Effects of electroacupuncture with "intestinal disease prescription" on NLRP3 inflammasome and intestinal mucosal barrier in rats with acute ulcerative colitis. / 电针"è‚ ç— æ–¹"å¯¹æ€¥æ€§æºƒç–¡æ€§ç»“è‚ ç‚Žå¤§é¼ NLRP3ç‚Žæ€§å°ä½“å’Œè‚ é»è†œå±éšœçš„å½±å“.

OBJECTIVES: To observe the effects of electroacupuncture (EA) with "intestinal disease prescription" on the intestinal mucosal barrier and NLRP3 inflammasome in rats with dextran sulfate sodium (DSS)-induced acute ulcerative colitis (UC), and explore the underlying mechanism of EA with "intestinal disease prescription" for the treatment of UC. METHODS: Thirty-two healthy male SPF-grade SD rats were randomly divided into a blank group, a model group, a medication group, and an EA group, with 8 rats in each group. Except for the blank group, the UC model was established by administering 5% DSS solution for 7 days. After modeling, the rats in the medication group were treated with mesalazine suspension (200 mg/kg) by gavage, while the rats in the EA group were treated with acupuncture at bilateral "Tianshu" (ST 25), "Shangjuxu" (ST 37) and "Zhongwan" (CV 12), with the ipsilateral "Tianshu" (ST 25) and "Shangjuxu" (ST 37) connected to the electrodes of the EA instrument, using disperse-dense wave, with a frequency of 10 Hz/50 Hz, and each intervention lasted for 20 minutes. Both interventions were performed once daily for 3 days. The general conditions of rats were observed daily. After intervention, the disease activity index (DAI) score was calculated; colon tissue morphology was observed using HE staining; serum levels of pro-inflammatory cytokines (interleukin [IL]-18, IL-1ß) were measured by ELISA; protein expression of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), and Caspase-1 in colon tissues was detected by Western blot; positive expression of zonula occludens-1 (ZO-1) and Occludin in colon tissues was examined by immunofluorescence. RESULTS: Compared with the blank group, the rats in the model group exhibited poor general conditions, slow body weight gain, shortened colon length (P<0.01), increased DAI score and spleen index (P<0.01), elevated serum IL-18 and IL-1ß levels, and increased protein expression of NLRP3, ASC, and Caspase-1 in colon tissues (P<0.01), along with decreased positive expression of ZO-1 and Occludin in colon tissues (P<0.01). Compared with the model group, the rats in the medication group and the EA group exhibited improved general conditions, accelerated body weight gain, increased colon length (P<0.05), reduced DAI scores and spleen indexes (P<0.05), decreased serum IL-18 and IL-1ß levels, and lower protein expression of NLRP3, ASC and Caspase-1 in colon tissues (P<0.05), as well as increased positive expression of ZO-1 and Occludin in colon tissues (P<0.05). There were no significant differences in the above indexes between the medication group and the EA group (P>0.05). Compared with the blank group, the rats in the model group exhibited disrupted colon mucosal morphology, disordered gland arrangement, and atrophy of crypts, along with significant inflammatory cell infiltration. Compared with the model group, the rats in both the medication group and the EA group showed relatively intact colon mucosal morphology, with restored and improved gland and crypt structures, and reduced inflammatory cell infiltration. CONCLUSIONS: EA with "intestinal disease prescription" has a significant therapeutic effect on DSS-induced UC, possibly by regulating the expression of NLRP3 inflammasome and proteins related to the intestinal mucosal barrier, thereby alleviating symptoms of ulcerative colitis.

Effects of moxibustion on intestinal barrier function and TLR4/NF-κB p65 signaling pathway in obese rats. / è‰¾ç¸å¯¹è‚¥èƒ–å¤§é¼ è‚ é“å±éšœåŠŸèƒ½åŠTLR4/NF-κB p65信号通路的影响.

