电针调节皮层NF-κB/NOD样受体蛋白3信号通路抑制脂多糖诱导的慢性神经炎症

Electroacupuncture inhibiting LPS-induced chronic neuroinflammation by regulating the cortical NF-κB/NOD-like receptor protein 3 signaling pathway

目的观察电针对脂多糖诱导的慢性神经炎症小鼠的NF-κB/NOD样受体蛋白3(NLRP3)信号通路及其小胶质细胞形态的影响,探究电针对小鼠脑保护作用机制。方法C57BL/6小鼠随机分为空白对照组(n=8)、模型组(n=12)、假电针组(n=6)、电针组(n=6)。除空白对照组外,其余组小鼠行脂多糖腹腔注射(0.25 mg/kg),连续7 d。第8天开始对假电针组和电针组小鼠进行针灸或电针足三里的治疗,连续7 d。在实验前、第7、14天分别对小鼠进行称重;第13天对小鼠进行高架十字迷宫实验;第14天对小鼠进行旷场实验;实验结束后应用免疫荧光法测定小鼠前额叶皮层区小胶质细胞离子钙接头蛋白分子1(Iba-1)的表达;Real-time PCR检测小鼠前额叶皮层核因子κB(NF-κB)、诱导型一氧化氮合酶(iNOS)、肿瘤坏死因子α(TNF-α)、Caspase-1、白细胞介素(IL)-18的mRNA表达;Western blotting检测小鼠前额叶皮层NF-κB、iNOS、NLRP3、凋亡相关斑点样蛋白(ASC)、Caspase-1、IL-1β、IL-18的蛋白表达。结果体重:第7天时,与对照组相比,模型组、假电针组、电针组小鼠体重均下降(P<0.0001);第14天时,与对照组相比,模型组小鼠体重下降(P<0.0001);与假电针组相比,电针组小鼠体重增加(P<0.05)。高架十字迷宫实验:与对照组相比,模型组小鼠总路程及开臂滞留时间减少,闭臂滞留时间增多(P<0.05)。旷场实验:与对照组相比,模型组小鼠总路程减少、运动速度减慢、中央区进入次数减少(P<0.001);与模型组相比,电针组小鼠总路程增加、运动速度增快、中央区进入次数增多(P<0.01)。免疫荧光法实验:与对照组相比,模型组小鼠前额叶皮层区Iba-1相对荧光度升高(P<0.05);与模型组和假电针组相比,电针组小鼠前额叶皮层区Iba-1相对荧光度下降(P<0.05)。Real-time PCR实验:与对照组相比,模型组NF-κB、iNOS、TNF-α、Caspase-1、IL-1β、IL-18的mRNA表达升高(P<0.05);与模型组相比,电针组NF-κB、iNOS、TNF-α、Caspase-1、IL-18的mRNA表达降低(P<0.05)。Western blotting实验:与对照组相比,模型组NF-κB、iNOS、Caspase-1、IL-1β、IL-18的蛋白表达升高(P<0.05);与模型组相比,电针组NF-κB、iNOS、NLRP3、ASC、Caspase-1、IL-1β、IL-18的蛋白表达降低(P<0.05);与假电针组相比,电针组IL-18的蛋白降低(P<0.05)。结论电针可以改善小鼠行为学表现,抑制小鼠皮层区小胶质细胞的激活,其可能是通过调节NF-κB/NLRP3通路发挥抗炎和保护作用。

Objective To observe the effect of electric stimulation on nuclear factor-κB(NF-κB)/NOD-like receptor protein 3(NLRP3)signaling pathway and microglial cell morphology in mice with lipopolysaccharide(LPS)induced chronic neuroinflammation,and to explore the protective mechanism of electric stimulation on brain of mice.Methods C57BL/6 mice were randomly divided into blank control group(n=8),model group(n=12),sham electroacupuncture group(n=6)and electroacupuncture group(n=6).Except blank control group,mice in other groups were injected intraperitoneally with LPS(0.25 mg/kg)for 7 consecutive days.On the 8th day,mice in the sham electroacupuncture group and electroacupuncture group were treated with acupuncture or Zusanli electroacupuncture for 7 consecutive days.The mice were weighed before the experiment,on the 7th and 14th days.On the 13th day,the elevated cross maze test was performed on the mice.The open field test was performed on the 14th day.After the experiment,immunofluorescence assay was used to determine the expression of microglial ionized calcium binding adaptor molecule-l(Iba-1)in prefrontal cortex region.The mRNA expression of NF-κB,inducible nitric oxide synthase(iNOS),tumor necrosis factor-α(TNF-α),Caspase-1 and interleukin(IL)-18 were detected by Real-time PCR.The protein expression levels of NF-κB,iNOS,NLRP3,apoptosis-associated speck-like protein containing a CARD(ASC),Caspase-1,IL-1βand IL-18 were detected by Western blotting.Results Weight change,On the 7th day,compared with the control group,the body weight of mice in model group,sham electroacupuncture group and electroacupuncture group decreased(P<0.0001),respectively;On the 14th day,compared with the control group,the weight of mice in the model group decreased(P<0.0001);Compared with the sham electroacupuncture group,the body weight of mice in the electroacupuncture group increased(P<0.05).Elevated cross maze experiment,compared with the control group,the total distance and open arm retention time of mice in model group decreased,while the closed arm retention time increased(P<0.05).The open field experiment showed that compared with the control group,the model group mice showed a decrease in total distance traveled,slower movement speed,and fewer entries into the central area(P<0.001);Compared with the model group,the electroacupuncture group showed an increase in all three indicators(P<0.01);Compared with the sham electroacupuncture group,the total distance and motion speed of mice in electroacupuncture group both increased(P<0.05).Immunofluorescence assay,compared with the control group,the relative fluorescence of Iba-1 in prefrontal cortex area of mice in model group increased(P<0.05).Compared with the model and sham electroacupuncture group,the relative fluorescence of Iba-1 in prefrontal cortex area of mice in electroacupuncture group decreased(P<0.05).Realtime PCR showed that compared with the control group,mRNA expressions of NF-κB,iNOS,TNF-α,Caspase-1 and IL-18 in the model group increased(P<0.05);Compared with the model group,mRNA expressions of NF-κB,iNOS,TNF-α,Caspase-1 and IL-18 in electroacupuncture group decreased(P<0.05).Western blotting indicated that compared with the control group,the protein expressions of NF-κB,iNOS,Caspase-1,IL-1βand IL-18 in model group increased(P<0.05);Compared with model group,the protein expressions of NF-κB,iNOS,NLRP3,ASC,Caspase-1,IL-1βand IL-18 in electroacupuncture group decreased(P<0.05);Compared with the sham electroacupuncture group,IL-18 protein in electroacupuncture group decreased(P<0.05).Conclusion Electroacupuncture can improve the behavioral performance of mice and inhibit the activation of microglia in the cortical region of mice,which may play an antiinflammatory and protective role by regulating NF-κB/NLRP3 pathway.