摘要
目的 探讨激活α7烟碱型乙酰胆碱受体(α7 nicotinic acetylcholine receptor,α7nAChR)对创伤性脑损伤(traumatic brain injury,TBI)大鼠认知功能及海马小胶质细胞极化的影响.方法 取36只6～8周雄性SD大鼠,按照随机数字表法分为假手术对照组(Sham组)(n=12)、TBI组(n=12)、TBI+α7nAChR激动剂组(n=6)、TBI+α7nAChR拮抗剂组(n=6).通过"自由落体撞击"建立TBI模型,造模后第4～6天,TBI+α7nAChR激动剂组大鼠腹腔注射α7nAChR激动剂PNU-282987(3mg/kg),TBI+α7nAChR拮抗剂组大鼠先腹腔注射α7nAChR拮抗剂甲基牛扁亭柠檬酸盐(5 mg/kg),45 min 后再注射 α7nAChR 激动剂 PNU-282987(3 mg/kg),TBI 组和 Sham 组大鼠腹腔注射等体积0.9％氯化钠溶液.采用Morris水迷宫实验评估大鼠的学习记忆功能,采用免疫荧光染色观察小胶质细胞标记蛋白离子钙结合接头蛋白1(ionized calcium binding adaptor molecule 1,Iba-1)及M2型小胶质细胞标志物精氨酸酶1(arginase 1,Arg-1),采用Western blot检测海马组织Arg-1水平.使用GraphPad Prism 9软件进行统计分析.两组间比较采用独立样本t检验,多组间比较采用单因素方差分析,采用Tukey进行多重检验.结果 水迷宫结果显示,造模后第7天,4组大鼠的逃避潜伏期差异有统计学意义(F=6.134,P＜0.05),TBI组和TBI+α7nAChR拮抗剂组大鼠逃避潜伏期差异无统计学意义(P＞0.05),但均高于Sham组(均P＜0.05),TBI+α7nAChR激动剂组大鼠逃避潜伏期[(31.87±9.01)s]短于 TBI 组[(56.75±2.62)s]和 TBI+α7nAChR 拮抗剂组[(60.00±0.00)s)](均P＜0.05).免疫荧光染色结果显示,4组大鼠Arg-1+/Iba-1+荧光强度和Arg-1+/Iba-1+细胞数均差异有统计学意义(F=17.37,9.33,均P＜0.05).TBI+α7nAChR激动剂组大鼠Arg-1+/Iba-1+细胞免疫荧光强度(0.27±0.03)和细胞数[(21.67±4.41)个]均高于 TBI 组[(0.14±0.03),(11.33±2.60)个]和TBI+α7nAChR 拮抗剂组[(0.10±0.03),(7.67±1.20)个](均 P＜0.05).Western blot 结果显示,4 组大鼠海马组织Arg-1蛋白表达水平差异有统计学意义(F=8.323,P=0.001).TBI组和TBI+α7nAChR拮抗剂组大鼠Arg-1蛋白表达差异无统计学意义(P＞0.05);TBI+α7nAChR激动剂组大鼠Arg-1蛋白表达(1.06±0.22)高于 TBI 组(0.60±0.13)和 TBI+α7nAChR 拮抗剂组(0.35±0.10)(均P＜0.05).结论 激活α7nAChR能够促进大鼠海马组织中M2型小胶质细胞表达,并改善TBI大鼠的学习记忆功能.
Abstract
Objective To explore the effects of activating α7 nicotinic acetylcholine receptor(α7nAChR)on cognitive function and polarization of hippocampal microglia in traumatic brain injury(TBI)rats.Methods Totally 36 male SD rats with 6-8 weeks old were randomly divided into Sham group(n=12),TBI group(n=12),TBI+α7nAChR agonist group(n=6)and TBI+α7nAChR antagonist group(n=6).The TBI model was established by the"free fall impact"method.From the 4th to 6th day after model-ing,mice in the TBI+α7nAChR agonist group were intraperitoneally injected with α7nAChR agonist PNU-282987(3 mg/kg).Rats in TBI+α7nAChR antagonist group were intraperitoneally injected withα7nAChR antagonist methyllycaconitine citrate(5 mg/kg)first,then 45 minutes later they were injected with α7nAChR agonist PNU-282987(3 mg/kg).Rats in the TBI group and Sham group were intraperitone-ally injected with an equal volume of 0.9％sodium chloride solution.Morris water maze test was used to e-valuate the learning and memory function of rats.Immunofluorescence staining was used to observe the ion-ized calcium binding adapter molecule 1(Iba-1)(a marker for microglia)and arginase 1(Arg-1)(a marker for M2 microglia).Western blot was used to detect the protein level of Arg-1 in hippocampal tissue.Statisti-cal analysis was performed using GraphPad Prism 9 software.Independent sample t test was used for com-parison between two groups,one-way ANOVA was used for comparison among multiple groups,and Tukey test was used for multiple comparison.Results The results of the water maze test showed that after 7 days of modeling,there was a statistical difference in the escape latency among the 4 groups of rats(F=6.134,P＜0.05).There was no statistical difference in the escape latency between the TBI group and the TBI+α7nAChR antagonist group(P＞0.05),but the both were higher than that of the Sham group(both P＜0.05).The escape latency of the TBI+α7nAChR agonist group((31.87±9.01)s)was shorter than that of the TBI group((56.75±2.62)s)and the TBI+α7nAChR antagonist group((60.00±0.00)s)(both P＜0.05).The results of immunofluorescence staining showed that there were statistical differences in the fluo-rescence intensity and cell numbers of Arg-1+/Iba-1+among the four groups(F=17.37,9.33,both P＜0.05).The immune fluorescence intensity(0.27±0.03)and cell numbers(21.67±4.41)of Arg-1+/Iba-1+in the TBI+α7nAChR agonist group were higher than those in the TBI group((0.14±0.03),(11.33±2.60))and TBI+α7nAChR antagonist group((0.10±0.03),(7.67±1.20))(all P＜0.05).The results of Western blot showed that there was a statistical difference in the level of Arg-1 protein in hippo-campus among the 4 groups(F=8.323,P=0.001).There was no significant difference in the level of Arg-1 protein between the TBI group and the TBI+α7nAChR antagonist group(P＞0.05),and the level of Arg-1 protein in the TBI+α7nAChR agonist group(1.06±0.22)was higher than that in the TBI group(0.60± 0.13)and TBI+α7nAChR antagonist group(0.35±0.10)(both P＜0.05).Conclusion Activatingα7nAChR can promote the polarization of M2 type microglia in rat hippocampal tissue and improve the learn-ing and memory function of TBI rats.
NETL
NSTL
万方数据