Preliminary Study On The Regulation Of Intestinal Microecology By Sanwei Ganjang Powders

ObjectiveSanwei Ganjang Powders（SWGJ）as a traditional Tibetan prescription,has been applied to liver diseases.Experiments also show that it has a protective effect on various liver injury models.Based on the theory of liver and intestine,this paper studies its effect on intestinal micro-ecology.Establishing the combined model of dysbacteriosis and liver injury in mice to observe the effect of SWGJ on liver and intestine.Replicating the intestinal microflora imbalance mouse model to observe the effect of SWGJ on the intestine.Reproducing a rat model of liver injury to observe the changes of intestinal micro-ecology in different stages of liver injury,to further explore the theory of liver and intestine,and the regulative effect of SWGJ.Methods1.SWGJ effect on liver and intestine in combined animal model.Dysbacteriosis combined liver injury model was estabalished with Clindamycin orally for 3 days.And mice were administered with SWGJ for 7 days,CCl₄was injected subcutaneously after the last administration.Serum levels of ALT and AST were detected to evaluate liver function.HE staining was used for pathological observation of liver and ileum.16Sr DNA sequencing was used to determine the changes of intestinal microbiota in vivo.2.SWGJ effect on intestinal function of ceftriaxone-induced dysbacteriosis in mice model.Free to drink 0.5mg/m L ceftriaxone sodium solution for 5 days to copy dysbacteriosis model.Continuous administration of SWGJ for 7 days.Scanning electron microscopy was used to observe the structure of intestinal tissue.16Sr DNA sequencing was used to detect the changes of the main genus of intestinal flora.3.Pharmacodynamics evaluation of liver protection of SWGJ.The model of chronic liver injury was induced by subcutaneous injection of 40%CCl₄oil solution for 8 weeks,twice a week.Starting from the 3th week,rats in treatment groups were given the appropriate doses of SWGJ,Silymarin and Bifico.after the modeling.In the administration of 2,4,6 weeks,a number of animals were processed randomly.The weight of rats was recorded weekly,and the hair color,drinking water,consumption and activity of rats were observed.The therapeutic effect of SWGJ were judged by the liver index,serum liver function enzyme ALT,AST level and liver pathological organization.4.SWGJ effect on intestinal microecology.HE staining was used for pathological observation of ileum.IHC was used to detect the level of Occludin and ZO-1 in ileum.16Sr DNA sequencing was used to determine the changes of intestinal microbiota in rats..The contents of LPS,TNF-αin intestinal homogenate were detected by ELISA.5.SWGJ effect on Nrf2 detoxification signal pathway.The contents of LPS,TNF-αin intestinal homogenate were dynamicly detected by ELISA.The total bile acids in the liver were detected by microplate method.The expression of Nrf2 and its downstream detoxifying enzymes NQO1,HO-1 and bile acid efflux transporter BSEP protein were detected by Western-Blot.Results1.SWGJ effect on liver and intestine in combined animal model.Liver enzyme results show the levels of ALT and AST of combined model were significantly increased（P<0.01）.According to HE staining show liver hepatic cord structure was disordered,hepatic lobular nuclei disappeared or pyknosis,and edema appeared in intestinal mucosa.Compared with the combined model group,SWGJ can reduce the levels of ALT,AST（P<0.01）,restore the structure of hepatic cord,alleviate the edema of liver cells,improve intestinal mucosal edema and inflammatory infiltration.Compared with the normal group（P<0.05）,16Sr DNA sequencing indicated that the level of Proteobacteria increased significantly（P<0.05）in the dysbacteriosis group,and the level of Tenericutes in the combined model increased significantly（P<0.05）.Compared with the combined model group,low dose of SWGJ can significantly reduce the levels of Proteobacteria,Bacteroides（P<0.01）.At the genus level,compared with the normal group,the abundance of Bacteroides and Parabacteroide in the dysbacteriosis group were significantly increased（P<0.05）.Compared with the combined model group,the level of Parabacteroide was significantly decreased（P<0.01）after the effect of SWGJ and Bifico.2.Effect of SWGJ on intestinal function of mice induced by ceftriaxone strain.In the model group,the intestinal villi were flat and sparsely arranged.The local villi were broken and lodging,and the goblet cell