OBJECTIVES: To observe the effects of moxibustion on intestinal barrier function and Toll-like receptor 4 (TLR4)/nuclear factor-κB p65 (NF-κB p65) signaling pathway in obese rats and explore the mechanism of moxibustion in the intervention of obesity. METHODS: Fifty-five Wistar rats of SPF grade were randomly divided into a normal group (10 rats) and a modeling group (45 rats). In the modeling group, the obesity model was established by feeding high-fat diet. Thirty successfully-modeled rats were randomized into a model group, a moxibustion group, and a placebo-control group, with 10 rats in each one. In the moxibustion group, moxibustion was applied at the site 3 cm to 5 cm far from the surface of "Zhongwan" (CV 12), with the temperature maintained at (46±1 ) ℃. In the placebo-control group, moxibustion was applied at the site 8 cm to 10 cm far from "Zhongwan" (CV 12), with the temperature maintained at (38±1) ℃. The intervention was delivered once daily for 8 weeks in the above two groups. The body mass and food intake of the rats were observed before and after intervention in each group. Using ELISA methool, the levels of serum triacylglycerol (TG), total cholesterol (TC) and lipopolysaccharide (LPS) were detected and the insulin resistance index (HOMA-IR) was calculated. HE staining was used to observe the morphology of colon tissue. The mRNA expression of zonula occludens-1 (ZO-1), Occludin, Claudin-1, TLR4 and NF-κB p65 in the colon tissue was detected by quantitative real-time PCR; and the protein expression of ZO-1, Occludin, Claudin-1, TLR4 and NF-κB p65 was detected by Western blot in the rats of each group. RESULTS: Compared with the normal group, the body mass, food intake, the level of HOMA-IR, and the serum levels of TC, TG and LPS were increased in the rats of the model group (P<0.01); those indexes in the moxibustion group were all reduced when compared with the model group and the placebo-control group respectively (P<0.01, P<0.05). Compared with the normal group, a large number of epithelial cells in the mucosa of colon tissue was damaged, shed, and the inflammatory cells were infiltrated obviously in the interstitium in the rats of the model group. When compared with the model group, in the moxibustion group, the damage of the colon tissue was recovered to various degrees and there were few infiltrated inflammatory cells in the interstitium, while, the epithelial injury of the colon tissue was slightly recovered and the infiltrated inflammatory cells in the interstitium were still seen in the placebo-control group. The mRNA and protein expressions of ZO-1, Occludin and Caudin-1 were decreased in the model group compared with those in the normal group (P<0.01). When compared with the model group and the placebo-control group, the mRNA and protein expressions of these indexes were increased in the moxibustion group (P<0.01, P<0.05). In the model group, the mRNA and protein expressions of TLR4 and NF-κB p65 were increased when compared with those in the normal group (P<0.01), and the mRNA and protein expressions of these indexes were reduced in the moxibustion group when compared with those in the model group and the placebo-control group (P<0.01). CONCLUSIONS: Moxibustion can reduce the body mass and food intake, regulate the blood lipid and improve insulin resistance in the rats of obesity. It may be related to alleviating inflammatory response through improving intestinal barrier function and modulating the intestinal TLR4/NF-κB p65 signaling pathway.

Occludin and collagen IV degradation mediated by the T9SS effector SspA contributes to blood-brain barrier damage in ducks during Riemerella anatipestifer infection.

Riemerella anatipestifer infection is characterized by meningitis with neurological symptoms in ducklings and has adversely affected the poultry industry. R. anatipestifer strains can invade the duck brain to cause meningitis and neurological symptoms, but the underlying mechanism remains unknown. In this study, we showed that obvious clinical symptoms, an increase in blood‒brain barrier (BBB) permeability, and the accumulation of inflammatory cytokines occurred after intravenous infection with the Yb2 strain but not the mutant strain Yb2ΔsspA, indicating that Yb2 infection can lead to cerebrovascular dysfunction and that the type IX secretion system (T9SS) effector SspA plays a critical role in this pathological process. In addition, we showed that Yb2 infection led to rapid degradation of occludin (a tight junction protein) and collagen IV (a basement membrane protein), which contributed to endothelial barrier disruption. The interaction between SspA and occludin was confirmed by coimmunoprecipitation. Furthermore, we found that SspA was the main enzyme mediating occludin and collagen IV degradation. These data indicate that R. anatipestifer SspA mediates occludin and collagen IV degradation, which functions in BBB disruption in R. anatipestifer-infected ducks. These findings establish the molecular mechanisms by which R. anatipestifer targets duckling endothelial cell junctions and provide new perspectives for the treatment and prevention of R. anatipestifer infection.

Whey protein supplementation reduced the liver damage scores of rats fed with a high fat-high fructose diet.

Different functional foods with bioactive nutrients are being explored for the management of NAFLD. Whey proteins are rich in bioactive peptides and are suggested to show antioxidant and anti-inflammatory effects. We aim to test the hypothesis that the whey protein supplementation following a high fat-high fructose (HFHF) diet would protect against liver damage, inflammation, endotoxemia and steatosis in male Wistar rats. 36 rats were randomized into four groups for 8 weeks as the HFHF diet group, HFHF diet and whey protein isolate (WPI-200mg/kg/day) group (HFHF+WPI), control (C) group, and C+WPI (200mg/kg/day) group. Rats fed with a HFHF diet had higher final body weight compared to C and C+WPI groups (p = 0.002). Thus, WPI showed no significant effects for the body weight of rats with a HFHF diet. On the other hand, the HFHF+WPI group had significantly lower abdominal circumference when compared with the HFHF group (p<0,001). Higher serum CRP levels were observed in the groups with a HFHF diet (p<0,001) and WPI supplementation showed no effects on CRP levels. Whey protein supplementation resulted with lower total liver damage score in HFHF+WPI group compared with the HFHF diet group (p<0,001). Conversely, higher liver damage scores were observed with the C+WPI group compared to C group (p<0,001). HFHF diet resulted with higher expression of TLR-4 in the liver meanwhile WPI supplementation showed no effects on liver TLR-4 expression. We observed higher colon Occludin expression in HFHF+WPI and C+WPI groups compared with HFHF and C groups (p<0,001). Our results showed that, whey protein supplementation might help improve liver damage associated with a high fat-high fructose diet and increase the expression of Occludin in the small intestine and colon.

