Inhibitory Activity Of Schisandrin B Against Hepatocellular Carcinoma HepG2 Cells And Its Protective Mechanism Against Acute Liver Failure In Mice

Hepatocellular carcinoma(HCC)is the fourth leading cause of cancer-related death and the most common primary malignant liver cancer worldwide.At the same time,the occurrence of the disease is directly related to liver cirrhosis.In recent years,the incidence of hepatocellular carcinoma is increasing,which endangers human health.Therefore,it is urgent to find drugs to treat HCC.Acute liver failure(ALF)is a life-threatening disease caused by a variety of factors such as drugs and viruses.Its clinical manifestations are acute and severe liver cell injury.Emergency liver transplantation can save patients’ lives,but it greatly increases the cost of resources.Therefore,there is an urgent need to explore drugs that can treat acute liver failure.Liver failure is easy to occur in patients with hepatocellular carcinoma,and the ultimate cause of death is mostly related to liver failure.Schisandra chinensis,as one of the raw materials of traditional Chinese medicine,is clinically used for the treatment of heart,liver and lung diseases.Schisandrin B(SSB)is the lignan with the highest content in Schisandra chinensis,which has pharmacological effects such as anticancer,anti-oxidation and anti-inflammation.In this study,based on the hepatoprotective activity of Schisandrin B,the inhibitory effect and mechanism of Schisandrin B on HCC and ALF were investigated in vitro and in vivo,respectively.Network pharmacology and molecular docking techniques were applied to investigate the inhibitory effect of Schisandrin B on HCC.The main research results are as follows:1.Schisandrin B induced autophagic-dependent cell death of HepG2 cells.With 5-fluorouracil and rapamycin as positive controls,MTT and CCK-8 cell viability assays and colony formation assay were used to prove the cytotoxicity and anti-proliferation activity of Schisandrin B on HepG2 cells.Cell morphology was observed by optical microscopy and crystal violet staining at 200 and 400 times field of view.Meanwhile,danoylglutarine fluorescence staining and adenoviride-Mcherry-GFP-LC3 b Adenovirus transfection showed that schisandryl ethyl greatly enhanced the cellular autophagy flow and intracellular autophagosome accumulation.Co-incubation with pathway inhibitors like 3-methyl adenosine(3-MA),dorsomorphin(Compound C),selisistat(EX527),and MRT68921(MRT)can protect HepG2 cells from SSB treatment and significantly reduce the quantity of autophagosomes and the expression of LC3B-II.2.Study on the mechanism of schisandrin B inducing apoptosis of HepG2 cells.In this study,the network experiments of network pharmacology and molecular docking were used to collect and analyze the overlapping protein targets enriched by schisandrin B and primary hepatocellular carcinoma respectively,and then three targets with the highest protein interaction degree among the 95 cross targets were selected for molecular docking,and the hydrogen bond and hydrophobic bond forces between the targets and schisandrin B were analyzed.Molecular docking experiments showed that schisandrin B had the strongest binding force and the most hydrogen bond numbers with the protein target Caspase3.Western Blot experiments showed that schisandrin B enhanced the expression of apoptosis-related genes,such as cleavedPARP and cleaved-Caspase3.Hoechst fluorescence staining showed that schisandrin B induced apoptosis in a small number of HepG2 cells,and cell viability experiments showed that pan-caspase inhibitor Z-VADFMK could partially inhibit schisandrin B-induced cell death.3.Study on the mechanism of schisandrin B inducing autophagy-dependent cell death in HepG2 cells.Western blotting analysis results showed that schisandrin B significantly enhanced the conversion trend of autophagy key protein LC3B-I to LC3B-II and increased the expression of protein LC3B-II in a dosedependent and time-dependent manner,thus activating autophagy.At the same time,the results also showed that the protein levels of autophagy-related proteins ATG5 and Beclin1 were significantly increased with the increase of schisandrin B dose,while the protein expression of p62 was significantly decreased.Western blotting was used to detect the protein expression in the upstream signaling pathway of autophagy-related proteins in HepG2 cells.The results showed that schisandrin B regulated the autophagy process by affecting the three upstream signaling pathways of AMPK/SIRT1,AMPK/ULK1/Beclin1 and Akt/m TOR in HepG2 cells.4.Schisandrin B exerted a hepatoprotective effect in LPS/D-Gal N-treated mice.In this study,we established a mouse model of ALF induced by LPS and D-Gal N.Therapeutic dosages of 50,100 mg/kg schisandrin B was used,with silibinin(200 mg/kg)as the positive control.The results showed that intraperitoneal injection of schisandrin B in corn oil once daily,seven days in advance,could prevent LPS/DGal N induced-fatal ALF,and significantly reduce liver cell injury and liver index increase caused by ALF,as well as the increase in serum ALT and AST content and SOD and MDA level in the liver.Meanwhile,HepG2 cells were pretreated with schisandrin B for 4 hours before 24-hour treatment with LPS(10 μg/m L),which significantly inhibited the LPS-induced increases in MDA,SOD and ROS levels.Hematoxylin and eosin staining assay showed that pretreatment with Schisandrin B(100 mg/kg)significantly reduced the acute liver injury caused by LPS and D-Gal N,including diffuse hepatic congestion,hepatocyte necrosis and cytoplasmic vacuolation.In this study,Western blot analysis confirmed that Schisandrin B significantly activated autophagy of mouse hepatocytes by up-regulating the protein level of LC3B-II.The results indicated that the protective mechanism of schisandrin B in mice with acute liver failure induced by LPS and D-Gal N might be through the activation of hepatocyte autophagy and the inhibition of oxidative stress.