| ObjectiveThe gut microbiota plays a vital role in maintaining the homeostasis of bile acids,so that considering gut microbiota as a new target is expected to become a new way to develop drugs for intrahepatic cholestasis.Since the aminoglycoside antibiotic gentamicin is not absorbed orally and can locally affect the intestine,it is often used to treat intestinal infections and is a good flora regulating drug.Based on above,this thesis indicates the therapeutic effect and mechanism of gentamicin on intrahepatic cholestasis in rats.MethodsThis thesis includes three parts of the research,of which the experiment content and methods are as follows:?1?Therapeutic effect of gentamicin on estrogen induced intrahepatic cholestasis in ratsMale Wistar rats were randomly divided into 5 groups,namely control group,model group,ursodeoxycholic acid group?UDCA?,25 mg/kg gentamicin group?25-GEN?,and 50 mg/kg gentamicin group?50-GEN?.Rats of the control group were injected subcutaneously with vehicles?propylene glycol solution,5 mL/kg?,and rats in the remaining four groups were injected subcutaneously with 17?-ethinylestradiol?5 mg/kg?for 7 consecutive days to construct the intrahepatic cholestasis model.From the third day of modeling,rats in the control group and the model group were orally administered with saline for 7 consecutive days.And rats in UDCA group,25-GEN group and 50-GEN group were orally treated with UDCA 60 mg/kg,25 mg/kg gentamicin or 50 mg/kg gentamicin separately for 7 consecutive days.On the 10th day,the serum,livers,kidneys,ileum tissues,ileum contents and cecum contents of rats were collected.The concentrations of serum total bile acid?TBA?,alanine aminotransferase?ALT?,aspartate aminotransferase?AST?,bilirubin,alkaline phosphatase?ALP?,?-glutamyltransferase?GGT?,cholesterol?CHOL?,blood urea nitrogen?BUN?,creatinine?CREA?,and cystatin-C?CYS-C?were determined by biochemical autoanalyser.UPLC/MS/MS were used to determine the concentrations of bile acids in serums,livers,ileum tissues,ileum contents and cecum contents.HPLC/MS/MS was used to determine the concentration of serum7?-hydroxy-4-cholesterol-3-one?C4?.Hematoxylin-eosin staining?HE staining?was used to investigate the pathological changes of livers,kidney tissues and ileum tissues.?2?Effects of gentamicin on bile acids levels in normal rats of different gendersFemale and male Wistar rats were separately divided into control group and50-GEN group.Saline or 50 mg/kg gentamicin were orally administered for 7consecutive days.After that,serum,livers,ileal tissues,ileal contents and cecum contents of rats were collected.The concentrations of TBA in serum and livers and the serum levels of ALT,AST,ALP,GGT,bilirubin,CHOL,BUN,CREA and CYS-C were determined.Furthermore,the concentrations of bile acids in serum,livers,ileum tissues,ileum contents,and cecum contents were analyzed.?3?Study on the mechanism of gentamicin in reducing the bile acids levels in rats?1?Male Wistar rats were orally administered with 50 mg/kg gentamicin for 7consecutive days.After 7 day repeated administration,Blood samples were collected through the right side of the femoral artery at the 10,20,40,60,90,120,180,240,360,480,and 600 minutes,and the maximum blood concentration and bioavailability of gentamicin were investigated.?2?Gentamicin was used to intervene Caco-2 cells for 72 hours,and the effects of gentamicin on ASBT and FXR protein expression were detected by Western Blotting technology.?3?Male Wistar rats were randomly divided into four groups,namely control group,gut microbiota loss group?3A?,25mg/kg gentamicin group?3A+25-GEN?,and 50 mg/kg gentamicin group?3A+50-GEN?.Rats in the control group were orally administered with saline for 7consecutive days,while which in the remaining three groups were treated a mixed antibiotic solution?streptomycin,neomycin and bacitracin,each 100 mg/kg/day?to construct the gut microbiota loss model.From the third day of modeling,the rats in control group and 3A group were orally administered with saline for 7 consecutive days.And the rats in the 3A+25-GEN group and the 3A+50-GEN group were orally administered 25 mg/kg gentamicin or 50 mg/kg gentamicin separately for 7consecutive days.On the 10th day,serum and livers were collected for TBA concentrations detection.This experiment aimed to investigate the effect of gut microbiota loss on the ability of gentamicin to reduce bile acids.?4?Male Wistar rats were divided into control group,25-GEN group and 50-GEN group.After administration of saline,25 mg/kg or 50 mg/kg gentamicin separately for 7consecutive days,the serum,livers,ileum tissues,ileum contents,cecum contents,biles and feces samples of rats were collected.The concentrations of TBA in serum,livers,biles,small intestines?the whole small intestine