Resveratrol ameliorates ulcerative colitis by upregulating Nrf2/HO­1 pathway activity: Integrating animal experiments and network pharmacology.

Ulcerative colitis (UC) is a chronic idiopathic inflammatory condition affecting the rectum and colon. Inflammation and compromisation of the intestinal mucosal barrier are key in UC pathogenesis. Resveratrol (Res) is a naturally occurring polyphenol that exhibits anti­inflammatory and antioxidant properties. Nuclear factor erythroid­2­related factor 2/heme oxygenase 1 (Nrf2/HO­1) pathway regulates occurrence and development of numerous types of diseases through anti­inflammatory and antioxidant activity. However, it is not clear whether Nrf2/HO­1 pathway is involved in the treatment of Res in UC. Therefore, the present study aimed to investigate whether Res modulates the Nrf2/HO­1 signaling pathway to attenuate UC in mice. Dextran sulfate sodium (DSS) was used to induce experimental UC in male C57BL/6J mice. Disease activity index (DAI) and hematoxylin eosin (H&E) staning was used to assessed the magnitude of colonic lesions in UC mice. ELISA) was utilized to quantify inflammatory cytokines (IL­6, IL­1ß, TNF­α and IL­10) in serum and colon tissues. Immunohistochemistry and Western blot were used to evaluate the expression levels of tight junction (TJ) proteins [zonula occludens (ZO)­1 and Occludin] in colon tissues. Pharmacokinetic (PK) parameters of Res were derived from TCMSP database. Networkpharmacology was employed to identify the biological function and pharmacological mechanism of Res in the process of relieving UC, and the key target was screened. The binding ability of Res and key target was verified by molecular docking. Finally, the effectiveness of key target was substantiated by Western blot. Res decreased DAI, ameliorated histopathological changes such as crypt loss, disappeatance of the mucosal epithelium, and inflammatory infiltration in mice. Additionally, Res decreased expression of pro­inflammatory cytokines IL­6, IL­1ß and TNF­α and increased anti­inflammatory factor IL­10 expression. Res also restored the decreased protein expression of ZO­1 and occludin after DSS treatment, increasing the integrity of the intestinal mucosal barrier. The PK properties of Res suggested that Res possesses the therapeutic potential for oral administration. Network pharmacology revealed that Res alleviated UC through anti­inflammatory and antioxidant pathways, and confirmed that Nrf2 has a high binding affinity with Res and is a key target of Res against UC. Western blotting demonstrated that Res treatment increased the protein levels of Nrf2 and HO­1. In conclusion, Res treatment activated the Nrf2/HO­1 pathway to decrease clinical symptoms, inflammatory responses, and intestinal mucosal barrier damage in experimental UC mice.

Protective effect of synbiotic combination of Lactobacillus plantarum SC-5 and olive oil extract tyrosol in a murine model of ulcerative colitis.

BACKGROUND: Ulcerative colitisis (UC) classified as a form of inflammatory bowel diseases (IBD) characterized by chronic, nonspecific, and recurrent symptoms with a poor prognosis. Common clinical manifestations of UC include diarrhea, fecal bleeding, and abdominal pain. Even though anti-inflammatory drugs can help alleviate symptoms of IBD, their long-term use is limited due to potential side effects. Therefore, alternative approaches for the treatment and prevention of inflammation in UC are crucial. METHODS: This study investigated the synergistic mechanism of Lactobacillus plantarum SC-5 (SC-5) and tyrosol (TY) combination (TS) in murine colitis, specifically exploring their regulatory activity on the dextran sulfate sodium (DSS)-induced inflammatory pathways (NF-κB and MAPK) and key molecular targets (tight junction protein). The effectiveness of 1 week of treatment with SC-5, TY, or TS was evaluated in a DSS-induced colitis mice model by assessing colitis morbidity and colonic mucosal injury (n = 9). To validate these findings, fecal microbiota transplantation (FMT) was performed by inoculating DSS-treated mice with the microbiota of TS-administered mice (n = 9). RESULTS: The results demonstrated that all three treatments effectively reduced colitis morbidity and protected against DSS-induced UC. The combination treatment, TS, exhibited inhibitory effects on the DSS-induced activation of mitogen-activated protein kinase (MAPK) and negatively regulated NF-κB. Furthermore, TS maintained the integrity of the tight junction (TJ) structure by regulating the expression of zona-occludin-1 (ZO-1), Occludin, and Claudin-3 (p < 0.05). Analysis of the intestinal microbiota revealed significant differences, including a decrease in Proteus and an increase in Lactobacillus, Bifidobacterium, and Akkermansia, which supported the protective effect of TS (p < 0.05). An increase in the number of Aspergillus bacteria can cause inflammation in the intestines and lead to the formation of ulcers. Bifidobacterium and Lactobacillus can regulate the micro-ecological balance of the intestinal tract, replenish normal physiological bacteria and inhibit harmful intestinal bacteria, which can alleviate the symptoms of UC. The relative abundance of Akkermansia has been shown to be negatively associated with IBD. The FMT group exhibited alleviated colitis, excellent anti-inflammatory effects, improved colonic barrier integrity, and enrichment of bacteria such as Akkermansia (p < 0.05). These results further supported the gut microbiota-dependent mechanism of TS in ameliorating colonic inflammation. CONCLUSION: In conclusion, the TS demonstrated a remission of colitis and amelioration of colonic inflammation in a gut microbiota-dependent manner. The findings suggest that TS could be a potential natural medicine for the protection of UC health. The above results suggest that TS can be used as a potential therapeutic agent for the clinical regulation of UC.

