| Red yeast rice is a kind of purplish red rice which is fermented by monascus fungus under certain conditions.Red yeast rice has many biological functions such as lowering blood lipid,lowering blood sugar,lowering blood pressure.In addition,it also has anti-inflammatory,antibacterial and other biological functions.Red yeast rice is rich in bioactive substances,such as polysaccharide,ergosterol,gamma-aminobutyric acid,unsaturated fatty acids and lovastatin.Among them,lovastatin is an important active ingredient in red yeast rice,which can effectively reduce blood cholesterol and lipoprotein levels.At present,in-depth studies have been conducted on the efficacy of lovastatin in lipid lowering.However,recent studies have found that lovastatin has potential anticancer effect,but the potential mechanism of lovastatin inducing cancer cell death is still not completely clear.In this study,lovastatin,the active ingredient extracted from red yeast rice,was used as the research object,hepatoma cell HepG2 was used as the cell model,and the anticancer efficacy of lovastatin was evaluated and the molecular mechanism of anticancer effect was explored,aiming to provide scientific theoretical basis for the development of anticancer active substances from safe foodborne natural products.In this study,lovastatin was extracted from red yeast rice by ultrasonically assisted method,and the content of Red yeast millovastatin was determined by high performance liquid phase.This method could accurately determine the content of Red yeast millovastatin in red yeast,and had a good linear relationship between 4～100ug/ml.The results showed that the purity of lovastatin was 96.7%after separation and purification by HPLC.Red yeast millovastatin can inhibit proliferative activity and cell migration of HepG2.Compared with the blank group,the addition of lovastatin can cause morphological changes of HepG2 cells in a certain concentration dependent manner.Flow cytometry showed that lovastatin(12.5 μg/ml)inhibited 73.3%of cells in G1 phase,an increase of 6.4%compared with blank control group.After Western Blot assay and RT-qRCR assay were used to detect the expressions of cycle-related proteins and genes,it was found that after lovastatin treatment,the expressions of p21 and p53 proteins forming the G1 checkpoint of cell cycle were up-regulated compared with the blank group,and the expressions of p16,p18 and p27 proteins were also up-regulated,indicating the same gene expression.Lovastatin also inhibited the phosphorylation of CDK2 and CDK6,and finally inhibited the protein expression of Cyclin D1 and Cyclin E1.Changes in these proteins and genes ultimately trigger cell cycle arrest.Red yeast millovastatin can promote apoptosis of HepG2.The results of Hoechst staining showed that with the increase of lovastatin concentration,the cell nucleus concentration increased and apoptosis became more obvious.It was confirmed that lovastatin induced premature dying of HepG2 cells.JC-1 staining determined that lovastatin caused the imbalance of mitochondrial membrane potential in HepG2.Quantitative analysis by flow cytometry showed that the survival rates of HepG2 cells in lovastatin groups of 0μg/ml,6.25 μg/ml,12.5 μg/ml and 25 μg/ml were 96.9%and 94.8%,respectively.Lovastatin induced apoptosis of HepG2 cells was determined by 91.2%and 89.1%.After Western Blot assay was used to detect the expression of apoptosis-related genes at the protein level,it was found that lovastatin up-regulated the ratio of Bax/Bcl-2 and promoted the expression of Cyt c protein.It stimulated the activation of Caspase family proteins,including Caspase 3,Caspase 8 and Caspase 9,thus promoting cell apoptosis.In order to further study the effect of lovastatin on gene changes of HepG2 cells,transcriptional sequencing was used to study the changes of lovastatin on RNA level of HepG2 cells,and 106 genes with significant changes were found.GO analysis and KEGG analysis showed that these genes were involved in a variety of biological processes and signal responses related to apoptosis,growth and migration.The protein interaction network prediction of these genes was found to form an interaction network centered on VEGFA.This suggests that VEGFA is a key regulator of angiogenesis during lovastatin inhibition of HepG2 cell growth,and targeting VEGFA may be a new regimen for inhibiting tumor growth. |
