Inhibitory Effect Of Emodin On PI3K/AKT Signaling Pathway In RA Fibroblast Like Synoviocytes

ObjectiveTo investigate the effect of emodin on the proliferation and migration of fibroblast like synoviocytes of rheumatoid arthritis,as well as the mechanism of PI3K/AKT signaling pathway and downstream molecules in MH7 A cell line.MethodUsing tissue extraction method and enzyme digestion method in vitro cultured synovial cells of rheumatoid arthritis,were added to different concentrations of Emodin(0?20?40?60?80?100?mol/L)and treatment with 6?12?24h training,change and the time effect relationship of proliferation ability was detected by CCK-8;Transwell method was used to detect the changes of the migration ability before and after treatment,and the apoptosis and proliferation of MH7 A cells 12h(40?60?80?mol/L)emodin were detected by flow cytometry;The expression of PI3 K and AKT in MH7 A cells was detected by immunofluorescence assay(TNF-a group,TNF-a+Emodin 60?mol/L group,LY294002 group)after 12 h treatment;Detection of(control group,TNF-a group,TNF-a+Emodin(40 ?mol/L)group,TNF-a+Emodin(60 ?mol/L)group,TNF-a+Emodin(80 ?mol/L)group,LY294002 group)after the treatment with 12 h,PI3K?AKT and the downstream molecules Bax?Bcl-2?Caspase-8 expression by Western Blot;Detection of(blank group,TNF-a Group,TNF-a+Emodin(40 ?mol/L)group,TNF-a+Emodin(60 ?mol/L)group,TNF-a+Emodin(80 ?mol/L)group,LY294002 group)after treatment of PI3 K and AKT mRNA expression by QRT-PCR;Result(1)Enzyme digestion method and tissue block method were used to obtain primary fibroblast like synoviocytes from 14 d and 21 d,respectively,and then treated with emodin in the expanded culture for the first time after 3 generations of RA.(2)CCK-8 detection of 6h after treatment,the inhibition rate of 20?40?60?80?100?mol/L emodin was followed by the order of 0.39±0.11,0.51± 0.04,0.55±0.07,0.72±0.18,0.98±0.11.And with the extension of treatment time,inhibition rate increased(P<0.05).(3)Transwell concentration detection method for 20?40?80?mol/L after emodin treatment,the number of cells through the cell membrane in the treatment group was significantly less than the control group,the migration test showed that the inhibition rates were 39.48%,?54.76%? 82.99%;invasion experiment showed that the inhibition rates were 25.38%,?60.41%?81.73% the difference from the control group were statistically significant(P<0.05).(4)Flow cytometric detection of emodin can induce apoptosis of MH7 A cells,in a concentration dependent manner,compared with the control group,the apoptosis rate increased with the increase of concentration(P<0.05);to detect different concentrations of emodin influence on MH7 A cell proliferation cycle results showed that compared with the control group,with the concentration of emodin increased.The percentage of S phase increased from 14.41% to 20.64%,while the percentage of G2 phase decreased from 11.52 per cent to 5.09%.(5)Immunofluorescence assay in MH7 A cells after treatment with TNF-a,PI3 K,AKT fluorescence expression was enhanced;after emodin treatment,PI3 K expression,AKT expression decreased significantly than that in the TNF-a treatment group;LY294002 treatment group,compared with other groups,the expression of PI3K? AKT was the weakest.(6)The expression of PI3 K,AKT protein and mRNA in MH7 A cells were significantly decreased(P<0.05)after emodin treatment.(7)The expression of Caspase-8 in the downstream of the PI3K/AKT signaling pathway was significantly lower and the expression of Bax/bcl-2 was significantly higher than the control group,and the difference was statistically significant(P<0.05).Conclusion(1)Different concentrations of emodin can inhibit the in vitro proliferation and migration of RA-FLSs,and has a dose-dependent and time-dependent manner;(2)Emodin can induce apoptosis of MH7 A cells,the proliferation of block in S phase.(3)The effect of emodin on PI3K/AKT signaling pathway to inhibit synoviocytes cell proliferation,in order to ease the change in patients with RA pathological synovial hyperplasia.