased intestinal permeability and inflammation in obesity with type 2 diabetes Cell Mol Life Sci 2024 81 77 38315242
22 Liu X Fang H Pan L Zhang P Lin H Gao H Ye C Mao D Luo Y S-amlodipine induces liver inflammation and dysfunction through the alteration of intestinal microbiome in a rat model Gut Microbes 2024 16 2316923 38400721
23 Allione A Pardini B Viberti C Giribaldi G Turini S Di Gaetano C Guarrera S Cordero F Oderda M Allasia M Gontero P Sacerdote C Vineis P Matullo G MMP23B expression and protein levels in blood and urine are associated with bladder cancer Carcinogenesis 2018 39 1254 1263 30052775
24 Pardini B Allione A Viberti C Giribaldi G Turini S Gaetano CD Guarrera S Cordero F Oderda M Allasia M Gontero P Sacerdote C Naccarati A Vineis P Matullo G Abstract 1258: MMP23B expression and protein levels in blood and urine are associated with bladder cancer risk Cancer Res 2018 78 1258
25 Choi JY Byeon HW Park SO Uyangaa E Kim K Eo SK Inhibition of NADPH oxidase 2 enhances resistance to viral neuroinflammation by facilitating M1-polarization of macrophages at the extraneural tissues J Neuroinflammation 2024 21 115 38698374
26 Li Y Guo X Zhan P Huang S Chen J Zhou Y Jiang W Chen L Lin Z TRPV1 Regulates Proinflammatory Properties of M1 Macrophages in Periodontitis Via NRF2 Inflammation 2024
27 Gonciarz W Piątczak E Chmiela M The influence of Salvia cadmica Boiss. extracts on the M1/M2 polarization of macrophages primed with Helicobacter pylori lipopolysaccharide in conjunction with NF-kappa B activation, production of cytokines, phagocytic activity and total DNA methylation J Ethnopharmacol 2023 310 116386 36921911
28 Mathiesen CBK Rudjord-Levann AM Gad M Larsen J Sellebjerg F Pedersen AE Cladribine inhibits secretion of pro-inflammatory cytokines and phagocytosis in human monocyte-derived M1 macrophages in-vitro Int Immunopharmacol 2021 91 107270 33360830
29 Honjoh K Nakajima H Hirai T Watanabe S Matsumine A Relationship of Inflammatory Cytokines From M1-Type Microglia/Macrophages at the Injured Site and Lumbar Enlargement With Neuropathic Pain After Spinal Cord Injury in the CCL21 Knockout (plt) Mouse Front Cell Neurosci 2019 13 525 31824269
30 Inoue Y Kamiya T Hara H Increased expression of ELOVL7 contributes to production of inflammatory cytokines in THP-1 cell-derived M1-like macrophages J Clin Biochem Nutr 2023 72 215 224 37251958
31 Jędrzejewski T Pawlikowska M Sobocińska J Wrotek S Protein-Bound Polysaccharides from Coriolus Versicolor Fungus Disrupt the Crosstalk Between Breast Cancer Cells and Macrophages through Inhibition of Angiogenic Cytokines Production and Shifting Tumour-Associated Macrophages from the M2 to M1 Subtype Cell Physiol Biochem 2020 54 615 628 32559360
32 Georgiadis A Tschernutter M Bainbridge JWB Balaggan KS Mowat F West EL Munro PMG Thrasher AJ Matter K Balda MS Ali RR Correction: The Tight Junction Associated Signalling Proteins ZO-1 and ZONAB Regulate Retinal Pigment Epithelium Homeostasis in Mice PLoS One 2023 18 e0295782 38060570
33 Schmidt A Finegan T Häring M Kong D Fletcher AG Alam Z Grosshans J Wolf F Peifer M Polychaetoid/ZO-1 strengthens cell junctions under tension while localizing differently than core adherens junction proteins Mol Biol Cell 2023 34 ar81 37163320
34 Wang K Hongyan Q Lassegue B Eaton DCC Song C Mu J Griendling KK Hernandes MS Abstract 12294: Poldip2 Mediates Brain Vascular Permeability by Regulating ZO-1 Phosphorylation and Localization at the Interendothelial Border Circulation 2023 148
35 Wu H Wu L Yu W Gu C Li Y Chen K Zhang L Qian F Veronica linariifolia subsp. dilatata ameliorates LPS-induced acute lung injury by attenuating endothelial cell barrier dysfunction via EGFR/Akt/ZO-1 pathway J Ethnopharmacol 2024 321 117545 38056533
36 Zhuang X Martin TA Ruge F Zeng JJ Li XA Khan E Dou Q Davies E Jiang WG Expression of Claudin-9 (CLDN9) in Breast Cancer, the Clinical Significance in Connection with Its Subcoat Anchorage Proteins ZO-1 and ZO-3 and Impact on Drug Resistance Biomedicines 2023 11
37 Li Y Wu L Yong Y Niu X Gao Y Zhou Q Xie H Liu X Li Y Yu Z Abd El-Aty AM Ju X Enhancing gut barrier integrity: Upregulation of tight junction proteins by chitosan oligosaccharide through the ERK1/2 signaling pathway Nutrition 2024 124 112428 38663127
38 Tu J Jiang Y Tu L Chen Y Pan L Fan X Tian J Li J Wang X Fu H Xu B Feng D Da-Cheng-Qi decoction improves severe acute pancreatitis capillary leakage syndrome by regulating tight junction-associated proteins Front Pharmacol 2024 15 1138251 38708079
39 Masuda D Nakanishi I Ohkubo K Ito H Matsumoto KI Ichikawa H Chatatikun M Klangbud WK Kotepui M Imai M Kawakami F Kubo M Matsui H Tangpong J Ichikawa T Ozawa T Yen HC St Clair DK Indo HP Majima HJ Mitochondria Play Essential Roles in Intracellular Protection against Oxidative Stress-Which Molecules among the ROS Generated in the Mitochondria Can Escape the Mitochondria and Contribute to Signal Activation in Cytosol? Biomolecules 2024 14
