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BACKGROUND:Endothelin has been found to be involved in the breakdown of the blood-spinal cord barrier after spinal cord injury,and stem cell-derived exosomes can reduce the permeability of the blood-spinal cord barrier and repair spinal cord injury. OBJECTIVE:To investigate whether exosomes produced by human umbilical cord mesenchymal stem cells can reduce the permeability of the blood-spinal cord barrier by inhibiting endothelin-1 expression,thus repairing spinal cord injury. METHODS:Exosomes were extracted from the cultured supernatant by the hyperspeed centrifugation method.The morphology of exosomes was observed by transmission electron microscope.The expression levels of tsg101 and CD63 were detected by western blot assay.Eighty SD rats were randomly divided into sham operation group,model group,exosome group,and endothelin-1 group(n=20).The modified Allen's method was used to create the rat model of spinal cord injury.In the endothelin-1 group,10 μL(1 μg/mL)endothelin-1 was injected directly into the injured area with a microsyringe.Immediately,1 day,2 days after operation,sham operation group and model group were injected with 200 μL PBS solution through the tail vein;the exosome group and endothelin-1 group were injected with 200 μL exosome(200 μg/mL)solution through the tail vein,respectively.Hind limb motor function scores were performed on days 1,3,7,14 and 21 after spinal cord injury.The blood-spinal cord barrier permeability was observed by Evans blue staining on day 7 after injury.The expression levels of tight junction proteins β-Catenin,ZO-1,Occludin and endothelin-1 in the spinal cord were detected by western blot assay. RESULTS AND CONCLUSION:(1)Basso-Beattie-Bresnahan score in the exosome group was significantly higher than that in the model group at 3-21 days after injury(P<0.05).Hematoxylin-eosin staining showed that spinal cord injury was greatly reduced in the exosome group compared with the model group.Basso-Beattie-Bresnahan score in the endothelin-1 group was significantly decreased compared with the exosome group(P<0.05).Spinal cord injury was more severe in the endothelin-1 group than that in the exosome group.(2)The expression of endothelin-1 in the model group was significantly increased compared with the sham operation group(P<0.05),and the expression of endothelin-1 in the exosome group was significantly decreased compared with the model group(P<0.05).(3)The blood-spinal cord barrier Evans blue exudate in the exosome group was significantly decreased compared with the model group(P<0.05).The expression levels of the tight junction proteins β-Catenin,Occludin and ZO-1 in the exosome group were increased(P<0.05);the Evans blue exudate in the endothelin-1 group was significantly increased compared with the exosome group(P<0.05).The expression level of tight junction protein was significantly decreased compared with the exosome group(P<0.05).(4)The results show that human umbilical cord mesenchymal cell-derived exosomes protect the permeability of the blood-spinal cord barrier by down-regulating the expression of endothelin-1 and play a role in the repair of spinal cord injury.