[Modulating gut microbiota improves neurological function and depressive symptoms in rats with post-stroke depression].

OBJECTIVE: To evaluate the effect of modulating gut microbiota for improving brain injury in rats with post-stroke depression. METHODS: Adult SD rats were randomized into normal control, middle cerebral artery occlusion (MCAO), post-stroke depression (PSD), PSD with fecal transplantation, PSD with antibiotics (rifaximin), PSD with probiotics (lactobacilli), and PSD with fluoxetine treatment groups (n=9). Neurological function scores of the rats were determined, and the changes in sugar water preference and immobility time in forced swimming test were observed; plasma levels of trimethylamine N-oxide (TMAO) and hydrogen sulfide (H2S) were detected with ELISA, Occludin, and the expressions of occludin, caudin-5 and IgG proteins Ⅰ the brain tissues were determined using Western blotting. RESULTS: Compared with those in the control group, the rats in MCAO and PSD groups had significantly increased neurological function scores, TMAO level, the ratio of TMAO/H2S, and immobility time in forced swimming test with a lowered level of H2S (P < 0.05). These changes were more obvious in PSD rats, which also exhibited a reduced sugar water preference with increased IgG protein and decreased occluding and caudin-5 expressions in the brain tissue (P < 0.05). TMAO/H2S ratio in PSD rats was positively correlated with neurological function score (R2=0.3235, P=0.0269) and immobility time in swimming (R2=0.6290, P=0.0004) and negatively with sugar water preference (R2=-0.4534, P=0.0059). Treatment with fecal transplantation, antibiotics, probiotics and fluoxetine all significantly reduced neurological function scores, immobility time in forced swimming, TMAO/H2S ratio, and IgG protein expression and increased sugar water preference and brain occludin and caudin-5 expressions of the PSD rats (P < 0.05). CONCLUSION: In PSD rats, TMAO/H2S ratio is correlated with neurological function score, immobility time in forced swimming and sugar water preference, and modulating intestinal flora can improve neurological function and depressive symptoms and improve the integrity of the blood-brain barrier.

[Mechanism of intestinal injury induced by acute diquat poisoning in rats].

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/ß-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/ß-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/ß-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/ß-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. CONCLUSIONS: 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.

Mitigation effect of hesperidin on X-ray radiation-induced intestinal barrier dysfunction in Caco-2 cell monolayers.

The tight junctions (TJs) and barrier function of the intestinal epithelium are highly sensitive to radiation. However, polyphenols can be used to reverse the effects of radiation. Here, we investigated the effects of hesperidin (hesperetin-7-rhamnoglucoside) on X-ray-induced intestinal barrier dysfunction in human epithelial Caco-2 monolayers. To examine whether hesperidin mitigated the effects of X-ray exposure (2 Gy), cell survival was evaluated and intestinal barrier function was assessed by measuring the transepithelial flux, apparent permeability coefficient (Papp), and barrier integrity. Hesperidin improved the survival of Caco-2 cell monolayers and attenuated X-ray exposure-induced intestinal barrier dysfunction. For fluorescein transport experiments, transepithelial flux and Papp of fluorescein in control group were significantly elevated by X-ray, but were restored to near control by 10 µM hesperidin pretreatment. Further, X-ray exposure decreased the barrier integrity and TJ interruption by reducing TJ-related proteins occludin and claudin-4, whereas cell monolayers pretreated with hesperidin before X-ray exposure were reinstated to control level. It was concluded that hesperidin treatment before X-ray exposure alleviated X-ray-induced intestinal barrier dysfunction through regulation of TJ-related proteins. These results indicate that hesperidin prevents and mitigates X-ray-induced intestinal barrier dysfunction.

Azilsartan improves urinary albumin excretion in hypertension mice.

