Effect Of 10-hydroxycamptothecine On On Inhibiting Fibroblast-like Synocytes In Patients With Rheumatoid Arthritis

Background and Objective Rheumatoid arthritis(RA) is a kind of autoimmune disease with the main feature is synovitis. The main reasons of bone struction in RA are the abnormal function of T lymphocytes and the abnormal proliferation of Fibroblast-like Synoviocytes(FLSs). RA has partly malignant tumor characteristics such as proliferative and destructive. 10-hydroxycamptothecin(10-HCPT) has significant effect on inhibiting cell proliferation, which is widely used in cancer treatment and has obtained better results. Whether from cell culture in vitro or by animal experiments has confirmed, 10-HCPT has significant effect on inhibiting the function of T lymphocytes invovled in the immune response in RA patients. However, there is no report about the role of 10-HCPT in RA FLSs. In this study, we will investigate the effects of 10-HCPT in the proliferation and functions in vitro FLSs from molecular and cell biology, provide a theoretical basis for 10-HCPT on the treatment of RA as an effective drug.Methods:1. FLSs were isolated from Synovial tissue of RA patients in Department of orthopaedics in People’s Hospital of Peking University and cultured in vitro. 2. 10-HCPT and Methotrexate(MTX) with different concentrations were used to treat FLSs for different time,and FLSs without 10-HCPT and MTX were served as the control group. 3. Cell Counting Kit-8(CCK-8) assay were applied to determine the proliferation of FLSs. 4. Annexin-V APC/7-AAD staining were used to detect the apoptosis of FLSs. 5. Measure the level of vascular endothelial growth factor(VEGF) in the supernatant fluid of FLSs by Enzyme-linked immunosorbent assay(ELISA). 6.The messenger Ribonucleic Acid(m RNA) expression of matrix metalloproteinase-3(MMP3) was measured by Reverse Transcription-Polymerase Chain Reaction(RT-PCR). 7.Use Spss16.0、Flowjo softwares for statistical analysis. Results: 1. 10-HCPT has equal effects with MTX in the proferation of FLSs,when the time is 24 hours and the concentration is 0.1μɡ/ml、1.0μɡ/ml(P>0.05). But 5.0μɡ/ml、10.0μɡ/ml 10-HCPT showed obvious inhibitory effect on FLSs compared with the same concentrations of MTX, when the time is 48 and 72 hours(P<0.05). 2. Compared with MTX group,10-HCPT showed higher effects on the induce apoptosis of FLSs(P<0.05). As the dose increases, the induction of apoptosis increases in a dose-dependent manner, the difference was statistically significant(P <0.05). 3.Compared with control group, high and low dose 10-HCPT groups showed significant values on the effect of inhibiting the expression of VEGF in FLSs. Compared with low-dose MTX group, both high and low dose 10-HCPT groups have significant difference(P<0.05); compared with high-dose MTX group, the high-dose 10-HCPT group have significant difference(P<0.05). 4.Compared with the control group, m RNA expression of MMP-3 markedly decreased in high concentration 10-HCPT and MTX groups(P<0.01). Compared with low concentration 10-HCPT group, m RNA expression of MMP-3 obviously decreased in high concentration 10-HCPT and MTX groups(P<0.01). But there was no statistical difference between 10-HCPT groups and MTX groups at the same concentration(P>0.05).Conclusions: 1.10-HCPT has significantly effects on inhibiting proliferation, inducing apoptosis, inhibiting the secretion of VEGF and the expression of MMP3 m RNA of FLSs. 2.10-HCPT has potential application in the treatment of RA.