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AIM:To investigate the effects of overexpressing α-Klotho(KL)in RAW264.7 cells stimulated by oxidative stress on the proliferation, migration, tube-formation and tight junction of human umbilical vein endothelial cells(HUVECs).METHODS:RAW264.7 cells were categorized into control, 4-hydroxynonenal(4HNE), and 4HNE+KL groups, with F4/80 expression assessed via immunofluorescence staining. Three groups of conditional media were prepared for HUVECs and culture divided into M&#xF8;-NC, M&#xF8;-4HNE, and M&#xF8;-4HNE+KL groups. Cell proliferation was evaluated using CCK8 assay, while scratch test and Transwell assays were employed to measure cell migration. Additionally, tube-formation assay was conducted to assess cell tubule formation, and Western blot assay was utilized to detect the protein expression levels of Claudin 5, Occludin and ZO 1.RESULTS:The results of immunofluorescence staining showed that the fluorescence intensity of F4/80 of RAW264.7 cells in the 4HNE group was significantly enhanced compared with the control group, while that of F4/80 in the 4HNE+KL group was significantly decreased compared with the 4HNE group(all P<0.05). The CCK8 assay results revealed a significant increase in the proliferation of HUVECs in the M&#xF8;-4HNE group compared with the M&#xF8;-NC group. Conversely, the proliferation of the M&#xF8;-4HNE+KL group exhibited a significant decrease compared with that in the M&#xF8;-4HNE group(all P<0.01). The results of scratch test and Transwell assays demonstrated a significant increase in the migration of HUVECs in the M&#xF8;-4HNE group compared with the M&#xF8;-NC group, while the migration of the M&#xF8;-4HNE+KL group exhibited a significant decrease compared with the M&#xF8;-4HNE group(all P<0.01). In the tube-formation assay, it was observed that the number of tubes formed by HUVECs in the M&#xF8;-4HNE group was significantly increased compared with the M&#xF8;-NC group, while that of tubes formed in the M&#xF8;-4HNE+KL group was significantly decreased compared with the M&#xF8;-4HNE group(all P<0.01). Additionally, the Western blot results revealed a significant decrease in the relative expression levels of Claudin 5, Occludin, and ZO 1 in the M&#xF8;-4HNE group compared with the M&#xF8;-NC group. Conversely, in the M&#xF8;-4HNE+KL group, there was a significant increase in the relative expression levels of Claudin 5, Occludin, and ZO 1 compared to the M&#xF8;-4HNE group(all P<0.01).CONCLUSIONS: KL inhibits the proliferation, migration, and tube-formation of HUVECs while enhancing the tight junction by changing the activation state of macrophages in the diabetic oxidative stress environment.
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Objective:To figure out the regulatory role of cimifugin in the inflammatory injury and epithelial barrier function in house dust mite(HDM)-induced human bronchial epithelial cells via NF-κB signaling.Methods:Human bronchial epithelial BEAS-2B cells were divided into blank control(Control)group,HDM group,HDM+cimifugin(0.01 μmol/L)group,HDM+cimifugin(0.1 μmol/L)group,HDM+cimifugin(1 μmol/L)group and positive drug(HDM+Dex)group.Cell viability and apoptosis were respectively estimated by MTT and TUNEL assays.Levels of inflammatory factors in cells were examined by ELISA.Transepithelial electrical resistance(TEER)and FITC-dextran 40 kD(FD-40)flux assessed the permeability of cell monolayers.Nuclear translocation of NF-κB in cells was detected by immunofluorescence assay.Western blot was used to analysis expressions of apoptosis,inflammatory,tight junction and NF-κB signaling-associated proteins.Results:HDM induced viability injury,apoptosis,inflammatory response,epithelial barrier damage and activated NF-κB signaling(all P<0.001)in BEAS-2B cells.Cimifugin treatment dose-dependently inhibited the viability injury(P<0.05),apoptosis(P<0.01),inflammatory response(P<0.05),epithelial barrier damage(P<0.05)and inactivated NF-κB signaling(P<0.05)in BEAS-2B cells exposed to HDM.Conclusion:Cimifugin significantly inhibits HDM-elicited inflammatory injury and epithelial barrier damage in bronchial epithelial cells.This finding may provide novel strategies for the prevention and treatment of allergic asthma.
