Study On The Mechanism Of Liver Injury Of Polygonum Multiflorum Based On Bioinformatics And The Effect Of Cholestasis In Rats

BACKGROUND&AIMS: The bioinformatics technique such as database/software was used to optimize the protein of Polygonum multiflorum Thunb(PMT)induced hepatotoxicity screened by ITRAQ,Gene Ontological(GO)annotation clustering analysis and protein-protein interaction analysis was performed.At the same time,the hepatic injury of the rat model of liver injury induced by lipopolysaccharide(LPS)was used to study the liver injury of the model.Further detect the relevant indexes of cholestasis,so as to provide a clue for clarifying the mechanism of hepatotoxicity of PMT and lay a theoretical foundation for the follow-up experiment.METHODS: GO annotation clustering analysis、protein-protein interaction analysis、KEGG pathway analysis and Meta Core network distribution of selected protein were performed on the differential protein.The male SD rats(180-200 g)were randomly divided into the following five groups: blank control group(Ctrl)、 LPS group(LPS)、 LPS+Acetaminophen(LPS+APAP)、Polygonum multiflorum group(AEP)and LPS + Polygonum multiflorum group(LPS + AEP).A dosage of 4 mg/kg LPS was injected to the caudal vein of rats in the LPS and the LPS+AEP groups.2 hours later,rats were administrated by intragastric route with AEP(12 g/kg,equivalent to 22 times the dose of clinical medication)respectively for 7 days according to each group ones a day.Since the liver-injury models of inflammation was successfully established,the general condition of rats such as body weight and clinical observation were observed,liver coefficient and histopathological examination were performed.bile was collected to determine flow rate 、 density and changes of major compositions on the Hour 2、Hour 14、Day 5 and Day 8,after getting the organ to body weight ratio for each rats.Liver samples were detected the expressions of hepatic bile salt export pump(BSEP),multidrug resistance protein 2(MRP2)and multidrug resistance protein 3(MRP3)were detected by real-time PCR.RESULTS: 1.The results showed that 17β-hydroxyol dehydrogenase 2 、 carbonic anhydrase isoenzyme 、 recombinant human protein tyrosine phosphatase non-receptor type 1 、 cytochrome P450 2B1(CYP2B1)、60S Ribosomal protein L13、estrogen sulfate transferase,alanine aminotransferase 2、mitochondrial ATP synthase complex subunit C1、superoxide dismutase 2(SOD2)and other dozens of protein as a potential biomarker of PMT liver injury by LPS induced,provides PMT liver toxicity research with detection and predictions methods.Further analysis of these proteins by protein-network interaction revealed that some important proteins such as: uridine diphosphate glucuronosyltransferase 2b17(UGT2B17)、CYP2B13、CYP2C6、estrogen sulfate transferase 2、17β-hydroxy steroid dehydrogenation Enzyme 2、60S ribosomal protein L10、40S ribosomal protein S23、superoxide dismutase 2、glutathione reductase、oleyl-Co A reductase、acyl-Co A thioesterase 5、zinc transporter 10 、 Zinc transporter 、 mitochondria into the receptor subunit TOM6 homology 、 mitochondrial ATP synthase complex subunit C1.2.Through the GO annotation clustering analysis of the differential proteins,there are 156 related to the cell composition,98 of which are related to the molecular function,and 321 related to the biological process.3.After KEGG pathway analysis,these differential proteins were mainly involved in steroid hormone biosynthetic pathway,ribosome pathway,chemical carcinogenic pathway,retinol metabolic pathway,primary immunodeficiency pathway,etc.4.Analyzed the distribution of the network by Meta Core,and we found that was ralated to the inflammatory system such as IL2/IL6 signaling pathway,immune-related signals such as MHC class 1 related antigen presentation pathway、antigen presentation phagocytic body signal、apoptotic mitochondria、DNA damage BER-NER repair,etc.5.Establish the rat model of liver injury induced by LPS successfully.6.The LPS + AEP group was significantly decreased in the LPS + AEP group compared with the control group and the AEP group(P<0.05).Serum biochemical indicators showed that there was no significant change in ALT and AST levels of LPS + AEP group.(P<0.05),and the bile density and bile flow rate were significantly decreased in LPS + AEP group(P<0.05).Analysis of bile composition showed a significant decrease in TCHO and a huge decrease in TBIL(P<0.05).Pathological report showed a slight degeneration of liver cells in the AEP group,while LPS + AEP group showed severe focal necrosis.(P<0.05).The level of BSEP,MRP2 and MRP3 was significantly increased(P<0.05),and the expression of BSEP and MRP2 was significantly increased(P<0.05).However,there was no significant effect of BSPS and MRP2 on the liver of rats after LPS + AEP administration,but the transcription level of MRP3 was increased(P<0.05).CONCLUSIONS: 1.By analyzing the GO annotation clustering analysis of the differential proteins,we found that these proteins mainly affect the body function in terms of metabolism,immunity,stress response,cell killing,etc.2.Through protein interaction analysis,we found some proteins that may be related to the hepatotoxicity mechanism,namely uridine diphosphate glucuronosyl transferase 2b17(UGT2B17)、CYP2B13、CYP2C6、estrogen sulfatase transferase 2、17β-hydroxy sterol Type dehydrogenase 2、60S ribosomal protein L10、40S ribosomal protein S23、superoxide dismutase 2、glutathione reductase、oleyl-Co A reductase、acyl-Co A thioesterase 5、zinc transport Protein 10、zinc transporter、mitochondria into receptor subunit TOM6 isoform、mitochondrial ATP synthase complex subunit C1.According to the results,it’s speculated that the toxicity mechanism may be related to bilirubin metabolism.3.The main metabolic pathways of these differential proteins were found by KEGG pathway analysis: steroid hormone biosynthetic pathway、ribosome pathway、chemical carcinogenic pathway、retinol metabolic pathway、primary immunodeficiency pathway.4.Analyzed the distribution of the network by Meta Core,and we found that was ralated to the inflammatory system such as IL2/IL6 signaling pathway,immune-related signals such as MHC class 1 related antigen presentation pathway and antigen presentation phagocytic body signal.In addition,it is also associated with bile acid metabolism regulation.5.LPS induced alcohol extract of PMT can significantly damage the rat liver cells and interfere with the bile secretion function,change the bile composition of rats caused by changes in related biochemical indicators.BSEP and MRP2 levels were not significantly affected,and MRP3 levels were compensatory increase,suggesting that there is indeed symptoms of cholestasis,but may be caused by other mechanisms.