tissues+contents?,feces and the levels of serum ALT,AST,ALP and other biochemical indicators were determined.The concentrations of serum C4 and bile acids in serums,livers,ileal tissues,ileal contents,cecum contents,biles and feces were tested.Then the pathological changes of the kidney and ileum tissue were observed.After that,the expression of relevant nuclear receptors,metabolic enzymes and transporters in the livers and ileum tissue were examined.Lastly 16SrDNA technology was used to analyze the gut microbiota abundance and diversity changes.Results?1?Therapeutic effect of gentamicin on intrahepatic cholestasis in rats:the results of serum biochemical indicator measurement showed that compared with control group,the serum concentrations of TBA,TBIL and IBIL in the model group were increased significantly?P<0.05?,the serum levels of ALT,ALP and DBIL were elevated greatly remarkably?P<0.01?.Compared with the model group,the concentrations of TBA,ALT,TBIL and IBIL in the 25-GEN group were decreased significantly?P<0.05?,and the levels of DBIL were reduced extremely evidently?P<0.01?,while the concentrations of ALT,ALP,TBIL and IBIL in the 50-GEN group were decreased significantly?P<0.05?,and the level of TBA and DBIL were reduced greatly remarkably?P<0.01?.The livers and ileum HE staining results showed that the degree of liver and intestinal injury in the 25-GEN group and 50-GEN group were significantly reduced compared with the model group.The kidney HE staining results showed no pathological changes in the kidneys of the 25-GEN group and the 50-GEN group.Analysis of the bile acids composition revealed that compared with the model group,the ratio of conjugated bile acids and unconjugated bile acids in serum,livers,and ileum contents of the 25-GEN group were evidently increased?P<0.05?,while that in livers of the 50-GEN group were remarkably elevated?P<0.05?,and that in serum,ileum tissues and ileum contents of the 50-GEN group were significantly increased?P<0.01?.In addition,compared with the model group,the ratios of primary bile acids and secondary bile acids in serum of the 25-GEN group were elevated evidently?P<0.05?,while that in livers,ileum tissues,ileum contents and cecum contents of the 25-GEN group were greatly significantly increased?P<0.01?.Moreover,the ratios of primary and secondary bile acids in ileum of the 50-GEN group were significantly higher than that in the model group?P<0.05?,while that in serum,livers,ileum and cecum contents were greatly significantly elevated?P<0.01?.In addition,compared with the control group,the serum C4 concentration in the model group was significantly increased,and there was no significant difference in serum C4 concentration between the 25-GEN group and the 50-GEN group compared with the model group?P>0.05?.?2?The effect of gentamicin on bile acids levels in normal rats of different genders:the biochemical indicator results showed that compared with the control group,the serum concentration of TBA in the male and female 50-GEN groups were significantly reduced?P<0.05?.The liver level of TBA in the male 50-GEN group was remarkably decreased?P<0.05?,but there was no statistical difference in females50-GEN group?P>0.05?.Compared with the control group,the ratios of conjugated bile acids and unconjugated bile acids in livers,ileum tissues and cecum contents of the male 50-GEN group were elevated evidently?P<0.05?,while that only in ileum contents?P<0.01?and cecum contents?P<0.05?of the female 50-GEN group were significantly increased.Moreover,compared with the control group,the ratio of primary bile acids and secondary bile acids in cecum contents of the male 25-GEN group was significantly elevated?P<0.05?,and that in serum,livers,ileum and ileum content of the male 25-GEN group were increasd extremely remarkably?P<0.01?,while that only in livers and cecum contents of the female 50-GEN group were evidently elevated?P<0.05?.?3?Study on the mechanism of gentamicin in reducing bile acids levels in rats:the results of pharmacokinetic tests showed that the maximum blood concentration of gentamicin was 6.49 ng/mL,and the AUC?0-t?was 3.55±0.27 mg/L·min.Histopathological staining of kidney and ileum showed that gentamicin did not cause kidney and intestinal damage.The serum concentrations of TBA in the 25-GEN group and 50-GEN group were significantly reduced compared with the control group?P<0.05?.The concentration of TBA in livers of the 25-GEN group was greatly significantly lower than that in the control group?P<0.01?,and that in liver of50-GEN group was decreased significantly?P<0.05?.Compared with the control group,the concentration of TBA in biles of the 25-GEN group had a decreased trend,but there was no significant difference?P>0.05?,while that in biles of the 50-GEN group was reduced