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Objective:To investigate the effects of diquat (DQ) on the expression of intestinal pyroptosis-related proteins and tight junction proteins in rats, and to analyze the role of pyroptosis in the intestinal injury of rats with acute DQ poisoning.Methods:A total of 36 Wistar male rats were randomly divided into control group, and 3 hours, 12 hours, 36 hours and 3 days exposure groups, with 6 rats in each group. Each exposure group was given 1/2 median lethal dose (LD50) of 115.5 mg/kg DQ by one-time gavage. The control group was given the same amount of normal saline by gavage. The control group was anesthetized at 3 hours after DQ gavage to take jejunal tissues; each exposure group was anesthetized at 3 hours, 12 hours, 36 hours, and 3 days after DQ gavage to take jejunal tissues, respectively. The general conditions of the rats were recorded. The pathological changes of jejunum tissue were observed by hematoxylin-eosin (HE) staining. The expression of intestinal pyroptosis-related proteins [NOD-like receptor protein 3 (NLRP3), cysteine aspartate-specific protease 1 (caspase-1), Gasdemin D (GSDMD)] in the intestinal tissues was observed by immunohistochemical staining. Western blotting was used to detect the expression of intestinal pyroptosis-related proteins and intestinal tight junction proteins (Occludin and Claudin-1).Results:Light microscopy showed that pathological changes occurred in jejunum tissue at the early stage of exposure (3 hours), and the injury was the most serious in the 12 hours exposure group, with a large number of inflammatory cells infiltrating in the tissue, and the damage was significantly reduced after 3 days exposure. Immunohistochemical results showed that NLRP3, caspase-1 and GSDMD were expressed in the jejunal mucosa of the control group and the exposure groups, and the positive cells in the control group were less expressed with light staining. The expression of the above proteins in the exposed group was increased significantly and the staining was deep. Western blotting results showed that compared with the control group, the expression of NLRP3 protein in jejunum tissues of all groups was increased, with the most significant increase in the 36 hours group (NLRP3/β-actin: 1.47±0.06 vs. 0.43±0.14, P < 0.01). Compared with the control group, the expression of GSDMD protein in the 3 hours, 12 hours and 36 hours exposure groups increased, and the expression of GSDMD protein in the 3 hours and 12 hours exposure groups increased significantly (GSDMD/β-actin: 1.04±0.40, 1.25±0.15 vs. 0.65±0.25, both P < 0.05). The expression of caspase-1 protein was increased in 36 hours exposure group compared with the control group (caspase-1/β-actin: 1.44±0.34 vs. 0.98±0.19, P > 0.05). Compared with the control group, the expression of Occludin and Claudin-1 proteins in each exposure group decreased, and the expression of Occludin proteins was significantly decreased in the 3 hours, 12 hours, and 36 hours exposure groups decreased significantly (Occludin/β-actin: 0.74±0.17, 0.91±0.20, 0.79±0.23 vs. 1.41±0.08, all P < 0.05). Although the protein expression of Claudin-1 decreased in each exposure group, the difference was not statistically significant. Conclusion:The intestinal injury caused by acute DQ poisoning may be related to the activation of pyroptosis pathway of small intestinal cells and the reduction of the density of intercellular junctions.
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Ulcerative colitis （UC）， a disease that affects the colon or rectum， is characterized by long-term recurrent inflammation and eventually leads to ulcers in the inner wall of the intestine. The disease has a high incidence and is difficult to be cured， which causes severe physical and mental discomfort and economic burden to the patients. Therefore， it is urgent to develop new therapies with high cure rate and low side effect. The pathological mechanism of UC is complex and involves multiple factors. The intestinal mucosal barrier damage is the main pathological basis of UC， which is a hot topic and a new research direction. Intestinal tight junction （TJ）， as the structural basis of the intestinal mucosal mechanical barrier， can actively regulate mucosal function and play a key role in the pathogenesis of UC. Traditional Chinese medicine （TCM） can regulate TJ protein via multiple pathways and multiple targets， repair the intestinal mucosal barrier， and thus block the progression of UC. Studies have demonstrated that Chinese herbal medicines and their components， Chinese medicine compound prescriptions， and Chinese medicine preparations can treat UC by regulating TJ protein to maintain the function and reduce the permeability of intestinal epithelium， providing a new therapeutic strategy for UC. Although TCM has unique advantages that western medicine cannot replace by mediating TJ protein expression in UC， a comprehensive review of this field remains to be carried out. Focusing on the status of UC and TCM syndrome differentiation and treatment， we retrieved relevant articles with ''ulcerative colitis''， ''tight junction''， and ''Chinese medicine'' as the keywords， and summarized the relationship of TJ and its key target proteins with UC to clarify the critical role of TJ in UC pathophysiology. Furthermore， we summarized the Chinese medicines regulating TJ in the treatment of UC in recent years， aiming to provide a theoretical basis for the development of drugs for this disease.
