Research On The Effect Of Diosmin On Inducing HepG2 Cell Apoptosis

Diosmin is a kind of flavonoid glycosides.Many years of research in this group have found that diosmin has anti-radiation and anti-cancer effects.In order to further study the pathways and targets of anti-cancer effects of natural active ingredients,this topic begins with the discussion of diosmin inhibiting HepG2 cell proliferation and inducing apoptosis.The main findings are as follows:In order to study the effects of diosmin on the morphology,proliferation,exercise capacity,population dependence and apoptosis of HepG2 cells,MTT colorimetry,cell scratch assay,cell clone formation assay and cell cycle assay are used.Get the following results:Diosmin can cause HepG2 cells to become larger,swollen and round,with blurred cell boundaries and poor refractive index;Diosmin inhibit the proliferation of HepG2cells in a concentration-dependent manner,and the inhibition rate of proliferation of hepatocellular carcinoma HepG2 cells is 56.32%with 300?g/mL diosmin;Diosmin is able to inhibit HepG2 cell migration,and the 48-hour migration inhibition rate of HepG2cells at 300?g/mL diosmin is 50.39%;Diosmin is able to inhibit the formation of HepG2single-cell clones,and the 15-day cell clone inhibition rate of HepG2 cells at 300?g/mL diosmin is 73.49%;After treatment with diosmin,the cells in G₀/G₁ phase increase significantly,and the cells in G₂/M phase decreased significantly?P<0.05?,and are dependent on concentration.The above results indicate that diosmin can induce apoptosis of HepG2 cells and inhibit HepG2 cell proliferation by causing G₀/G₁ phase arrest in HepG2 cells,predicting a signal pathway Ras/Raf/MEK/ERK and a key target Akt that may inhibit cell cycle.To investigate the mechanism of apoptosis induced by diosmin and inhibit cell proliferation,bioinformatics analysis of Ras/Raf/MEK/ERK and PI3K/Akt signaling pathways in 1003 HCC patients is performed by cBioPortal,and then these pathways are performed by Real-time PCR.The gene differential expression is verified to obtain the following results:Several high frequency mutation sites are explored:PIK3CA?H1047R/L?,PIK3CA?E545K/A?,AKT1?E17K?,KRAS?G12C/D?on HCC patients,and Kaplan-Meier and Log are used.The-rank test plots the survival rate and survival time of HCC patients after mutations in related genes;Real-time PCR results show that the relative expression of Bax mRNA in HepG2 cells is significantly up-regulated,the relative expression of Bcl-2 mRNA is significantly down-regulated,and the ratio of relative expression of Bax/Bcl-2 mRNA is increased with the increase of diosmin concentration.Concentration-dependent;as the concentration of diosmin increase,the relative expression of Caspase-3 mRNA in HepG2 cells is down-regulated in a concentration-dependent manner.When the concentration of diosmin is in the range of 0-200?g/mL,the relative expression of mRNA of MEK,ERK and Akt is significantly down-regulated,and the relative expression of mRNA of Ras and Raf do not change significantly.When the concentration of diosmin is between 200?g/mL and 300?g/mL,the relative expression of mRNA of Ras,Raf,MEK,ERK and Akt don't change significantly.The above experimental results indicate that diosmin can induce apoptosis of hepatocellular carcinoma HepG2 cells through mitochondrial pathway,and other pathways need to further detect phosphorylation of corresponding proteins.To further investigate the mechanism by which diosmin induces apoptosis and inhibits cell proliferation,Western Blot is used to determine the relative expression of the above pathway proteins,and the following results are obtained:With the increase of diosmin concentration,the relative expression of Bax protein in HepG2 cells is significantly up-regulated,Bcl-2 protein is significantly down-regulated,and the ratio of Bax/Bcl-2 is significantly down-regulated.With the increase of diosmin concentration,the relative expression of cleaved Caspase-3 protein in HepG2 cells is significantly up-regulated in a concentration-dependent manner.When the concentration of diosmin is in the range of 0-200?g/mL,the protein expression of KRas is significantly down-regulated,Raf,MEK and ERK proteins is not significantly changed,and p-MEK and p-ERK protein is not significantly changed.Significantly down-regulated.When the concentration of diosmin is between 200?g/mL and 300?g/mL,KRas,Raf,MEK and ERK protein don't change significantly,and p-MEK and p-ERK protein continued to decrease.The relative expression of Akt protein in HepG2 cells doesn't change significantly with the increase of diosmin concentration,while the relative expression of p-Akt protein is significantly up-regulated in a concentration-dependent manner.The above experimental results indicate that diosmin can induce apoptosis of hepatoma HepG2 cells through the mitochondrial pathway.In addition,high concentration of diosmin can inhibit the proliferation of HepG2 cells.It speculates that diosmin may act as a MEK inhibitor to inhibit the phosphorylation of MEK and reduce the transmission of downstream signals,thereby inhibiting the proliferation of HepG2 cells.