remarkably?P<0.05?.The concentrations of TBA in small intestine?the entire small intestine tissue+contents?of the 25-GEN group and the50-GEN group were not significantly changed compared with the control group?P>0.05?.Compared with the control group,the total bile acids pool in the 25-GEN group was significantly reduced?P<0.05?,but there was no significant difference in the 50-GEN group?P>0.05?,while the numbers of bile acid cycles in the 25-GEN group and the 50-GEN group were lower than that in the control group,but there were no statistical difference?P>0.05?.And,compared with the control group,the concentration of feces TBA evidently elevated after the 3 days of administration in the25-GEN group?P<0.05?,while the feces TBA concentrations in the 25-GEN group after the 1 and 7 day of administration had no significant difference?P>0.05?.The concentrations of feces TBA in the 50-GEN group after the 1st,3rd and 7th day of administration were lower than that in the control group,but had no significant difference?P>0.05?.Western Blotting results showed that compared with the control group,gentamicin had no effect on the expression of ASBT and FXR proteins in Caco-2 cells?P>0.05?,but could affect significantly the expression of relevent transporters,metabolizing enzymes and nuclear receptors in rats.Compared with the control group,the expression of Ntcp in livers of 25-GEN group was remarkably increased?P<0.05?,the expression of Bsep was extremely significantly elevated?P<0.01?,the expression of Mrp2 was evidenty reduced?P<0.05?,the expression of Fxr and Fgf15 in ileum were significant decreased?P<0.05?,and the expression of Asbt was remarkably increased?P<0.05?.Moreover,the expression of Fxr and Ntcp in livers of the 50-GEN group were significantly higher than that in the control group?P<0.05?,Cyp7a1 and Bsep expression were greatly evidently increased?P<0.01?,Mrp2 expression was remarkably reduced?P<0.05?,the expression of Fxr and Fgf15in ileum were significantly decreased?P<0.05?,and the expression of Asbt was evidently increased?P<0.05?.In terms of changes in bile acids composition,compared with the control group,the ratios of conjugated and unconjugated bile acids in livers of the 25-GEN group and that in biles and cecum contents of 50-GEN group were significantly elevated?P<0.05?.And compared with the control group,the ratio of primary and secondary bile acids in livers of the 25-GEN group was increased remarkably?P<0.05?,and that in ileum tissue,cecum contents,feces and biles were increased extremely evidently?P<0.01?.Moreover,the ratios of primary and secondary bile acids in serum,livers,bile and ileum contents of the 50-GEN group were elevated significantly?P<0.05?,while that in ileum tissues,cecum contents and feces increased greatly remarkably?P<0.01?.Compared with the control group,the concentrations of secondary bile acids in biles of the 25-GEN group and the 50-GEN group were significantly reduced?P<0.05?,and that in serum,livers,cecum contents and feces were extremely significantly decreased?P<0.01?.In addition,compared with the control group,the serum concentration of primary bile acids in the 25-GEN group was evidently reduced?P<0.05?,and that in feces and cecum contents were significantly increased?P<0.05?.The concentrations of primary bile acids in serum and biles of the 50-GEN group were significantly lower than that in control group?P<0.05?,while that in cecum contents?P<0.05?and feces?P<0.01?were increased remarkably.The results of gut microbiota loss experiment showed that the concentrations of TBA in serum and livers of the 3A+25-GEN and the 3A+50-GEN groups were not significantly different from that of the 3A group?P>0.05?.The16SrDNA results showed that gentamicin had little effect on the diversity of the gut microbiota,but it could reduce the abundance of Lactobacillus in a dose-dependent manner.ConclusionsGentamicin could effectively improve the liver function-related biochemical indicators in cholestatic rats,relieve liver and ileum tissue injures caused by estrogen disorders and has no renal toxicity.The effect of gentamicin on bile acids levels in normal rats had a gender difference,but it could reduce the serum levels of TBA,increase the proportion of conjugated bile acids and reduce the ratio of secondary bile acids in both male and female rats in different degrees.The mechanism of gentamicin reducing bile acids in rats may be related to reducing the abundance of Lactobacillus,decreasing the production of secondary bile acids,increasing fecal excretion of primary bile acids,elevating the proportion of hydrophilic bile acids,and reducing the concentration of hepatotoxic bile acids,thereby activating the expression of Bsep and promoting bile acids efflux. |
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