The Role And Mechanism Of Intestinal Microbiota In Monocrotaline Induced Hepatic Sinusoidal Obstruction Syndrome In Rat

BackgroundHepatic sinus obstruction syndrome(HSOS)is a serious hepatic vascular disease with a high mortality rate.In China,the major causes of HSOS is the intake of herbals containing pyrrolizidine alkaloids(PAs).Among them,the traditional Chinese herbal medicine gynura segetum is the most common.Monocrotaline(MCT)is a kind of PAs derived from the leguminous plants of the genus Pectoris.The pathological changes of rat HSOS constructed by MCT are most similar to those of human HSOS.Some studies suggest that PAs may produce hepatotoxicity by forming highly electrophilic pyrrole metabolites after liver dehydrogenation.The active pyrrole metabolite binds to glutathione(GSH)to form pyrrole and glutathione conjugate and is detoxified.When GSH depletion occurs,active pyrrole metabolites interact with proteins to form pyrrole-protein adducts(PPAs),which are considered to be a key pathogenic factor in PAs induced HSOS.A recent study showed that intestinal flora is disturbed in PAs induced mouse HSOS model.Pretreatment of intestinal flora with antibiotics significantly improved HSOS damage,suggesting that intestinal flora is involved in the occurrence of HSOS.A large number of studies have confirmed that changes in microbial composition will lead to changes in related metabolites,such as tryptophan metabolism.Currently,a variety of microbial metabolites have been detected in circulation and in the liver.Some of these metabolites,such as the tryptophan microbial metabolite indole and indole derivatives,are ligands for host receptors,such as aromatic hydrocarbon receptor(Ah R).Ah R is a ligand-dependent transcription factor that can be activated by tryptophan microbial metabolites.The activation of Ah R can activate a variety of signaling pathways,including the activation of Nrf2 antioxidant stress pathway.Studies have shown that Ah R is highly expressed in the liver.In addition,the activation of Nrf2 was inhibited in HSOS rat model,and the level of liver oxidative stress was increased.Activation of Nrf2 antioxidant pathway significantly improved HSOS damage.However,in rat HSOS induced by MCT,the composition of intestinal flora and the expression profile of microbialrelated metabolites have not been reported yet.In addition,the expression of Ah R in HSOS and the role of Ah R-Nrf2,an antioxidant pathway,in HSOS have not been studied.In view of this,this paper will carry on the in-depth discussion to the above issues.Methods1.To investigate the role of intestinal flora in MCT induced HSOS1.1 Evaluation of liver damage induced by 90mg/kg MCTThe study was divided into two groups:(1)control group;(2)HSOS model group.After 48 hours of MCT intervention,(1)liver specimens were collected and gross liver morphology was observed.The body weight and liver weight of the rats were weighed to evaluate liver congestion.(2)Portal vein serum was collected,and the expression levels of ALT and AST were detected by ELISA to evaluate the damage of liver cells.(3)The expression of matrix metalloproteinase-9(MMP-9),a marker of hepatic sinus endothelial cell damage,was detected by q RT-PCR and Western blot.(4)The hepatic tissue was stained with H&E,and the dilatation and bleeding of hepatic sinuses,the damage of central venous endothelium,and the coagulant necrosis of hepatic cells were assessed,and the HSOS severity score was calculated.1.2 Evaluation of liver damage induced by small dose MCTSmall doses of MCT(45mg/kg,60mg/kg,or 75mg/kg)were intragastric administered,respectively.Liver samples were collected 48 h later,and the detection indexes were the same as 1.1.1.3 To observe the effect of faecal microbiota transplantation(FMT)on liver damage induced by MCT1.3.1 The intestinal flora was pretreated with a broad-spectrum antibiotic mixture(ABX)by gavage for 7 days to establish the intestinal flora clearance model.The feces of HSOS model rat(MCT 90mg/kg)were collected and then transplanted into the intestinal flora clearance model by