基于TLR9/MyD88/NF-κB信号通路探讨针刺预处理对运动性骨骼肌损伤大鼠炎性反应的影响

Effect of acupuncture pretreatment on inflammatory response of exercise-induced skeletal muscle damage in rats based on TLR9/MyD88/NF-κB signaling pathway

目的:观察针刺预处理对运动性骨骼肌损伤(EIMD)大鼠toll样受体9/髓样分化蛋白88/核因子-κB(TLR9/MyD88/NF-κB)信号通路及炎性反应的影响,探讨针刺预处理干预EIMD的作用机制。方法:将88只雄性SD大鼠随机分为空白组(8只)、模型组(40只)和针刺预处理组(40只)。模型组及针刺预处理组随机分为造模后0、12、24、48、72 h 5个亚组,每亚组8只。造模前,针刺预处理组于双侧“足三里”及“关元”行针刺干预,每次20 min,每日1次,连续干预7 d。模型组和针刺预处理组采用动物实验跑台进行1次间歇性下坡跑离心运动,建立EIMD模型。透射电镜观察大鼠腓肠肌超微结构;ELISA法检测大鼠血清白细胞介素(IL)-6、肿瘤坏死因子-α(TNF-α)含量;Western blot法及实时荧光定量PCR法检测大鼠腓肠肌组织TLR9、MyD88、NF-κB p65蛋白及mRNA表达。结果:与空白组比较,模型组大鼠各时点腓肠肌超微结构呈现不同程度损伤,肌丝紊乱、扭曲,线粒体明显肿胀,部分线粒体嵴缺损;与模型组比较,针刺预处理组损伤较轻,肌纤维大多排列整齐,线粒体数量明显增多。与空白组比较,模型组大鼠造模后0、12、24、48 h血清IL-6含量升高(P<0.01),造模后各时点血清TNF-α含量升高(P<0.05,P<0.01);与模型组同时点比较,针刺预处理组大鼠造模后12、24、48 h血清IL-6含量降低(P<0.01,P<0.05),造模后24、48、72 h血清TNF-α含量降低(P<0.01)。模型组和针刺预处理组血清IL-6、TNF-α含量整体均呈现先升高后下降的趋势。与空白组比较,模型组大鼠造模后12、24、48、72 h腓肠肌组织TLR9、NF-κB p65蛋白及mRNA表达增加(P<0.01,P<0.05),造模后各时点腓肠肌组织MyD88蛋白及mRNA表达增加(P<0.05,P<0.01);与模型组同时点比较,针刺预处理组造模后12、24、48、72 h腓肠肌组织TLR9蛋白表达减少(P<0.05,P<0.01),造模后24、48、72 h腓肠肌组织TLR9 mRNA表达减少(P<0.01,P<0.05),造模后24、48、72 h腓肠肌组织MyD88蛋白及mRNA表达减少(P<0.01,P<0.05),造模后24、48 h腓肠肌组织NF-κB p65蛋白及mRNA表达减少(P<0.01)。模型组和针刺预处理组腓肠肌组织TLR9、MyD88、NF-κB p65蛋白及mRNA表达量整体均呈现先升高后下降的趋势。结论:针刺预处理可以减轻运动性骨骼肌损伤,其作用机制可能与调控TLR9/MyD88/NF-κB信号通路表达从而抑制炎性反应有关。

Objective To observe the effects of acupuncture pretreatment on toll-like receptor 9/myeloid differentiation factor 88/nuclear factor-κB(TLR9/MyD88/NF-κB)signaling pathway and inflammatory response in the rats with exerciseinduced skeletal muscle damage(EIMD)and explore the underlying mechanism of this pretreatment for EIMD.Methods A total of 88 male SD rats were randomly divided into a blank group(8 rats),a model group(40 rats)and an acupuncture pretreatment group(40 rats).In the model group and the acupuncture pretreatment group,5 subgroups were randomized according to the sampling time of 0,12,24,48 and 72 h of modeling,with 8 rats in each one,respectively.Before modeling,in the acupuncture pretreatment group,acupuncture was delivered at bilateral"Zusanli"(ST 36)and"Guanyuan"(CV 4)for 20 min,once daily for consecutive 7 days.By one-time intermittent downhill centrifugal exercise on animal experimental treadmill,EIMD model was established in the model group and the acupuncture pretreatment group.The ultrastructure of gastrocnemius muscle was observed under transmission electron microscope.The levels of interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)in the serum were detected by ELISA.The protein and mRNA expression of TLR9,MyD88 and NF-κB p65 in the gastrocnemius tissue of rats was detected by the Western blot and real-time PCR,respectively.Results Compared with the blank group,the gastrocnemius ultrastructure in the model group showed the damage of different degrees,with myofilaments disarranged and twisted,mitochondria obviously swollen and mitochondrial crista partially defected.Compared with the model group,the injury was mild,most of muscle fibers were arranged neatly and the number of mitochondria increased remarkably in the acupuncture pretreatment group.Compared with the blank group,in the model group,the serum IL-6 levels increased at 0,12,24 and 48 h after modeling in the rats(P<0.01),and TNF-αlevels were elevated at each time point after modeling(P<0.05,P<0.01).In the acupuncture pretreatment group,the serum IL-6 levels were reduced at 12,24 and 48 h after modeling(P<0.01,P<0.05),and the TNF-αlevels decreased at 24,48 and 72 h after modeling(P<0.01)when compared with those in the model group at the same time points separately.The serum IL-6 and TNF-αlevels ascended and then tended to decline in the model group and the acupuncture pretreatment group.Compared with the blank group,the protein and mRNA expression of TLR9 and NF-κB p65 in the gastrocnemius tissue increased at 12,24,48 and 72 h after modeling(P<0.01,P<0.05),and the protein and mRNA expression levels of MyD88 in the gastrocnemius tissue at each time point after modeling were elevated(P<0.05,P<0.01)in the model group.When compared with the model group at the same time points,in the acupuncture pretreatment group,the protein expression of TLR9 in the gastrocnemius tissue decreased at 12,24,48 and 72 h after modeling(P<0.05,P<0.01),and the mRNA expression of TLR9 was declined at 24,48 and 72 h after modeling(P<0.01,P<0.05);the protein and mRNA expression of MyD88 in the gastrocnemius decreased at 24,48 and 72 h after modeling(P<0.01,P<0.05),and that of NF-κB p65 was reduced at 24 h and 48 h after modeling(P<0.01).The protein and mRNA expression of TLR9,MyD88 and NF-κB p65 in the gastrocnemius tissue showed a trend of decrease after increase in the model group and the acupuncture pretreatment group.Conclusion Acupuncture pretreatment can alleviate exercise-induced skeletal muscle damage,which may be related to modulating the expression of TLR9/MyD88/NF-κB signaling pathway to inhibit the inflammatory response.