Protective Effect And Mechanism Of Schisandrae Chinensis Fructus On Acetaminophen-induced Drug-induced Liver Injury

Objective: Drug-induced Liver Injury(DILI)is one of the most common adverse reactions based on pharmacological effects of drugs.Excessive or long-term use of Acetaminophen(APAP),a kind of drug of analgesic and antipyretic,can cause DILI.As the ideal traditional Chinese medicine(TCM)to treat DILI by APAP,Schisandrae Chinensis Fructus(SCF)has the advantages of obvious curative effect and little side effect.This study aimed to explore the protective effects,mechanism of action of SCF treating DILI by APAP.The results had far-reaching significance for the clinical application of SCF of TCM.Methods: 1.Established the mouse DILI model with three APAP doses of 200 mg/kg,300 mg/kg,and 400 mg/kg,determined the AST and ALT in the plasma,and chose the optimal APAP modeling dose.2.The mice were pre-protected with different doses of SCF extract,and the mouse DILI model was established by APAP.The traditional biochemical indicators of AST,ALT and ALP in mice plasma were measured,as well as early indicators of liver injury(PNG,GLDH,Arg-1,?-GST),inflammation levels(TNF-?,IL-1? and IL-6),oxidative stress and lipid peroxidation indicators(MDA,SOD,GSH,GSH-Px).3.After oral administration of rats,UPLC-Q-TOF/MS technology was used to identify the prototype products and metabolites of SCF in rat 's plasma.4.The prototype products and metabolites of SCF combined the TCMSSP database and the Swiss Target Prediction database were used to obtain the targets of SCF active ingredients.Then the OMIN database and Gene Cards database were used to screen the potential targets of SCF treating DILI.And the DAVID database was used to obtain the information about targets and pathways.5.Based on the plasma metabolomics profiling with UPLC-Q-TOF/MS approach,mechanism of action of SCF treating DILI by APAP was explored according to the perspective of endogenous metabolic changes.PCA and OPLS-DA analysis methods were used.And differential metabolites were screened according to VIP-values and P-values,and metabolite identification was performed by using databases such as HMDB.Finally,the biomarkers of SCF treating DILI by APAP were determined.6.The metabolomics and network pharmacology were used to screen targets.Targets were experimentally validated by Quantitative Real-time PCR analysis.Then we speculated the possible pathways and mechanisms of SCF treating DILI by APAP.Results: 1.The DILI model was successfully established by APAP at the doses of 200 mg / kg,300 mg / kg and 400 mg / kg.The levels of AST and ALT were significantly higher than those of the blank group mice(P <0.01).2.SCF extracted groups could significantly reduce the level of AST,ALT,TNF-?,IL-1?,PNP,and MDA,significantly increased the levels of of SOD and GSH,which treated APAPinduced liver damage.3.UPLC-Q-TOF/MS technology was used to identify 10 metabolites and 9 blood prototype components which included Gomisin T,Gomisin J,Schisandrol A,Gomisin D,Schisandrol B,Schisandrin B,Angeloylgomisin Q,Schisandrin A,and Schisandrin.4.Research indicated 9 blood prototype and 10 metabolic components in SCF mainly act on 16 key targets such as SRC,EGFR,TNF,and PTGS2,which through the regulation of apoptosis,regulation of cell death and protein amino acid phosphorylation to regulate SCF treating DILI..And it was used to enrich 15 signal pathways such as Pathway in cancer,VEGF,Bladder cancer,Erb B signaling pathway and Gn RH signaling pathway et al.5.Based on the plasma metabolomics profiling with UPLC-Q-TOF/MS approach,32 kinds of metabolites related to the process of liver injury were identified.These metabolites were enriched in 13 metabolic pathways,including retinol pathway,arachidonic acid pathway,glycerophospholipid metabolism,porphyrin chlorophyll pathway and amino acid pathway.6.Combined with the targets screened by metabonomics and network pharmacology,35 targets were used to identify,included PTGS2,SRC,TNF,PLA2G4 A and EGFR et al,and 23 target genes were finally identified for Quantitative Real-time PCR analysis.Conclusion: 1.In the pharmacodynamic study of SCF treating APAP-induced DILI,it was found that a dose of 200 mg/kg APAP was optimum.SCF had a significant protective effect on APAPinduced DILI,and mainly reduced liver enzymes in plasma,againsted inflammation levels?combated oxidative stress and lipid peroxidation indicators to treat APAP-induced DILI in mice.2.9 blood prototype and 10 metabolic components in SCF blood were identified by UPLCQ-TOF/MS technology.It is speculated that these components were the effect components in SCF which played a role in preventing or treating diseases.3.Based on network pharmacology,the research was preliminarily verified the molecular mechanism of the pharmacological effects about SCF treatment on DILI,which provided a theoretical basis for further in-depth study of its mechanism of action,and also fully demonstrated the multi-component,multi-target and multi-pathway characteristics of SCF.4.The metabolomics technology was used to further verify the protective effect of SCF on APAP-induced DILI,and preliminary explored that the liver-protective effect of SCF on DILI may be related to metabolism of retinol metabolism,arachidonic acid metabolism,and glycerophospholipid metabolism.5.Based on the plasma metabolomics profiling and Quantitative Real-time PCR analysis,we speculated that SCF treating APAP-induced DILI might be affected by EGFR/TNF/NF-k B pathway?EGFR/MDM2/p53 pathway?EGFR/AA pathway?Keap1-NRF2/ARE/HO-1-NQO1 pathway?PLD1/p21 pathway and LPCAT1/PI3K/AKT metabolic pathways.And the result of study would lay the foundation for the treatment of APAP-induced DILI research and development of Chinese medicine.