gavage,once a day,for consecutive 7 days.Then a small dose of MCT was administered(according to the experimental results of 1.2).Liver samples were collected 48 h later,and the test indexes were the same as 1.1.1.3.2 The feces of normal rats were collected and then transplanted to rats in the low-dose MCT intervention group by gavage,once a day,for consecutive 5 days.Then,liver samples were collected and the test indexes were the same as 1.1.2.To detect changes of intestinal microflora in rat HSOS model induced by 90mg/kg MCTThe feces of control rat and HSOS rat were collected for 16 S r DNA sequencing of intestinal flora.Rarefaction dilution curve,sample species composition analysis,? diversity analysis(ACE,Chao1,Shannon,Simpson),ANOSIM similarity analysis,and ?-diversity analysis(PCA analysis,PCo A analysis)were performed to evaluate the changes of intestinal microflora composition.KEGG metabolic pathway analysis was further performed to evaluate the differences in metabolic pathways of functional genes in the microbial community between the groups.3.To detect changes of fecal metabolomics in rat HSOS model induced by 90mg/kg MCTThe feces of control rat and HSOS model rat were collected for non-targeted fecal metabolomics analysis based on ultra-high performance liquid chromatographyquadrupole time-of-flight mass spectrometry(UHPLC-QEMS).Principal component analysis(PCA),orthogonal partial least squares discriminant analysis(OPLS-DA),screening of differential metabolites,and metabolic pathway analysis of differential metabolites were performed to evaluate the changes of microbial metabolites in rat MCTHSOS model.4.To evaluate the effect of activated AhR-Nrf2 antioxidant pathway on MCT-HSOS4.1 Effects of microbial metabolites of tryptophan on MCT-HSOSAccording to the results of fecal metabolomics,to determine whether the tryptophan metabolic pathway is the most critical metabolic pathway among differential metabolites.The tryptophan metabolites with reduced metabolic level and identified as Ah R ligand were further screened out.Then the related metabolites were supplemented by gavage,and after this the rats were treated with MCT(90mg/kg).Livers were collected 48 hours later.The detection indexes are the same as 1.1.4.2 Effects of supplemental AHR agonist Ficz on MCT-HSOSFicz was injected intraperitoneally for 3 consecutive days,pre-intervention of normal rats,and then treated with 90mg/kg MCT,and liver specimens were collected 48 hours later.The detection indicators were the same as 1.1.4.3 Detection of the level of oxidative stress in the liverThe ELISA method was used to detect the liver oxidative stress levels of rats in control group,HSOS model group,tryptophan microbial metabolism intervention group,and Ficz intervention group,including malondialdehyde(MDA),reactive oxygen species(ROS),GSH/GSSG,and Glutathione S-transferase(GST).4.4 Detection of Ah R-Nrf2 antioxidant pathway4.4.1 Ah R expression level and activation detectionThe q RT-PCR method was used to detect the expression of total Ah R in the liver in each group.The liver nucleoprotein was extracted and the expression of Ah R in the cell nucleus was detected by Western blot.The expression level of CYP1A1,an Ah R activation indicator,was detected by q RT-PCR and Western blot.4.4.2 Nrf2 expression level and activation detectionThe q RT-PCR method was used to detect the expression of total Nrf2 in the liver in each group;the liver nucleoprotein was extracted,and the expression of Nrf2 at the nuclear level was detected by western blot to evaluate the activation level of Nrf2.4.4.3 Nrf2 downstream antioxidant molecule detectionThe q RT-PCR method was used to detect the expression of liver GCLC,GCLM,NQO1,and HO-1 in each group.Results1.In the rat HSOS model induced by MCT(90mg/kg),there was severe liver damage: Compared with the control group,the liver of HSOS model rats(1)was obvious congestion and swelling,and the liver weight and liver weight/body weight ratio were significantly increased;(2)The ALT and AST levels in the HSOS group were significantly increased;(3)HE