促甲状腺激素通过抗炎蛋白CTRP3促进软骨细胞分化

Thyroid stimulating hormone promotes chondrocyte differentiation via anti-inflammatory protein CTRP3

目的探讨促甲状腺激素(TSH)对软骨细胞(PMCs)分化的作用及相关机制。方法利用qRT-PCR、Western blotting、免疫荧光检测PMCs细胞外基质Ⅱ型胶原(ColⅡ)、Ⅹ型胶原(ColⅩ)以及基质金属蛋白酶3和13(MMP3,MMP13)的表达变化。采用qRT-PCR、ELISA检测TSH刺激PMCs后炎症因子白介素-1β(IL-1β)、白介素-6(IL-6)、肿瘤坏死因子-α(TNFα)的表达水平。应用高通量RNA-seq技术筛选TSH调控PMCs分化的差异表达基因,通过在原代PMCs中过表达CTRP3,探索TSH调控PMCs分化的机制。结果qRT-PCR、Western blotting结果显示,TSH刺激下PMCs中MMP3(P=0.016,P<0.001)和MMP13(P均<0.001)的表达显著增加。qRT-PCR、ELISA结果显示,IL-1β(P=0.007,P=0.002)、IL-6(P均<0.001)、TNFα(P=0.006,P<0.001)的表达上调。通过RNA-seq筛选出差异表达基因CTRP3。CTRP3过表达后PMCs中ColⅡ表达增高(F=76.062,F=77.085,F=190.115)、ColⅩ表达降低(F=33.494,F=38.424,F=43.351);同时与单纯TSH刺激相比,MMP3(F=88.607,F=214.145,F=135.60)、MMP13(F=116.561,F=138.674,F=86.865)表达均显著降低(P均<0.001),IL-1β、IL-6、TNFα的表达则均降低(P均<0.001)。结论CTRP3对TSH诱导的PMCs分化具有保护作用,有望成为亚临床甲状腺功能减退症(SCH)患者关节损伤的潜在治疗靶点。

Objective To investigate the role of thyroid stimulating hormone(TSH)in the differentiation of primary mouse chondrocytes(PMCs)and the underlying mechanism.Methods The expressions of chondrocyte differentiation markers,including extracellular matrix collagen typeⅡ(ColⅡ)and collagen typeⅩ(ColⅩ),and matrix metalloproteinase 3(MMP3)and matrix metalloproteinase 13(MMP13)were detected on both mRNA and protein levels with qRT-PCR,Western blotting and immunofluorescent staining.The changes of interleukin-1β(IL-1β),interleukin-6(IL-6)and tumor necrosis factor-α(TNFα)were detected in TSH stimulated group and control group with qRT-PCR and ELISA.The differentially expressed genes of PMCs treated with TSH were screened with high-throughput RNA-Seq.PMCs were transfected with C1 q/tumor necrosis factor-related protein 3(CTRP3)overexpression plasmid o explore the mechanism of TSH regulating PMCs differentiation.Results TSH upregulated the expressions of MMP3(P=0.016,P<0.001),MMP13(P<0.001),IL-1β(P=0.007,P=0.002),IL-6(P<0.001)and TNFα(P=0.006,P<0.001).The differentially expressed gene CTRP3 was screened out with RNA-seq.After CTRP3 overexpression,the expression of ColⅡin PMCs was increased(F=76.062,F=77.085,F=190.115,P<0.001),while the expression of ColⅩwas decreased(F=33.494,F=38.424,F=43.351).Compared with simple TSH stimulation,after CTRP3 overexpression,the expressions of MMP3(F=88.607,F=214.145,F=135.60),MMP13(F=116.561,F=138.674,F=86.865),IL-1β,IL-6 and TNFαwere significantly decreased(P<0.001).Conclusion CTRP3 has a protective effect on TSH-induced differentiation of PMCs,and it may become a promising anti-inflammatory therapeutic target for joint injury in patients with subclinical hypothyroidism(SCH).