丹参酮Ⅱ-A调控Th17/Treg免疫平衡影响骨关节炎软骨退变大鼠中TLR4相关通路表达的机制研究

Effect of TanshinoneⅡ-A on Th17/Treg Balance and TLR4 Related Pathways in Osteoarticular Cartilage Degeneration Rats

目的探讨丹参酮Ⅱ-A(tanshinoneⅡ-A,TSⅡ-A)对骨关节炎(osteoarthritis,OA)软骨退变大鼠中调节性T细胞(regulatory T,Treg)/辅助性T细胞17(Th17)的影响,以及其对Toll样受体4(Tolllike receptor 4,TLR4)信号的调控。方法以关节腔注射木瓜蛋白酶构建大鼠OA模型。造模成功后,分为TSⅡ-A低剂量组(TSⅡ-A-L)、TSⅡ-A高剂量组(TSⅡ-A-H)、阳性对照药物硫酸氨基葡萄糖(glucosamine sulfate,DGS)组进行药物干预;酶联免疫吸附试验检测大鼠血清中肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和白细胞介素-6(interleukin-6,IL-6)水平;脱氧核糖核苷酸末端转移酶介导的缺口末端标记试剂盒(TUNEL)检测大鼠软骨组织中的细胞凋亡;流式细胞仪检测脾组织中Treg、Th17的比重,实时荧光定量聚合酶链式反应(qRT-PCR)检测大鼠膝关节软骨组织中TLR4、髓样分化因子88(myeloid differentiation factor 88,MyD88)、以及核转录因子(nuclear factorκB,NF-κB)、蛋白聚糖(aggrecan)和Ⅱ型胶原(CollagenⅡ)、叉状头/翅膀状螺旋转录因子3(forkhead/winged helix transcription factor 3,Foxp3)、IL-17、B淋巴细胞瘤-2(B cell lymphoma-2,Bcl-2)和Bcl-2相关X蛋白(Bcl2-associated X protein,Bax)的mRNA的表达;蛋白质印迹法检测软骨组织中TLR4、MyD88、p-NF-κB、Bcl-2和Bax的表达。结果与Sham组相比,其余各组大鼠脾组织中Treg细胞的比重、膝关节软骨组织中aggrecan和CollagenⅡ以及Foxp3、Bcl-2的表达明显下降,血清中TNF-α和IL-6的含量、软骨组织中的细胞凋亡率、脾组织中Th17细胞的比重、膝关节软骨组织中IL-17、TLR4、MyD88、p-NF-κB和Bax的表达明显升高;与OA组相比,TSⅡ-A-L组、TSⅡ-A-H组、DGS组大鼠脾组织中Treg细胞的比重、膝关节软骨组织中aggrecan和CollagenⅡ以及Foxp3、Bcl-2的表达明显升高,血清中TNF-α和IL-6的含量、软骨组织中的细胞凋亡率、脾组织中Th17细胞的比重、膝关节软骨组织中IL-17、TLR4、MyD88、p-NF-κB和Bax的表达明显下降;与TSⅡ-A-L组相比,TSⅡ-A-H组大鼠脾组织中Treg细胞的比重、膝关节软骨组织中aggrecan和CollagenⅡ以及Foxp3、Bcl-2的表达明显升高,血清中TNF-α和IL-6的含量、软骨组织中的细胞凋亡率、脾组织中Th17细胞的比重、膝关节软骨组织中IL-17、TLR4、MyD88、p-NF-κB和Bax的表达明显下降,差异均具有统计学意义(P均<0.05),TSⅡ-A-H组与DGS组间各指标的差异不具有统计意义(P>0.05)。结论丹参酮Ⅱ-A(TSⅡ-A)能明显抑制OA大鼠关节软骨组织中TLR4、MyD88、p-NF-κB的表达,降低软骨组织中的细胞凋亡率,这可能与改善Treg/Th17免疫失衡有关。

Objective To investigate the effect of tanshinoneⅡ-A(TSⅡ-A)on regulatory T(Treg)/helper T cell 17(Th17)in osteoarthritis(OA)cartilage degeneration rats,and its regulation on the expression of Toll like receptor 4(TLR4)signaling pathway related genes.Methods The rat OA model was constructed by injecting papain into the joint cavity,rats then were divided into 3 groups for drug intervention:TSⅡ-A low-dose group(TSⅡ-A-L),TSⅡ-A high-dose group(TSⅡ-A-H),and glucosamine sulfate(DGS)group as positive control.Enzyme linked immunosorbent assay was used to detect the levels of tumor crossing factor-α(TNF-α)and interleukin-6(IL-6)in serum.TUNEL assay was used to detect the cell apoptosis in rat cartilage tissues.Flow cytometry was used to detect the proportion of Treg and Th17 in spleen tissues.qRT-PCR was used to detect the mRNA expression levels of TLR4,myeloid differentiation factor 88(MyD88),nuclear factorκB(NF-κB),aggrecan,CollagenⅡ,forkhead/winged helix transcription factor 3(Foxp3),IL-17,B cell lymphoma-2(Bcl-2),and Bcl-2 associated x protein(Bax)in rat knee joint cartilage tissues.Western blotting assay was used to detect the expression levels of TLR4,MyD88,p-NF-κB,Bcl-2 and Bax in the cartilage tissues.Results The proportion of Treg cells,and the expression levels of aggrecan,CollagenⅡ,Foxp3,and Bcl-2 in the OA group were significantly decreased,and the levels of TNF-αand IL-6,the cell apoptosis rate,the proportion of Th17 cells,and the expression levels of IL-17,TLR4,MyD88,p-NF-κB and Bax were significantly increased when compared with those in the Sham group.The proportion of Treg cells,and the expression levels of aggrecan,CollagenⅡ,Foxp3,and Bcl-2 in the TSⅡ-A-L group,TSⅡ-A-H group,and DGS group were significantly increased,and the levels of TNF-αand IL-6,the cell apoptosis rate,the proportion of Th17 cells,and the expression levels of IL-17,TLR4,MyD88,p-NF-κB and Bax were significantly decreased when compared with those in the OA group.The proportion of Treg cells and the expression levels of aggrecan,CollagenⅡ,Foxp3,and Bcl-2 in the TSⅡ-A-H group and the DGS group were significantly increased,and the levels of TNF-αand IL-6,the cell apoptosis rate,the proportion of Th17 cells,and the expression levels of IL-17,TLR4,MyD88,p-NF-κB and Bax were significantly decreased when compared with those in the TSⅡ-A-L group(all P<0.05).No statistically significance in those above indexes were found between the TSⅡ-A-H group and the DGS group(P>0.05).Conclusion TanshinoneⅡ-A(TSⅡ-A)could significantly inhibit the expression of TLR4,MyD88,p-NF-κB and reduce the cell apoptosis rate in the articular cartilage tissues of OA rats,which may be related to the improvement of Treg/Th17 imbalance in rats.