二烯丙基三硫化物对人类风湿关节炎成纤维样滑膜细胞的作用

Effects of diallyl trisulfide in human fibroblast-like synoviocytes from rheumatoid arthritis

目的探讨二烯丙基三硫化物(DATS)对TNF-α诱导的人RA-成纤维样滑膜细胞(FLS)增殖以及炎症因子、MMPs和血管内皮生长因子(VEGF)分泌的影响。方法体外培养RA-FLS,采用不同浓度DATS处理细胞,通过CCK-8法测定细胞增殖的活性,加用TNF-α刺激后,通过实时荧光定量(q)-PCR、ELISA检测IL-1β、MMP-1、VEGF的mRNA和蛋白的表达水平,采用单因素方差分析比较多组定量资料,采用LSD-t法比较组间均数。结果分离出的RA-FLS中CD90、CD29表达的阳性率>90%。经100 μmol/L、200 μmol/L、300 μmol/L DATS处理,细胞增殖活力分别为[(98.92±0.40)%、(95.91±0.32)%、(94.05±0.24)%],与正常对照组比较增殖活性均下降,加入200 μmol/L、300 μmol/L DATS的实验组差异有统计学意义(t=4.46,P<0.05;t=7.98,P<0.05)。加用TNF-α刺激后,与实验对照组相比,加入100 μmol/L DATS实验A组中的IL-6 mRNA相对表达量升高[(1.14±0.04)倍,t=5.74,P<0.05],但蛋白变化差异无统计学意义,MMP-1、VEGF蛋白及mRNA变化差异无统计学意义。加入200 μmol/L DATS实验B组中IL-6、MMP-1、VEGF蛋白水平分别为[(108.0±4.7)ng/L,t=-63.79,P<0.05;(26.0±1.0)ng/L,t=-9.68,P<0.05;(57.9±0.7),t=-34.59,P<0.05]、mRNA较实验对照组的相对表达量分别为[(0.42±0.06)倍,t=-23.47,P<0.05;(0.14±0.03)倍,t=-36.59,P<0.05;(0.36±0.09)倍,t=-13.1,P<0.05];加入300 μmol/L DATS实验C组中IL-6、MMP-1、VEGF蛋白水平[(24.2±2.3)ng/L,t=-88.69,P<0.05;(22.7±1.0)ng/L,t=-14.13,P<0.05;(34.5±1.7),t=-48.45,P<0.05],mRNA较实验组对照组的相对表达量为[(0.041±0.027)倍,t=-38.48,P<0.05;(0.027±0.027)倍,t=-41.22,P<0.05;(0.131±0.047)倍,t=-17.74,P<0.05],均较实验对照组明显降低,且2组蛋白水平间比较差异有统计学意义(t=-24.89,P<0.05;t=-4.45,P<0.05;t=-13.87,P<0.05)。结论DATS浓度≥200 μmol/L时能够抑制RA-FLSs的增殖,并抑制其分泌IL-6、MMP-1、VEGF,而且呈剂量依赖关系。

Objective To study the proliferation and inflammatory phenotypes of human fibroblast-like synoviocytes (FLS) induced by tumor necrosis factor-α(TNF-α). Methods The rheumatoid arthritis (RA)-FLS were cultured in vitro, then treated with different concentrations of diallyl trisulfide (DATS). The proliferation activity was detected by CCK-8 method. Then TNF-α was used to stimulate the RA-FLS, mRNA and protein expression of interleukin (IL)-6, matrix metalloproteinases (MMP)-1 and vascular endothelial growth&nbsp;factor (VEGF) were detected by quantitative real time polymerase chain reaction (qPCR) and enzyme linked immunosorbent assay (ELISA). Differences among groups were determined by one-way analysis of variance (ANOVA), LSD-t test was used for comparison between 2 groups. Results RA-FLS was successfully is-olated and cultured in vitro. The positive rate of CD90 and CD29 in the RA-FLS was more than 90%. The proli-feration activity of RA-FLS treated with 100 μmoL/L, 200 μmol/L and 300 μmol/L DATS was (98.92 ± 0.40)%, (95.91±0.32)%, (94.05±0.24)%, respectively. As Compared with the normal control group, the pro-liferation activity of RA-FLS was lower, and the statistically significant difference is between normal control group 200 μmol/L and 300 μmol/L DATS (t=-4.46, P<0.05;t=-7.98, P<0.05). After TNF-α stimulation, the expression of IL-6's mRNA in experiment group (100μmol/L DATS) is higher compared with the model control group (t=5.74, P<0.05), but the change of IL-6's protein is no significant difference (t=-0.49, P=0.627). The differences of mRNA and protein expression levels of MMP-1 and VEGF between the experiment group (100μmol/L DATS) and the model control group were not statistically significant. The relative mRNA level [(0.42 ± 0.06), t=-23.47, P<0.05;(0.14±0.039), t=-36.59, P<0.05;(0.36±0.09), t=-13.1, P<0.05)] and the protein levels [(108.0±4.7) ng/L, t=-63.79, P<0.05, (26.0±1.0) ng/L, t=-9.68, P<0.05;(57.9±0.7), t=-34.59, P<0.05] of IL-6, MMP-1, VEGF in experiment group (200μmol/L DATS) were significantly decreased. And the relative mRNA level [(0.041 ±0.027), t=-38.48, P<0.05;(0.027 ±0.027), t=-41.22, P<0.05;(0.131 ±0.047), t=-17.74, P<0.05] and the protein levels [(24.2 ±2.3) ng/L , t=-88.69, P<0.05;(22.7 ±1.0) ng/L , t=-14.13, P<0.05;(34.5 ±1.7), t=-48.45, P<0.05] of IL-6, MMP-1, VEGF in the experiment group (300 μmol/L DATS) were also significantly decreased. The difference between the two groups was significant (t=-24.89, P<0.05;t=-4.45, P<0.05;t=-13.87, P<0.05). Conclusion DATS can inhibit the proliferation and the effect of TNF-αinduced secretion of IL-6, MMP-1 and VEGF in RA-FLS. The effect is dose-dependent.