| ObjectiveMaintaining the function of the gut-liver axis is an important adjuvant therapy for the treatment of liver diseases.Both traditional Chinese medicine and western medicine focus on the function of the gut-liver axis in the pathogenesis and treatment of liver diseases,while bile acid enterohepatic and enterotoxin effect play an important role in maintaining the function of the gut-liver axis.Sanwei Ganjiang powder(SWGJ)derived from traditional Tibetan prescription,has long demonstrated its effectiveness in long-term liver damage therapy.With the deepening of research,we found that SWGJ has a significant effect on protecting liver and gut in chronic liver injury rats,and since gut maintain close ties with liver in biological functions,we speculated that SWGJ plays an important role in maintaining the function of gut-liver axis.To verify this hypothesis,this study observed the regulating effect of SWGJ on the gut-liver axis of Intestinal dysbacteriosis mouse,and preliminarily explored the specific mechanism from the perspective of bile acid enterohepatic and enterotoxin effect.Methods1.Dynamic effects of antibiotic-induced intestinal dysbacteriosis on intestinal mucosa barrier and liver functionIntestinal dysbacteriosis mouse model was caused by ceftriaxone sodium.After administration of ceftriaxone sodium for 3,6 and 10 days,the changes of the main genus in intestinal flora was detected by 16S rDNA sequencing,hematoxylin-eosin(HE)staining was to observe the ileum pathological changes,transmission electron microscope(TEM)was used to observe the tight junction conditions of ileum,immunohistochemical(IHC)was used to detect the positive expression of zonula occluden-1(ZO-1)in ileum tissues.The contents of TNF-α and LPS in intestinal homogenate were detected by enzyme-linked immunosorbent assay(ELISA),liver homogenate level of ALT and AST were detected to evaluate liver function.HE staining was used for pathological observation of liver.2.The regulation of SWGJ on the of gut-liver axis in intestinal dysbacteriosis mouseIntestinal dysbacteriosis mouse model was administrated by 12g/kg ceftriaxone sodium for 10 days.During this time,SWGJ,bifendate and bifico were sequentially administered for 7d,intestinal flora(16S rDNA analysis)of cecal contents,histology(H&E staining),tight connections(Immunohistochemistry,IHC),ultrastructure(Transmission electron microscopy,TEM)were assessed,and liver function was also evaluated by kit including ALT,AST and H&E staining.3.The regulation of SWGJ on enterotoxin effect in intestinal dysbacteriosis mouse and IEC-6 cells①Inflammatory cytokines(TNF-α,IL-1β and LPS were assessed by enzyme-linked immunosorbent assay(ELISA)in intestinal tract of intestinal dysbacteriosis mouse.② IEC-6 were stimulated with 10μg/mL LPS,the major components of pathogen-associ ated molecule pattern,or 25ng/mL TNF-α.At the same time,treated with SWGJ containing serum(5%,10%,20%)or 6-ginger(2,4 and 8 μM)for 24h.The cell vitality was determined by MTT,cell migration ability was tested by Wound Healing.4.The regulation of SWGJ on bile acid enterohepatic in intestinal dysbacteriosis mouseTC and TBA were evaluated by kit,immunohistochemical(IHC)was used to detect the positive expression of FGF-15 in ileum tissues,and the protein expression related bile acid synthesis and reabsorption such as FXR,CYP7A1,NTCP,the hepatic BA efflux transporters such as BSEP and MRP2 in intestinal dysbacteriosis mouse liver were detected by western blot.5.The effect of SWGJ on Nrf2/Bachl signal pathway in intestinal dysbacteriosis mouseThe nucleoprotein in liver tissue was extracted to detect the expression of Nrf2/Bach1.Immunofluorescence assay(IFA)was used to measured the nuclear Nrf2 and Bach1.In addition,The protein expression of antioxidation enzyme HO-1 and detoxification enzyme NQO1 in intestinal dysbacteriosis mouse liver were detected by western blot.Results1.After treatment with ceftriaxone sodium,the relative abundance of Bacillus,Lotus and Staphylococcus decreased