摘要
目的:探讨新生大鼠缺氧缺血性脑损伤模型中TRPC3通路介导的神经自噬作用。方法:7日龄Sprague-Dawley(SD)雄性幼鼠随机分为4组,每组20只,分别为Sham组、HIBD组、HIBD+Vector组和HIBD+TRPC3组。HIBD+Vector组和HIBD+TRPC3组在诱导HIBD模型前24 h,分别将Vector或TRPC3注射到左侧脑室中。评估各组幼鼠脑梗死面积、神经系统评分、脑水肿体积、神经元凋亡和线粒体自噬。体外将小鼠海马神经元细胞系(HT22)分为以下6组:Control组、OGD/R组、OGD/R+TRPC3组、OGD/R+NC组、OGD/R+si-Pink1组和OGD/R+TRPC3+si-Pink1组。除Control组外,其他组使用氧气-葡萄糖剥夺/再灌注(Oxygen-glucose deprivation/reperfusion,OGD/R)方法建立体外脑缺血再灌注模型。通过线粒体膜电位的检测线粒体损伤情况。结果:与Sham组相比,HIBD组和HIBD+Vector组显示出较大的梗死面积、脑含水量、神经学评分和神经元凋亡(P<0.05)。HIBD+TRPC3组的梗死面积、脑含水量、神经学评分和神经元凋亡较HIBD+Vector组明显降低(P<0.05)。与Sham组相比,HIBD组和HIBD+Vector组线粒体自噬相关蛋白PTEN诱导假定激酶1(PTEN induced putative kinase 1,Pink1)、E3泛素连接酶(Parkin RBR E3 ubiquitin protein ligase,Parkin)和微管相关蛋白轻链3(light chain 3,LC3)Ⅱ的表达水平增加(P<0.05)。与HIBD+Vector组相比,HIBD+TRPC3组Pink1、Parkin和Lc3Ⅱ的表达水平进一步增加(P<0.05)。体外实验显示,TRPC3上调可以强烈增加Pink1、Parkin的表达和Lc3Ⅱ/Lc3Ⅰ比值,以及减少选择性自噬接头蛋白(sequestosome-1,p62)的表达。Pink1沉默可以部分阻止TRPC3对线粒体自噬的影响。TRPC3上调增加了健康线粒体的数量,减少了受损线粒体的数量,提高了HT-22细胞的生存率。然而,Pink1沉默阻止了TRPC3对线粒体功能和增殖能力的恢复。结论:TRPC3通过激活Pink1/Parkin通路介导的线粒体自噬,在HIBD早期阶段中发挥了重要的保护作用。
Objective:To explore neuro-autophagy mediated by the transient receptor potential channel 3(TRPC3)pathway in a neonatal rat model of hypoxic-ischemic brain damage(HIBD).Methods:Seven-day-old male Sprague-Dawley(SD)mice were randomly divided into four groups,with 20 rats in each group:sham group,HIBD group,HIBD+vector group,and HIBD+TRPC3 group.At 24 hours before the induction of the HIBD models,vector and TRPC3 were injected into the left ventricle of the young mice in the HIBD+vector and HIBD+TRPC3 groups,respectively.The mice in each group were assessed for brain infarct area,neurological score,cerebral edema volume,and neuronal apoptosis and mitophagy.The in vitro mouse hippocampal neuronal cell line(HT22)was divided into the following six groups:control group,OGD/R group,OGD/R+TRPC3 group,OGD/R+NC group,OGD/R+si-Pink1 group,and OGD/R+TRPC3+si-Pink1 group.The oxygen-glucose deprivation/reperfusion(OGD/R)method was used to establish an in vitro cerebral ischemia-reperfusion model in all groups except the control group.Mitochondrial damage was measured based on the mitochondrial membrane potential.Results:Compared with the sham group,the HIBD and HIBD+vector groups had greater infarct area,brain water content,neurological score,and neuronal apoptosis(P<0.05).The infarct area,brain water content,neurological score,and neuronal apoptosis were significantly reduced in the HIBD+TRPC3 group compared with the HIBD+vector group(P<0.05).The expression levels of mitophagy-related proteins PTEN-induced putative kinase 1(Pink1),Parkin RBR E3 ubiquitin-protein ligase(Parkin),and light chain 3(LC3)Ⅱwere significantly higher in the HIBD and HIBD+vector groups than in the sham group(P<0.05).The expression levels of Pink1,Parkin,and LC3Ⅱwere further increased in the HIBD+TRPC3 group compared with the HIBD+vector group(P<0.05).In vitro experiments showed that TRPC3 upregulation significantly increased Pink1 and Parkin expression and the LC3Ⅱ/LC3Ⅰratio,and reduced sequestosome-1(p62)expression.Silencing Pink1 partially blocked the effect of TRPC3 on mitophagy.TRPC3 upregulation increased the number of healthy mitochondria,reduced the number of damaged mitochondria,and increased HT-22 cell survival rate.However,silencing Pink1 prevented TRPC3 from restoring mitochondrial function and proliferative capacity.Conclusion:TRPC3 plays an important protective role in the early stages of HIBD by activating the Pink1/Parkin pathway-mediated mitophagy.
作者
薛梅
杨丽
樊晓艳
李玲
钱金明
Xue Mei;Yang Li;Fan Xiaoyan;Li Ling;Qian Jinming(Department of Neonatology,The Affiliated Taizhou People’s Hospital of Nanjing Medical University)
出处
《重庆医科大学学报》
CAS
CSCD
北大核心
2024年第8期978-985,共8页
Journal of Chongqing Medical University
基金
江苏省卫生健康委员会资助项目(编号:20212343)。
关键词
新生儿
缺血缺氧性脑病
典型的瞬时受体电位3
线粒体自噬
neonatal
hypoxic-ischemic brain damage
typical transient receptor potential channels 3
mitophagy