Hypertension is one of the most important risk factors for chronic kidney diseases, leading to hypertensive nephrosclerosis, including excessive albuminuria. Azilsartan, an angiotensin II type 1 receptor blocker, has been widely used for the treatment of hypertension. However, the effects of Azilsartan on urinary albumin excretion in hypertension haven't been reported before. In this study, we investigated whether Azilsartan possesses a beneficial property against albuminuria in mice treated with angiotensin II and a high-salt diet (ANG/HS). Compared to the control group, the ANG/HS group had higher blood pressure, oxidative stress, and inflammatory response, all of which were rescued by Azilsartan dose-dependently. Importantly, the ANG/HS-induced increase in urinary albumin excretion and decrease in the expression of occludin were reversed by Azilsartan. Additionally, it was shown that increased fluorescence intensity of FITC-dextran, declined trans-endothelial electrical resistance (TEER) values, and reduction of occludin and krüppel-like factor 2 (KLF2) were observed in ANG/HS-treated human renal glomerular endothelial cells (HrGECs), then prevented by Azilsartan. Moreover, the regulatory effect of Azilsartan on endothelial monolayer permeability in ANG/HS-treated HrGECs was abolished by the knockdown of KLF2, indicating KLF2 is required for the effect of Azilsartan. We concluded that Azilsartan alleviated diabetic nephropathy-induced increase in Uterine artery embolization (UAE) mediated by the KLF2/occludin axis.

Myricetin alleviates diabetic cardiomyopathy by regulating gut microbiota and their metabolites.

BACKGROUND: The gut microbiota is involved in the pathogenesis of diabetic cardiomyopathy (DCM). Myricetin protects cardiac function in DCM. However, the low bioavailability of myricetin fails to explain its pharmacological mechanisms thoroughly. Research has shown that myricetin has a positive effect on the gut microbiota. We hypothesize that myricetin improves the development of DCM via regulating gut microbiota. METHODS: DCM mice were induced with streptozotocin and fed a high-fat diet, and then treated with myricetin by gavage and high-fat diet for 16 weeks. Indexes related to gut microbiota composition, cardiac structure, cardiac function, intestinal barrier function, and inflammation were detected. Moreover, the gut contents were transplanted to DCM mice, and the effect of fecal microbiota transplantation (FMT) on DCM mice was assessed. RESULTS: Myricetin could improve cardiac function in DCM mice by decreasing cardiomyocyte hypertrophy and interstitial fibrosis. The composition of gut microbiota, especially for short-chain fatty acid-producing bacteria involving Roseburia, Faecalibaculum, and Bifidobacterium, was more abundant by myricetin treatment in DCM mice. Myricetin increased occludin expression and the number of goblet cells in DCM mice. Compared with DCM mice unfed with gut content, the cardiac function, number of goblet cells, and expression of occludin in DCM mice fed by gut contents were elevated, while cardiomyocyte hypertrophy and TLR4/MyD88 pathway-related proteins were decreased. CONCLUSIONS: Myricetin can prevent DCM development by increasing the abundance of beneficial gut microbiota and restoring the gut barrier function.

Mannose enhances intestinal immune barrier function and dextran sulfate sodium salt-induced colitis in mice by regulating intestinal microbiota.

Background: Inflammatory bowel disease (IBD) greatly affects human quality of life. Mannose has been reported to be used to treat IBD, but the mechanism is currently unknown. Methods: C57/BL mice were used as research subjects, and the mouse acute colitis model was induced using dextran sulfate sodium salt (DSS). After oral administration of mannose, the body weights and disease activity index (DAI) scores of the mice were observed. The colon lengths, histopathological sections, fecal content microbial sequencing, colon epithelial inflammatory genes, and tight junction protein Occludin-1 expression levels were measured. We further used the feces of mice that had been orally administered mannose to perform fecal bacterial transplantation on the mice with DSS-induced colitis and detected the colitis-related indicators. Results: Oral administration of mannose increased body weights and colon lengths and reduced DAI scores in mice with DSS-induced colitis. In addition, it reduced the expression of colon inflammatory genes and the levels of serum inflammatory factors (TNF-α, IL-6, and IL-1ß), further enhancing the expression level of the colonic Occludin-1 protein and alleviating the toxic response of DSS to the intestinal epithelium of the mice. In addition, gut microbial sequencing revealed that mannose increased the abundance and diversity of intestinal flora. Additionally, after using the feces of the mannose-treated mice to perform fecal bacterial transplantation on the mice with DSS-induced colitis, they showed the same phenotype as the mannose-treated mice, and both of them alleviated the intestinal toxic reaction induced by the DSS. It also reduced the expression of intestinal inflammatory genes (TNF-α, IL-6, and IL-1ß) and enhanced the expression level of the colonic Occludin-1 protein. Conclusion: Mannose can treat DSS-induced colitis in mice, possibly by regulating intestinal microorganisms to enhance the intestinal immune barrier function and reduce the intestinal inflammatory response.

Effect of High Sodium Intake on Gut Tight Junctions' Structure and Permeability to Bacterial Toxins in a Rat Model of Chronic Kidney Disease.

