基于TLRs/NF-κB信号通路观察不同滑膜细胞来源炎性外泌体对家兔膝关节的影响及其作用机制

Effect of inflammatory exosomes derived from different synoviocytes on rabbit knee joint and its mechanism based on TLRs/NF-κB signal pathway

目的基于TLRs/NF-κB信号通路观察不同滑膜细胞来源炎性外泌体对家兔膝关节的影响及其作用机制。方法将两种滑膜细胞用脂多糖诱导炎症后,分别提取其外泌体。将20只实验用家兔用随机数字表法将其分为5组:正常组、盐水组、A组(注射A细胞外泌体混悬液)、B组(注射B细胞外泌体混悬液)、A+B组(注射两种外泌体混合混悬液),每组4只。正常组不作处理,盐水组注射生理盐水,分别在三组外泌体组家兔关节腔内注射对应外泌体混悬液(浓度为1×107/ml)。干预2周后,记录各组家兔行为学变化,酶联免疫吸附试验法检测各组家兔血清中肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、IL-6及基质金属蛋白酶13(MMP-13)表达水平,蛋白质印迹法检测各组滑膜组织中Toll样受体2(TLR2)、P65、髓样分化因子88(MyD88)、TNF受体相关因子2(traf2)。结果正常组和盐水组干预前后家兔行为学Lequesne MG指数比较,差异无统计学意义(P>0.05);干预后,A、B组及A+B组家兔行为学Lequesne MG指数高于干预前,且高于正常组(P<0.05);A、B组及A+B组家兔血清中TNF-α、IL-1β、IL-6及MMP-13水平,滑膜组织中TLR2、P65、MyD88、traf2蛋白表达均高于正常组(P<0.05);A+B组家兔血清中TNF-α、IL-1β、IL-6及MMP-13水平,滑膜组织中TLR2、P65、MyD88、traf2高于A组和B组(P<0.05);A组家兔滑膜组织中TLR2、P65、MyD88、traf2蛋白表达量均高于B组(P<0.05)。结论炎症环境下的两种滑膜细胞外泌体均可激活滑膜TLRs/NF-κB信号通路,加重滑膜炎症。

Objective To observe the effect of inflammatory exosomes derived from different synoviocytes on rabbit knee joint and its mechanism based on TLRs/NF-κB signal pathway.Methods Two synoviocytes were treated with lipopolysaccharide to induce inflammation,the exosomes were extracted separately.Twenty experimental rabbits were divided into five groups by random number table method:normal group,saline group,group A(injected with exosome suspension of A cell),group B(injected with exosome suspension of B cell),and group A+B(injected with two kinds of exosome mixed suspension),with four rabbits in each group.The normal group was not treated,the saline group was injected with normal saline,and the corresponding exosome suspension was injected into the joint cavity of rabbits in the exosome group(the concentration was 1×107/ml).After two weeks of intervention,the behavioral changes of rabbits in each group were recorded,and the expression levels of tumor necrosis factorα(TNF-α),interleukin-1β(IL-1β),IL-6 and matrix metalloproteinase 13(MMP-13)in serum were detected by enzyme-linked immunosorbent assay,Toll-like receptor 2(TLR2),P65,myeloid differentiation factor 88(MyD88)and TNF receptor-associated factor 2(traf2)were detected by Western blot.Results There was no significant difference in behavioral Lequesne MG index between normal group and saline group before and after intervention(P>0.05).The behavioral Lequesne MG index of rabbits in groups A,B and A+B after intervention was higher than that before intervention, and higher than that in normal group (P<0.05);the levels of TNF-α, IL-1β, IL-6, and MMP-13 in serum and the expressions of TLR2, P65, MyD88, and traf2 in synovium tissue of rabbits in groups A, B and A+B were higher than those in normal group (P<0.05);the levels of TNF-α, IL-1β, IL-6, and MMP-13 in serum of rabbits , and the levels of TLR2, P65, MyD88, and traf2 in synovium in group A+B were higher than those in groups A and B (P<0.05). The protein expressions of TLR2, P65, MyD88, and traf2 in synovium tissue of rabbits in group A were higher than those in group B (P<0.05). Conclusion Both kinds of synoviocytes secretions in inflammatory environment can activate synovial TLRs/NF-κB signal pathway aggravates synovial inflammation.