基于线粒体动力学探讨电针改善实验性近视豚鼠视网膜细胞凋亡的作用机制

Study on the mechanism of electroacupuncture relieving retinal cell apoptosis in experimental myopic guinea pigs based on mitochondrial dynamics

目的基于线粒体动力学探讨电针改善实验性近视视网膜细胞凋亡的作用机制。方法2周龄三色豚鼠140只随机分为正常对照(NC)组、透镜诱导型近视(LIM)组、透镜诱导型近视+假穴(LIM+SHAM)组和透镜诱导型近视+电针干预(LIM+EA)组,每组35只;其中NC组正常饲养,LIM组、LIM+SHAM组和LIM+EA组豚鼠右眼均配戴-6.0 D透镜以建立实验性近视模型,LIM+EA组豚鼠在合谷穴与太阳穴给予电针刺激,LIM+SHAM组豚鼠在假穴位进行干预。造模2周和4周后测量豚鼠的近视屈光度和眼轴长度,采用实时荧光定量PCR(q-PCR)和Western blot检测各组豚鼠视网膜组织中线粒体动力相关蛋白(DRP1)、视神经萎缩蛋白(OPA1)、Bcl-2相关X蛋白(BAX)和B细胞淋巴瘤-2(BCL-2)的mRNA和蛋白表达情况,HE染色观察各组豚鼠视网膜组织的形态改变,TUNEL染色检测视网膜组织的细胞凋亡情况。结果造模2周和4周后,与NC组相比,LIM组、LIM+SHAM组豚鼠右眼与左眼屈光度和眼轴长度的差值均明显增大(均为P<0.001);与LIM组相比,LIM+EA组豚鼠右眼与左眼屈光度和眼轴长度的差值均明显减小(均为P<0.01)。造模4周后,HE染色结果显示,NC组豚鼠视网膜排列均匀、内外核层细胞形态正常;与NC组相比,LIM组和LIM+SHAM组豚鼠视网膜厚度明显变薄,排列紊乱;与LIM组相比,LIM+EA组豚鼠视网膜厚度略有增加,排列相对规则。造模4周后,TUNEL染色结果显示,与NC组相比,LIM组和LIM+SHAM组豚鼠视网膜的绿色荧光明显增强;与LIM组相比,LIM+EA组豚鼠视网膜的绿色荧光明显减弱。造模2周和4周后,与NC组相比,LIM组和LIM+SHAM组豚鼠视网膜中DRP1、BAX的mRNA和蛋白相对表达量均明显升高(均为P<0.05),OPA1、BCL-2的mRNA和蛋白相对表达量均明显降低(均为P<0.05);与LIM组相比,LIM+EA组豚鼠视网膜中DRP1、BAX的mRNA和蛋白相对表达量均明显降低(均为P<0.05),OPA1、BCL-2的mRNA和蛋白相对表达量均明显升高(均为P<0.05)。结论实验性近视可明显增加视网膜线粒体分裂;电针干预通过抑制视网膜线粒体分裂,增加线粒体融合来减轻视网膜细胞凋亡,改善视网膜的形态结构,进而延缓近视的发展。

Objective To investigate the mechanism of electroacupuncture in relieving the apoptosis of retinal cells in experimental myopia based on mitochondrial dynamics.Methods In the study,140 two-week-old tricolor guinea pigs were divided into the normal control(NC)group,lens-induced myopia(LIM)group,LIM+sham acupoint(LIM+SHAM)group,and LIM+electroacupuncture intervention(LIM+EA)group,with 35 guinea pigs in each group.Guinea pigs in the NC group were fed normally,and those in the LIM group,LIM+SHAM group and LIM+EA group wore a-6.0D lens on the right eye to induce myopia.Guinea pigs in the LIM+EA group were treated at Hegu and Taiyang acupoints,while those in the LIM+SHAM group were treated at sham acupoints.After 2-and 4-week myopia induction,the diopter and axial length of the guinea pigs were measured,and the messenger ribonucleic acid(mRNA)and protein levels of dynamin-related protein 1(DRP1),optic atrophy 1(OPA1),Bcl-2 associated X protein(BAX)and B cell lymphoma-2(BCL-2)in the retinas of the guinea pigs in each group were detected by quantitative PCR and Western blot,respectively.Meanwhile,HE staining was taken to observe the morphological changes in the retina of the guinea pigs in each group,and TUNEL staining was used to detect apoptosis.Results After 2-and 4-week myopic induction,compared with the NC group,the differences in diopter and axial length between the right and left eyes of guinea pigs in the LIM and the LIM+SHAM groups significantly increased(all P<0.001).Compared with the LIM group,the differences in diopter and axial length between both eyes of guinea pigs in the LIM+EA group significantly decreased(all P<0.01).At 4 weeks after myopic induction,HE staining results showed that the retinas of the guinea pigs in the NC group were evenly arranged,and the morphology of inner and outer nuclear layer cells was normal.Compared with the NC group,the retinas of the guinea pigs in the LIM and the LIM+SHAM groups were significantly thinner and disorderly arranged.Compared with the LIM group,the retinal thickness of guinea pigs in the LIM+EA group slightly increased,and the arrangement was relatively regular.The TUNEL staining results showed that compared with the NC group,the green fluorescence of the guinea pig retina in the LIM group and LIM+SHAM group was significantly enhanced;compared with the LIM group,the green fluorescence of the guinea pig retina in the LIM+EA group was significantly weaken.After 2-and 4-week myopic induction,the mRNA and protein expression levels of DRP1 and BAX in the LIM and the LIM+SHAM groups were significantly higher than those in the NC group(all P<0.05),while the mRNA and protein expression levels of OPA1 and BCL-2 were significantly lower(all P<0.05).The mRNA and protein expression levels of DRP1 and BAX in the LIM+EA group were significantly lower than those in the LIM group(all P<0.05),while the mRNA and protein expression levels of OPA1 and BCL-2 were significantly higher(all P<0.05).Conclusion Experimental myopia can enhance retinal mitochondrial fission.Electroacupuncture intervention can alleviate retinal cell apoptosis and improve the morphological structure of the retina by inhibiting retinal mitochondrial fission and increasing mitochondrial fusion,thereby delaying myopia development.