gradually,and the relative abundance of Chryseobacterium,Pseudomonas and Psychrobacter increased gradually.The positive expression of ZO-1 and tight justion decreased with the prolongation of treatment time.There was no significant change in TNF-α and IL-1β at pre-dysbacteriosis,but the increase was significant after the continued dysbacteriosis(P<0.01).At the same time,the persistent imbalance of the intestinal flora would injure the liver,which is manifested by the significant increase of ALT and AST in the liver(P<0.01),and HE staining showed liver hepatic cord structure was disordered and liver cells were swelling.2.Compared with control group,in model group intestinal flora imbalance,intestinal mucosal epithelium edema with enlarged interstit in ileum tissue,the positive expression of ZO-1 and tight justion decreased,and the liver function was impaired;Compared with the model group,SWGJ could improved the symptoms of intestinal flora imbalance,increase the positive expression of ZO-1 and Occludin,improve the histopathological changes in the ileum,In addition,SWGJ significantly improved the pathological changes of liver tissue,the structural disorder of hepatic lobules,and the increased expression of ALT and AST in the model group(P<0.01).3.①Compared with control group,the expression of TNF-α,IL-1β and LPS were significant increased in moel group(P<0.01 or P<0.05):Compared with the model group,SWGJ significantly down-regulate the expression of TNF-α,IL-1β and LPS in the intestinal tract(P<0.01 or P<0.05).②Compared with control group,the cell activity of IEC-6 under LPS or TNFstimulation was significantly decreased(P<0.01),and the cell migration ability was significantly reduced(P<0.01).Both the SWGJ containing serum and 6-gingerol can significantly improve the cell activity of IEC-6 cells(P<0.01)and the cell migration ability(P<0.01).4.Compared with control group,TBA and TC were significantly increased(P<0.01 or P<0.05),the positive expression of FGF-15 was decreased,the expressions of CYP7A1,FXR,BSEP,MRP2 and NTCP were significantly decreased(P<0.01 or P<0.05);Compared with model group,TBA and TC were significantly decreased(P<0.01 or P<0.05),the positive expression of FGF-15 was increased,the expression of CYP7A1,FXR,BSEP,MRP2 and NTCP were significantly increased(P<0.01).5.Compared with control group,the nuclear input of Nrf2 and Bach1 in the model group showed an upward trend,but the nuclear input expression of Bach1 increased significantly(P<0.01),while the nuclear input expression of Nrf2 showed no statistical significance,lead to the ratio of Nrf2 to Bach1 significante increase(P<0.01).NQO1 and HO-1 were significantly down-regulated in the total protein expression(P<0.01).Compared with model group,the expressions of Nrf2,HO-1 and NQO1 increased significantly after the treatment of SWGJ(P<0.01),the expressions of Bach1 decreased significantly(P<0.01),and the ratio of Nrf2 to Bach1 in nucleus decreased significantly(P<0.01).Conclusion1.With continuous intestinal flora imbalance,intestinal microecological is damaged,the destruction of intestinal mucosal epithelial barrier is aggravated,the expression of inflammatory factors are gradually increased,and liver function is affected.2.SWGJ can improve the level of bacteria imbalance in intestinal dysbacteriosis mouse,and it also has a dual regulating effect on intestinal and liver injury caused by dysbacteriosis.3.In vivo,SWGJ could reduce the enterotoxin effect in intestinal dysbacteriosis mouse.in vitro also suggested that SWGJ could reduce the enterotoxin effect caused by LPS and TNF-α,which is manifested in reducing intestinal inflammation and promoting intestinal mucosa repair,etc.However,the vitro studies on bile acid enterohepatic are still under exploration.4.SWGJ can up-regulate the ratio of Nrf2 to Bach1,and has a certain regulatory effect on bile acid enterohepatic-related proteins,which may be the mechanism of maintaining the gut-liver axis homeostasis. |
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