Role Of Genistein On The Proliferation And Apoptosis Of Fibroblast-like Synoviocytes From Rats With Collagen â…¡ Induced Arthritis

Objectives:To investigate the effects of Genistein(abbreviated Gen) on the proliferation and apoptosis of fibroblast-like synoviocytes(abbreviated FLS) from rats with collagenⅡinduced arthritis(abbreviated CIA).Subject:Fibroblast-like synoviocytes from rats with CIA.Methods:At first, the rats were scored with the arthritis index(AI)once a week after the CIA was induced with collagen typeⅡand complete Freund's adjuvant(CFA). At the sixth week, the X-ray of joints were taken out. After the rats were sacrificed, synovial tissue from knee joint were detected by pathological examination. The primary fibroblast-like synoviocytes were separated from the synovial tissue with collagenase digestion and cultured. The Trypan exclusion assay was used to analyze the viability of FLS. MTT assay was performed to study the effect of Gen on FLS proliferation. Different concentrations of Gen(50μM, 100μM, 200μM)were given to the incubation with FLS and Western blotting assay was used to test the effect of Gen on the expression of bcl-2,bax,ERK,AKT,p-ERK,p-AKT in FLS.Results:1,The rats with CIA were induced with collagen typeⅡand complete Freund's adjuvant. The rats caught arthrocele apparently three days later. After detected by the arthritis index (AI), the degree of paw swelling, X-ray and pathological detection, it indicated that the CIA was well construceted. After the primary synoviocytes were cultured to 4th generation, the Cells were highly homogeneous.2,The Trypan blue exclusion test showed that the viability of FLS was more than 90%, which was suitable for our experiment. MTT assay showed that the proliferation of FLS in CIA rats was increased when compared with that in the normal control group. Compared with model group, the proliferation of FLS in CIA rats treated with different concentrations of Gen (50μM, 100μM, 200μM) was inhibited significantly (P<0.05) in a dose-dependent manner. The proliferation of FLS of CIA rats in the Gen3(200uM)group was lower than that in normal group and the difference between them had statistical significance in 72 h after treatment with Gen (P<0.05). Western blot analysis showed that FLS in model group significantly decreased levels of bax protein and increased expression of bcl-2 protein, reliable apoptosis mediating factors, relative to normal group (P<0.05). Compared with model group, the different concentrations of Gen increased expression of bax protein in a dose-dependent manner, and significant differences were shown at concentrations of 100μM (Gen2), 200μM (Gen3) and the model groups (P <0.05), while no significant differences were found at concentrations of 50μM (Gen1) and 100μM (Gen2) groups (P> 0.05). Meanwhile, the different concentrations of Gen (50μM, 100μM, 200μM) also significantly reduced expression of bcl-2 protein (P<0.05).3,The activation of MAPK and PI3K/AKT signaling pathways were evaluated by Western blotting assay. Although the expression of EKR and AKT in FLS was not significantly different in normal group, model group and treatment group (P>0.05), significant increases of p-ERK and p-AKT in FLS in model group were observed relative to normal control group (P<0.05). The treatment of cells with Gen in different concentrations (50μM, 100μM, 200μM) significantly down-regulated the expression of p-ERK in FLS of CIA rats in a dose-dependent manner (P<0.05). The results also shown that the different concentrations of Gen (50μM, 100μM, 200μM) down -regulated the expression of p-AKT, and significant differences of p-AKT levels in Gen1 (50μM), Gen2 (100μM) and normal group (P<0.05) and nonsignificant differences of p-AKT levels in Gen3 (200μM) and normal group (P>0.05) were observed, respectively. Compared with model group, the expression of ERK and AKT protein in Gen treatment FLS groups were no significant difference (P>0.05). The expression of p-ERK and p-AKT protein in Gen treatment groups were lower than them in model group (P<0.05) in a dose-dependent inhibition with Gen concentrations.Conclusion:1,The CIA model was successfully constructed, the primary FLS of rats'joint were separated and cultured well.2,Gen could inhibit the proliferation of FLS in CIA rats, and regulate the expression of apoptosis mediating proteins which consequently promote FLS apoptosis in CIA rats. These findings demonstrate that Gen improved the excessive proliferation of synovium and the inhibition of apoptosis in CIA rats via regulating the homeostatic balance between synovial cells proliferation and apoptosis in CIA rats.3,Gen inhibited the activation of MAPK and PI3K-AKT signal transduction pathways in FLS of CIA rats. Consequently, Gen may regulate the balance of proliferation and apoptosis in FLS of CIA rats through adjusting signal transduction pathways in FLS.