The Effects And Mechanism Of Pterocarya Hupehensis Skan Extract On Proliferation And Apoptosis Of MH7A Cells

Objective: To observe the different extracts of Pterocarya hupehensis Skan on proliferation and apoptosis of human rheumatoid arthritis fibroblast-like synovial MH7 A cells,for finding out the best active extract for inducing apoptosis,and then to explore the possible mechanism of induced apoptosis of MH7 A cells.Methods: To prepare different extracts of Pterocarya hupehensis Skan with ethanol,petroleum ether,chloroform,ethyl acetate,andn-butyl alcohol;and then to co-culture MH7 A cells with each Pterocarya hupehensis Skan extract with different concentrations and different time.The proliferation of MH7 A cells was detected by CCK-8;The MH7 A cells were treated with extracts of the active site with the best inhibitory effect on proliferation,and then to detect their apoptosis by Annexin V-FITC/PI double staining;the enzyme activity of Caspase-3 and Casaspase-9 were detected by colorimetry;the protein levels of Cyt-C,Cleaved Caspase-3,Cleaved Caspase-9,Bcl-2 and Bax were detected by Western blotting;the mRNA levels of P53,Bax,Bak,Bcl-2 and Bcl-xL were detected by real-time fluorescence quantitative PCR.Results: Compared with the negative control group,neither different concentration of Pterocarya hupehensis Skan petroleum ether extract nor n-butanol extract could inhibit the proliferation of MH7 A cells at 6h,12 h,24h.Pterocarya hupehensis Skan chloroform extract obviously inhibited the proliferation of MH7 A cells from 500?g/ml at 6h,12 h,24h(P<0.01).Pterocarya hupehensis Skan ethyl acetate extract obviously inhibited the proliferation of MH7 A cells begin from 1mg/ml at 6h,12 h,24h(P<0.05 or P<0.01).The ethanol extract of Pterocarya hupehensis Skan inhibited the cell proliferation from low dosage of 3.125?g/ml.Compared with the negative control group,Pterocarya hupehensis Skan ethanol extract induced MH7 A cells apoptosis with 25?g/ml,50?g/ml,and100?g/ml(P<0.01)at 24 h,in a dose-dependent manner(P<0.05).The enzyme activity of Caspase-3 and Casaspase-9 were increased in different concentrations(25?g/ml,50?g/ml,100?g/ml)of the Pterocarya hupehensis Skan ethanol extract after24 h.The protein expression of Bcl-2 was decreased while the level of Cyt-C,Cleaved Caspase-3,cleaved Caspase-9 and Bax were increased in cell.The mRNA levels of P53,Bax,and Bak were increased.Oppositely,the mRNA levels of Bcl-2 and Bcl-xL were decreased.Conclusion: Compared with extracts of Pterocarya hupehensis Skan with petroleum ether,chloroform,ethyl acetate and n-butanol,ethanol extract could obviously inhibit the proliferation of MH7 A cells andinduce apoptosis of MH7 A cells.The ethanol extract of Pterocarya hupehensis Skan could regulate and control the protein expression ofCyt-C,P53,Caspase family and Bcl-2 family.These results sugest that ethanol extract of Pterocarya hupehensis Skan could induce the apoptosis of human rheumatoid arthritis fibroblast-like synovial MH7 A cell by activating the mitochondrial apoptotic pathways.