INTRODUCTION: Uremic toxicity changes the gut structure and permeability, allowing bacterial toxins to translocate from the lumen to the blood during chronic kidney failure (CKD). Clinical fluid overload and tissue edema without uremia have similar effects but have not been adequately demonstrated and analyzed in CKD. AIMS: To investigate the effect of sodium intake on the plasma concentration of gut-derived uremic toxins, indoxyl sulfate (IS), and p-cresyl sulfate (pCS) and the expression of genes and proteins of epithelial gut tight junctions in a rat model of CKD. METHODS: Sham-operated (control group, CG) and five-sixths nephrectomized (5/6Nx) Sprague-Dawley rats were randomly assigned to low (LNa), normal (NNa), or high sodium (HNa) diets., Animals were then sacrificed at 8 and 12 weeks and analyzed for IS and pCS plasma concentrations, as well as for gene and protein expression of thigh junction proteins, and transmission electron microscopy (TEM) in colon fragments. RESULTS: The HNa 5/6Nx groups had higher concentrations of IS and pCS than CG, NNa, and LNa at eight and twelve weeks. Furthermore, HNa 5/6Nx groups had reduced expression of the claudin-4 gene and protein than CG, NNa, and LNa. HNa had reduced occludin gene expression compared to CG. Occludin protein expression was more reduced in HNa than in CG, NNa, and LNa. The gut epithelial tight junctions appear dilated in HNa compared to NNa and LNa in TEM. CONCLUSION: Dietary sodium intake and fluid overload have a significant role in gut epithelial permeability in the CKD model.

Gegen Qinlian decoction ameliorates TNBS-induced ulcerative colitis by regulating Th2/Th1 and Tregs/Th17 cells balance, inhibiting NLRP3 inflammasome activation and reshaping gut microbiota.

ETHNOPHARMACOLOGICAL RELEVANCE: Chinese herbal medicine Gegen Qinlian Decoction (GQD) has been clinically shown to be an effective treatment of ulcerative colitis (UC) in China. However, the underlying mechanism of GQD's anti-ulcerative colitis properties and its effect on gut microbiota still deserve further exploration. AIM OF THE STUDY: This study observed the regulatory effects of GQD on Th2/Th1 and Tregs/Th17 cells balance, the NOD-like receptor family pyrin domain containing 3 (NLRP3) infammasome and gut microbiota in TNBS-induced UC in BALB/c mice. MATERIALS AND METHODS: 61 main chemical compounds in the GQD were determined by UPLC-Q-TOF/MS. The UC BALB/c model was established by intrarectal administration of trinitrobenzene sulfonic acid (TNBS), and GQD was orally administered at low and high dosages of 2.96 and 11.83 g/kg/day, respectively. The anti-inflammatory effects of GQD for ulcerative colitis were evaluated by survival rate, body weight, disease activity index (DAI) score, colonic weight and index, spleen index, hematoxylin-eosin (HE) staining and histopathological scores. Flow cytometry was used to detect the percentage of CD4, Th1, Th2, Th17 and Tregs cells. The levels of Th1-/Th2-/Th17-/Tregs-related inflammatory cytokines and additional proinflammatory cytokines (IL-1ß, IL-18) were detected by CBA, ELISA, and RT-PCR. The expressions of GATA3, T-bet, NLRP3, Caspase-1, IL-Iß, Occludin and Zonula occludens-1 (ZO-1) on colon tissues were detected by Western blot and RT-PCR. Transcriptome sequencing was performed using colon tissue and 16S rRNA gene sequencing was performed on intestinal contents. Fecal microbiota transplantation (FMT) was employed to assess the contribution of intestinal microbiota and its correlation with CD4 T cells and the NLRP3 inflammasome. RESULTS: GQD increased the survival rate of TNBS-induced UC in BALB/c mice, and significantly improved their body weight, DAI score, colonic weight and index, spleen index, and histological characteristics. The intestinal barrier dysfunction was repaired after GQD administration through promoting the expression of tight junction proteins (Occludin and ZO-1). GQD restored the balance of Th2/Th1 and Tregs/Th17 cells immune response of colitis mice, primarily inhibiting the increase in Th2/Th1 ratio and their transcription factor production (GATA3 and T-bet). Morever, GQD changed the secretion of Th1-/Th2-/Th17-/Tregs-related cytokines (IL-2, IL-12, IL-5, IL-13, IL-6, IL-10, and IL-17A) and reduced the expressions of IL-1ß, IL-18. Transcriptome results suggested that GQD could also remodel the immune inflammatory response of colitis by inhibiting NOD-like receptor signaling pathway, and Western blot, immunohistochemistry and RT-PCR further revealed that GQD exerted anti-inflammatory effects by inhibiting the NLRP3 inflammasome, such as down-regulating the expression of NLRP3, Caspase-1 and IL-1ß. More interestingly, GQD regulated gut microbiota dysbiosis, suppressed the overgrowth of conditional pathogenic gut bacteria like Helicobacter, Proteobacteria, and Mucispirillum, while the probiotic gut microbiota, such as Lactobacillus, Muribaculaceae, Ruminiclostridium_6, Akkermansia, and Ruminococcaceae_unclassified were increased. We further confirmed that GQD-treated gut microbiota was sufficient to relieve TNBS-induced colitis by FMT, involving the modulation of Th2/Th1 and Tregs/Th17 balance, inhibition of NLRP3 inflammasome activation, and enhancement of colonic barrier function. CONCLUSIONS: GQD might alleviate TNBS-induced UC via regulating Th2/Th1 and Tregs/Th17 cells Balance, inhibiting NLRP3 inflammasome and reshaping gut microbiota, which may provide a novel strategy for patients with colitis.

