臭氧关节腔注射对胶原诱导性关节炎大鼠趋化因子水平的影响

Effects of intra-articular ozone injection on chemokine levels in collagen-induced arthritis rats

目的探究臭氧(O_(3))关节腔注射对胶原诱导性关节炎(collagen-induced arthritis,CIA)大鼠血清及关节滑膜、淋巴结、脾脏中相关趋化因子的影响,阐明O_(3)治疗RA炎症反应的潜在作用机制。方法将40只Wistar大鼠随机分为正常对照组(n=8)和模型组(n=32)。模型组采用注射完全弗氏佐剂+牛Ⅱ型胶原蛋白的方法建立CIA大鼠模型。将造模成功后的大鼠随机分为:CIA组、甲氨蝶呤组(MTX组)和臭氧组(O_(3)组)。治疗结束后1周,流式多因子检测技术测定各组大鼠血清中人巨噬细胞趋化蛋白-1(human macrophage chemoattractant protein-1,MCP-1)、调节激活正常T细胞表达和分泌因子(reduced upon activation,normal T cell expressed and secreted,RANTES)、C-X-C配体10(C-X-C ligand10,CXCL10)的表达水平;免疫组化法检测滑膜组织中C-X-C趋化因子受体3(C-X-C chemokine receptor 3,CXCR3)的表达水平;逆转录-聚合酶链反应(RT-PCR)检测滑膜、淋巴结、脾脏中CXCL10、CXCR3的mRNA表达水平。结果与正常对照组相比,CIA组大鼠血清MCP-1、RANTES、CXCL10水平显著升高(P<0.05),滑膜组织内可见明显滑膜增生,CXCR3蛋白表达水平升高,滑膜组织和淋巴结中CXCL10、CXCR3 mRNA水平显著升高(P<0.05),脾脏组织中CXCL10 mRNA水平也显著升高(P<0.05)。与CIA组相比,O_(3)组和MTX组大鼠血清CXCL10浓度明显降低(P<0.05),滑膜增生明显减轻,滑膜组织中CXCR3蛋白表达水平降低(P<0.05);与CIA组相比,O_(3)组和MTX组大鼠滑膜组织中CXCL10、CXCR3 mRNA水平显著降低(P<0.05);与MTX组相比,O_(3)组大鼠血清中CXCL10浓度、滑膜组织中CXCL10和CXCR3 mRNA表达水平的差异无统计学意义(P>0.05)。结论O_(3)关节腔注射能够显著减轻CIA大鼠的滑膜增生,其作用机制可能与O_(3)抑制CIA大鼠体内CXCL10/CXCR3轴有关,这为O_(3)治疗RA提供了实验依据。

Objective To investigate the effect of ozone(O_(3))intra-articular injection on collagen-induced arthritis(CIA)-related chemokines in serum and joint synovium,lymph node and spleen,and elucidate the potential mechanism of O_(3) in the treatment of RA inflammatory response.Methods Forty Wistar rats were randomly divided into normal control group(n=8)and model group(n=32).CIA rat models were established by injection of Freund’s complete adjuvant plus bovine typeⅡcollagen,and the successful model rats were randomly divided into three groups:CIA model group(CIA group),methotrexate group(MTX group)and ozone group(O_(3) group).One week after treatment,the expression levels of human macrophage chemoattractant protein-1(MCP-1),reduced upon activation,normal T cell expressed and secreted(RANTES)and C-X-C ligand 10(CXCL10)in the serum of rats in each group were detected by flow cytometry.The expression level of C-X-C chemokine receptor 3(CXCR3)in synovial tissue was detected by immunohistochemistry.And the mRNA expression levels of CXCL10 and CXCR3 in synovium,lymph node and spleen were detected by reverse transcription-polymerase chain reaction(RT-PCR).Results Compared with normal control group,the serum concentrations of MCP-1,RANTES and CXCL10 were significantly increased in CIA group(P<0.05),obvious synovial hyperplasia was observed in synovial tissue and the expression level of CXCR3 protein was increased(P<0.05).Compared with normal control group,the mRNA levels of CXCL10 and CXCR3 in lymph nodes were significantly increased in CIA group(P<0.05),and the level of CXCL10 mRNA in spleen tissue was also significantly increased(P<0.05).Compared with CIA group,the serum CXCL10 concentration was significantly decreased in O_(3) group and MTX group(P<0.05),synovial hyperplasia was significantly reduced and the expression level of CXCR3 protein in synovial tissue was decreased(P<0.05).Compared with CIA group,the mRNA levels of CXCL10 and CXCR3 in synovial tissue in O_(3) group and MTX group were significantly decreased(P<0.05).There was no significant difference in serum concentration of CXCL10 and the expression levels of CXCL10 and CXCR3 mRNA in synovial tissue between MTX group and O_(3) group(P>0.05).Conclusion O_(3) intra-articular injection can significantly reduce the synovial hyperplasia in CIA rats by inhibiting the CXCL10/CXCR3 axis,which can provide an experimental basis for O_(3) in treatment of RA.