Tong-Xie-Yao-Fang Modulates Mucosal Repair via CRH-TLR4- Mediated Autophagy in Stress-Induced Colitis

Tong-Xie-Yao-Fang Modulates Mucosal Repair via CRH-TLR4- Mediated Autophagy in Stress-Induced Colitis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Tong-Xie-Yao-Fang Modulates Mucosal Repair via CRH-TLR4- Mediated Autophagy in Stress-Induced Colitis Menglin Li, Rong Huang, Mingxu Zheng, Wanting Cao, Bin Lv, Jing Zhao, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5426177/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Background: Autophagy plays an essential role in inflammatory processes and mucosal repair, with Corticotropin-Releasing Hormone (CRH) and Toll-Like Receptor 4 (TLR4) also playing significant roles in regulating cellular autophagy. We hypothesized that disruptions in the CRHR2-mediated CRH-TLR4 signaling axis are associated with altered autophagy in intestinal epithelial cells (IECs) in ulcerative colitis. Furthermore, we investigated how these disruptions impact the mucosal repair mechanisms that are enhanced by Tong-Xie-Yao-Fang(TXYF). Methods: A 3% dextran sodium sulfate (DSS) solution was administered to mice for five days to induce a colitis model. Restraint stress was applied for 2 hours daily. while the CRHR2 antagonist Astressin 2B (Ast2B) at a dose of 20 μg/(kg·day) was given intraperitoneally, the CRHR2 agonist UCN2 at 30 μg/(kg·day) was administered peritoneally. TXYF at 5.6 g/(kg·day) was delivered by gavage, all for nine consecutive days. Body weight, fecal characteristics, and occult blood were monitored across all groups of mice throughout the experiment. The mice were euthanized by cervical dislocation, and the entire colon distal to the ileum was immediately dissected for further analysis. This included histological scoring of the colon, ELISA to detect changes in CRH and TLR4 levels in the colonic mucosa, Western blot analysis to assess autophagy through the LC3II/LC3I ratio and P62 levels, and in situ detection of apoptosis in intestinal epithelial cells using the TUNEL method. Results: Compared to the DSS group, DSS mice subjected to daily restraint stress exhibited increased TLR4 expression (P=0.0241<0.05), greater weight loss (P=0.0343<0.05), higher histological scores (P=0.0171<0.05), and an elevated apoptosis index (P=0.0375<0.05). Additionally, these mice had higher serum IL-6 levels than those given DSS alone (P=0.0004<0.05). The DSS+Ast2B group showed a higher LC3BⅡ/LC3BⅠ ratio compared to the DSS+NS group (P=0.0417<0.05). In contrast, the DSS+Ucn2 group had lower LC3BⅡ/LC3BⅠ (P=0.0011<0.05) and TLR4 (P=0.0025<0.05) protein expression, along with higher p62 protein levels (P=0.0129<0.05) compared to the DSS+NS group. Correlation analysis revealed a significant association between TLR4 and autophagy-related proteins (LC3 and p62) (P<0.05). Compared to the DSS+STRESS+NS group, the DSS+STRESS+TXYF group showed lower expression of CRH (P=0.0031<0.05) and TLR4 (P=0.0058<0.05), with reduced autophagy levels (all P<0.05). Additionally, the DSS+STRESS+TXYF group demonstrated increased colon length (P=0.0004<0.05) and reduced DAI scores (P=0.0384<0.05), histopathological scores (P=0.0015<0.05), and apoptosis levels (P=0.0125<0.05). Compared to the DSS+STRESS+Ast2B group, the DSS+STRESS+TXYF+Ast2B group showed lower expression of CRH (P=0.0109<0.05) and TLR4 (P=0.0057<0.05), along with reduced levels of the autophagy-related protein LC3 (P=0.0146<0.05). Furthermore, the DSS+STRESS+TXYF+Ast2B group exhibited increased colon length (P<0.0001) and reduced DAI scores (P<0.0001), histopathological scores (P<0.0001), and apoptosis levels (P=0.0002<0.05). Conclusion: Restraint stress enhances the expression of TLR4, thereby exacerbating colitis. CRHR2 serves to inhibit TLR4 protein expression, consequently reducing autophagy levels in intestinal epithelial cells. TXYF mediates the effects of CRHR2 on the CRH-TLR4 signaling axis, diminishing apoptosis in intestinal epithelial cells, inhibiting autophagy, preserving the integrity of the intestinal mucosal barrier, and exerting anti-inflammatory effects. Health sciences/Gastroenterology Health sciences/Pathogenesis Tong-Xie-Yao-Fang Ulcerative colitis autophagy stress Corticotropin-Releasing Hormone Receptor 2 CRH-TLR4 axis Figures Figure 1 Figure 2 Figure 3 1. Introduction Inflammatory Bowel Disease (IBD) is a chronic, recurring inflammatory condition of the intestines influenced by a complex interplay of neural, epigenetic, and environmental factors. It encompasses two main types: Ulcerative Colitis (UC) and Crohn's Disease (CD). The precise etiology and pathogenesis of IBD remain unclear. Globally, the incidence of Ulcerative Colitis (UC) is rising, with common symptoms including bloody stools, diarrhea, urgency, incontinence, fatigue, increased bowel frequency, and mucus in the stool [ 1 ] . Up to 15% of UC patients may require a colectomy [ 2 ] . Awareness of UC is growing in Asia, and with the rapid pace of urbanization and migration from rural to urban areas, along with increasing population density, the incidence of the disease in Asia is expected to continue increasing [ 3 , 4 ] . Recent studies have shown that autophagy is crucial in the inflammatory process and mucosal healing. Autophagy is considered an essential catabolic pathway in cells. Research indicates that the knockout of autophagy-related genes—such as Atg4B/autophagin-1, Atg16L1, and IRGM—significantly exacerbates the severity of IBD in animal models, suggesting that baseline autophagy is essential for maintaining intestinal homeostasis and the intestinal defense barrier [ 5 – 7 ] . However, other reports have noted that excessive activation of intestinal autophagy may worsen IBD [ 8 ] . Consequently, the role of autophagy in UC remains a topic of debate and requires further investigation. It has been confirmed that varying degrees of stress and associated psychosocial responses, such as anxiety and depression, contribute to the onset and progression of UC by damaging the intestinal mucosal barrier [ 9 , 10 ] . In the central nervous system, stress-induced physiological responses are triggered by the hypothalamic-pituitary-adrenal (HPA) axis, leading to the release of corticotropin-releasing hormone (CRH), which regulates neuroendocrine functions and immune responses in visceral organs. CRH is also present in peripheral tissues, including digestive organs and cells, where it directly modulates gastrointestinal inflammation. Psychosocial stress may further exacerbate intestinal autophagy by influencing gut microbiota and inflammation, worsening IBD [ 11 ] . TLR4 is expressed in immune and intestinal epithelial cells, functioning as an environmental sensor for autophagy. In both human and murine macrophages, autophagy and autophagy-mediated cell death can be triggered by lipopolysaccharide (LPS). Tong-Xie-Yao-Fang (TXYF), originating from the Jingyue Complete Book, is composed of four traditional Chinese herbs: stir-fried Atractylodes, stir-fried White Peony Root, stir-fried Tangerine Peel, and Siler Root. It is commonly used to treat Irritable Bowel Syndrome (IBS). There is a correlation and overlap in pathogenesis, genetic factors, gut microbiota dysbiosis, and immune dysfunction between IBS and Inflammatory Bowel Disease (IBD), with evidence of cross-treatment efficacy [ 13 , 14 ] . TXYF is effective for diarrhea-predominant IBS (IBS-D) and has been shown in animal studies to improve intestinal permeability and strengthen the intestinal mucosal barrier, likely by inhibiting inflammatory cascades and the NF-κB and Notch signaling pathways [ 15 ] . Our previous research found that TXYF downregulates CRHR1 and upregulates CRHR2, acting similarly to UCN2 by intervening through CRHR2 to regulate epithelial cell migration, proliferation, and apoptosis, thereby exerting anti-inflammatory effects and aiding mucosal healing in colitis mice [ 16 ] . We hypothesize that CRHR2-mediated CRH-TLR4 signaling axis disruptions are associated with autophagy in intestinal epithelial cells in ulcerative colitis. Furthermore, these disruptions may be linked to the mucosal repair mechanisms facilitated by the traditional Chinese medicine formula Tong-Xie-Yao-Fang (TXYF). The findings of this study will clarify whether stress-related CRHR2 regulation of the CRH-TLR4 signaling axis disruption is linked to mucosal repair and autophagy in intestinal epithelial cells (IECs) during dextran sodium sulfate (DSS)-induced colitis in mice. Additionally, the study will explore the mechanisms by which TXYF promotes mucosal repair. 