The Role Of Autophagy In The Induction Of Hepatocellular Carcinoma Cell Apoptosis By Sulfated Ginkgo Biloba Leaves Polysaccharide In Vitro

Liver cancer is a common malignant tumor disease in humans and animals.Among them,hepatocellular carcinoma(HCC)has the highest clinical incidence,accounting for about 70%-90% of the total incidence of liver cancer.At present,the treatment of HCC mainly adopts palliative surgical treatment,chemical treatment and other methods.Although these methods have certain therapeutic effects,they also have disadvantages such as high risk,large toxic and side effects,and serious damage to the body.Therefore,the development of natural new drugs for treating HCC and improving the sensitivity of the drugs to HCC are the focus of current development of anti-tumor drugs.Ginkgo biloba polysaccharide(GBLP)is an active ingredient extracted from ginkgo biloba leaves,which has various biological activities such as regulating immunity,anti-tumor,anti-oxidation and so on.Studies have shown that the antitumor activity of many traditional Chinese medicine polysaccharides is significantly enhanced after being modified by sulfated molecules.Therefore,in this study,sulfonic acid-pyridine method was used to prepare sulfated ginkgo biloba polysaccharides(SGBLP).Hepatocellular carcinoma HepG2 cells were used as test models to detect cell activity,major apoptotic proteins and autophagy proteins and signal transduction of SGBLP on HepG2 cells.Effects of the pathway PI3 K / AKT / mTOR.The main findings are as follows:1.The crude polysaccharide of Ginkgo biloba was extracted from the dry powder of Ginkgo biloba by water extraction and alcohol precipitation method,and purified Ginkgo biloba polysaccharide was obtained through a series of processes such as deproteinization,pigment removal and dialysis.The polysaccharide content measured by phenol-sulfuric acid method was 84.66%.The protein content was determined by the Maslan blue method to be0.561%,and then GBLP was sulfated with a chlorosulfonic acid-pyridine method to modify the sulfated molecule.The barium chloride-gelatin turbidimetric method was used todetermine the degree of substitution of sulfate by 2.7.2.Incubate different concentrations of GBLP solution with HepG2 for 24 h,48h,and 72 h.The results of MTT test showed that with the increase of SGBLP concentration and prolonged action time,the viability of HepG2 cells gradually decreased,and it showed a time-dose relationship.The results showed that SGBLP had a significant inhibitory effect on HepG2 proliferation.3.Western Blot was used to detect the expression levels of autophagy-related proteins LC3,p62,and Beclin1,the expression of apoptosis-related proteins Bax,Bcl-2,and signaling pathways after SGBLP was applied to HepG2 cells at different concentrations for 48 h.Expression of proteins p-PI3 K,AKT,p-AKT,mTOR,and p-mTOR.The test results show that as the concentration of SGBLP increases,the expression of autophagy-related proteins Beclin1 and LC3 increases,and the expression of p62 decreases;the expression of apoptosis-related proteins Bax increases,and the expression of Bcl-2 decreases;signaling pathway proteins The expression of total AKT and mTOR protein did not change significantly,while the expression of p-PI3 K,p-AKT and p-mTOR protein decreased with the increase of SGBLP concentration.The results indicate that SGBLP can induce autophagy and apoptosis in HepG2 cells,and the PI3K-AKT-mTOR signaling pathway is involved in this process.4.Before giving HepG2 cells different concentrations of SGBLP,hepG2 cells were pretreated with autophagy inhibitor 3-MA for 1h to inhibit the occurrence of autophagy in HepG2 cells.MTT method was used to detect HepG2 cell viability,and Western Blot technology was used to detect changes in the expression of autophagy-related proteins LC3,p62,Beclin1,and apoptosis-related proteins Bax and Bcl-2.The results showed that after3-MA pretreatment,the SGBLP group was added,compared with the SGBLP group alone,the cell activity was significantly reduced,the expression of anti-apoptotic proteins was reduced,and the expression of pro-apoptotic proteins was increased.This indicates that inhibition of autophagy can enhance the inhibition of SGBLP on the proliferation activity of HepG2 cells and promote the apoptosis of HepG2 cells.