摘要
目的探讨电针通过激活lncRNA XmiR-146aNav 1.7轴抑制卫星胶质细胞(SGC)炎症并改善卒中大鼠抑郁的机制。方法应用随机数字法将SD大鼠分为对照组、模型组和电针组,各15只。离体培养的SGC的分组:从Genepharma(中国上海)构建了携带miR-146a抑制剂(LV-anti-miR-146a,150 nM)或阴性对照(LV-nc,100 nM)的重组慢病毒载体,感染SGC 24 h。NC为正常培养。通过蔗糖偏好和运动活动比较大鼠的抑郁样行为。蛋白质印迹检测大鼠海马组织TNF-α、IL-1β和IL-6 mRNA表达。通过PCR检测大鼠脑组织中lncRNAXIST/miR-146a/Nav 1.7的mRNA表达。通过双荧光素酶报告实验检测miR-146a与XIST的3’-UTR,miR-146a与Nav1.7间的相互作用。结果模型组大鼠的蔗糖偏好指数和运动计数较对照组减少(P<0.05),电针组大鼠的蔗糖偏好指数和运动计数较模型组增加(P<0.05),模型组神经损伤评分较对照组升高(P<0.05),电针组神经学损伤评分较模型组降低(P<0.05)。模型组TNF-α、IL-1β和IL-6蛋白表达较对照组升高(P<0.05),电针组TNF-α、IL-1β和IL-6蛋白表达较模型组降低(P<0.05)。模型组XIST和Nav1.7 mRNA表达较对照组升高(P<0.05),电针组XIST和Nav1.7mRNA表达较模型组降低(P<0.05);模型组miR-146a的mRNA表达较对照组降低(P<0.05),电针组miR-146a的mRNA表达较模型组升高(P<0.05)。与Mut-XIST相比,LV-anti-miR-146a与Wt-XIST结合位点的荧光素酶活性降低(P<0.05),miR-146a抑制XIST的荧光素酶活性,RNA pull-down分析显示Bio-XIST可下调miR-146,而Bio-XIST-mut不能(P<0.05)。miR-146a抑制Nav1.7的荧光素酶活性(P<0.05),LV-miR-146a感染引起的miR-146a过表达抑制了Nav1.7编码基因SCN9A的转录(P<0.05)。结论电针通过激活lncRNA XmiR-146aNav 1.7轴抑制SGC炎症,从而有效缓解脑卒中后抑郁的抑郁样行为。XIST作为miR-146a海绵促进miR-146a靶基因Nav1.7的表达,从而调节SGC激活。
Objective To explore the mechanism of electroacupuncture inhibiting satellite glial cell(SGC)inflammation and improving depression in stroke rats by activating lncRNA XmiR-146aNav 1.7 axis.Methods SD rats were randomly divided into 3 groups by random number method:control group(n=15),model group(n=15),EA group(n=15).Grouping of in vitro cultured SGC:Recombinant lentiviral vectors carrying miR-146a inhibitor(LV-anti-miR-146a,150 nM)or negative control(LV-nc,100 nM)were constructed from Genepharma(Shanghai,China)and infected with SGC for 24 hours.NC was normal culture.The depression-like behaviors of rats were compared by sucrose preference and exercise activity.The mRNA expressions of TNF-α,IL-1βand IL-6 in hippocampus were detected by Western blotting.The mRNA expression of lncRNAXIST/miR-146a/Nav 1.7 in rat brain tissue was detected by PCR.The interactions between miR-146a and the 3’-UTR of XIST,between miR-146a and Nav1.7 were detected by dual luciferase reporter assay.Results Rats in model group showed lower sucrose preference index and exercise count than those in control group and EA group(P<0.05).Rats in model group showed higher neurological injury score than those in control group and EA group(P<0.05).The protein expressions of TNF-α,IL-1βand IL-6 in model group were significantly higher than those in control group and EA group(P<0.05).The mRNA expressions of XIST and Nav1.7 in model group were significantly higher than those in control group and EA group(P<0.05).And the mRNA expression of miR-146a in model group was significantly lower than those in control group and EA group(P<0.05).Compared with Mut-XIST,the luciferase activity of LV-anti-miR-146a and Wt-XIST binding site was decreased(P<0.05).miR-146a inhibited the luciferase activity of XIST.RNA pull-down analysis showed that Bio-XIST could down-regulate miR-146,and Bio-XIST-mut could not(P<0.05).miR-146a inhibited the luciferase activity of Nav1.7(P<0.05),and the overexpression of miR-146a caused by LV-miR-146a infection inhibited the transcription of Nav1.7 encoding gene SCN9A(P<0.05).Conclusion Electroacupuncture inhibits SGC inflammation by activating the lncRNA XmiR-146aNav 1.7 axis,thereby effectively alleviating the depression-like behavior of post-stroke depression.XIST acts as a miR-146a sponge to promote the expression of miR-146a target gene Nav1.7,thereby regulating SGC activation.
作者
王婵
郑苏
左薇
许明军
张刚
严文彦
骆文仙
朱雪萍
WANG Chan;ZHENG Su;ZUO Wei(Shiyan Taihe Hospital,Affiliated Hospital of Hubei University of Medicine,Shiyan 442000,China)
出处
《精神医学杂志》
2022年第3期239-244,共6页
Journal of Psychiatry
基金
湖北省卫生健康委中医药科研项目(青年人才项目)(编号:ZY2021Q015)
十堰市引导性科研项目(编号:21Y37)。