2. Materials and methods 2.1 Animals experiments Male SPF-grade CD-1 (ICR) mice, aged 8–10 weeks and weighing 22g ± 2g, were acclimated for one week under controlled conditions of 22–24°C, 50–60% humidity, and a 12-hour light/dark cycle. All mice were purchased from Shanghai BK Laboratory Animal Co., Ltd. The mice (n = 110) were randomly divided into 11 groups: Control group (n = 10), DSS group (n = 10), DSS + Ast2B group (n = 10), DSS + UCN2 group (n = 10), DSS + TXYF group (n = 10), DSS + Ast2B + TXYF group (n = 10), DSS + Restraint Stress group (n = 10), DSS + Restraint Stress + Ast2B group (n = 10), DSS + Restraint Stress + UCN2 group (n = 10), DSS + Restraint Stress + TXYF group (n = 10), and DSS + Restraint Stress + Ast2B + TXYF group (n = 10). Dextran sodium sulfate (DSS) powder was dissolved in drinking water to prepare a 3% DSS solution, which was provided to the mice ad libitum for five consecutive days to induce a colitis model. From day 6 to day 15, the mice were switched to regular drinking water. TXYF formula consists of 15g of stir-fried Atractylodes, 12g of stir-fried White Peony Root, 10g of Siler, and 6g of dried Tangerine Peel, totaling 43g. The herbs were soaked in an appropriate amount of distilled water for 1 hour, then an additional eight times the volume of distilled water was added. The mixture was simmered for 30 minutes, filtered, and the filtrate from two extractions was combined. After vacuum filtration, the combined filtrate was concentrated by rotary evaporation to yield a 100% herbal solution (1mL contains 1g of raw herbs). The solution was stored at four°C and diluted with distilled water to the desired concentration before experiment use. CRHR2 antagonist Astressin 2B (Ast2B) was administered intraperitoneally at a dose of 20 µg/(kg·d), CRHR2 agonist UCN2 at 30 µg/(kg·d) was given intraperitoneally, restraint stress was applied for 2 hours per day, and TXYF was administered by gavage at 5.6 g/(kg·d) for nine days. Following the treatment period, mice were anesthetized by CO 2 , followed euthanized by cervical dislocation, and the entire colon distal to the cecum was immediately excised for subsequent analysis. All animal experiments were conducted according to the ethical principles adopted by the Animal Experimentation Center of Zhejiang Chinese Medical University and were approved by the Animal Ethics Committee of Zhejiang Chinese Medical University(Approval No. IACUC-20190401-12). All methods for animals were performed in accordance with the relevant guidelines and regulations. This study is reported in accordance with ARRIVE guidelines ( https://arriveguidelines.org ). 2.2Colon Disease Activity Index and Histologic Score The mice were evaluated using the Disease Activity Index (DAI) and histological scoring criteria. The general condition, body weight changes, stool consistency, and occult or gross blood in the stool were observed and recorded daily for all mice. The DAI score was computed based on the percentage of body weight loss, stool consistency, and the presence of occult or gross blood in the stool. The DAI score was determined as follows: DAI = (body weight loss score + stool consistency score + stool occult/gross blood score) / 3. Histological assessment was conducted in a double-anonymized manner. Mice were euthanized by cervical dislocation, and the abdomen was opened to isolate the colon for further analysis quickly. The colon length was measured, and it was then opened along the mesenteric axis. The lumen was rinsed with cold saline to remove debris, and the colon was examined macroscopically for signs of congestion and ulcers. Approximately 1 cm of colon tissue from the area with the most noticeable lesions was collected, fixed in 10% formalin, and processed for routine HE staining to assess morphological changes in the colonic mucosa. Histopathological scoring of the colon tissue was conducted, including assessments of inflammation, injury depth, crypt damage, and the extent of inflammation (%). The colon pathology score was calculated as the sum of the inflammation score, injury depth score, crypt damage score, and inflammation extent (%) score. 2.3 ELISA for CRH, TLR-4 Frozen colon tissue samples were sectioned, weighed, homogenized, and centrifuged (5810R tabletop high-speed refrigerated centrifuge, Eppendorf, Germany) to collect the supernatant using a double-antibody sandwich ABC-ELISA method. ELISA kits for mouse corticotropin-releasing hormone (CRH) and mouse Toll-like receptor 4 (TLR-4) (both from Shanghai Westang) were used. The assays were performed using a DENLEY DRAGON Wellscan MK 3 automatic microplate reader (Thermo, Finland). All tests were conducted according to the manufacturer's instructions. 2.4 Western Blot Using the Bicinchoninic Acid (BCA) protein quantification method, the tissue samples were ground in liquid nitrogen, and a protease inhibitor (Protease et al., cOmplet tabelet, TMEDTA-free Protease Inhibitor Cocktail Therapy) was added. The tissue was lysed with lysis buffer (RIPA et al., Ltd.), homogenized and centrifuged. Protein quantification was performed using a BCA Protein Assay Kit (Procell et al., Ltd.) according to the manufacturer's instructions. SDS-PAGE gels were prepared, and the samples were denatured before electrophoresis. A PVDF membrane (Millipore, USA) was used as the solid-phase support, and wet transfer was performed. The membrane was then incubated with primary antibodies (LC3A, LC3B, P62 from Cell Signaling Technology; β-actin from Proteintech Group) and HRP-conjugated secondary antibodies (Jackson ImmunoResearch). Detection was carried out using the FluorChem HD2 imaging system for direct imaging. The target molecular weight and grayscale values were analyzed using a gel image processing system and software. The LC3II/LC3I ratio and P62 levels were calculated to assess the level of autophagy. 2.5 Apoptosis of intestinal epithelial cells was detected in situ using the TUNEL assay Apoptosis of intestinal epithelial cells was assessed in situ utilizing the TUNEL assay (terminal deoxynucleotidyl transferase dUTP nick-end labeling), adhering to the protocol provided by the TUNEL Cell Apoptosis Detection Kit (Beyotime et al. C1088, Lot No. 030719190621). Cells with brown-yellow granules in their nuclei were classified as positive for apoptosis. Under an optical microscope, five random fields of view (×400) were selected, and the number of positive cells was counted to calculate the apoptosis index (AI), defined as AI = (number of positive cells / total number of cells) × 100%. 2.6 Statistic Analysis All analyses were conducted using SPSS 25.0 statistical software (IBM et al., USA). One-way analysis of variance (ANOVA) was used to compare groups, followed by multiple LSD comparisons. All data are presented as mean ± standard deviation. A P-value of < 0.05 was considered statistically significant. 3. Results 3.1 Restraint stress increases TLR4 expression and exacerbates inflammation in DSS-induced colitis mice. We initially evaluated the role of endogenous CRH in mouse colitis by applying restraint stress after DSS-induced colitis. Following the cessation of DSS treatment, the mice were subjected to 2 hours of restraint stress daily for nine days. Throughout the study, we monitored body weight loss, disease activity index (DAI), colon length, and histological scores. Compared to the DSS group, the DSS mice subjected to daily restraint stress showed increased TLR4 expression (P = 0.0241 < 0.05) (Fig. 1 G) and greater body weight loss (P = 0.0343 < 0.05) (Fig. 1 B), with no significant difference in colon length (Fig. 1 A). The severity of colitis, assessed by DAI scores, histological scores, and apoptosis levels, was greater in the restraint-stressed DSS mice. These mice showed higher histological scores (P = 0.0171 < 0.05) (Fig. 1 D, H) and a higher apoptosis index (P = 0.0375 < 0.05) (Fig. 1 E, I), though DAI scores did not differ significantly (Fig. 1 C). Additionally, serum IL-6 levels, measured by ELISA, were significantly elevated in the restraint-stressed DSS mice compared to the DSS group alone (P = 0.0004 < 0.05) (Fig. 1 F). 3.2 TLR4 Involvement in Cellular Autophagy, CRHR2 Inhibits TLR4 Protein Expression and Reduces the Extent of Autophagy in Intestinal Epithelial Cells After inducing colitis with DSS, we ceased DSS treatment and administered intraperitoneal injections of normal saline (NS), a CRHR2 antagonist (Ast2B), and a CRHR2 agonist (Ucn2) to the mice to assess alterations in TLR4 expression and autophagy levels. Compared to the DSS + NS group, the DSS + Ast2B group exhibited a higher LC3BⅡ/LC3BⅠ ratio (P = 0.0417 < 0.05) (Figs. 2 A-B), although there were no statistically significant differences in p62 or TLR4 levels (Figs. 2 A, C-D). In contrast, the DSS + Ucn2 group showed lower levels of LC3BⅡ/LC3BⅠ (P = 0.0011 < 0.05) and TLR4 (P = 0.0025 < 0.05) protein expression (Figs. 2 A-B, D), as well as higher levels of p62 protein expression (P = 0.0129 < 0.05) (Figs. 2 A, C) compared to the DSS + NS group. Furthermore, a correlation analysis between TLR4 and autophagy-related proteins (LC3 and p62) revealed a significant correlation between TLR4 and autophagy (P < 0.05) (Fig. 2 E). 3.3 TXYF mediates CRHR2 to influence the CRH-TLR4 signaling axis, thereby reducing autophagy in intestinal epithelial cells and alleviating colitis. Finally, after inducing colitis with DSS, we subjected the mice to restraint stress and administered the corresponding interventions to evaluate the targeted effect of TXYF on the CRH-TLR4 signaling axis and its therapeutic effects on colitis. Following the cessation of DSS treatment, the mice were subjected to 2 hours of daily restraint stress for nine days while also receiving intraperitoneal injections of normal saline (NS), intraperitoneal injections of Ast2B, or TXYF by gavage. Compared to the DSS + STRESS + NS group, the DSS + STRESS + TXYF group exhibited significantly lower expression of CRH (P = 0.0031 < 0.05) (Fig. 3 B) and TLR4 (P = 0.0058 < 0.05) (Fig. 3 C), along with reduced levels of autophagy (all P < 0.05) (Figs. 3 D-E, J). Additionally, the DSS + STRESS + TXYF group showed increased colon length (P = 0.0004 < 0.05) (Figs. 3 A, F) and reductions in DAI scores (P = 0.0384 < 0.05) (Fig. 3 G), histological scores (P = 0.0015 < 0.05) (Figs. 3 H, K), and apoptosis levels (P = 0.0125 < 0.05) (Figs. 3 I, L). Our previous experiments demonstrated that TXYF can mimic the effects of UCN2, enhancing the anti-inflammatory action of CRHR2. Using a CRHR2 antagonist, we found that compared to the DSS + STRESS + Ast2B group, the DSS + STRESS + TXYF + Ast2B group had significantly lower expression of CRH (P = 0.0109 < 0.05) (Fig. 3 B) and TLR4 (P = 0.0057 < 0.05) (Fig. 3 C), as well as reduced levels of the autophagy-related protein LC3 (P = 0.0146 < 0.05) (Figs. 3 D-E, J). However, p62 levels did not show a significant difference. Additionally, the DSS + STRESS + TXYF + Ast2B group exhibited increased colon length (P < 0.0001) (Figs. 3 A, F) and reductions in DAI scores (P < 0.0001) (Fig. 3 G), histological scores (P < 0.0001) (Figs. 3 H, K), and apoptosis levels (P = 0.0002 < 0.05) (Figs. 3 I, L). 