Study On Active Compounds Of Schisandra Chinensis In The Treatment Of Nonalcoholic Fatty Liver Disease And Preliminary Exploration Of Mechanism

Non-alcoholic fatty liver disease(NAFLD)is a metabolic dysfunction disease characterized by the deposition of lipids in liver cells induced by factors other than alcohol and other specific liver damage factors.This disease is a progressive disease that may further develop into non-alcoholic steatohepatitis(NASH),and in severe cases may develop into cirrhosis and liver cancer.It is the main reason of end-stage liver transplantation and liver disease.In recent years,with the epidemic of metabolic syndrome such as obesity and type 2 diabetes,the global incidence rate of NAFLD has reached 25-30%,which has become a worldwide public health problem.In China,NAFLD is also in a rapid growth stage and has become the second largest chronic liver disease after viral hepatitis.So far,the pathogenesis of NAFLD has not been clearly elucidated.There are no FDA approved specific drugs for NAFLD in clinical practice,and only insulin sensitizers,lipid-lowering drugs,antioxidant stress drugs,and liver protective drugs can alleviate symptoms,which cannot effectively reverse its course of disease.Therefore,it is urgent to explore effective and safe therapeutic drugs.Traditional Chinese medicine(TCM)has special superiority in treating liver diseases,and small molecules of TCM have become an important source of new drug discovery due to their structural diversity and drug like properties.Schisandra chinensis fruit,sourced from Schisandra chinensis(Turcz.)Baill.,which has the effect of astringent,tonifying kidney and calming heart.Clinical studies have found that Schisandra chinensis and its its related products,such as WZ capsule,fuzheng huayu capsule,bifendate and bicyclol,have shown good preventive and therapeutic effects in the treatment of liver diseases including viral hepatitis,drug-induced liver injury,and liver failure.Recently,studies found that Schisandra chinensis also shows good application prospects in the treatment of NAFLD.For example,the extract of Schisandra chinensis can relieve symptoms in the NAFLD mouse model induced by tunicamycin(100 mg/kg for 2 days)and HFD(300 mg/kg for 16 weeks),which through inhibiting the ER stress response;Schisandra chinensis alcohol extract(100-400 mg/kg)can also improve liver lipid deposition in HFD induced NAFLD model mice by activating two key lipid metabolism signaling pathways,AMPK/SREBPs and PPAR.However,these reports mainly focus on the effect studies of extracts,their active ingredients and mechanisms of action are still unclear.Therefore,it is of great significance to explore the active ingredients and mechanisms of Schisandra chinensis against NAFLD.The specific study contents and results are as follows:1.Screening of the most effective anti-NAFLD fractionRipe S.chinensis fruits were collected in September 2020 in Liaoning,China.Dried samples(500 g)were powdered and extracted using 10-fold volume 95%,80%,and 65%aqueous EtOH sequentially,each for 2 h.The EtOH extract was concentrated under reduced pressure to obtain a residue.The residue suspended in H2O was successively partitioned with PE and EtOAc to yield the PE-,EtOAc,and H2O soluble fractions(PET,ETA,and HOF,respectively).The anti-NAFLD effects of the PET,ETA,and HOF fractions were evaluated using the FFA-induced HepG2 cell model.The results showed that PET treatment significantly improved intracellular lipid accumulation and reduced triglyceride(TG)and total cholesterol(TC)levels,while ETA and HOF were inactive.The above results indicate that PET was the active fraction of the EtOH extract of S.chinensis fruit.On this basis,the activities of PET,ETA and HOF were screened using the HFD-induced NAFLD mouse model,and the results were consistent with the cell experiment.PET significantly improved the lipid droplet accumulation,globular degeneration and necrosis of model liver,and reduced TG and TC levels in mice.Thus,PET was regarded as the target fraction for further elucidation of anti-NAFLD active components.2.Chemical characterization of the active fraction PET based on UPLC-Q-TOF/MSTo further explore the anti NAFLD active components of the PET fraction of Schisandra chinensis,ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF/MS)technology was used to describe the chemical composition.The mass accuracy of all measured compounds was within 5 ppm.Based on the fragmentation pattern(information on fragment ion peaks)and retention time of compounds,37 compounds were preliminarily characterized,including 36 dibenzocyclooctene-type and 1 diarylbutane-type lignans.23 compounds were further confirmed by comparing with the reference standard.Compounds 8 and 37 have been identified for the first time in Schisandra chinensis.3.Core compounds,targets and pathways prediction of active fraction PET based on network pharmacologyBased on the identification of 37 chemical components using UPLC-Q-TOF/MS technology,network pharmacology methods were applied to predict and analyze the potential active components,key targets,and main pathways of Schisandra chinensis.432 targets related to compounds in Schisandra chinensis PET and 968 genes related NAFLD were identified.By intersecting the two,112 potential targets for PET treatment of NAFLD were obtained,and a protein interaction network(PPI)was constructed.PPI data was inputed into Cytoscape 3.7.1 to construct a weighted compound-target network.The results showed that compounds 3,24,and 27 had the highest weight,while GSK3β、PIK3CA、EGFR、TNF-α、MTOR and JNK1 are core target genes.Gene Ontology(GO)function and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis showed that the PET may play a role in alleviating NAFLD through insulin resistance.Integrating compound,target and pathway information to obtain the mechanism diagram of the active component exerting relieving NAFLD activity based on the insulin resistance signaling pathway.Among these,compound 3,considered to be the core component involved in NAFLD therapy,which overlapped with most of the targets contained in the insulin resistance signaling pathway.4.Efficacy verification and mechanism exploration of PET treatment NAFLDTo validate the anti-NAFLD effects of PET and compound 3 predicted based on network pharmacology analysis,a NAFLD mouse model was constructed using HFD feeding.The mice were orally administered PET(100 and 200 mg/kg)and compound 3(30 and 60 mg/kg)for another 4 weeks.Pioglitazone(PGZ)(10 mg/kg)was used as a positive control.In vivo experiments demonstrated that PET and core active compound 3 treatment significantly attenuated hepatic steatosis and reduced the levels of serum alanine transaminase(ALT),aspartate transaminase(AST),insulin,malondialdehyde(MDA),hepatic triglyceride(TG),and total cholesterol(TC)in HFD-induced mice.Moreover,treatment with PET and compound 3 alleviated glucose tolerance and insulin resistance.To further explore the mechanism of PET and compound 3 in alleviating NAFLD,quantitative real time PCR(qRT PCR)was used to analyze the core target genes predicted by network pharmacology.The results showed that Schisandra chinensis PET and compound 3 can regulate Pik3ca,Gsk3β,Jnkl and Tnf-α.In summary,this study indicates that the active fraction of PET from the EtOH extract of S.chinensis fruit has potent NAFLD treatment activities in vitro and in vivo.By integrating UPLC-Q-TOF/MS analysis,network pharmacology,and in vivo experiments,we found that the anti-NAFLD effects were attributable to the three compounds(3,24,and 27)contained in PET.The potential mechanism is associated with the insulin resistance pathway,which can regulate the expression of Pik3ca,Gsk3β,Jnk1,and Tnf-α.Our study provides scientific evidence for the development of the active fraction PET from S.chinensis fruit as a novel natural therapeutic agent against NAFLD and important hints for further mechanistic studies.