Study On Apoptosis Of Human Hepatoma Cell Line HepG2 Induced By Ethanol Extract Of Moringa Oleifera Leaves

Hepatocellular carcinoma（HCC）is one of the highest proportion of primary liver cancer,with the characteristics of long course of disease,high recurrence rate,easy spread,and difficult treatment.At present,its treatment methods mainly include surgical treatment,radiotherapy and chemotherapy,immunotherapy,targeted therapy,natural compound therapy,etc.Moringa oleifera Lam.has good edible and medicinal value because it contains a variety of bioactive ingredients such as phenols,polysaccharides,vitamins,and proteins.The natural compounds contained in Moringa oleifera leaves have gradually attracted attention in the treatment of HCC due to their advantages of low toxic side effects,good tumor inhibition effect,and wide sources.Therefore,ethanol extract of Moringa oleifera leaves was used as the research object to study its anti-liver cancer activity and mechanism.Firstly,the active ingredients in the leaves of Moringa oleifera were extracted using an ethanol ultrasound assisted method,and the ethanol extract of Moringa oleifera leaves was obtained after rotary steaming and freeze-drying.The qualitative and quantitative analysis of the bioactive components in the ethanol extract of Moringa oleifera leaves was carried out by liquid chromatography-mass spectrometry（LC-MS）.The results showed that the ethanol extract of Moringa oleifera leaves contained 5-caffeoylquinic acid,isoquercitrin,astragaloside,quercetin,kaempferol-7-O-glucoside and p-coumarin and other anti-cancer active components.By screening the cell line,drug treatment concentration,and drug action time using the CCK-8 method,it was found that after 24 hours of treatment,without affecting the survival of human normal liver cell L02,the IC₅₀ value of human liver cancer cell HepG2 was the lowest(IC₅₀=269.5μg/m L),therefore HepG2 cells were ultimately selected as the research object,with 100,200μg/m L is the drug treatment concentration,and 24 hours is the drug treatment time.On this basis,the effects of ethanol extract from Moringa oleifera leaves on the proliferation and migration ability of HepG2 cells were evaluated through colony formation,EdU detection,scratching,and Transwell experiments.It was found that after treatment with ethanol extract,the proliferation and migration ability of HepG2cells were significantly reduced.By detecting the aerobic glycolysis in HepG2 cells before and after the ethanol extract treatment,it was found that the ethanol extract of Moringa oleifera leaves could significantly increased the intracellular glucose content and ROS level,and reduce the content of pyruvic acid,lactic acid and the activity of glucose-6-phosphate dehydrogenase（G6PDH）.In order to further observe the inhibitory effect of the ethanol extract of Moringa oleifera leaves on the aerobic glycolysis of HepG2 cells,the mRNA and protein expression levels of key regulatory enzymes were detected by qPCR and Western Blot.The results showed that the ethanol extract of Moringa oleifera leaves could effectively reduce the expression of hexokinase 2（HK2）,pyruvate kinase isozyme type M2（PKM2）,phosphofructokinase-1（PFK1）and lactate dehydrogenase（LDHA）in cells,and reduce the level of aerobic glycolysis in tumor cells.In this study,the effects of ethanol extract from Moringa oleifera leaves on apoptosis of HepG2 cells were further observed through live and dead cell staining,DAPI staining and TUNEL apoptosis detection.It was found that ethanol extract treatment could significantly induce apoptosis of HepG2 cells.It was found that ethanol extract treatment can significantly induce cell apoptosis.Afterwards,the expression levels of apoptosis related genes and proteins were detected through qPCR and Western Blot experiments.The results showed that the mRNA expression level of BCL-2 was significantly reduced in HepG2 cells after treatment,while the mRNA expression level of Caspase-3/8/9 was significantly increased.The expression levels of BCL-2 protein and activated Caspase-3/9 protein showed the same trend as mRNA.In addition,the expression levels of cleaved PARP and Caspase-8 total protein were increased.The above results indicated that the ethanol extract of Moringa oleifera leaves can promote HepG2 cell apoptosis.In order to explore the regulatory mechanism of the ethanol extract of Moringa oleifera leaves promoting apoptosis of HepG2 cells,this paper detected the changes of mitochondrial membrane potential of HepG2 cells before and after the ethanol extract of Moringa oleifera leaves treatment.It was found that the mitochondrial membrane potential of HepG2 cells significantly decreased after the action of ethanol extract.The expression of genes related to mitochondrial apoptosis pathway was detected by qPCR.The results showed that the ethanol extract of Moringa oleifera leaves could increase the mRNA expression levels of HUWE1,BIM,BID,BAX,Smac/Diablo,APAF1,and AIF,while reducing the expression levels of MCL1 and XIAP.This indicates that the ethanol extract of Moringa oleifera leaves can induce HepG2 cell apoptosis by activating the mitochondrial apoptosis pathway.In conclusion,the ethanol extract of Moringa oleifera leaves has a good inhibitory effect on HepG2 cells,can significantly reduce the level of intracellular aerobic glycolysis,and can induce HepG2 cell apoptosis through the endogenous apoptosis pathway of cell mitochondria.This study will lay a certain research foundation for the development of natural compound anti-liver cancer drugs and the utilization of spicy wood leaves.