电针对局灶性脑缺血大鼠miR-218及HMGB1/TLR4通路表达的影响

Effects of electroacupuncture on expression of miR-218 and HMGB1/TLR4 signaling pathway in rats with focal cerebral ischemia

目的:观察电针对局灶性脑缺血大鼠miR-218及高迁移率族蛋白B1(HMGB1)/Toll样受体4(TLR4)通路的影响,探讨电针对脑缺血后炎性反应的调控机制。方法:雄性SD大鼠随机分为假手术组、栓塞组、电针组,每组18只。采用Longa法制备右侧大脑中动脉阻塞模型。电针组于造模后24 h电针“百会”“风府”及左侧“内关”“心俞”,每日1次,每次20 min,治疗1周。采用改良神经功能缺损评分(mNSS)评估大鼠神经功能;HE染色观察缺血区脑组织形态学变化;ELISA法检测缺血区脑组织肿瘤坏死因子-α(TNF⁃α)和白细胞介素(IL)-6含量;免疫荧光染色法观察脑缺血区HMGB1、TLR4的阳性表达;实时荧光定量PCR法检测HMGB1、TLR4 mRNA和miR-218的表达。结果:与同时点假手术组比较,栓塞组mNSS评分升高(P<0.01),脑组织缺血区有神经元坏死迹象,缺血区TNF-α和IL-6含量显著升高(P<0.01),HMGB1、TLR4阳性表达及mRNA表达均升高(P<0.01),miR-218表达降低(P<0.01)。与同时点栓塞组比较,治疗7 d电针组mNSS评分显著降低(P<0.01),电针组组织变性坏死程度轻于同时点栓塞组,电针7 d时大鼠脑组织缺血区IL-6和TNF-α含量降低(P<0.01),HMGB1和TLR4的阳性表达及mRNA表达降低(P<0.01,P<0.05),miR-218表达升高(P<0.05)。结论:电针可减轻脑缺血诱导的脑组织炎性损伤,改善局灶性脑缺血后大鼠神经功能的缺损,其效应可能与激活miR-218表达进而抑制HMGB1/TLR4通路及其下游促炎细胞因子水平相关。

Objective To observe the effect of electroacupuncture(EA)on high mobility group box 1(HMGB1)/toll-like receptor 4(TLR4)signaling pathway and inflammatory injury in rats with focal cerebral ischemia(FCI),so as to explore its mechanisms underlying amelioration of ischemic stroke.Methods Male SD rats were randomly divided into sham operation(n=18),model(n=18)and EA(n=18)groups.The FCI model was established according to Longa’s method.In the EA group,24 h after modeling,EA stimulation was applied to“Baihui”(GV20),“Fengfu”(GV16),and“Neiguan”(PC6)and“Xinshu”(BL15)on the left side for 20 min,once a day for 1 week.The neurological deficit severity was evaluated by the modified neurological severity score(mNSS),and the morphological changes of neurons were observed after H.E.staining.The expression of HMGB1 and TLR4 proteins were detected by immunofluorescence staining.The contents of tumor necrosis factor(TNF)-αand interleukin(IL)-6 in the ischemic area tissue of the brain were detected using ELISA.The mRNA expressions of HMGB1 and TLR4 as well as the expression of miR-218 in the ischemic cortex were detected by PCR.Results Compared with the sham operation group,the mNSS at 2 h,48 h and 7 day(d),IL-6 and TNF-αcontents,HMGB1 and TLR4 immunoactivity and mRNA expression at 48 h and 7 d were significantly increased(P<0.01),while miR-218 expression at 48 h and 7 d was obviously downregulated(P<0.01)in the model group.In comparison with the model group,the mNSS at 7 d,IL-6 and TNF-αcontents,HMGB1 and TLR4 immunoactivity and mRNA expression at 7 d not at 48 h were significantly decreased(P<0.01,P<0.05),while the expression of miR-218 at 7 d was apparently up-regulated(P<0.05)in the EA group.H.E.staining showed that there were signs of neuronal necrosis including blurred outline of the cells,disordered internal structure,dense cytoplasm,shrunk nuclei,etc.in the ischemic area of the brain at 48 h and 7 d in the model group,which was evidently milder on day 7 in the EA group.Conclusion EA can reduce the inflammatory injury of brain tissue and improve the neurological impairment in rats with FCI,which may be associated with its effects in inhibiting HMGB1/TLR4 signaling and down-regulating the level of pro-inflammatory cytokines.