Effect and mechanism of Prunella vulgaris L. extract on alleviating lipopolysaccharide-induced acute mastitis in protecting the blood-milk barrier and reducing inflammation.

ETHNOPHARMACOLOGICAL RELEVANCE: According to ancient literature, Prunella vulgaris L. (P vulgaris) alleviates mastitis and has been used in China for many years; however, there are no relevant reports that confirm this or the mechanism of its efficacy. AIM OF THE STUDY: To explore the anti-acute mastitis effect and potential mechanism of P vulgaris extract. MATERIALS AND METHODS: First, the active ingredients and targets of P vulgaris against mastitis were predicted using network pharmacology. Next, the relevant active ingredients were enriched using macroporous resins and verified using UV and UPLC-Q-TOF-MS/MS. Lastly, a mouse model of acute mastitis was established by injecting lipopolysaccharides into the mammary gland and administering P vulgaris extract by oral gavage. The pathological changes in mammary tissue were observed by HE staining. Serum and tissue inflammatory factors were measured by ELISA method. MPO activity in mammary tissue was measured using colorimetry and MPO expression was detected by immunohistochemistry. The expression of tight junction proteins (ZO-1, claudin-3, and occludin) in mammary tissue was detected by immunofluorescence and Western blot. iNOS and COX-2 in mammary tissue were detected by Western blot. MAPK pathway and NF-κB pathway related proteins were also detected by Western blot. RESULTS: Network pharmacology predicted that phenolic acids and flavonoids in P vulgaris had anti-mastitis effects. The contents of total flavonoids and total phenolic acids in P vulgaris extract were 64.5% and 29.4%, respectively. UPLC-Q-TOF-MS/MS confirmed that P vulgaris extract contained phenolic acids and flavonoids. The results of animal experiments showed that P vulgaris extract reduced lipopolysaccharide-induced inflammatory edema, inflammatory cell infiltration, and interstitial congestion of mammary tissue. It also reduced the levels of serum and tissue inflammatory factors TNF-α, IL-6, and IL-1ß, and inhibited the activation of MPO. Furthermore, it downregulated the expression of MAPK and NF-κB pathway-related proteins. The expressions of ZO-1, occludin, and claudin-3 in mammary gland tissues were upregulated. CONCLUSIONS: P vulgaris extract can maintain the integrity of mammary connective tissue and reduce its inflammatory response to prevent acute mastitis. Its mechanism probably involves regulating NF-κB and MAPK pathways.

Transcriptional Levels of Intercellular Junction Proteins in an Alveolar Epithelial Cell Line Exposed to Irradiation or Bleomycin.

We performed a comparative study of the effects of X-ray irradiation and bleomycin on the mRNA levels of E-cadherin and tight junction proteins (claudin-3, claudin-4, claudin-18, ZO-2, and occludin) in an alveolar epithelial cell line L2. Irradiation decreased claudin-4 levels and increased occludin levels, while the levels of other mRNAs remained unchanged. Bleomycin increased the expression levels of all proteins examined except claudin-3. Irradiation and bleomycin have different effects on the expression level of intercellular junction proteins, indicating different reactions triggered in alveolar epithelial cells and a great prospects of further comparative studies.

Xylooligosaccharides ameliorate insulin resistance by increasing Akkermansia muciniphila and improving intestinal barrier dysfunction in gestational diabetes mellitus mice.

Little is known regarding the effects of xylooligosaccharides (XOS) on insulin resistance (IR) in gestational diabetes mellitus (GDM). We aimed to investigate this issue and its mechanism. Sixty female mice were randomly allotted to 4 groups (n = 15): control, high fat diet (HFD), GDM, and GDM + XOS. The control mice were fed an AIN-93 diet, while the mice in the other groups were fed 45% HFD. After pregnancy, mice in GDM and GDM + XOS groups were intraperitoneally injected with 30 mg kg-1 streptozocin for 3 days from the first day of pregnancy. Mice in the GDM + XOS group were then fed an HFD containing 2% XOS. Fasting glucose and insulin levels were monitored. The fecal Akkermansia muciniphila (Akk. muciniphila) and Bifidobacterium were measured by qPCR. The Chiu scores were calculated from hematoxylin-eosin (HE)-stained ileal tissues. Phosphorylated Akt in the liver and occludin and ZO-1 in the intestinal tissues were determined by western blotting. XOS reduced (p < 0.05) fasting blood glucose and insulin and HOMA-IR, and increased (p < 0.05) Akt phosphorylation in the livers of GDM mice. Moreover, XOS decreased (p < 0.05) TNFα, IL-1ß, IL-15 and LPS in the serum, increased (p < 0.05) fecal Akk. muciniphila abundance, lowered (p < 0.05) Chiu's scores, and enhanced (p < 0.05) occludin and ZO-1 expression. XOS ameliorate IR by increasing Akk. muciniphila and improving intestinal barrier dysfunction in GDM mice.