structure was destroyed.After treatment with the high-dose SWGJ group and the Bifico group,the intestinal villi became morphologically plump and arranged closely and neatly,goblet cell can be seen.Compared with the normal group,the relative contents of Enterococcus and Robinsoniella in the model group increased significantly（P<0.05 or P<0.01）.Compared with the model group,the relative contents of Enterococcus and Robinsoniella in the SWGJ group decreased significantly（P<0.01）.3.The effect of SWGJ on the general condition and liver function of CCl₄-induced liver injury in rats.The body weight of rats in each group increased with time,the normal group had the fastest growth,and the model group had the slowest growth.The hepatic index of the model group increased significantly with time（P<0.05）,and there was no significant change with time after SWGJ treatment.The ALT and AST levels in the model group increased with time,and there was a more significant difference in ALT（P<0.05）.The ALT and AST levels decreased significantly with time after SWGJ treatment（P<0.05）.Liver lobular structure disorder,inflammatory cell infiltration and balloon-like lesions were observed in liver tissue of model group at different time points.With the prolongation of model time,hepatic steatosis,hepatic lobular necrosis deepened,and nuclear condensation or disappeared.The leaflets were destroyed,and the proliferation of collagen fibers in the portal area was clearly visible.SWGJ can improve the degree of inflammatory cell infiltration and fibrosis in liver tissue after the action of the SWGJ.With the extension of the administration time,the degree of improvement of the liver injury caused by the drug gradually increases.4.Effect of SWGJ on intestinal microecology in rats with CCl₄-induced liver injury.At each time point,intestinal mucosal epithelial edema,lodging or abscission of villi were observed in the intestine tissue of the model group,and the intestinal space became larger.As the progress of the modeling process,there was an increasing trend.SWGJ can improve the mucous membrane oedema and the loss of villi in ileum.The positive expression levels of the tight junction proteins ZO-1 and Occludin in the model group decreased with the prolongation of the modeling time,and the positive expression level was significantly increased after the action of SWGJ.16Sr DNA sequencing showed that the intestinal flora diversity and abundance decreased in liver injury model,and the diversity and abundance increased after treatment with SWGJ;the level of Firmicutes in the model group decreased significantly（P<0.05）,and the level of Bacteroidetes significantly elevated（P<0.05）,the level of Actinobacteria increased,but there was no significant difference.Compared with the model group,the level of Actinobacteria significantly decreased after the action of SWGJ（P<0.05）,and the level of the Firmicutes increased significantly（P<0.05）.5.Effects of SWGJ on Nrf2 detoxification enzymes and bile acids in rats with hepatic injury.The expression of Nrf2 in the model group was significantly decreased（P<0.01）,the expression of downstream detoxifying enzymes HO-1 and NQO1 was significantly increased（P<0.01）,and the total bile acid in the model group was significantly increased（P<0.01）.The expression of transporter BSEP was significantly decreased（P<0.05）.The expression of Nrf2 and NQO1 was significantly increased after the action of SWGJ（P<0.05 or P<0.01）,the total bile acid content in the liver was significantly decreased（P<0.05）,and the expression of BSEP was significantly increased（P<0.05）.Conclusion1.SWGJ can improve the degree of liver injury and the level of flora imbalance in the combined mouse model.SWGJ has dual effects of protecting liver and regulating intestinal flora.2.SWGJ can improve the intestinal pathology and imbalance of intestinal flora in mice with dysbacteriosis.SWGJ has the function of regulating intestinal function.3.SWGJ can reduce the damage degree of liver function,intestinal pathology and intestinal epithelial barrier damage in different course of liver injury,reduce the levels of intestinal LPS and TNF-α,and regulate the structure and diversity of intestinal microflora in chronic liver injury rats.SWGJ can protect the liver and regulate the intestinal micro-ecology.4.The effect of SWGJ on intestinal micro-ecology may be achieved by affecting the detoxification function of Nrf2,upregulating the level of toxic enzymes,and increasing the efflux of bile acid.