| OBJECTIVE:The purpose of this experiment is to isolate different strains from the patients with obstructive jaundice and screen them with emodin,then implant them into the rats with obstructive jaundice to study the effect of the strain on the rats with obstructive jaundice and the effect and significance of emodin on the screened strains.METHODS:1.We collected 50 preoperative feces from patients with obstructive jaundice who underwent minimally invasive surgery in the treatment of cholecystolithiasis complicated with common bile duct stones from March 2018 to March 2019.The obtained samples were separated and cultured in different media,and different strains in the bile and feces of the patients were isolated.The emodin was added to the isolated strain to observe the effect of emodin on different strains,to screen the strains which were most affected.To determine the effect of emodin on the strain,we detected the growth curve of the strain,the MIC of emodin and the CFU value of the strain under different MICs.2.Forty male rats were randomly divided into 5 groups each group 8 rats,there are the control group,the common bile duct ligation group,the emodin group,the strain group,and the emodin plus strain group.Every groups were underwent common bile duct ligation except the control group.The feces of rats in each group were collected 7 days after modeling,and the rats in each group were killed 14 days later to collect serum,liver tissue,kidney tissue and feces.It is used to detect the biochemical indexes of serum and the contents of various bile acids to analysis the pathological damage of liver and kidney,the expression of bile acid related receptor and the change of transcriptome,as well as the macro gene of fecal flora.RESULTS: 1.Results of flora screeningAs of March 2019,a total of 22 samples from 50 patients have been isolated and cultured,and more than 1400 strains and 200 strains have been isolated.Emodin(0.3g/L)was added to the isolated strains by using the Alamar Blue chromogenic assay,and the difference in OD600 values after color change was observed.After the Alamar Blue color development experiment was repeated more than 3 times the selected strain XD114(Lysinibacillus sp.,LY.SP)was found as the most susceptible to emodin inhibition.The growth curve and the MIC of emodin were examined.The MIC of the effect of the emodin on the strain was finally determined to be(0.3g/L)and the effect of CFU on the 4-fold MIC was most pronounced.The strain was isolated from OJ patients for the first time,and little is known about the relationship between the strain and human metabolism.This experiment was the first to explore the role of LY.SP in OJ disease and the effect of emodin on the role of ly.sp in OJ.2.Results of serum biochemistry and bile acid in ratsSerum biochemical tests were carried out for BUN,SCR,ALT,ASL,TBIL and DBIL.Compared with the control group,the above indexes of the operation group and the strain group were increased(P < 0.05),and after the administration of emodin,the indexes of the emodin group were decreased compared with the operation group(P < 0.05),and the indexes of the emodin plus strain group were also decreased compared with the operation group(P < 0.05).The collected serum was detected by bile acid spectrum.The contents of LCA,CA,CDCA,DCA,GCA,GCDCA,TCA,TCDCA,CDCA,DCA,TCA,TCDCA,TCDCA,TCDCA,BDCA,DCA were detected.In BDL group,TDCA,CA,GCA,GCDCA,TCA and TCDCA were significantly increased compared with CON(P < 0.01),while emodin treatment could significantly reverse the changes of serum bile acid caused by surgery(P < 0.01).The results showed that LY.SP could significantly increase TCA,TCDCA and TUDCA in serum,while rhubarb could significantly reduce the increase of serum bile acid caused by ly.sp(P < 0.05).LY.SP strain significantly aggravated the deterioration of serum biochemical indexes caused by common bile duct ligation,even more than three times of BDL group in TBIL and DBIL indexes.However,the inhibition of emodin on LY.SP significantly reduced the increase of blood biochemical indexes caused by LY.SP and common bile duct ligation.3.Histopathological changes in liver and expression of bile acid-related receptors FXR and Gpbar-1 in rats.The pathological sections of rats in each group were observed under microscope.In BDL group,there were large areas of small bile duct like hyperplasia and fibrosis,accompanied by inflammatory cell infiltration,small bile duct like epithelial cell proliferation,capillary bile duct expansion,small bile duct and fibrous tissue proliferation,and fibrous septum formation.LY.SP group had similar performance with BDL group.However,after intragastric administration of emodin,the liver fibrosis in EMO group and EMO + LY.SP group was significantly less than that in BDL group and LY.SP group.In terms of the changes of FXR and Gpbar-1 gene expression in liver.RT-PCR results showed that compared with CON group,BDL group and LY.SP group were significantly lower(P < 0.01),while EMO group and EMO + LY.SP group were significantly higher than BDL and LY.SP group.At the same time,the immunohistochemistry results of liver FXR and Gpbar-1 were consistent with the trend of RT-PCR.4.Histopathological changes of kidney and expression of bile acid related receptors FXR and Gpbar-1 in ratsThe pathological sections of rat kidney can be observed under 400 times microscope.We found that in BDL group and LY.SP group,there were some of the glomerulosclerosis,some of the epithelial cells exfoliated and necrotic,accompanied by interstitial inflammatory cell infiltration and cytoplasmic disintegration.There was no significant fibrosis in EMO group and EMO + LY.SP group.In terms of the expression of FXR and Gpbar-1 genes in kidney,RT-PCR showed that compared with CON group,the expression of BDL group and LY.SP group decreased significantly(P < 0.01);compared with BDL group and LY.SP group,EMO group and EMO + LY.SP group significantly increased the expression of FXR and Gpbar-1.The same trend was found in the immunohistochemistry results of FXR and Gpbar-1 genes.In the aspect of bile acid receptor related expression in liver and kidney,we found that LY.SP was more able to inhibit the expression of Gpbar-1 in liver and kidney than BDL.5.Transcriptomics changes in rat liver and kidney tissuesThe similarity of gene expression in liver group L was higher,followed by that in kidney group K,and there was a significant difference in gene expression between groups L and K.In the liver tissue,the gene expression of each group was close to no significant difference.In the kidney tissue,the expression of con group was similar to that of emo group,but the difference was greater than the other three groups.Choledochal ligation affects the metabolism of fat and bile acid in rats by affecting the gene expression in this pathway,while emodin intervention can adjust the genes affected by BDL,thus making the overall gene expression more similar to con Conclusion: 1.After the occurrence of obstructive jaundice,it can cause the change of intestinal flora,including the increase of lysinibacillus sp.2.The changes of lysinibacillus sp.in intestinal tract were correlated with the changes of serum bile acid,liver and kidney function,bile acid related receptor fxrand Gpbar-1 expression and transcriptome in obstructive jaundice.3.Emodin can regulate the expression of bile acid related receptors fxrand Gpbar-1 in obstructive jaundice,the changes of transcriptome and serum bile acid profile,and improve the liver and kidney function and pathological damage.4.Emodin can improve the liver and kidney function of obstructive jaundice by inhibiting the abnormal increase of lysinibacillus sp. |
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