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@#Tight junction(TJ) is complex dynamic system involved in protein interactions in the paracellular secretory pathway, with both barrier and fence functions. Claudin family, the main section of tight junction strands, will be abnormal in expression pattern in the circumstances of radiation injuries, inflammation, Sj?gren's syndrome, diabetes and other pathological conditions in salivary glands. This change leads to abnormal structure and function of tight junctions, indirectly manifested as salivary gland dysfunction. In addition, the difference of Claudin expression in salivary gland tumors can also be used as an indicator of tumor type and prognosis. This review focuses on the progress of research on common Claudin in salivary glands, including the structure, function, expression patterns of related diseases and their applications. It is believed that the review may provide new ideas for clinical and basic research on Claudin protein-related diseases.
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Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with unclear etiology and limited treatment options. The median survival time for IPF patients is approximately 2-3 years and there is no effective intervention to treat IPF other than lung transplantation. As important components of lung tissue, endothelial cells (ECs) are associated with pulmonary diseases. However, the role of endothelial dysfunction in pulmonary fibrosis (PF) is incompletely understood. Sphingosine-1-phosphate receptor 1 (S1PR1) is a G protein-coupled receptor highly expressed in lung ECs. Its expression is markedly reduced in patients with IPF. Herein, we generated an endothelial-conditional S1pr1 knockout mouse model which exhibited inflammation and fibrosis with or without bleomycin (BLM) challenge. Selective activation of S1PR1 with an S1PR1 agonist, IMMH002, exerted a potent therapeutic effect in mice with bleomycin-induced fibrosis by protecting the integrity of the endothelial barrier. These results suggest that S1PR1 might be a promising drug target for IPF therapy.
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Objective:To investigate the effect of Xuanbai Chengqi Decoction (XBCQT) on lung-gut injury and intestinal function, and analyze its effect on intestinal flora in sepsis mice.Methods:C57 male mice were randomly divided into three groups with 12 mice in each group: control group, model group and treatment group. The sepsis model was prepared by intra-peritoneal injection of lipopolysaccharide (LPS) 5 mg/kg. XBCQT was administered by gavage 24 h before, 0.5 h after and 12 h after modeling. The lung, colon and blood samples were collected at 24 h after modeling. The pulmonary and intestinal inflammatory cytokine content of interleukin-1β (IL-1β), IL-6, tumor necrosis factor α (TNF-α) and monocyte chemoattractant protein (MCP-1) was measured by real-time fluorescence quantitative PCR. HE staining was used to evaluate the structural damage and changes of lung and gut, and Western blot and Immunohistochemistry methods were used to analyze the expression of occludin and claudin-1 in intestinal epithelium. Finally, the plasma endotoxin content of each group was tested by Limulus test kit. Fecal DNA of mice was extracted and the changes of intestinal flora in sepsis mice were detected by 16S rDNA quantitative PCR. The measurement data among the three groups were compared by one-way analysis of variance.Results:(1) XBCQT significantly reduced the pulmonary inflammatory cytokine IL-1β, IL-6, TNF-α and MCP-1 expression (all P<0.05), and attenuated lung injury. (2) Compared to the model group, the treatment group exhibited a reduction in intestinal damage and a decrease in the intestinal inflammatory cytokines (all P<0.05). XBCQT increased the expression of epithelial tight junction and mucin of colon, and improved the intestinal epithelium barrier function. (3) XBCQT treatment decreased the content of endotoxin in plasma of sepsis mice ( P<0.05), promoted the growth of beneficial bacteria Akkermansia muciniphila and reduced the expression of Enterococcus in the intestine of sepsis mice (all P<0.05). Conclusions:XBCQT can significantly improve the intestinal inflammatory injury, regulate the intestine epithelium barrier and improve the intestinal function in sepsis mice.