4. Discussion This study aims to investigate how CRHR2-mediated disruption of the CRH-TLR4 signaling axis affects autophagy in intestinal epithelial cells in ulcerative colitis (UC) and to explore the potential mechanisms of the traditional Chinese medicine formula TXYF in mucosal repair. By using a DSS-induced colitis model in mice and applying various interventions, including restraint stress, the CRHR2 antagonist Astressin 2B (Ast2B), the CRHR2 agonist UCN2, and TXYF treatment, we systematically evaluated the inflammation levels, autophagy activity, and intestinal mucosal repair across different experimental groups. 4.1 The Regulatory Role of CRHR2 in Autophagy and Inflammation The experimental results revealed that DSS mice subjected to restraint stress experienced more significant weight loss, higher histological scores in intestinal tissues, and an increased apoptosis index compared to the DSS group. Additionally, TLR4 expression was notably elevated. These findings suggest that restraint stress exacerbates colitis through the TLR4 pathway. This observation is consistent with previous research, which indicates that stress can aggravate intestinal inflammation by enhancing the release of inflammatory mediators [ 9 – 11 ] . Further analysis revealed that restraint stress significantly increased serum IL-6 levels in the mice, suggesting that stress induces an inflammatory response through IL-6. The underlying mechanism may be related to TLR4-induced abnormal autophagy in the intestines, as our results showed a correlation between TLR4 and autophagy-related proteins (LC3 and p62), indicating a positive correlation between TLR4 expression and autophagy levels. Previous studies have demonstrated that in both in vivo and in vitro models of acute kidney injury, lipopolysaccharide (LPS) can induce autophagy in renal tubular epithelial cells through the TLR pathway, which helps counteract endotoxin-induced kidney damage and regulates downstream signaling pathways of TLR [ 17 ] . In contrast, TLR4 gene knockout mice in a model of isoproterenol-induced myocardial fibrosis showed reduced levels of autophagy [ 18 ] . However, other studies have indicated that the TLR4 signaling pathway can enhance the interaction between MyD88 or TRIF and the autophagy-related protein Beclin-1; this interaction inhibits the association between Beclin-1 and Bcl-2, with Bcl-2 interfering with the formation of the Beclin-1/Vps34 complex, thereby suppressing autophagy [ 19 ] . Additionally, TLR4 activation has been reported to lead to mTOR activation, which interacts with MyD88, IRF5, and IRF7 to regulate TNFα, IL-10, IL-12, and type I interferons, consequently exacerbating autophagy [ 20 ] . The state of autophagy in UC may be dynamically regulated, exhibiting varying levels of activity at different stages of the disease and across different regions of the intestine. A decrease in autophagy can result in the accumulation of damaged organelles and proteins within intestinal epithelial cells, which leads to increased cell death and compromised intestinal barrier function. This impairment facilitates the penetration of pathogens and toxins into the intestinal tissues, thereby triggering inflammation. The persistent inflammation in UC can activate autophagy through reactive oxygen species (ROS) to respond to cellular damage. A study by Felix Clemens Richteri and colleagues found that autophagy occurs in adipose tissue during the onset of colitis in mice. In autophagy-deficient adipose tissue, the NRF2-mediated P450-EPHX pathway is activated, leading to altered levels of lipid peroxides and a reduction in the production of the anti-inflammatory cytokine IL-10, thereby exacerbating intestinal inflammation [ 21 ] . Meanwhile, research by Maria Naama and colleagues suggests that in mice, autophagy is activated through the Beclin 1 protein gene, which helps inhibit endoplasmic reticulum stress in the intestine and promotes excessive mucus secretion. This mucus overproduction is influenced by the microbiota and involves interaction with the Nod2 protein, which reduces endoplasmic reticulum stress [ 22 ] . Our study shows that TLR4 is involved in cellular autophagy, and the high expression of TLR4 in UC intestinal epithelial cells may exacerbate intestinal autophagy. The study results indicate that the CRHR2 antagonist Ast2B increased the LC3BⅡ/LC3BⅠ ratio in DSS mice, suggesting elevated levels of autophagy. Conversely, the CRHR2 agonist UCN2 reduced TLR4 expression and the LC3BⅡ/LC3BⅠ ratio while increasing p62 protein levels, implying that CRHR2 inhibits abnormal autophagy in colonic epithelial cells by reducing TLR4 expression. Thus, restraint stress, through endogenous CRH, may promote TLR4 expression and worsen intestinal inflammation. In contrast, upregulating CRHR2 can mitigate abnormal autophagy in intestinal epithelial cells by inhibiting TLR4 expression, thereby reducing intestinal inflammation. 4.2 The Role of TXYF in the Treatment of Colitis In traditional Chinese medicine theory, Tongxie Yaofang (TXYF) is believed to have the effects of benefiting Qi, strengthening the spleen, clearing heat, eliminating dampness, and promoting blood circulation to remove stasis. Previous research indicates that TXYF significantly improves intestinal barrier dysfunction [ 23 , 24 ] . In studies involving an IBS rat model, TXYF was found to downregulate CRF-R1 and upregulate CRF-R2 expression, which helps reduce visceral hypersensitivity [ 23 ] . Further proteomics analysis indicated that TXYF participates in the reconstruction of the intestinal barrier by downregulating cytokeratin 8 (CK8) and upregulating transgelin protein levels [ 24 ] . It has been confirmed that intestinal barrier dysfunction exists in IBS [ 25 ] , and increasing research suggests that IBS and IBD share similarities in their pathogenesis, such as common genetic factors, similar gut microbiota dysbiosis, immune dysregulation, and the effectiveness of overlapping treatments [ 13 ] . Therefore, we used the DSS-induced ulcerative colitis mouse model to investigate the therapeutic effects of TXYF. The results showed that TXYF targets CRH-R2, enhances intestinal epithelial cell migration and proliferation, reduces apoptosis, suppresses immune-inflammatory responses in the intestinal mucosa, and promotes mucosal repair. However, the specific mechanisms remain to be further explored [ 16 ] . In this study, we further discovered that, compared to the DSS + STRESS + NS group, the DSS + STRESS + TXYF group exhibited reduced CRH and TLR4 expression, decreased autophagy levels, increased colon length, lower DAI scores, histological scores, and apoptosis levels. These findings suggest that TXYF exerts a protective effect on the intestinal mucosal barrier by modulating the CRH-TLR4 signaling axis, thereby reducing autophagy and apoptosis in intestinal epithelial cells. Notably, the DSS + STRESS + TXYF + Ast2B group exhibited even more significant effects, further confirming the critical role of CRHR2 in the anti-inflammatory action mediated by TXYF. The addition of the CRHR2 antagonist significantly reduced CRH and TLR4 expression, as well as levels of the autophagy-related protein LC3, leading to further improvements in colon length, DAI scores, histological scores, and apoptosis levels. These findings suggest that TXYF exerts its anti-inflammatory and mucosal repair effects by targeting CRHR2 to inhibit CRH-TLR4 axis-mediated abnormal autophagy in the intestine. 4.3 Study Limitations and Future Directions Although this study highlights the crucial role of CRHR2 in regulating autophagy and inflammation, as well as the potential mechanisms of TXYF in promoting mucosal repair, there are still some limitations. Firstly, the study primarily used a mouse model. While the DSS-induced colitis model is commonly employed in UC research, it does not fully replicate the pathological mechanisms of human UC. Therefore, future studies should further validate these findings in human intestinal cells from UC patients and clinical UC populations to confirm their translational relevance. Secondly, this study focused primarily on the regulatory effects of the CRHR2 and TLR4 signaling axis on autophagy and inflammation. However, CRHR2 may also participate in the regulation of inflammatory responses and cellular autophagy through other pathways. Future research should explore the role of CRHR2 in other signaling pathways to gain a comprehensive understanding of its regulatory mechanisms in UC. Furthermore, while TXYF exhibited significant protective effects, the specific components and mechanisms of action have not been fully elucidated. TXYF is a compound formula consisting of multiple traditional Chinese medicines, and its active ingredients may interact through various pathways. Future research should focus on isolating and identifying the active components of TXYF and investigating their specific mechanisms of action. This study will provide a theoretical foundation for the modernization and standardization of traditional Chinese medicine. 5. Conclusion This study underscores the crucial role of CRHR2 in regulating autophagy and inflammation, especially under stress conditions. By modulating the CRH-TLR4 signaling axis, CRHR2 can inhibit TLR4 expression, reduce autophagy and apoptosis in intestinal epithelial cells, and alleviate colitis symptoms. Additionally, TXYF enhances its anti-inflammatory and mucosal repair effects through CRHR2 mediation, providing new theoretical support for the use of traditional Chinese medicine in the treatment of ulcerative colitis. Declarations Conflict of Interest Statement The authors declare that there are no conflicts of interest regarding the publication of this paper. The authors have not received any financial support, nor do they have any personal or institutional affiliations that could influence the content or outcomes of this research. Author Contribution A wrote the main manuscript text; B-D prepared figures 1-3; A-D has completed animal and molecular experiments；All authors reviewed the manuscript. Data Availability All data are available from the first author ( [email protected] ). 