The FAK/occludin/ZO-1 complex is critical for cadmium-induced testicular damage by disruption of the integrity of the blood-testis barrier in chickens.

Cadmium (Cd) is a well-known testis toxicant. The blood-testis barrier (BTB) is a crucial component of the testis. Cd can disrupt the integrity of the BTB and reproductive function. However, the mechanism of Cd-induced disruption of BTB and testicular damage has not been fully elucidated. Here, our study investigates the effects of Cd on BTB integrity and testicular dysfunction. 80 (aged 1 day) Hy-Line white variety chickens were randomly designed into 4 groups and treated for 90 days, as follows: control group (essential diet), 35 Cd, 70 Cd and 140 Cd groups (35, 70 and 140 mg/kg Cd). The results found that Cd exposure diminished volume of the testes and induced histopathological lesions in the testes. Exposure to Cd induced an inflammatory response, disrupted the structure and function of the FAK/occludin/ZO-1 protein complex and disrupted the tight junction and adherens junction in the BTB. In addition, Cd exposure reduced the expression of steroid-related proteins and inhibited testosterone synthesis. Taken together, these data elucidate that Cd disrupts the integrity of the BTB and further inhibits spermatogenesis by dissociating the FAK/occludin/ZO-1 complex, which provides a basis for further investigation into the mechanisms of Cd-induced impairment of male reproductive function and pharmacological protection.

Gingipain-carrying outer membrane vesicles from Porphyromonas gingivalis cause barrier dysfunction of Caco-2 cells by releasing gingipain into the cytosol.

Ingestion of Porphyromonas gingivalis, a periodontal pathogen, disrupts the intestinal barrier in mice. However, the involvement of outer membrane vesicles (OMVs) secreted from P. gingivalis in the destruction of the intestinal barrier remains unclear. In this study, we tested the hypothesis that OMVs carrying gingipains, the major cysteine proteases produced by P. gingivalis, affects the intestinal barrier function. OMVs increased the permeability of the Caco-2 cell monolayer, a human intestinal epithelial cell line, accompanied by degradation of the tight junction protein occludin. In contrast, OMVs prepared from mutant strains devoid of gingipains failed to induce intestinal barrier dysfunction or occludin degradation in Caco-2 cells. A close histological examination revealed the intracellular localization of gingipain-carrying OMVs. Gingipain activity was detected in the cytosolic fraction of Caco-2 cells after incubation with OMVs. These results suggest that gingipains were internalized into intestinal cells through OMVs and transported into the cytosol, where they then directly degraded occludin from the cytosolic side. Thus, P. gingivalis OMVs might destroy the intestinal barrier and induce systemic inflammation via OMV itself or intestinal substances leaked into blood vessels, causing various diseases.

Huperzine A ameliorates neurological deficits after spontaneous subarachnoid hemorrhage through endothelial cell pyroptosis inhibition.

Spontaneous subarachnoid hemorrhage (SAH) is a kind of hemorrhagic stroke which causes neurological deficits in survivors. Huperzine A has a neuroprotective effect, but its role in SAH is unclear. Therefore, we explore the effect of Huperzine A on neurological deficits induced by SAH and the related mechanism. In this study, Evans blue assay, TUNEL staining, immunofluorescence, western blot analysis, and ELISA are conducted. We find that Huperzine A can improve neurological deficits and inhibit the apoptosis of nerve cells in SAH rats. Huperzine A treatment can improve the upregulation of brain water content, damage of blood-brain barrier, fibrinogen and matrix metalloprotein 9 expressions and the downregulation of ZO-1 and occludin expressions induced by SAH. Huperzine A inhibit the expressions of proteins involved in pyroptosis in endothelial cells in SAH rats. The increase in MDA content and decrease in SOD activity in SAH rats can be partly reversed by Huperzine A. The ROS inducer H 2O 2 can induce pyroptosis and inhibit the expressions of ZO-1 and occludin in endothelial cells, which can be blocked by Huperzine A. In addition, the increase in the entry of p65 into the nucleus in endothelial cells can be partly reversed by Huperzine A. Huperzine A may delay the damage of blood-brain barrier in SAH rats by inhibiting oxidative stress-mediated pyroptosis and tight junction protein expression downregulation through the NF-κB pathway. Overall, Huperzine A may have clinical value for treating SAH.