results in HSOS rat liver tissue showed obviously dilation and bleeding of the liver the HSOS severity score increased significantly.At the same time,the expression of the LSEC injury marker MMP-9 increased significantly.2.FMT aggravated the liver damage induced by MCT(60mg/kg).Compared with the liver injury induced by 60mg/kg MCT,FMT +MCT 60mg/kg significantly increased the degree of liver injury,ALT and AST were significantly increased,and the expression of MMP-9 was increased.Hepatic pathology showed the dilatation and hemorrhage of hepatic sinuses,the damage of central venous endothelium,and the coagulation necrosis of hepatic cells were more serious,and the HSOS severity score was significantly increased.3.Transplantation of fecal bacteria liquid from normal rats can alleviate liver damage induced by MCT(60mg/kg).Compared with the liver injury induced by MCT 60mg/kg,MCT 60mg/kg + FMT could significantly alleviate the degree of liver injury,ALT and AST were significantly decreased,MMP-9 expression was down-regulated,liver pathology indicated the dilatation and hemorrhage of hepatic sinuses,the damage of central venous endothelium,and the coagulation necrosis of hepatic cells were alleviated,and the HSOS severity score was significantly reduced.4.In MCT(90mg/kg)-induced rat HSOS,intestinal flora was disturbed.Compared with the control group,(1)flora ? diversity in HSOS model group was significantly decreased.(2)The ?-diversity indicated that the HSOS model group had obvious microbial community changes.At the phylum level,MCT significantly reduced the relative abundance of Firmicutes and increased the relative abundance of Bacteroidetes in rats.At the family level,compared with the normal control group,Ruminococcaceae and Lachnospiraceae from Firmicutes decreased in model group,while Muribaculaceae from Bacteroidetes also decreased.However,the number of Prevotellaceae in Bacteroidetes was significantly increased.At the genus level,Alloprevotella,Muribaculaceae and Prevotellaceae in the HSOS model were decreased,and importantly,flora that could metabolize tryptophan,such as Bacteroides,Bifidobacterium,Lactobacillus,and Clostridium were also decreased.However,Prevotella-1 and Prevotella-9 were significantly elevated.The prediction of KEGG metabolic pathway suggested changes in several pathways such as tryptophan metabolism.5.Disturbance of intestinal flora significantly reduces microbial tryptophan metabolites.In the analysis of metabolic pathways,tryptophan metabolic pathway had the largest influence factor in topological analysis,and the P value of enrichment analysis showed significant differences,suggesting that tryptophan metabolic pathway played an important role in MCT induced HSOS in rats.Further analysis of differential metabolites showed that the expression of tryptophan metabolites was decreased,including indole-3--carboxylic Acid,indole-3-acetamide,indole-3-acetamide(IAAld),indole-3-propanoic Acid(IAA),indole-3-propanoic Acid,indole-3-methyl Acetate,and indole-3-carboxylic Acid.6.IAAld,or IAA,or Ficz can activate AhR-Nrf2 antioxidant pathway and improve 90mg/kg MCT induced liver damage.The intestinal supplement IAAld?or IAA,or Ficz abdominal intervention,can significantly reduce the oxidative stress damage induced by MCT,reduce the expression level of MDA and ROS,increase GSH/GSSG,and increase the activity of GST.Significantly improve liver congestion,reduce liver weight and liver weight/weight ratio;reverse elevated ALT and AST.IAAld,or IAA,or Ficz can activate Ah R and promote the nuclear transfer of Ah R.Meanwhile,Nrf2 nuclear transfer was promoted,the expression of Nrf2 nuclear protein was increased,and the expression of downstream antioxidant molecules GCLC,GCLM,and NQO1 were also increased.ConclusionsIntestinal flora is involved in the occurrence and development of HSOS.IAAld,IAA or Ficz can activate the liver Ah R-Nrf2 antioxidant pathway,alleviate the level of liver oxidative stress in HSOS and thus improve liver damage.Clinically,Ah R ligand supplementation may be a new therapeutic approach for HSOS.