Cytotoxicity Of Total Flavonoids From Peony Shells On HepG2 Cells

Flavonoids are widely distributed in the nature and they have various biological activities such as anti-inflammatory,antibacterial,anti-viral,anti-tumor,and anti-oxidation.Therefore,the research heat of flavonoid drugs for cancer therapy is also constantly improving.Peony has great medicinal value and also ornamental value.Peony and rhizome are rich in paeoniflorin,which have antibacterial and anti-inflammatory,anti-tumor,anti-cardiovascular disease,anti-oxidation,and enhancing body immunity efficacy.There are many medicinal ingredients such as paeoniflorin and albiflorin contained in peony,and in the near recent years,flavonoids are also contained in this peony,which has attracted much attation in anti-cancer of flavonoids.In this study,the total flavonoids from the peony shell were used for toxicity test on the human hepatocarcinoma cells?HepG2?to determine and evaluate the toxicity of flavonoids in order to further enrich and improve the mechanism of action of flavonoids on cancer cells and also to evaluate the harmful effect of flavonoids abuse on humans.This study would be valuable for studying the effects of flavonoids on human health and its protection.The following are the research contents and the main experimental results obtained:?1?Toxicity of total flavonoids from peony shells on HepG2 cellsCell viability was firstly detected by MTT assay and the LC₅₀0 of total flavonoids on HepG2cells was obtained by conducting acute toxicity test.Then the cell cycle and apoptosis were detected by flow cytometry,cell and organelle morphology were detected by inverted fluorescence microscopy to evaluate the toxic effect of total flavonoids on HepG2 cells.The results showed that flavonoids exposure at the 0.1 mM had slight effect on the viability of HepG2 cells while exposure at the 0.2 mM had significant effect on the viability and apoptosis of HepG2 cells when compared to the control group.The experimental data showed that the high concentration exposure of total flavonoids?0.1mM-0.4 mM?significantly inhibited cell viability of HepG2 cells and showed a pattern of dose-response relationship.HepG2 cells were exposed to total flavonoids of 0.1 mM-0.4 mM peony shell.The inverted fluorescein microscopy observation revealed that the high concentration of flavonoids changed the morphology of HepG2 cells and caused cell damage.The result of Hoechst 33342 staining the nucleus of HepG2 cells showed that flavonoids exposure at high concentrations?0.2 mM-0.4 mM?altered the nucleus form and induced apoptosis of HepG2cells.The result of acridine orang staining revealed that obvious damage to lysosomes of HepG2cells was observed when treated by high concentrations of flavonoids?0.2 mM-0.4 mM?,which was in a pattern of dose-response relationship.Rhodamine-123 staining showed that flavonoids exposure at high concentrations caused obvious damage to the mitochondria of HepG2cells.These results indicate that flavonoids exposure at lower concentration only had slight effect on HepG2 cells,but high concentration exposure caused cellullar damages and apoptosis of HepG2cells.The apoptosis and cycle detected by the cytometry showed that the apoptosis of HepG2 cells treated with 0.1 mM low concentration of peony shell flavonoids was not obvious;0.2 mM and0.4 mM higher concentrations of peony flavonoids treated HepG2 cells,the apoptotic condition was significantly elevated and presented in a dose-dependent manner.The cell cycle test showed that the cell cycle of HepG2 treated with 0.1 mM and 0.2 mM flavonoids did not change significantly;The cell cycle of HepG2 treated with 0.4 mM higher concentration of peony flavonoids had a certain change.?2?Apoptosis induced by HepG2 cells exposed to total flavonoids from peony shell and its biochemical mechanismThe result of cytotoxocity test on HepG2 cells showed that flavonoids exposure at lower concentration induced ROS generation which was not different with the control cells,however,flavonoids exposure at higher concentrations caused excessive ROS and aopoptosis of HepG2cells.To explore the pathway of the aopoptosis of HepG2 cells induced by flavonoids,the cells were exposed to 0.1 mM-0.4 mM of total flavonoids from peony shells to determine the response of the antioxidant system of HepG2 cells.The results showed that ROS content significantly increased while the activities of SOD and CAT deacreased in the flavonoids-treated cells when compared to that of control cells.Moreover,the GHS content decreased,but MDA level significantly increased.This result indicates that flavonoids exposure induces excessive ROS,which causes oxidative stress and lipid peroxidation of HepG2 cells.The result of qPCR determination showed that the expressions of p53,p21/WAF1,and fas were up-regulated in HepG2 cells exposed to the total flavonoids?0.1 mM-0.4 mM?in comparasion with the control cells.Futhermore,total flavonoids exposure also promoted the transcriptional expressions of Bax and Bcl-2 and their ratio.In addtion,it was found that flavonoids exposure increased the transcriptions of c-fos,c-jun,and caspase-8.The main conclusions of this study:?1?The exposure concentration of total flavonoids in peony shells affected the toxicity on HepG2 cells.Flavonoids exposure at high concentrations?0.2 mM-0.4 mM?could induce apoptosis of HepG2 cells in which mitochondria and lysosomes may be closely involved.?2?Flavonoids exposure at high concentrations could induce apoptosis of HepG2 cells by inducing excessive ROS and activating mitochondrial apoptosis signaling pathway in HepG2 cells.?3?Exposure to total flavonoids at high concentration may also activate the Fas/FasL death receptor pathway and mediate apoptosis.?4?Abnormal expressions of p53,p21,Bax and Bcl-2 may be involoved in the apoptosis of HepG2 cells induced by total flavonoids at high concentration.The results of this study may be valuable for revealing the causes of hepatitis/hepatocarcinoma in susceptible populations from toxicological and molecular levels,and also for providing theoretical support for the safety risk assessment and human health protection of flavonoid drug abuse.