Effects of rapamycin on proliferation and apoptosis of fibroblast synovial cells of rheumatoid arthritis by regulating the AKT/mTORC1 pathway / 中华风湿病学杂志

ABSTRACT

Objective:To investigate the effect of rapamycin on the proliferation and apoptosis of rheumatoid arthritis synovial fibroblasts (RA-FLS) and its mechanism.Methods:Synovial tissues were collected from patients with RA during joint replacement surgery, and primary synovial fibroblasts were extracted by trypsin digestion. The effect of rapamycin on the proliferation of RA-FLS was detected by cell counting kit (CCK-8) method. RA-FLS were divided into the control group and the rapamycin group (10 nmol/L). The effect of rapamycin on apoptosis of RA-FLS cells was detected by flow cytometry. The mRNA expres-sion levels of mammalian target of rapamycin (mTOR), serine/threonine-protein kinase AKT, B lymphocy-toma-2 (Bcl-2) associated X gene (Bax) and Bcl-2 were detected by RT-PCR. The protein expression levels of Bax, Bcl-2, mTOR, p-mTOR (2448), AKT, p-AKT and mTORC1 downstream related molecules protein S6 kinase 1(S6K1), p-S6K1, eukaryotic translation initiation factor-binding protein 1 (4EBP1) and p-4EBP1 were detected by Western blot. Differences between the two groups were compared using two independent samples t-test. Results:The results showed that the proliferation efficiency of RA-FLS treated with rapamycin was significantly weaker than that of the control group, and the drug inhibition rate of rapamycin increased with the increase of rapamycin concentration. The apoptosis rate of rapamycin group was significantly higher than that of the control group (5.31±0.59)% vs (3.49±0.40)%, t=7.83, P=0.001). The expression of Bax mRNA in rapamycin group was significantly increased (1.35±0.04 vs 1.00±0.00, t=15.60, P=0.004), while the expression of Bcl-2 mRNA (0.790±0.003 vs 1.000±0.000, t=85.50, P=0.007), mTOR mRNA (0.41±0.08 vs 1.00±0.00, t=14.37, P=0.044) and AKT mRNA (0.59±0.08 vs 1.00±0.00, t=7.54, P=0.017) were decreased, and the differences were statistically significant when compared with the control group. Compared with the control group, the protein expression of Bax in rapamycin group was significantly increased (0.75±0.10 vs 0.48±0.09, t=4.04, P=0.007), and the expression levels of Bcl-2 (0.632±0.055 vs 0.758±0.020, t=7.35, P=0.002), p-AKT/AKT(0.61±0.07 vs 0.88±0.04, t=5.61, P=0.005), p-mTOR/mTOR(0.92±0.12 vs 1.28±0.09, t=5.05, P=0.002), p-S6K1/S6K1(0.884±0.020 vs 1.023±0.058, t=4.52, P=0.004) and p-4EBP1/4EBP1 were decreased(0.86±0.05 vs 1.11±0.05, t=6.00, P=0.004). Conclusion:Rapamycin may inhibit the proliferation and induce apoptosis of RA-FLS cells by inhibiting AKT/mTORC1 pathway.