“夹脊”电针调控HMGB1/TLR4/NF-κB信号通路抑制脊髓损伤大鼠炎性反应的机制研究

Electroacupuncture of“Jiaji”(EX-B2)inhibits inflammatory response by regulating HMGB1/TLR4/NF-κB signaling pathway in rats with spinal cord injury

目的:观察“夹脊”电针对脊髓损伤(SCI)大鼠高迁移率族蛋白1(HMGB1)/Toll样受体4(TLR4)/核转录因子-κB(NF-κB)通路相关蛋白及炎性因子表达的影响,探讨“夹脊”电针抑制SCI大鼠炎性反应促进神经功能恢复的可能机制及时间动态规律。方法:54只SD大鼠随机分为假手术组、模型组、电针组,每组分为3、7、14 d 3个亚组,每个亚组6只。采用Allen’s法制备SCI大鼠模型。电针组于造模后1 d取双侧胸(T)9、T11“夹脊”行电针干预,密波(100 Hz),电流强度1 mA,1次/d,每次30 min,分别治疗3、7、14 d。采用运动功能(BBB)评分量表观察各组大鼠后肢运动功能;HE染色观察脊髓组织病理形态;ELISA法检测脊髓组织中诱导型一氧化氮合酶(iNOS)、环氧合酶-2(COX-2)的含量;Western blot及免疫荧光染色法分别检测脊髓组织HMGB1、TLR4、NF-κB p65的蛋白相对表达量和荧光阳性表达。结果:与假手术组比较,模型组大鼠造模后和治疗后各时间点BBB评分降低(P<0.05),神经元数量大幅减少、炎性浸润严重,脊髓组织iNOS、COX-2含量升高(P<0.05),HMGB1、TLR4、NF-κB p65的蛋白表达及荧光阳性表达升高(P<0.05)。与模型组比较,分别治疗3、7、14 d后电针组大鼠的BBB评分升高(P<0.05),神经元丢失及炎性浸润情况得到改善,脊髓组织iNOS、COX-2含量降低(P<0.05),HMGB1、TLR4、NF-κB p65的蛋白表达及荧光阳性表达减少(P<0.05)。结论:“夹脊”电针通过调节HMGB1/TLR4/NF-κB信号通路,下调iNOS和COX-2的表达,从而抑制SCI后神经炎性反应,促进SCI神经功能恢复。

Objective To observe the effect of electroacupuncture(EA)of“Jiaji”(EX-B2)on expression of high mobility group box 1(HMGB1)/Toll-like receptor 4(TLR4)/nuclear factor-κB(NF-κB)related proteins and inflammation-related factors in spinal cord injury(SCI)rats,so as to explore the dynamic process of inhibiting inflammatory response in rats with SCI,and to provide a new idea and theoretical basis of molecular biology for the treatment of SCI.Methods Fifty-four SD rats were randomly divided into sham operation,model,and EA groups(n=18 in each group),which were further divided into 3 d,7 d and 14 d subgroups,with 6 rats at each time point.The SCI model was established according to the Allen’s method.Rats in EA group received EA(1 mA,100 Hz)intervention at EX-B2 of T9 and T1124 hours after modeling for 30 min,once a day for 3 d,7 d,or 14 d,respectively.The motor function of rats’hind limbs was evaluated using BBB scale,the morphological structure of rats’spinal cord tissue was observed by H.E.staining.The contents of inducible nitric oxide synthase(iNOS)and cyclooxygenase-2(COX-2)in spinal cord tissue were detected by ELISA.Western blot and immunofluorescence staining were used to detect the relative protein expression and fluorescence positive expression of HMGB1,TLR4 and NF-κB p65 in spinal cord tissue,respectively.Results Compared with the sham operation group,the BBB scores after modeling and on day 3,7 and 14 were all decreased(P<0.05),while the contents of iNOS and COX-2 in spinal cord tissue,the protein expression and fluorescence positive expression of HMGB1,TLR4,and NF-κB p65 were increased(P<0.05)in the model group.In comparison with the model group,the BBB scores on day 3,7 and 14 were obviously increased(P<0.05),while the contents of iNOS and COX-2,the protein expression and fluorescence positive expression of HMGB1,TLR4,and NF-κB p65 were significantly down-regulated in the EA group(P<0.05).Conclusion EA of“Jiaji”can promote the recovery of SCI neurological function,which may be related to its function in regulating the HMGB1/TLR4/NF-κB signaling pathway,down-regulating the expression of iNOS and COX-2,and inhibiting the neuroinflammatory response after SCI.