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Tong-Xie-Yao-Fang improves intestinal permeability in diarrhoea-predominant irritable bowel syndrome rats by inhibiting the NF-κB and notch signalling pathways[J]. BMC complementary and alternative medicine, 2019, 19(1): 337. Gong S-S, Fan Y-H, Wang S-Y, et al. Mucosa repair mechanisms of Tong-Xie-Yao-Fang mediated by CRH-R2 in murine, dextran sulfate sodium-induced colitis[J]. World Journal of Gastroenterology, 2018, 24(16): 1766–1778. Leventhal J S, Ni J, Osmond M, et al. Autophagy Limits Endotoxemic Acute Kidney Injury and Alters Renal Tubular Epithelial Cell Cytokine Expression[J]. PloS One, 2016, 11(3): e0150001. Dong R, Wang Z, Zhao C, et al. Toll-like receptor 4 knockout protects against isoproterenol-induced cardiac fibrosis: the role of autophagy[J]. Journal of Cardiovascular Pharmacology and Therapeutics, 2015, 20(1): 84–92. Shi C-S, Kehrl J H. MyD88 and Trif target Beclin 1 to trigger autophagy in macrophages[J]. The Journal of Biological Chemistry, 2008, 283(48): 33175–33182. Schmitz F, Heit A, Dreher S, et al. Mammalian target of rapamycin (mTOR) orchestrates the defense program of innate immune cells[J]. European Journal of Immunology, 2008, 38(11): 2981–2992. Richter F C, Friedrich M, Kampschulte N, et al. Adipocyte autophagy limits gut inflammation by controlling oxylipin and IL-10[J]. The EMBO journal, 2023, 42(6): e112202. Naama M, Telpaz S, Awad A, et al. Autophagy controls mucus secretion from intestinal goblet cells by alleviating ER stress[J]. Cell Host & Microbe, 2023, 31(3): 433-446.e4. Guanqun Chao, Bin Lv, Lina Meng, et al. The effect of Tongxie Yaofang on the expression of CRF in the brain and spinal cord of rats with visceral hypersensitivity [J]. China Journal of Chinese Materia Medica, 2010, 35(15): 2012–2016.[in Chinese] Ying Ding, Bin Lv, Lina Meng, et al. The effect of Tongxie Yaofang on the protein expression profile of colonic mucosa in rats with visceral hypersensitivity [J]. Gastroenterology, 2012, 17(11): 660-664. [in Chinese] Stanisic V, Quigley E M. The overlap between IBS and IBD - what is it and what does it mean?[J]. Expert review of gastroenterology & hepatology, 2014, 8(2): 139–145. Additional Declarations No competing interests reported. Supplementary Files 2aand3j.pdf Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 26 Apr, 2025 Reviewers agreed at journal 26 Apr, 2025 Reviewers invited by journal 24 Apr, 2025 Submission checks completed at journal 24 Apr, 2025 First submitted to journal 07 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5426177","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":447502801,"identity":"11efad53-031f-4b13-b17a-692f8aded316","order_by":0,"name":"Menglin Li","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhejiang Chinese Medical University (Zhejiang Provincial Hospital of Traditional Chinese Medicine)","correspondingAuthor":false,"prefix":"","firstName":"Menglin","middleName":"","lastName":"Li","suffix":""},{"id":447502802,"identity":"34255933-2aaa-4f40-bc8e-de6ed3a3b939","order_by":1,"name":"Rong Huang","email":"","orcid":"","institution":"Jiaxing Hospital of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Rong","middleName":"","lastName":"Huang","suffix":""},{"id":447502803,"identity":"22440016-1e0c-449c-9c2b-fe682ee21000","order_by":2,"name":"Mingxu Zheng","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhejiang Chinese Medical University (Zhejiang Provincial Hospital of Traditional Chinese Medicine)","correspondingAuthor":false,"prefix":"","firstName":"Mingxu","middleName":"","lastName":"Zheng","suffix":""},{"id":447502804,"identity":"921b5cc9-c02f-4232-b16d-7a5ff00eb97b","order_by":3,"name":"Wanting Cao","email":"","orcid":"","institution":"Beilun District Hospital of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Wanting","middleName":"","lastName":"Cao","suffix":""},{"id":447502805,"identity":"720bf263-1cf1-40ed-97d8-8ca67f2ff6e5","order_by":4,"name":"Bin Lv","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhejiang Chinese Medical University (Zhejiang Provincial Hospital of Traditional Chinese Medicine)","correspondingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Lv","suffix":""},{"id":447502806,"identity":"bfbc70ac-4b6c-417f-b168-2871860b2fc2","order_by":5,"name":"Jing Zhao","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhejiang Chinese Medical University (Zhejiang Provincial Hospital of Traditional Chinese Medicine)","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Zhao","suffix":""},{"id":447502807,"identity":"c471609f-fa9b-4e7b-bdbc-1976e2b6ae4b","order_by":6,"name":"Yihong Fan","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhejiang Chinese Medical University (Zhejiang Provincial Hospital of Traditional Chinese Medicine)","correspondingAuthor":false,"prefix":"","firstName":"Yihong","middleName":"","lastName":"Fan","suffix":""},{"id":447502808,"identity":"d9e4f54a-6601-4779-b1ea-cc1840f916ed","order_by":7,"name":"Yi Xu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIie3PMWvCUBDA8YNAdDia9QRJv8JB4E2CX+UekUwtuJnBwRJJhlZc/RiOHRuE5/LcM0b6BSpdOgj1A7T44ubwfvP9uTsAz7tDYVTsv888wvG+qlvJ5+7kgUwGOM1isDbl1hp3EsOTAvzaJdCIGhyXQYfDwKqWONAvG8lyvQghql7lehKUM2YOdYFH0+j3IZA9bB1bdlsSRl32JGu0DYHp2ZUI0weTfgNRU10GXZKJGiyYE+qLgm4JmTQBlpjRpiTWoPOXx3VRf8L5F7lX1aeffB5H1ep68gfeNu55nuf96wICXkomdfLDUwAAAABJRU5ErkJggg==","orcid":"","institution":"The First Affiliated Hospital of Zhejiang Chinese Medical University (Zhejiang Provincial Hospital of Traditional Chinese Medicine)","correspondingAuthor":true,"prefix":"","firstName":"Yi","middleName":"","lastName":"Xu","suffix":""}],"badges":[],"createdAt":"2024-11-10 13:38:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5426177/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5426177/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81355600,"identity":"c20a3332-13b2-41fc-9ce7-4141933faac8","added_by":"auto","created_at":"2025-04-25 07:32:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1531640,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRestraint stress increases TLR4 expression in DSS-induced mice and exacerbates colitis symptoms. \u003c/strong\u003eA: Colon length; B: Body weight changes over 16 days for each group of mice; C: DAI; D: Histological scores; E: Apoptosis index; F: Serum IL-6 levels; G: TLR4 expression in intestinal tissues; H: Representative hematoxylin-eosin (HE) stained histological images; I: Representative TUNEL-stained images. Data are presented as mean ± standard deviation, with n = 6–10 per group.\u003c/p\u003e","description":"","filename":"figture1.png","url":"https://assets-eu.researchsquare.com/files/rs-5426177/v1/973d3a84195068c9226a7064.png"},{"id":81355603,"identity":"98b9e596-de00-4e7c-ba23-2d2741eb0053","added_by":"auto","created_at":"2025-04-25 07:32:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":189863,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCRHR2 inhibits TLR4 protein expression to reduce abnormal autophagy in intestinal epithelial cells.\u003c/strong\u003e A: Representative images of autophagy-related protein expression from Western blot analysis; B: Bar graph showing LC3BⅡ/LC3BⅠ/β-actin expression levels in colon tissue across different groups of mice; C: Bar graph showing p62/β-actin expression levels in colon tissue across different groups of mice; D: Bar graph showing TLR4 expression levels in colon tissue across different groups of mice; E: Correlation analysis between TLR4 and autophagy-related proteins. Data are presented as mean ± standard deviation, with n = 6–10 per group.\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5426177/v1/602e9214af74378bf7c085dd.png"},{"id":81355606,"identity":"94057760-50e4-43f6-bcff-dec56c2abe8b","added_by":"auto","created_at":"2025-04-25 07:32:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4261390,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTXYF mediates CRHR2 to influence the CRH-TLR4 signaling axis, reducing autophagy in intestinal epithelial cells and improving colitis. \u003c/strong\u003eA: Representative images of colons from different groups of mice; B: Bar graph showing CRH expression levels; C: Bar graph showing TLR4 expression levels; D: Bar graph showing LC3BⅡ/LC3BⅠ/β-actin expression levels in colon tissue from different groups of mice; E: Bar graph showing p62/β-actin expression levels in colon tissue from different groups of mice; F: Colon length; G: DAI scores; H: Histological scores; I: Apoptosis index; J: Representative images of autophagy-related proteins from Western blot analysis; K: Representative hematoxylin-eosin (HE) stained histological images; L: Representative TUNEL-stained images. Data are presented as mean ± standard deviation, with n = 6–10 per group.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5426177/v1/8d3699c6bc6854b9a42244fb.png"},{"id":81358071,"identity":"25e3c592-b48b-4df0-b1fe-2e32e3f6717f","added_by":"auto","created_at":"2025-04-25 07:56:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6270860,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5426177/v1/e11b4ddd-9f76-4536-b6c0-0cb18e855ead.pdf"},{"id":81356637,"identity":"45d78a87-80b1-4960-b4c1-1aaef8b4a0f5","added_by":"auto","created_at":"2025-04-25 07:40:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":211260,"visible":true,"origin":"","legend":"","description":"","filename":"2aand3j.