甜茶树苷H对高糖致小鼠肾足细胞损伤的作用研究  

Effect of Cyclocarioside H on the Damage of Renal Podocytes in Mice Stimulated by High Glucose

作　　者：陈毓[1] 孙伟翔 李昊达 张婷 杨海峰[1] 陈晓兰[1] 李锋涛 CHEN Yu;SUN Weixiang;LI Haoda;ZHANG Ting;YANG Haifeng;CHEN Xiaolan;LI Fengtao(Jiangsu Agri-animal Husbandry Vocational College,Taizhou 225300,China;Jiangsu Key Laboratory for High-Tech Research and Development of Veterinary Biopharmaceuticals,Taizhou 225300,China;Jiangsu Health Vocational College,Nanjing 211800,China)

机构地区：[1]江苏农牧科技职业学院,泰州225300 [2]江苏省兽用生物制药高技术研究重点实验室,泰州225300 [3]江苏卫生健康职业学院,南京211800

出　　处：《中国畜牧兽医》2024年第8期3256-3266,共11页China Animal Husbandry & Veterinary Medicine

基　　金：西藏自治区科技计划项目(XZ202101YD0001C);2022年度江苏省中医药科技发展计划项目(MS2022143);2022年泰州市科技支撑计划发展(指导性)项目(泰科技[2022]14号);江苏高校“青蓝工程”优秀教学团队培养对象资助项目(苏教师函[2022]29号)。

摘　　要：[目的]探究甜茶树苷H抑制NLRP3/ASC/Caspase1通路从而改善高糖导致的小鼠肾足细胞(MPC-5)的损伤作用。[方法]试验采用不同浓度(0、3.25、7.5、15、30、60、120μmol/L)甜茶树苷H分别孵育MPC-5细胞,通过CCK-8法检测细胞活力,判定安全给药浓度。将MPC-5细胞分为空白对照组(CON)、D(+)-无水葡萄糖等渗对照组(MA)、高糖组(HG)以及不同浓度(3.25、7.5、15μmol/L)甜茶树苷H组(CY3.25、CY7.5和CY15组)。HG和不同浓度甜茶树苷H组分别加入30μmol/L D(+)-无水葡萄糖,MA组加入5.5 mmol/L D(+)-无水葡萄糖+24.5 mmol/L甘露醇,CON组加入不含FBS的DMEM培养基,孵育细胞24 h后,CY3.25、CY7.5和CY15组培养基更换为不同浓度甜茶树苷H,并再次孵育细胞24 h。收集细胞,采用CCK-8法和乳酸脱氢酶(lactate hydrogenase,LDH)法分别检测细胞活力和损伤程度;利用碘化丙啶(PI)荧光染色法检测细胞凋亡率;通过免疫荧光法观察MPC-5细胞中NOD样受体热蛋白结构域相关蛋白3(NLRP3)和凋亡相关斑点样蛋白(ASC)共定位情况;采用ELISA法检测细胞上清液中白细胞介素-1β前体(Pro-IL-1β)和白细胞介素-1β(IL-1β)含量;通过Western blotting检测NLRP3、ASC、半胱氨酸天冬酶1(Caspase1)/半胱氨酸天冬酶1前体(Pro-Caspase1)以及肾结蛋白(Desmin)、肾病蛋白(Nephrin)、线粒体裂变因子(MFF)、胶原Ⅳ(CollagenⅣ)蛋白表达量。[结果]与0μmol/L甜茶树苷H组相比,7.5和15μmol/L甜茶树苷H组细胞活力显著升高(P<0.05),30、60和120μmol/L甜茶树苷H组细胞活力显著降低(P<0.05)。因此,后续选择3.25~15μmol/L为甜茶树苷H的安全作用浓度。采用以上安全作用浓度甜茶树苷H处理细胞,结果显示,与HG组相比,CY3.25、CY7.5和CY15组细胞活力显著升高(P<0.05),细胞上清液中LDH活力显著降低(P<0.05),且呈现剂量依赖性;CY7.5和CY15组细胞凋亡率显著降低(P<0.05)。免疫荧光法检测结果显示,与CON组相比,HG组细胞中NLRP3、ASC荧光增强[Objective]This experiment was to investigate the effect of cyclocarioside H on the damage of renal podiocytes(MPC-5)induced by high glucose by inhibiting the NLRP3/ASC/Caspase1 pathway.[Method]MPC-5 cells were incubated with different concentrations(0,3.25,7.5,15,30,60 and 120μmol/L)of cyclocarioside H,and the cell viability was detected by CCK-8 method to determine the safe concentration.MPC-5 cells were divided into blank control(CON),D(+)-anhydrous glucose isoosmotic control(MA),high glucose(HG)and cyclocarioside H with different concentrations(3.25,7.5 and 15μmol/L)groups(CY3.25,CY7.5 and CY15).HG and CY3.25,CY7.5 and CY15 groups were added with 30μmol/L D(+)-anhydrous glucose,MA group was added with 5.5 mmol/L D(+)-anhydrous glucose+24.5 mmol/L mannitol,CON group was added with DMEM medium without FBS.After incubating the cells for 24 h,the culture medium of CY3.25,CY7.5 and CY15 groups was changed to different concentrations of cyclocarioside H,and the cells were incubated again for 24 h.The cells were collected,and CCK-8 and lactate hydrogenase(LDH)assay were used to determine cell viability and damage degree,respectively.The apoptosis rate was detected by fluorescent staining with propyl iodide(PI).The co-localization of NOD-like receptor heat protein domain associated protein 3(NLRP3)and apoptosis-associated speck-like proteins(ASC)in MPC-5 cells was observed by immunofluorescence.The contents of Pro-IL-1βand IL-1βin the supernatant were detected by ELISA.Western blotting was used to detect the expression of NLRP3,ASC,Caspase1/Pro-Caspase1,Desmin,Nephrin,MFF and CollagenⅣproteins.[Result]Compared with 0μmol/L cyclocarioside H group,the cell viability of 7.5 and 15μmol/L cyclocarioside H groups was significantly increased(P<0.05),and that of 30,60 and 120μmol/L cyclocarioside H groups was significantly decreased(P<0.05).Therefore,3.25-15μmol/L was subsequently selected as the safe concentration of cyclocarioside H.The cells were treated with the above safe concentration of cyclocarioside H,and

关 键 词：甜茶树苷H 高糖 足细胞 损伤作用 

分 类 号：S859.7[农业科学—临床兽医学]