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Objective:To analyze the correlation between the severity of acute pancreatitis (AP) and the levels of zonulin, zonula occludens protein-1 (ZO-1), tumor necrosis factor -α (TNF -α) in the peripheral blood of patients with acute pancreatitis (AP), and the value of predicting moderate and severe AP.Methods:The clinical data of 115 AP patients admitted to the Second Affiliated Hospital of Anhui Medical University from June 2020 to January 2022 were retrospectively analyzed. They were divided into mild group (69 cases) and moderate severe group (46 cases). The blood levels of zonulin, ZO-1, and TNF-α were measured for all patients on the 1st, 3rd, and 7th day after admission, and the results of the two group tests were compared. The correlation between zonulin, ZO-1, TNF -α and Acute Physiology and Chronic Health Evaluation Ⅱ (APACHE Ⅱ) scores on the 1st day was and the value of various indicators for predicting moderate to severe AP were analyzed.Results:The C-reactive protein (CRP) levels of AP patients in the moderate to severe group were higher than those in the mild group, and the difference was statistically significant (all P<0.05). The levels of zonulin, ZO-1, and TNF -α in AP patients in the moderate to severe group showed an upward trend on the 1st, 3rd, and 7th days after admission. The levels of zonulin, ZO-1, and TNF -α in AP patients in the moderate to severe group were higher than those in the mild group at the same time point, and the differences were statistically significant (all P<0.05). The APACHE Ⅱ score of AP patients on the first day of admission was positively correlated with the levels of zonulin, ZO-1, and TNF -α ( r=0.736, 0.552, 0.621, all P<0.05). Zonulin had the highest area under the curve (AUC) for predicting moderate to severe AP, at 0.892, with an optimal threshold of 2.075 pg/ml. Zonulin had the highest sensitivity, at 0.804, and ZO-1 had the highest specificity, at 0.926. Using zonulin ≥2.075 pg/ml, ZO-1≥399.4 ng/ml, and TNF -α≥40.88 pg/ml as thresholds; the sensitivity and specificity obtained from parallel experiments were 0.976 and 0.710, respectively; The sensitivity and specificity obtained from the series of experiments were 0.326 and 0.999, respectively. Conclusions:There is a correlation between the serum levels of zonulin, ZO-1, and TNF -α in AP patients and the severity of AP. Zonulin, ZO-1, and TNF -α have certain clinical value in predicting moderate to severe AP.
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Objective:To investigate the protective effect of water-soluble dietary fiber fructooligosaccharides(FOS) on intestinal mucosal barrier in mice with ulcerative colitis(UC), and to find a new drug option for the treatment of patients with UC.Methods:This study used 4% dextran sodium sulfate(DSS)to induce a 7-day UC mouse model.Male C57BL/6 mice aged 6-8 weeks were randomly divided into three groups: the control group drank distilled water; The model group was given 4% DSS; The oligofructose group was administered 20 mg/mL FOS by gavage simultaneously with DSS induction.The body weight, fecal characteristics, and fecal blood status of mice daily, and the disease activity index(DAI)score were monitored.After the experiment, HE staining was used to observe the pathological changes in the colon tissue of mice.Real time fluorescence quantitative PCR, Western Blot, and immunofluorescence staining were used to detect the expression levels of tight junction proteins ZO-1, Claudin-1, and Occludin in the intestines of each group of mice.Results:Compared with control group, the weight of mice decreased and the DAI score increased in model group( P<0.05). However, the weight of mice in FOS group was higher than that in model group, and the DAI score was lower than that in model group ( P<0.05). Compared with control group, the colon length of mice in model group was shortened[(7.52±0.41)cm vs.(5.48±0.19)cm], and the histopathological score was increased (0.53±0.38 vs.3.51±0.18). However, the colon length of mice in FOS group[(6.82±0.63)cm] was longer than that in model group, and the hispathological score(2.33±0.63) was lower than that in model group.All the differences were statistically significant ( P<0.05). The expression levels of ZO-1, Claudin-1, Occludin protein and its mRNA levels in model group were lower than those in control group ( P<0.05). However, those of FOS group were higher than those in model group ( P<0.05). Conclusion:FOS could alleviate the symptoms of weight loss in UC mice, reduce their DAI score, improve inflammation of colon tissue, upregulate the expression of tight junction proteins ZO-1, Claudin-1, and Occludin, and protect the intestinal mucosal barrier.