The Material Basis Of Liver Injury Induced By Polygonum Multiflorum Based On HLA-B*35:01 Allele

Background and objective:Polygonum multiflorum(PM),a traditional Chinese medicine,has been widely used in the treatment of premature graying hair,neurasthenia,hypercholesterolemia,hyperlipidemia,constipation,aging,inflammation,tumor,neurodegenerative diseases,and in the field of diet and health care.PM-induced liver injury have a negative impact on the clinical use of PM,and have also attracted the attention of scholars at home and abroad.Relevant research has also made positive progress,but the material basis and mechanism of PM-induced liver injury are still inconclusive.Our previous study confirmed that PM induced liver injury is closely related to genetic polymorphism,and the HLA-B*35:01 allele is a specific biomarker of PM-induced liver injury.This study aimed to find the material basis for the liver injury caused by PM based on HLA-B*35:01 allele,and provide support for further elucidation of the mechanism of PM-induced liver injury.Methods:1.The chemical constituents of P.multiflorum and its metabolites were summarized though the Pub Med,Pub Chem,CNKI database and Wanfang database.The candidate small molecule ligand library was constructed.The receptor molecule of HLA-B*35:01 protein was prepared from the RCSB PDB database.Auto Dock vina was used for molecular docking.And the order was arranged according to the binding energy from the smallest to the largest.The components that might interact with the receptor binding domain were selected by binding energy.2.The five main components of PM(emodin,emodin-8-O-β-D-glucoside,physcion,2,3,5,4′-tetrahydroxystilbene-2-Ο-β-D-glucoside,catechin)were metabolized in vitro.Co-incubation allows the compound to bind to HLA-B*35 protein.Immunoaffinity chromatography is used to purify HLA-B*35 protein.The compounds were removed from the HLA-B*35 protein and determined by the UPLC-QTRAP MS.3.Humanized HLA-B*35:01 transgenic C57BL/6 mice(Tg)and wild-type C57BL/6 mice(WT)were randomly divided into eight groups:WT solvent control group(0.1%CMC-Na),WT low-dose emodin group(5 mg/kg/day),WT mid-dose emodin group(50 mg/kg/day)and WT high-dose emodin group(100 mg/kg/day),Tg solvent control group(0.1%CMC-Na),Tg low-dose emodin group(5 mg/kg/day),Tg middle-dose emodin group(50 mg/kg/day)and Tg high-dose emodin group(100mg/kg/day),n=5.Mice were administered with the emodin or solvent for 5 weeks.The growth state of the mice in each group was regularly observed,and the levels of ALT and AST were detected.Finally,the pathological changes of the mouse liver were evaluated by observing the appearance of the liver and H&E staining,the proliferation of T lymphocytes was detected by flow cytometry.Results:1.There were 132 chemical compositions in the small molecule ligand library.The screening resulted in 10 compounds forecasted to bind HLA-B*35:01.And the phase II metabolite of emodin had lower binding energies with HLA-B*35:01 molecular.The phase II metabolite and HLA-B*35:01 molecular were linked by hydrogen bond.2.Emodin,physcion,emodin-8-O-β-D-glucoside,2,3,5,4′-tetrahydroxystilbene-2-Ο-β-D-glucoside,and catechin can be metabolized into glucuronidation products by in vitro incubation in the presence of UDPGA.The above five components of PM and their metabolites were incubated with C1R-B*35:01 cells,purified and isolated,and identified by UPLC-QTRAP MS.Only emodin and emodin-8-O-β-D-glucoside and Emodin glucuronide were found.Relative quantitative analysis showed that compared with the C1R-B*35:03 PM group,the C1R-B*35:01 PM group had higher levels of emodin,emodin-8-O-β-D-glucoside,and emodin glucuronide,among which emodin glucuronide was statistically different(P<0.05).3.To verify the hepatotoxicity of emodin,WT mice and humanized HLA-B*35:01Tg C57BL/6 mice were treated with emodin.Five weeks later,there was no statistical difference in ALT and AST levels(P>0.05).H&E staining showed that the hepatocytes of the solvent control group in the WT mice were neatly arranged,while the low-,mid-and high-dose emodin group occasionally saw punctate hepatocyte necrosis and a small amount of lymphocyte infiltration;the hepatocytes in the solvent group of the Tg had a complete and clear structure,while the low-,mid-and high-dose emodin group showed obvious hepatocyte necrosis,accompanied by lymphocyte infiltration.The results of flow cytometry showed that the proportion of CD8~+T lymphocyte subsets of HLA-B*35:01 Tg mice was significantly increased(P<0.05),while emodin had no significant effect on CD8~+T lymphocytes in WT mice,indicating that emodin-induced HLA-B*35:01 Tg mice had more severe liver damage,and emodin may promote the proliferation of CD8~+T lymphocytes and cause liver damage.Conclusions:Emodin,emodin-8-O-β-D-glucoside and emodin glucuronide can bind to HLA-B*35:01 protein,and emodin may mediate liver injury by stimulating the proliferation and activation of CD8~+T lymphocytes and inducing adaptive immune responses,which rely on the presentation of HLA-B*35:01 protein.