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5426177/v1/691ab960bada38c765b8b8d5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Tong-Xie-Yao-Fang Modulates Mucosal Repair via CRH-TLR4- Mediated Autophagy in Stress-Induced Colitis","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eInflammatory Bowel Disease (IBD) is a chronic, recurring inflammatory condition of the intestines influenced by a complex interplay of neural, epigenetic, and environmental factors. It encompasses two main types: Ulcerative Colitis (UC) and Crohn's Disease (CD). The precise etiology and pathogenesis of IBD remain unclear. Globally, the incidence of Ulcerative Colitis (UC) is rising, with common symptoms including bloody stools, diarrhea, urgency, incontinence, fatigue, increased bowel frequency, and mucus in the stool\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Up to 15% of UC patients may require a colectomy\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Awareness of UC is growing in Asia, and with the rapid pace of urbanization and migration from rural to urban areas, along with increasing population density, the incidence of the disease in Asia is expected to continue increasing\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRecent studies have shown that autophagy is crucial in the inflammatory process and mucosal healing. Autophagy is considered an essential catabolic pathway in cells. Research indicates that the knockout of autophagy-related genes\u0026mdash;such as Atg4B/autophagin-1, Atg16L1, and IRGM\u0026mdash;significantly exacerbates the severity of IBD in animal models, suggesting that baseline autophagy is essential for maintaining intestinal homeostasis and the intestinal defense barrier\u003csup\u003e[\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. However, other reports have noted that excessive activation of intestinal autophagy may worsen IBD\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Consequently, the role of autophagy in UC remains a topic of debate and requires further investigation.\u003c/p\u003e \u003cp\u003eIt has been confirmed that varying degrees of stress and associated psychosocial responses, such as anxiety and depression, contribute to the onset and progression of UC by damaging the intestinal mucosal barrier\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. In the central nervous system, stress-induced physiological responses are triggered by the hypothalamic-pituitary-adrenal (HPA) axis, leading to the release of corticotropin-releasing hormone (CRH), which regulates neuroendocrine functions and immune responses in visceral organs. CRH is also present in peripheral tissues, including digestive organs and cells, where it directly modulates gastrointestinal inflammation. Psychosocial stress may further exacerbate intestinal autophagy by influencing gut microbiota and inflammation, worsening IBD\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTLR4 is expressed in immune and intestinal epithelial cells, functioning as an environmental sensor for autophagy. In both human and murine macrophages, autophagy and autophagy-mediated cell death can be triggered by lipopolysaccharide (LPS).\u003c/p\u003e \u003cp\u003eTong-Xie-Yao-Fang (TXYF), originating from the Jingyue Complete Book, is composed of four traditional Chinese herbs: stir-fried Atractylodes, stir-fried White Peony Root, stir-fried Tangerine Peel, and Siler Root. It is commonly used to treat Irritable Bowel Syndrome (IBS). There is a correlation and overlap in pathogenesis, genetic factors, gut microbiota dysbiosis, and immune dysfunction between IBS and Inflammatory Bowel Disease (IBD), with evidence of cross-treatment efficacy\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. TXYF is effective for diarrhea-predominant IBS (IBS-D) and has been shown in animal studies to improve intestinal permeability and strengthen the intestinal mucosal barrier, likely by inhibiting inflammatory cascades and the NF-κB and Notch signaling pathways\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Our previous research found that TXYF downregulates CRHR1 and upregulates CRHR2, acting similarly to UCN2 by intervening through CRHR2 to regulate epithelial cell migration, proliferation, and apoptosis, thereby exerting anti-inflammatory effects and aiding mucosal healing in colitis mice\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe hypothesize that CRHR2-mediated CRH-TLR4 signaling axis disruptions are associated with autophagy in intestinal epithelial cells in ulcerative colitis. Furthermore, these disruptions may be linked to the mucosal repair mechanisms facilitated by the traditional Chinese medicine formula Tong-Xie-Yao-Fang (TXYF).\u003c/p\u003e \u003cp\u003eThe findings of this study will clarify whether stress-related CRHR2 regulation of the CRH-TLR4 signaling axis disruption is linked to mucosal repair and autophagy in intestinal epithelial cells (IECs) during dextran sodium sulfate (DSS)-induced colitis in mice. Additionally, the study will explore the mechanisms by which TXYF promotes mucosal repair.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Animals experiments\u003c/h2\u003e \u003cp\u003eMale SPF-grade CD-1 (ICR) mice, aged 8\u0026ndash;10 weeks and weighing 22g\u0026thinsp;\u0026plusmn;\u0026thinsp;2g, were acclimated for one week under controlled conditions of 22\u0026ndash;24\u0026deg;C, 50\u0026ndash;60% humidity, and a 12-hour light/dark cycle. All mice were purchased from Shanghai BK Laboratory Animal Co., Ltd. The mice (n\u0026thinsp;=\u0026thinsp;110) were randomly divided into 11 groups: Control group (n\u0026thinsp;=\u0026thinsp;10), DSS group (n\u0026thinsp;=\u0026thinsp;10), DSS\u0026thinsp;+\u0026thinsp;Ast2B group (n\u0026thinsp;=\u0026thinsp;10), DSS\u0026thinsp;+\u0026thinsp;UCN2 group (n\u0026thinsp;=\u0026thinsp;10), DSS\u0026thinsp;+\u0026thinsp;TXYF group (n\u0026thinsp;=\u0026thinsp;10), DSS\u0026thinsp;+\u0026thinsp;Ast2B\u0026thinsp;+\u0026thinsp;TXYF group (n\u0026thinsp;=\u0026thinsp;10), DSS\u0026thinsp;+\u0026thinsp;Restraint Stress group (n\u0026thinsp;=\u0026thinsp;10), DSS\u0026thinsp;+\u0026thinsp;Restraint Stress\u0026thinsp;+\u0026thinsp;Ast2B group (n\u0026thinsp;=\u0026thinsp;10), DSS\u0026thinsp;+\u0026thinsp;Restraint Stress\u0026thinsp;+\u0026thinsp;UCN2 group (n\u0026thinsp;=\u0026thinsp;10), DSS\u0026thinsp;+\u0026thinsp;Restraint Stress\u0026thinsp;+\u0026thinsp;TXYF group (n\u0026thinsp;=\u0026thinsp;10), and DSS\u0026thinsp;+\u0026thinsp;Restraint Stress\u0026thinsp;+\u0026thinsp;Ast2B\u0026thinsp;+\u0026thinsp;TXYF group (n\u0026thinsp;=\u0026thinsp;10).\u003c/p\u003e \u003cp\u003eDextran sodium sulfate (DSS) powder was dissolved in drinking water to prepare a 3% DSS solution, which was provided to the mice ad libitum for five consecutive days to induce a colitis model. From day 6 to day 15, the mice were switched to regular drinking water.\u003c/p\u003e \u003cp\u003eTXYF formula consists of 15g of stir-fried Atractylodes, 12g of stir-fried White Peony Root, 10g of Siler, and 6g of dried Tangerine Peel, totaling 43g. The herbs were soaked in an appropriate amount of distilled water for 1 hour, then an additional eight times the volume of distilled water was added. The mixture was simmered for 30 minutes, filtered, and the filtrate from two extractions was combined. After vacuum filtration, the combined filtrate was concentrated by rotary evaporation to yield a 100% herbal solution (1mL contains 1g of raw herbs). The solution was stored at four\u0026deg;C and diluted with distilled water to the desired concentration before experiment use.\u003c/p\u003e \u003cp\u003eCRHR2 antagonist Astressin 2B (Ast2B) was administered intraperitoneally at a dose of 20 \u0026micro;g/(kg\u0026middot;d), CRHR2 agonist UCN2 at 30 \u0026micro;g/(kg\u0026middot;d) was given intraperitoneally, restraint stress was applied for 2 hours per day, and TXYF was administered by gavage at 5.6 g/(kg\u0026middot;d) for nine days. Following the treatment period, mice were anesthetized by CO\u003csub\u003e2\u003c/sub\u003e, followed euthanized by cervical dislocation, and the entire colon distal to the cecum was immediately excised for subsequent analysis. All animal experiments were conducted according to the ethical principles adopted by the Animal Experimentation Center of Zhejiang Chinese Medical University and were approved by the Animal Ethics Committee of Zhejiang Chinese Medical University(Approval No. IACUC-20190401-12). All methods for animals were performed in accordance with the relevant guidelines and regulations. This study is reported in accordance with ARRIVE guidelines (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://arriveguidelines.org\u003c/span\u003e\u003cspan address=\"https://arriveguidelines.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2Colon Disease Activity Index and Histologic Score\u003c/h2\u003e \u003cp\u003eThe mice were evaluated using the Disease Activity Index (DAI) and histological scoring criteria. The general condition, body weight changes, stool consistency, and occult or gross blood in the stool were observed and recorded daily for all mice. The DAI score was computed based on the percentage of body weight loss, stool consistency, and the presence of occult or gross blood in the stool. The DAI score was determined as follows: DAI = (body weight loss score\u0026thinsp;+\u0026thinsp;stool consistency score\u0026thinsp;+\u0026thinsp;stool occult/gross blood score) / 3. Histological assessment was conducted in a double-anonymized manner. Mice were euthanized by cervical dislocation, and the abdomen was opened to isolate the colon for further analysis quickly. The colon length was measured, and it was then opened along the mesenteric axis. The lumen was rinsed with cold saline to remove debris, and the colon was examined macroscopically for signs of congestion and ulcers. Approximately 1 cm of colon tissue from the area with the most noticeable lesions was collected, fixed in 10% formalin, and processed for routine HE staining to assess morphological changes in the colonic mucosa. Histopathological scoring of the colon tissue was conducted, including assessments of inflammation, injury depth, crypt damage, and the extent of inflammation (%). The colon pathology score was calculated as the sum of the inflammation score, injury depth score, crypt damage score, and inflammation extent (%) score.