驻景丸加减方含药血清调控PINK1/Parkin介导的线粒体自噬对加压诱导大鼠RGCs损伤模型的影响  

Effects of the modified Zhujing Pill-medicated serum to modulate PINK1/Parkin-mediated mitochondrial autophagy on the pressure-induced retinal ganglion cell injury model in rats

作　　者：高佳 刘红佶 张丹 汪伟 GAO Jia;LIU Hongji;ZHANG Dan;WANG Wei(Chengdu University of Traditional Chinese Medicine,Chengdu 610000,Sichuan Province,China;Mianyang Hospital,School of Medicine,University of Electronic Science and Technology,Mianyang 621000,Sichuan Province,China;The Third Affiliated Hospital of Yunnan University of Traditional Chinese Medicine,Kunming 650000,Yunnan Province,China)

机构地区：[1]成都中医药大学,四川省成都市610000 [2]电子科技大学医学院附属绵阳医院,四川省绵阳市621000 [3]云南中医药大学第三附属医院,云南省昆明市650000

出　　处：《眼科新进展》2025年第5期342-347,364,共7页Recent Advances in Ophthalmology

基　　金：国家自然科学基金项目(编号:82305324);云南省科学技术厅项目(编号:202101AZ070001-090,202301AZ070001-100)。

摘　　要：目的观察驻景丸加减方含药血清对加压诱导大鼠视网膜神经节细胞(RGCs)损伤模型线粒体自噬及相关蛋白的影响。方法分别使用驻景丸加减方(剂量为10 mL·kg^(-1))和等量生理盐水灌胃雄性SD大鼠各10只制备驻景丸加减方含药血清和空白血清。分别予0、20、40、60、80 mmHg(1 kPa=7.5 mmHg)的压力体外加压培养大鼠RGCs损伤模型,根据加压实验结果,最终选取80 mmHg体外加压培养的大鼠RGCs损伤模型用于后续实验。将大鼠RGCs随机分为对照组(NG组,细胞不做任何处理)、模型组(MG组,损伤模型细胞且不做后续血清干预)、空白血清组(KB组,损伤模型细胞且使用空白大鼠血清干预)以及驻景丸加减方血清组(LVP组,损伤模型细胞且使用驻景丸加减方含药血清干预)。采用流式细胞仪检测干预前后大鼠RGCs凋亡情况;透射电镜观察各组大鼠RGCs线粒体形态;Western blot实验检测各组大鼠RGCs的PINK1、parkin、LC3 II/I以及p62蛋白表达情况。结果透射电镜观察显示,NG组细胞线粒体双膜结构完整,线粒体嵴分布均匀;MG组和KB组细胞可见线粒体双膜结构破坏,线粒体内大量空泡;LVP组细胞线粒体可见双膜结构,线粒体内空泡较MG组和KB组减少,形态基本和NG组细胞一致。流式细胞仪检测结果显示,NG组、MG组、KB组以及LVP组大鼠RGCs凋亡率分别为(4.25±0.55)%、(20.79±3.06)%、(30.07±3.23)%以及(11.42±0.16)%。与NG组相比,MG组大鼠RGCs凋亡率上升,差异有统计学意义(P<0.01);与MG组及KB组相比,LVP组大鼠RGCs凋亡率均下降,差异均有统计学意义(均为P<0.05)。Western blot检测结果显示,与NG组相比,MG组大鼠RGCs的PINK1、Parkin以及LC3 II/I蛋白相对表达水平均上升,p62蛋白相对表达水平下降,差异均有统计学意义(均为P<0.01);与MG组及KB组相比,LVP组大鼠RGCs的PINK1、Parkin以及LC3 II/I蛋白相对表达水平均下降,p62蛋白相对表达水平均上升,差异均有统计学意Objective To investigate the effects of modified Zhujing Pill(ZJP)-medicated serum on mitochondrial autophagy and related proteins in a pressure-induced retinal ganglion cell(RGC)injury model in rats.Methods The modified ZJP-medicated serum and blank serum were prepared by the intragastric administration of modified ZJP(10 mL·kg^(-1))to 10 male Sprague-Dawley(SD)rats and equivalent normal saline to another 10 male SD rats.The RGC injury models of rats were constructed under the in vitro pressure of 0 mmHg,20 mmHg,40 mmHg,60 mmHg,and 80 mmHg(1 kPa=7.5 mmHg),respectively.According to the pressurization experiment results,the RGC injury model established under the in vitro pressure of 80 mmHg was selected for subsequent experiments.The RGCs of rats were divided into the normal group(NG group,untreated RGCs),model group(MG group,injured RGCs without serum intervention),blank serum group(KB group,injured RGCs treated with blank serum),and modified ZJP-medicated serum group(LVP group,injured RGCs treated with modified ZJP-medicated serum).The apoptosis of RGCs was detected by flow cytometry;the mitochondrial ultrastructure was observed via transmission electron microscopy;the protein expression level of PINK1,Parkin,LC3 II/I,and p62 was measured by Western blotting.Results Transmission electron microscopy revealed the intact double-membrane structure of mitochondria and evenly distributed mitochondrial crista in the NG group,while the MG and KB groups exhibited disrupted mitochondrial membranes,accompanied by a large number of vacuoles in the mitochondria.The mitochondria of the LVP group had a double-membrane structure and the vacuoles in the mitochondria decreased compared with those of the MG and KB groups,and the mitochondrial morphology was basically the same as that of the NG group.The flow cytometry results demonstrated the apoptosis rate of RGCs was(4.25±0.55)%,(20.79±3.06)%,(30.07±3.23)%,and(11.42±0.16)%in the NG,MG,KB,and LVP groups,respectively.Compared with the NG group,the apoptosis rate of RGCs in ra

关 键 词：驻景丸加减方 视网膜神经节细胞 含药血清 线粒体自噬 PINK1/Parkin 

分 类 号：R775[医药卫生—眼科]