硫化氢通过激活ATP敏感性钾通道保护小鼠在体缺血损伤视网膜和原代缺氧视网膜神经节细胞  

Hydrogen sulfide protects against retinal ischemic injury in mice through activation of ATP-sensitive potassium channels

作　　者：张玉娥 张君[2] 高秀娟[1] 张迪[1] 庄金珠 孙晖[1] 苏云 ZHANG Yu’e;ZHANG Jun;GAO Xiujuan;ZHANG Di;ZHUANG Jinzhu;SUN Hui;SU Yun(Binzhou Medical University,Yantai 264003,China;BinZhou Polytechnic College,Binzhou 256603,China;Yantai Central Blood Station,Yantai 264030,China)

机构地区：[1]滨州医学院,山东烟台264003 [2]滨州职业学院,山东滨州256603 [3]烟台中心血站,山东烟台264030

出　　处：《中国病理生理杂志》2023年第10期1859-1867,共9页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81971281);山东省高校科技计划(No.J18KA165)。

摘　　要：目的:研究硫化氢(H_2S)是否能够通过激活ATP敏感性钾通道(K_(ATP)通道)减轻小鼠原代缺氧视网膜神经节细胞及在体缺血视网膜损伤。方法:(1)培养分离纯化的小鼠原代视网膜神经节细胞(RGCs),给予缺氧处理或缺氧+硫氢化钠(NaHS)处理,利用Hoechst 33258染色和ELISA测定培养液中乳酸脱氢酶(LDH)浓度观察细胞死亡率变化;给予K_(ATP)通道激动剂或阻断剂,利用膜片钳、Western blot和钙成像技术观察硫化氢是否通过K_(ATP)通道减轻缺氧导致的细胞损伤;(2)在体建立小鼠视网膜缺血模型,模型动物分为5组(每组8只小鼠),分别为溶剂对照组、NaHS组、NaHS+K_(ATP)通道阻断剂组、K_(ATP)通道激动剂组和K_(ATP)通道激动剂+K_(ATP)通道断剂组,组织学切片观察视网膜各层厚度及RGCs层细胞密度变化。结果:(1)与氧糖剥夺(OGD)组相比,100μmol/L和300μmol/L NaHS可以显著减轻缺氧导致的原代RGCs细胞死亡率(P<0.01),同时也减少LDH释放量(1.5±0.3和1.3±0.2, P<0.01);与OGD组相比,NaHS和K_(ATP)通道激动剂可使细胞显著超极化(P<0.05, n=26),诱发放电显著减少(P<0.01, n=29),显著减少钙内流(P<0.01, n=14)和cleaved caspase-3表达量(P<0.05, n=7),显著减轻氧糖剥夺导致的原代RGCs损伤;而K_(ATP)通道阻断剂使细胞去极化,诱发放电增多,增加钙离子内流和cleaved caspase-3表达,逆转了这种保护作用;(2)在体实验中,给予NaHS或K_(ATP)通道激动剂可以显著逆转缺血损伤导致的视网膜厚度降低(P<0.05,n=8);与缺血组比较,RGCs层细胞密度也显著增高(P<0.05, n=8),而K_(ATP)通道阻断剂能够抑制这种保护作用。结论:NaHS可通过激活K_(ATP)通道减轻原代缺氧小鼠RGCs和小鼠在体缺血视网膜导致的损伤。AIM:To investigate the protective effect of hydrogen sulfide(H2S)against ischemic retinal injury in mice,and to explore its underlying mechanism.METHODS:Cultured and purified primary mice retinal ganglion cells(RGCs)were subjected to oxygen-glucose deprivation(OGD)treatment,alone or in combination with NaHS treatment.Cell death rates and lactate dehydrogenase(LDH)concentrations in the culture medium were assayed using Hoechst 33258 staining and ELISA.Resting potential,the number of induced action potentials,calcium influx and cleaved cas‐pase-3 expression were measured by patch clamp technique,calcium imaging techniques and Western blot to evaluate the protective effect of H2S in OGD-induced cellular damage by administration of ATP-sensitive potassium channel(KATP chan‐nel)agonist(pinacidil)or inhibitor(glibenclamide).Furthermore,forty healthy SPF male C57BL/6 mice were selected,and retinal ischemia models were induced by elevating intraocular pressure.These models were randomly divided into five groups,each consisting of 8 mice:vehicle group,exogenous H2S donor(NaHS)group,NaHS+KATP channel inhibitor group,KATP channel agonist group,and KATP channel agonist+inhibitor group.The cell density of RGC layer and retinal thickness were assayed by histological sections to assess the protective effect of H2S in ischemic retinal damage.RE-SULTS:(1)Compared with OGD group,the death rates of primary RGCs treated with OGD+NaHS(100 and 300µmol/L)were significantly reduced(P<0.01),while the release of LDH in RGCs culture medium was significantly decreased in OGD+NaSH group(P<0.01).Furthermore,after treatment with NaHS and KATP channel agonist,the resting potential was significantly hyperpolarized(P<0.05),the number of induced action potentials was significantly reduced(P<0.01),the calcium influx was significantly decreased(P<0.01),and the expression of cleaved caspase-3 was significantly reduced(P<0.05).(2)Compared with the ischemia group,the cell density of RGC layer and retinal thickness were significantly increased in i

关 键 词：缺血 缺氧 视网膜神经节细胞 硫化氢 ATP敏感性钾通道 

分 类 号：R774[医药卫生—眼科] R363[医药卫生—临床医学]