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 ELISA for CRH, TLR-4\u003c/h2\u003e \u003cp\u003eFrozen colon tissue samples were sectioned, weighed, homogenized, and centrifuged (5810R tabletop high-speed refrigerated centrifuge, Eppendorf, Germany) to collect the supernatant using a double-antibody sandwich ABC-ELISA method. ELISA kits for mouse corticotropin-releasing hormone (CRH) and mouse Toll-like receptor 4 (TLR-4) (both from Shanghai Westang) were used. The assays were performed using a DENLEY DRAGON Wellscan MK 3 automatic microplate reader (Thermo, Finland). All tests were conducted according to the manufacturer's instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Western Blot\u003c/h2\u003e \u003cp\u003eUsing the Bicinchoninic Acid (BCA) protein quantification method, the tissue samples were ground in liquid nitrogen, and a protease inhibitor (Protease et al., cOmplet tabelet, TMEDTA-free Protease Inhibitor Cocktail Therapy) was added. The tissue was lysed with lysis buffer (RIPA et al., Ltd.), homogenized and centrifuged. Protein quantification was performed using a BCA Protein Assay Kit (Procell et al., Ltd.) according to the manufacturer's instructions. SDS-PAGE gels were prepared, and the samples were denatured before electrophoresis. A PVDF membrane (Millipore, USA) was used as the solid-phase support, and wet transfer was performed. The membrane was then incubated with primary antibodies (LC3A, LC3B, P62 from Cell Signaling Technology; β-actin from Proteintech Group) and HRP-conjugated secondary antibodies (Jackson ImmunoResearch). Detection was carried out using the FluorChem HD2 imaging system for direct imaging. The target molecular weight and grayscale values were analyzed using a gel image processing system and software. The LC3II/LC3I ratio and P62 levels were calculated to assess the level of autophagy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Apoptosis of intestinal epithelial cells was detected in situ using the TUNEL assay\u003c/h2\u003e \u003cp\u003eApoptosis of intestinal epithelial cells was assessed in situ utilizing the TUNEL assay (terminal deoxynucleotidyl transferase dUTP nick-end labeling), adhering to the protocol provided by the TUNEL Cell Apoptosis Detection Kit (Beyotime et al. C1088, Lot No. 030719190621). Cells with brown-yellow granules in their nuclei were classified as positive for apoptosis. Under an optical microscope, five random fields of view (\u0026times;400) were selected, and the number of positive cells was counted to calculate the apoptosis index (AI), defined as AI = (number of positive cells / total number of cells) \u0026times; 100%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Statistic Analysis\u003c/h2\u003e \u003cp\u003eAll analyses were conducted using SPSS 25.0 statistical software (IBM et al., USA). One-way analysis of variance (ANOVA) was used to compare groups, followed by multiple LSD comparisons. All data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. A P-value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Restraint stress increases TLR4 expression and exacerbates inflammation in DSS-induced colitis mice.\u003c/h2\u003e \u003cp\u003eWe initially evaluated the role of endogenous CRH in mouse colitis by applying restraint stress after DSS-induced colitis. Following the cessation of DSS treatment, the mice were subjected to 2 hours of restraint stress daily for nine days. Throughout the study, we monitored body weight loss, disease activity index (DAI), colon length, and histological scores.\u003c/p\u003e \u003cp\u003eCompared to the DSS group, the DSS mice subjected to daily restraint stress showed increased TLR4 expression (P\u0026thinsp;=\u0026thinsp;0.0241\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG) and greater body weight loss (P\u0026thinsp;=\u0026thinsp;0.0343\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), with no significant difference in colon length (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The severity of colitis, assessed by DAI scores, histological scores, and apoptosis levels, was greater in the restraint-stressed DSS mice. These mice showed higher histological scores (P\u0026thinsp;=\u0026thinsp;0.0171\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, H) and a higher apoptosis index (P\u0026thinsp;=\u0026thinsp;0.0375\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, I), though DAI scores did not differ significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Additionally, serum IL-6 levels, measured by ELISA, were significantly elevated in the restraint-stressed DSS mice compared to the DSS group alone (P\u0026thinsp;=\u0026thinsp;0.0004\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.2 TLR4 Involvement in Cellular Autophagy, CRHR2 Inhibits TLR4 Protein Expression and Reduces the Extent of Autophagy in Intestinal Epithelial Cells\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAfter inducing colitis with DSS, we ceased DSS treatment and administered intraperitoneal injections of normal saline (NS), a CRHR2 antagonist (Ast2B), and a CRHR2 agonist (Ucn2) to the mice to assess alterations in TLR4 expression and autophagy levels.\u003c/p\u003e \u003cp\u003eCompared to the DSS\u0026thinsp;+\u0026thinsp;NS group, the DSS\u0026thinsp;+\u0026thinsp;Ast2B group exhibited a higher LC3BⅡ/LC3BⅠ ratio (P\u0026thinsp;=\u0026thinsp;0.0417\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B), although there were no statistically significant differences in p62 or TLR4 levels (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, C-D). In contrast, the DSS\u0026thinsp;+\u0026thinsp;Ucn2 group showed lower levels of LC3BⅡ/LC3BⅠ (P\u0026thinsp;=\u0026thinsp;0.0011\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and TLR4 (P\u0026thinsp;=\u0026thinsp;0.0025\u0026thinsp;\u0026lt;\u0026thinsp;0.05) protein expression (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B, D), as well as higher levels of p62 protein expression (P\u0026thinsp;=\u0026thinsp;0.0129\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, C) compared to the DSS\u0026thinsp;+\u0026thinsp;NS group. Furthermore, a correlation analysis between TLR4 and autophagy-related proteins (LC3 and p62) revealed a significant correlation between TLR4 and autophagy (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.3 TXYF mediates CRHR2 to influence the CRH-TLR4 signaling axis, thereby reducing autophagy in intestinal epithelial cells and alleviating colitis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFinally, after inducing colitis with DSS, we subjected the mice to restraint stress and administered the corresponding interventions to evaluate the targeted effect of TXYF on the CRH-TLR4 signaling axis and its therapeutic effects on colitis. Following the cessation of DSS treatment, the mice were subjected to 2 hours of daily restraint stress for nine days while also receiving intraperitoneal injections of normal saline (NS), intraperitoneal injections of Ast2B, or TXYF by gavage.\u003c/p\u003e \u003cp\u003eCompared to the DSS\u0026thinsp;+\u0026thinsp;STRESS\u0026thinsp;+\u0026thinsp;NS group, the DSS\u0026thinsp;+\u0026thinsp;STRESS\u0026thinsp;+\u0026thinsp;TXYF group exhibited significantly lower expression of CRH (P\u0026thinsp;=\u0026thinsp;0.0031\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) and TLR4 (P\u0026thinsp;=\u0026thinsp;0.0058\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), along with reduced levels of autophagy (all P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-E, J). Additionally, the DSS\u0026thinsp;+\u0026thinsp;STRESS\u0026thinsp;+\u0026thinsp;TXYF group showed increased colon length (P\u0026thinsp;=\u0026thinsp;0.0004\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, F) and reductions in DAI scores (P\u0026thinsp;=\u0026thinsp;0.0384\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG), histological scores (P\u0026thinsp;=\u0026thinsp;0.0015\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH, K), and apoptosis levels (P\u0026thinsp;=\u0026thinsp;0.0125\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI, L).\u003c/p\u003e \u003cp\u003eOur previous experiments demonstrated that TXYF can mimic the effects of UCN2, enhancing the anti-inflammatory action of CRHR2. Using a CRHR2 antagonist, we found that compared to the DSS\u0026thinsp;+\u0026thinsp;STRESS\u0026thinsp;+\u0026thinsp;Ast2B group, the DSS\u0026thinsp;+\u0026thinsp;STRESS\u0026thinsp;+\u0026thinsp;TXYF\u0026thinsp;+\u0026thinsp;Ast2B group had significantly lower expression of CRH (P\u0026thinsp;=\u0026thinsp;0.0109\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) and TLR4 (P\u0026thinsp;=\u0026thinsp;0.0057\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), as well as reduced levels of the autophagy-related protein LC3 (P\u0026thinsp;=\u0026thinsp;0.0146\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-E, J). However, p62 levels did not show a significant difference. Additionally, the DSS\u0026thinsp;+\u0026thinsp;STRESS\u0026thinsp;+\u0026thinsp;TXYF\u0026thinsp;+\u0026thinsp;Ast2B group exhibited increased colon length (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, F) and reductions in DAI scores (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG), histological scores (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH, K), and apoptosis levels (P\u0026thinsp;=\u0026thinsp;0.0002\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI, L).