针刺对宫内窘迫导致的缺氧缺血脑损伤大鼠海马炎性反应的影响

Effect of acupuncture on hippocampal inflammation in rats with intrauterine distress induced hypoxic-ischemic brain injury

目的:观察针刺对宫内窘迫缺氧缺血脑损伤(HIBD)大鼠的行为学、血清炎性细胞因子、海马组织NOD样受体蛋白3(NLRP3)炎性小体相关蛋白及小胶质细胞激活状态的影响,探讨针刺通过调控海马炎性反应干预HIBD的机制。方法:夹闭孕鼠子宫动脉10 min延迟剖宫产法建立HIBD大鼠模型。将HIBD大鼠随机分为模型组和针刺组,每组12只,以正常分娩的12只大鼠为正常组。针刺大鼠"本神",每次10 min,每日1次,连续14 d。采用HE染色法观察大鼠海马组织病理变化,三箱社交实验观察大鼠的社交能力,用ELISA法检测大鼠血清白细胞介素(IL)-1β、IL-18含量,用Western blot法检测大鼠海马组织NLRP3、凋亡相关斑点样蛋白(ASC)、半胱氨酸天冬氨酸蛋白酶-1(Caspase-1)蛋白相对表达量,用免疫荧光染色法检测海马组织小胶质细胞阳性标记物离子钙接头蛋白(IBA-1)的阳性表达。结果:与正常组比较,模型组大鼠海马神经元排列松散,出现细胞固化萎缩和整个细胞深染的现象,模型组大鼠探索陌生鼠时间明显减少(P<0.01),血清IL-1β、IL-18含量升高(P<0.01,P<0.05),海马NLRP3、ASC、Caspase-1蛋白相对表达量升高(P<0.05,P<0.01),海马CA1、CA3区IBA-1阳性表达增多(P<0.001,P<0.01);针刺组大鼠海马神经元结构较模型组有所改善,出现细胞固化、萎缩、深染的情况较少,与模型组比较,针刺组大鼠探索陌生鼠时间增加(P<0.01),血清IL-1β、IL-18含量降低(P<0.01,P<0.05),海马NLRP3、ASC、Caspase-1蛋白相对表达量降低(P<0.01),海马CA1、CA3区IBA-1阳性表达减少(P<0.01)。结论:针刺可有效改善宫内窘迫HIBD大鼠社交行为障碍,其机制可能与降低海马炎性反应蛋白NLRP3、ASC、Caspase-1的表达,减少海马小胶质细胞激活,降低血清IL-1β、IL-18含量,从而减轻炎性反应有关。

Objective To observe the effects of acupuncture on the behavior,serum inflammatory cytokines,hippocampal Nod-like receptor protein 3(NLRP3)inflammasome-related proteins and microglia activation in rats with intrauterine distress induced ischemia-hypoxic brain injury(HIBD),so as to explore the mechanism of acupuncture underlying treatment of HIBD by regulating hippocampal inflammation.Methods The HIBD model was established by clipping bilateral uterine arteries for 10 min and delaying caesarean delivery.HIBD rats were randomly divided into model group and acupuncture group according to body weight and litter factors,with 12 rats in each group.Twelve rats delivered normally were regarded as the normal group.Acupuncture was applied to bilateral"Benshen"(GB13)for 10 min,once a day for 14 consecutive days.H.E.staining was used to observe the pathological changes of hippocampus.The three chamber sociability test was used to determine the rat’s social ability.The serum IL-1β and IL-18 levels,the hippocampal expressions of NLRP3,apoptosis associated speck like protein containing CARD(ASC)and cysteinyl aspartate specific proteinase-1(Caspase-1),andionized calcium bindingadaptor molecule-1(IBA-1)were detected by ELISA,Western blot and Immunofluorescence,separately.Results Compared with the normal group,The hippocampal neurons in the model group are arranged loosely,and the cells are solidified and shrunk and the whole cells are deeply stained.The time of exploring the stranger rat in the model group was significantly reduced(P<0.01).The contents of IL-1β and IL-18,the expressions of NLRP3,ASC,Caspase-1 and IBA-1 of the model group were higher than those of the normal group(P<0.01,P<0.05,P<0.001).Following acupuncture intervention,the cell structure of hippocampal neurons was improved,and there were fewer cases of cell solidification,atrophy,and deep staining.In comparison with the model group,the time of exploring stranger rat in the acupuncture group was significantly increased(P<0.01).The contents of IL-1β and IL-18,the expressions of NLRP3,ASC and Caspase-1,and IBA-1 of the acupuncture group were significantly down-regulated than those of the model group(P<0.01,P<0.05).Conclusion Acupuncture can effectively improve the social behavior deficits of HIBD rats,which may be related to inhibition of the expression of inflammatory response proteins NLRP3,ASC and Caspase-1 in the hippocampus,decrease the activation of microglia in the hippocampus,and reduce serum IL-1βand IL-18,thereby alleviating inflammation.