Hepaticprotective Effect And Mechanisms Of Oridonin On Bisphenol A-induced Liver Injury In Rats Based On Metabolomics

Background:Bisphenol A(BPA),one of the most produced chemicals in the world,has been widely used in the manufacture of packaging materials for various daily necessities.As an endocrine disrupting chemicals,long-term exposure to BPA may induce neurotoxicity,reproductive toxicity,metabolic diseases,etc.BPA is mainly metabolized by the liver,and its metabolites are potentially more toxic.Notably,the liver tissue,compared with other organs,is more vulnerable as the major organ responsible for BPA metabolism.Accumulating reports have revealed the damage caused by BPA to the liver,kidney,and other organs by generating oxidative stress.Oridonin(Ori),a diterpenoid from Isodon species,is the main chemical composition of Chinese medicinal material Rabdosia rubescens Hara.Previous studies have documented that oridonin has many biological activities,including anti-inflammatory,suppression of oxidative stress and anti-tumor.However,the potential action of oridonin against BPA-induced liver injury from the perspective of molecular biology still needs to be elucidated.Objectives:This research aimed at investigating the protective effect of oridonin against BPAinduced liver injury in rats.The protective mechanism was systematically explored by using metabonomics technology at the metabolic level,which might provide a theoretical basis for the clinical effect of oridonin and the development of liver protection drugs.Methods:1.48 SD male rats were randomly divided into 6 groups(n=8): Control group,Vehicle group,Oridonin group(10 mg/kg),Bisphenol A group(500 mg/kg),Bisphenol A + Oridonin group(500+10 mg/kg),Bisphenol A + Diammonium glycyrrhizinate group(500+40 mg/kg).Rats were continuously administered every day for 14 days.2.Automatic biochemical analyzer was used to analyze the serum levels of aspartate aminotransferase(AST),alanine aminotransferase(ALT)and alkaline phosphatase(ALP).The liver tissues of rats were stained by hematoxylin-eosin(H(5)E)and TUNEL for histopathological and cellular apoptosis evaluation,respectively.3.UPLC-MS/MS metabolomics technology was used to analyze endogenous small molecule metabolites in rat liver tissue samples of each group.We used SIMCAP 14.1 software to performe Orthogonal partial least squares discriminant analysis(OPLS-DA).The compounds,meeting the screening conditions(P < 0.05 and the variable importance in projection(VIP)value > 1),were selected as differential compounds for metabolic pathway analysis and enrichment analysis by Metaboanalyst website.4.A quantitative analysis method for purine metabolites based on LC-MS/MS was established,which was used to determine the content of hypoxanthine,xanthine and uric acid in liver tissue of rats.5.The determination of XOD activity and molecular docking simulation experiment were performed to explore the effect of oridonin on XOD.6.The production of ROS in rat hepatocytes was analyzed by frozen section immunofluorescence experiment.The levels of glutathione peroxidase(GPX),catalase(CAT),superoxide dismutase(SOD),total antioxidant capacity(T-AQC),and the content of malondialdehyde(MDA),total glutathione(T-GSH),oxidized glutathione(GSSG)in the liver tissue were determined using the assay kits.Furthermore,the m RNA levels of GPX,CAT,SOD and NF-E2-related factor 2(Nrf2)were measured by Real-time quantitative PCR(RT-q PCR)to analyze the effect of oridonin on the antioxidant capacity of BPA-exposed rats.7.The immunocytochemistry(IHC)was used to analyze the expression of F4/80 and Myeloperoxidase(MPO)in the liver tissues,and the RT-q PCR experiment was performed to detected the m RNA levels of TNF-α,IL-1β,and IL-6,aimed at analyzing the effect of oridonin on the anti-inflammatory ability of BPA-exposed rats.Results:1.The protective effect of oridonin against BPA-induced liver injury in rats:Compared with the Vehicle group,BPA could significantly increase the serum levels of AST,ALT and ALP(P < 0.05),and the levels of AST and ALT were significantly reduced after oridonin treatment(P < 0.05).The histopathological changes of liver tissue in rats were investigated by staining H&E,in which obvious hepatocyte damage were observed in BPA group.Concurrently,administration of oridonin significantly reduced the liver injuries,and the histopathological morphology basically returned to the state of the Control group.TUNEL staining results showed that oridonin could significantly reduce the increase of liver apoptotic cells in BPA-induced rats.2.Hepatoprotective effects of oridonin against BPA-induced liver injury in rats via inhibiting purine metabolism: A total of 28 significant differential metabolites were screened with meeting the screening conditions(P < 0.05 and VIP > 1)in the liver tissue,including: uric acid,hypoxanthine,xanthine nucleoside,guanine,guanine nucleotide and adenine,etc.The results of pathway enrichment and analysis depicted that purine metabolism,acyl-t RNA biosynthesis,spermidine and spermine metabolism were reprogrammed,especially purine metabolism.3.Oridonin could exert a protective effect on the liver via inhibiting the activity of XOD and regulating the disorded purine metabolism: The quantitative analysis results in the liver tissue samples showed that the contents of hypoxanthine and xanthine in BPA group were lower than that of Vehicle group,respectively,while and the level of uric acid was markedly elevated.The co-administration of BPA with Ori significantly reversed the change trend of compound content.Combined with XOD activity determination and molecular docking simulation results,we found that oridonin could inhibit the activity of XOD by binding to cavity 5 of protein 2CKJ through hydrogen bonding.Further studies have found that oridonin could remarkably lower the level of ROS,enhance the activities and m RNA expression levels of antioxidant enzymes(GPX,CAT and SOD),reduce the content of MDA and GSSG,and enhance the total antioxidant capacity.In addition,oridonin could significantly decrease the m RNA expression levels of TNF-α,IL-1β,and IL-6,and down-regulate the expression of MPO and F4/80.Conclusions:1.Oridonin could reduce the levels of serum liver function enzymes,improve ameliorated the abnormal histopathological changes and reduce hepatic apoptosis,thus playing a protective effect on BPA-induced liver injury in rats.2.Oridonin could inhibit the activity of XOD,reduce the conversion of hypoxanthine to uric acid,regulate purine metabolism,enhance the antioxidant capacity and anti-inflammatory effects,thus playing a protective role in BPA-induced liver injury in rats.