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study aims to investigate how CRHR2-mediated disruption of the CRH-TLR4 signaling axis affects autophagy in intestinal epithelial cells in ulcerative colitis (UC) and to explore the potential mechanisms of the traditional Chinese medicine formula TXYF in mucosal repair. By using a DSS-induced colitis model in mice and applying various interventions, including restraint stress, the CRHR2 antagonist Astressin 2B (Ast2B), the CRHR2 agonist UCN2, and TXYF treatment, we systematically evaluated the inflammation levels, autophagy activity, and intestinal mucosal repair across different experimental groups.\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4.1 The Regulatory Role of CRHR2 in Autophagy and Inflammation\u003c/h2\u003e \u003cp\u003eThe experimental results revealed that DSS mice subjected to restraint stress experienced more significant weight loss, higher histological scores in intestinal tissues, and an increased apoptosis index compared to the DSS group. Additionally, TLR4 expression was notably elevated. These findings suggest that restraint stress exacerbates colitis through the TLR4 pathway. This observation is consistent with previous research, which indicates that stress can aggravate intestinal inflammation by enhancing the release of inflammatory mediators\u003csup\u003e[\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Further analysis revealed that restraint stress significantly increased serum IL-6 levels in the mice, suggesting that stress induces an inflammatory response through IL-6. The underlying mechanism may be related to TLR4-induced abnormal autophagy in the intestines, as our results showed a correlation between TLR4 and autophagy-related proteins (LC3 and p62), indicating a positive correlation between TLR4 expression and autophagy levels. Previous studies have demonstrated that in both in vivo and in vitro models of acute kidney injury, lipopolysaccharide (LPS) can induce autophagy in renal tubular epithelial cells through the TLR pathway, which helps counteract endotoxin-induced kidney damage and regulates downstream signaling pathways of TLR\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn contrast, TLR4 gene knockout mice in a model of isoproterenol-induced myocardial fibrosis showed reduced levels of autophagy \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. However, other studies have indicated that the TLR4 signaling pathway can enhance the interaction between MyD88 or TRIF and the autophagy-related protein Beclin-1; this interaction inhibits the association between Beclin-1 and Bcl-2, with Bcl-2 interfering with the formation of the Beclin-1/Vps34 complex, thereby suppressing autophagy\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Additionally, TLR4 activation has been reported to lead to mTOR activation, which interacts with MyD88, IRF5, and IRF7 to regulate TNFα, IL-10, IL-12, and type I interferons, consequently exacerbating autophagy\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe state of autophagy in UC may be dynamically regulated, exhibiting varying levels of activity at different stages of the disease and across different regions of the intestine. A decrease in autophagy can result in the accumulation of damaged organelles and proteins within intestinal epithelial cells, which leads to increased cell death and compromised intestinal barrier function. This impairment facilitates the penetration of pathogens and toxins into the intestinal tissues, thereby triggering inflammation. The persistent inflammation in UC can activate autophagy through reactive oxygen species (ROS) to respond to cellular damage. A study by Felix Clemens Richteri and colleagues found that autophagy occurs in adipose tissue during the onset of colitis in mice. In autophagy-deficient adipose tissue, the NRF2-mediated P450-EPHX pathway is activated, leading to altered levels of lipid peroxides and a reduction in the production of the anti-inflammatory cytokine IL-10, thereby exacerbating intestinal inflammation\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMeanwhile, research by Maria Naama and colleagues suggests that in mice, autophagy is activated through the Beclin 1 protein gene, which helps inhibit endoplasmic reticulum stress in the intestine and promotes excessive mucus secretion. This mucus overproduction is influenced by the microbiota and involves interaction with the Nod2 protein, which reduces endoplasmic reticulum stress\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Our study shows that TLR4 is involved in cellular autophagy, and the high expression of TLR4 in UC intestinal epithelial cells may exacerbate intestinal autophagy.\u003c/p\u003e \u003cp\u003eThe study results indicate that the CRHR2 antagonist Ast2B increased the LC3BⅡ/LC3BⅠ ratio in DSS mice, suggesting elevated levels of autophagy. Conversely, the CRHR2 agonist UCN2 reduced TLR4 expression and the LC3BⅡ/LC3BⅠ ratio while increasing p62 protein levels, implying that CRHR2 inhibits abnormal autophagy in colonic epithelial cells by reducing TLR4 expression. Thus, restraint stress, through endogenous CRH, may promote TLR4 expression and worsen intestinal inflammation. In contrast, upregulating CRHR2 can mitigate abnormal autophagy in intestinal epithelial cells by inhibiting TLR4 expression, thereby reducing intestinal inflammation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.2 The Role of TXYF in the Treatment of Colitis\u003c/h2\u003e \u003cp\u003eIn traditional Chinese medicine theory, Tongxie Yaofang (TXYF) is believed to have the effects of benefiting Qi, strengthening the spleen, clearing heat, eliminating dampness, and promoting blood circulation to remove stasis. Previous research indicates that TXYF significantly improves intestinal barrier dysfunction\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. In studies involving an IBS rat model, TXYF was found to downregulate CRF-R1 and upregulate CRF-R2 expression, which helps reduce visceral hypersensitivity\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Further proteomics analysis indicated that TXYF participates in the reconstruction of the intestinal barrier by downregulating cytokeratin 8 (CK8) and upregulating transgelin protein levels\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. It has been confirmed that intestinal barrier dysfunction exists in IBS\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e, and increasing research suggests that IBS and IBD share similarities in their pathogenesis, such as common genetic factors, similar gut microbiota dysbiosis, immune dysregulation, and the effectiveness of overlapping treatments\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Therefore, we used the DSS-induced ulcerative colitis mouse model to investigate the therapeutic effects of TXYF. The results showed that TXYF targets CRH-R2, enhances intestinal epithelial cell migration and proliferation, reduces apoptosis, suppresses immune-inflammatory responses in the intestinal mucosa, and promotes mucosal repair. However, the specific mechanisms remain to be further explored\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. In this study, we further discovered that, compared to the DSS\u0026thinsp;+\u0026thinsp;STRESS\u0026thinsp;+\u0026thinsp;NS group, the DSS\u0026thinsp;+\u0026thinsp;STRESS\u0026thinsp;+\u0026thinsp;TXYF group exhibited reduced CRH and TLR4 expression, decreased autophagy levels, increased colon length, lower DAI scores, histological scores, and apoptosis levels. These findings suggest that TXYF exerts a protective effect on the intestinal mucosal barrier by modulating the CRH-TLR4 signaling axis, thereby reducing autophagy and apoptosis in intestinal epithelial cells.\u003c/p\u003e \u003cp\u003eNotably, the DSS\u0026thinsp;+\u0026thinsp;STRESS\u0026thinsp;+\u0026thinsp;TXYF\u0026thinsp;+\u0026thinsp;Ast2B group exhibited even more significant effects, further confirming the critical role of CRHR2 in the anti-inflammatory action mediated by TXYF. The addition of the CRHR2 antagonist significantly reduced CRH and TLR4 expression, as well as levels of the autophagy-related protein LC3, leading to further improvements in colon length, DAI scores, histological scores, and apoptosis levels. These findings suggest that TXYF exerts its anti-inflammatory and mucosal repair effects by targeting CRHR2 to inhibit CRH-TLR4 axis-mediated abnormal autophagy in the intestine.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Study Limitations and Future Directions\u003c/h2\u003e \u003cp\u003eAlthough this study highlights the crucial role of CRHR2 in regulating autophagy and inflammation, as well as the potential mechanisms of TXYF in promoting mucosal repair, there are still some limitations. Firstly, the study primarily used a mouse model. While the DSS-induced colitis model is commonly employed in UC research, it does not fully replicate the pathological mechanisms of human UC. Therefore, future studies should further validate these findings in human intestinal cells from UC patients and clinical UC populations to confirm their translational relevance.