The Cytotoxic Effect Of Osthole On L02 Cells And Its Preliminary Mechanism

Objective:Osthole is a natural coumarin derivative extracted from Cnidium monnieri（L.）Cuss.with a wide range of biological and pharmacological activities.In this experiment,different concentrations of osthole were used to establish a cytotoxicity model on L02 cells,in order to clarify the hepatic toxicity effect of osthole and its possible mechanism of action,provide experimental basis for the toxicological research of osthole.Methods:1.L02 cells were treated with different concentrations of Ost（0,50,100,150,200,250 and 300μM）,using MTT method to detect the survival rate;the morphological changes of the cells were observed under an optical microscope;Hoechst 33342 staining method was used to detect the nuclear morphology after different concentrations of Ost（0,50,100 and 200μM）for 24 h.2.L02 cells were treated with different concentrations of Ost（0,50,100,150and 200μM）for 24 h.The Lactate dehydrogenase（LDH）release rate was measured with a LDH assay kit to observe the cell membrane injury.The Superoxide Dismutase（SOD）assay kit and Malondialdehyde（MDA）assay kit were used to measure the total SOD activity and MDA content in the cells,and the intracellular redox environment was observed.3.L02 cells were treated with different concentrations of Ost（0,50,100 and 200μM）for 24 h,stained with PI and detected by flow cytometry.The cycle results were analyzed by Mod Fit software.The percentage changes of G0/G1,G2/M,and S phase cells were observed;Annexin V/PI double staining was detected by flow cytometry to observe cell apoptosis.4.L02 cells were treated with different concentrations of Ost（0,50,100 and 200μM）for 24 h（or 48 h）.The expression level of apoptosis-related proteins（a.Mitochondrial pathway apoptosis-related proteins:Bcl-2,Bax;b.Caspase family proteins:pro-caspase3,cleaved-Caspase 3（p17）;c.Endoplasmic reticulum stress pathway related proteins:Bip,CHOP）and proliferation-associated protein p-Histon H3（Ser10）were detected by Western-blot.To explore the toxic effects of different concentrations of Ost on L02 cells at different time and possible toxic damage mechanisms.Results:1.L02 cells were exposed to 0-300μM Ost,and the IC₅₀s were 363.90±31.15μM,290.30±17.32μM and 134.47±10.11μM at 12,24 and 48 h,respectively.Compared with the control group,the cell survival rate of the Ost-treated group was significantly decreased（P<0.05）.Compared with the 12-hour and 24-hour groups,the viability of the cells in the 48-hour group was significantly decreased.Observed under an inverted microscope,the number of cells decreased as the concentration increased,and the shape of cell was shrinking and rounding,and the cells were partially detached.After staining with Hoechst 33342,the cells in the treated groups were pyknosis,partial breakdown,and cytoplasmic condensation.2.After L02 cells were exposed to Ost for 24 h,the LDH release rate increased with the increase of drug concentration.Compared with the control group,150 and200μM groups had a significant difference（P<0.01）.In the high-concentration group,the activity of SOD decreased and the MDA content increased.This showed that Ost could produce some toxic damage to L02 cells,and changed the redox environment in cells,which increased the intracellular oxidative stress.3.The results of cycle detection showed that the percentage of cells in G2/M phase increased after administration,and compared with the control group,150 and200μM groups had a significant difference（P<0.05）;Annexin V/PI double staining assay detected the apoptosis of L02 cells treated with 50,100,and 200μM Ost for 24h.The results showed that with the increase of the concentration of Ost,the apoptosis rate of cells increased and there was a significant difference in each group compared with the control group（P<0.05）.4.Compared with the control group,the relative expression levels of Bcl-2 and pro-Caspase 3 decreased with increasing of Ost,and the expression of Bax clecleaved-Caspase 3（p17）,CHOP and Bip（48 h）increased.It suggested that Ost can induce apoptosis.It is probably through the mitochondrial pathway and the endoplasmic reticulum pathway.The expression levels of p-Histon H3（Ser10）were decreased in the 100 and 200μM groups,indicating that Ost inhibited L02 cell proliferation at a certain concentration.Conclusion:1.Ost has toxic effects on L02 cells,which is time and concentration dependent.2.Ost can reduce the expression of Bcl-2 and pro-Caspase 3 in L02 cells and increase the expression of Bax,cleaved-Caspase3,CHOP and Bip.This suggests that Ost induces apoptosis,possibly through the mitochondrial pathway and the endoplasmic reticulum pathway.