断藤益母汤含药血清对人类风湿关节炎滑膜成纤维细胞增殖及TLR4表达的影响

目的探讨断藤益母汤(DYD)含药血清对体外培养的类风湿关节炎(rheumatoid arthritis,RA)患者滑膜成纤维细胞(fibrolast-like synoviocytes,FLS)增殖以及TLR4(Toll like receptor 4)mRNA表达的影响和作用机制。方法体外培养RA-FLS,取第3~5代细胞,分别加入来氟米特和DYD高、中、低剂量家兔含药血清,空白组加入正常家兔血清,MTT法检测24 h、48 h、72 h后各组含药血清对RA-FLS的影响。取RA-FLS,空白组不加脂多糖(LPS),阳性对照组加LPS20μg.mL-1,实验组在加入LPS后分两组,分别加入来氟米特和细胞抑制率最高的DYD含药血清。Realtime-PCR法检测以上4组细胞TLR4 mRNA的表达。结果来氟米特和DYD高剂量组含药血清作用48 h、72 h,二者均能显著抑制RA-FLS增殖(Ρ<0.05);对LPS诱导的TLR4的mRNA表达也有显著抑制作用(Ρ<0.05);来氟米特和DYD药效相当,两者比较差异无统计学意义。结论断藤益母汤高、中剂量含药血清作用48 h、72 h后能显著抑制RA-FLS增殖,且高剂量血清可下调LPS诱导的TLR4mRNA表达,这可能是该方治疗RA的机制之一。

Effects of Serum Containing Duanteng Yimu Decoction on Proliferation of Fibroblast-like Synoviocytes (FLS)and TLR4 Expression in Rheumatoid Arthritis Patients

Objective To investigate the effects of serum containing Duanteng Yimu Decoction(DYD) on the proliferation of fibroblast-like synoviocytes and TLR4(Toll-like receptor 4) expression in rheumatoid arthritis(RA) patients.Methods Synovial specimens obtained from the joint of RA patients.In the first experiment,the third to fifth generations of subcultured RA-FLS were co-cultured with leflunomide or serum containing high-,moderate and low-dosage DYD.RA-FLS of control group was cultured with the serum of normal rabbit.After 24,48 and 72 hours,the morphological features of RA-FLS were observed under inverted microscope,Proliferation was measured by MTT method.In the second experiment,RA-FLS of control group was cultured without lipopolysaccharide(LPS) added,the expeviment group was added with LPS 20 μg·mL-1,and the experiment group was added with leflunomide or DYD-containing serum with the highest cell inhibiting rate after treatment with LPS.Realtime-PCR was used for the observation of TLR4 mRNA in RA-FLS.Results Compared to the control group,leflunomide and serum containing high-dosage DYD obviously inhibited the proliferation of RA-FLS(Ρ < 0.05) after culturing for 48 and 72 hours,and had obvious inhibitory effect on TLR4 mRNA after stimulation with LPS(Ρ < 0.05).The effect of serum containing DYD and leflunomide.Conclusion Serum containing high-and moderate-dosage DYD can inhibit RA-FLS proliferation,and serum containing high-dosage DYD can down-regulate the expression of TLR4 mRNA,which may be one of the therapeutic mechanisms of DYD for RA.