\u003c/p\u003e \u003cp\u003eSecondly, this study focused primarily on the regulatory effects of the CRHR2 and TLR4 signaling axis on autophagy and inflammation. However, CRHR2 may also participate in the regulation of inflammatory responses and cellular autophagy through other pathways. Future research should explore the role of CRHR2 in other signaling pathways to gain a comprehensive understanding of its regulatory mechanisms in UC.\u003c/p\u003e \u003cp\u003eFurthermore, while TXYF exhibited significant protective effects, the specific components and mechanisms of action have not been fully elucidated. TXYF is a compound formula consisting of multiple traditional Chinese medicines, and its active ingredients may interact through various pathways. Future research should focus on isolating and identifying the active components of TXYF and investigating their specific mechanisms of action. This study will provide a theoretical foundation for the modernization and standardization of traditional Chinese medicine.\u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study underscores the crucial role of CRHR2 in regulating autophagy and inflammation, especially under stress conditions. By modulating the CRH-TLR4 signaling axis, CRHR2 can inhibit TLR4 expression, reduce autophagy and apoptosis in intestinal epithelial cells, and alleviate colitis symptoms. Additionally, TXYF enhances its anti-inflammatory and mucosal repair effects through CRHR2 mediation, providing new theoretical support for the use of traditional Chinese medicine in the treatment of ulcerative colitis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of Interest Statement\u003c/h2\u003e \u003cp\u003eThe authors declare that there are no conflicts of interest regarding the publication of this paper. The authors have not received any financial support, nor do they have any personal or institutional affiliations that could influence the content or outcomes of this research.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eA wrote the main manuscript text; B-D prepared figures 1-3; A-D has completed animal and molecular experiments；All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data are available from the first author ([email protected]).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eUngaro R, Mehandru S, Allen P B, et al. Ulcerative colitis[J]. Lancet (London, England), 2017, 389(10080): 1756\u0026ndash;1770.\u003c/li\u003e\n\u003cli\u003eFumery M, Singh S, Dulai P S, et al. Natural History of Adult Ulcerative Colitis in Population-based Cohorts: A Systematic Review[J]. 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The protection role of Atg16l1 in CD11c+dendritic cells in murine colitis[J]. Immunobiology, 2017, 222(7): 831\u0026ndash;841.\u003c/li\u003e\n\u003cli\u003eHu X, Deng J, Yu T, et al. ATF4 Deficiency Promotes Intestinal Inflammation in Mice by Reducing Uptake of Glutamine and Expression of Antimicrobial Peptides[J]. Gastroenterology, 2019, 156(4): 1098\u0026ndash;1111.\u003c/li\u003e\n\u003cli\u003eSu W, Chen Y, Cao P, et al. Fusobacterium nucleatum Promotes the Development of Ulcerative Colitis by Inducing the Autophagic Cell Death of Intestinal Epithelial[J]. Frontiers in Cellular and Infection Microbiology, 2020, 10: 594806.\u003c/li\u003e\n\u003cli\u003eGuerrero-Alba R, Valdez-Morales E E, Jimenez-Vargas N N, et al. Stress activates pronociceptive endogenous opioid signalling in DRG neurons during chronic colitis[J]. Gut, 2017, 66(12): 2121\u0026ndash;2131.\u003c/li\u003e\n\u003cli\u003eGracie D J, Ford A C. A Bidirectional Relationship Between Symptom Reporting and Perceived Stress, But Not Disease Activity, in Inflammatory Bowel Disease: More Questions Than Answers?[J]. Gastroenterology, 2017, 153(5): 1444\u0026ndash;1445.\u003c/li\u003e\n\u003cli\u003eWang S-L, Shao B-Z, Zhao S-B, et al. Intestinal autophagy links psychosocial stress with gut microbiota to promote inflammatory bowel disease[J]. Cell Death \u0026amp; Disease, 2019, 10(6): 391.\u003c/li\u003e\n\u003cli\u003eXu Y, Jagannath C, Liu X-D, et al. Toll-like receptor 4 is a sensor for autophagy associated with innate immunity[J]. Immunity, 2007, 27(1): 135\u0026ndash;144.\u003c/li\u003e\n\u003cli\u003eQuigley E M M. Overlapping irritable bowel syndrome and inflammatory bowel disease: less to this than meets the eye?[J]. Therapeutic Advances in Gastroenterology, 2016, 9(2): 199\u0026ndash;212.\u003c/li\u003e\n\u003cli\u003eHalpin S J, Ford A C. 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Expert review of gastroenterology \u0026amp; hepatology, 2014, 8(2): 139\u0026ndash;145.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Tong-Xie-Yao-Fang, Ulcerative colitis, autophagy, stress, Corticotropin-Releasing Hormone Receptor 2, CRH-TLR4 axis","lastPublishedDoi":"10.21203/rs.3.rs-5426177/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5426177/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eAutophagy plays an essential role in inflammatory processes and mucosal repair, with Corticotropin-Releasing Hormone (CRH) and Toll-Like Receptor 4 (TLR4) also playing significant roles in regulating cellular autophagy. We hypothesized that disruptions in the CRHR2-mediated CRH-TLR4 signaling axis are associated with altered autophagy in intestinal epithelial cells (IECs) in ulcerative colitis. Furthermore, we investigated how these disruptions impact the mucosal repair mechanisms that are enhanced by Tong-Xie-Yao-Fang(TXYF).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eA 3% dextran sodium sulfate (DSS) solution was administered to mice for five days to induce a colitis model. Restraint stress was applied for 2 hours daily. while the CRHR2 antagonist Astressin 2B (Ast2B) at a dose of 20 μg/(kg·day) was given intraperitoneally, the CRHR2 agonist UCN2 at 30 μg/(kg·day) was administered peritoneally. TXYF at 5.6 g/(kg·day) was delivered by gavage, all for nine consecutive days. Body weight, fecal characteristics, and occult blood were monitored across all groups of mice throughout the experiment. The mice were euthanized by cervical dislocation, and the entire colon distal to the ileum was immediately dissected for further analysis. This included histological scoring of the colon, ELISA to detect changes in CRH and TLR4 levels in the colonic mucosa, Western blot analysis to assess autophagy through the LC3II/LC3I ratio and P62 levels, and in situ detection of apoptosis in intestinal epithelial cells using the TUNEL method.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eCompared to the DSS group, DSS mice subjected to daily restraint stress exhibited increased TLR4 expression (P=0.0241\u0026lt;0.05), greater weight loss (P=0.0343\u0026lt;0.05), higher histological scores (P=0.0171\u0026lt;0.05), and an elevated apoptosis index (P=0.0375\u0026lt;0.05). Additionally, these mice had higher serum IL-6 levels than those given DSS alone (P=0.0004\u0026lt;0.05). The DSS+Ast2B group showed a higher LC3BⅡ/LC3BⅠ ratio compared to the DSS+NS group (P=0.0417\u0026lt;0.05). In contrast, the DSS+Ucn2 group had lower LC3BⅡ/LC3BⅠ (P=0.0011\u0026lt;0.05) and TLR4 (P=0.0025\u0026lt;0.05) protein expression, along with higher p62 protein levels (P=0.0129\u0026lt;0.05) compared to the DSS+NS group. Correlation analysis revealed a significant association between TLR4 and autophagy-related proteins (LC3 and p62) (P\u0026lt;0.05). Compared to the DSS+STRESS+NS group, the DSS+STRESS+TXYF group showed lower expression of CRH (P=0.0031\u0026lt;0.05) and TLR4 (P=0.0058\u0026lt;0.05), with reduced autophagy levels (all P\u0026lt;0.05). Additionally, the DSS+STRESS+TXYF group demonstrated increased colon length (P=0.0004\u0026lt;0.05) and reduced DAI scores (P=0.0384\u0026lt;0.05), histopathological scores (P=0.0015\u0026lt;0.05), and apoptosis levels (P=0.0125\u0026lt;0.05). Compared to the DSS+STRESS+Ast2B group, the DSS+STRESS+TXYF+Ast2B group showed lower expression of CRH (P=0.0109\u0026lt;0.05) and TLR4 (P=0.0057\u0026lt;0.05), along with reduced levels of the autophagy-related protein LC3 (P=0.0146\u0026lt;0.05). Furthermore, the DSS+STRESS+TXYF+Ast2B group exhibited increased colon length (P\u0026lt;0.0001) and reduced DAI scores (P\u0026lt;0.0001), histopathological scores (P\u0026lt;0.0001), and apoptosis levels (P=0.0002\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eRestraint stress enhances the expression of TLR4, thereby exacerbating colitis. CRHR2 serves to inhibit TLR4 protein expression, consequently reducing autophagy levels in intestinal epithelial cells. TXYF mediates the effects of CRHR2 on the CRH-TLR4 signaling axis, diminishing apoptosis in intestinal epithelial cells, inhibiting autophagy, preserving the integrity of the intestinal mucosal barrier, and exerting anti-inflammatory effects.\u003c/p\u003e","manuscriptTitle":"Tong-Xie-Yao-Fang Modulates Mucosal Repair via CRH-TLR4- Mediated Autophagy in Stress-Induced Colitis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-25 07:32:31","doi":"10.21203/rs.3.rs-5426177/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2025-04-26T13:29:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"125473350309234407107516826179400921372","date":"2025-04-26T13:27:25+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-24T13:18:31+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-24T04:48:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-04-07T19:41:44+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5be9f443-ae97-4698-b1a6-d6cf6a180a23","owner":[],"postedDate":"April 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":47632549,"name":"Health sciences/Gastroenterology"},{"id":47632550,"name":"Health sciences/Pathogenesis"}],"tags":[],"updatedAt":"2025-05-21T04:38:24+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-25 07:32:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5426177","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5426177","identity":"rs-5426177","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}