清肾颗粒对大鼠NRK-52E细胞转分化模型miR-23b和PINK1/Parkin通路的影响  

Effect of Qingshen granules on miR-23b and PINK1/Parkin pathwayin rat NRK-52E cell transdifferentiation model

作　　者：金华[1,2] 张叶青 呼琴 张磊 陈诺[3] 韩燕全 王亿平[1] JIN Hua;ZHANG Ye-qing;HU Qin;ZHANG Lei;CHEN Nuo;HAN Yan-quan;WANG Yi-ping(the First Affiliated Hospital,Anhui University of Chinese Medicine,Hefei 230031,China;Center for Xin′an Medicine and Modernization of Traditional Chinese Medicine of IHM,Anhui University of Chinese Medicine,Hefei 230012,China;Graduate School,Anhui University of Chinese Medicine,Hefei 230012,China;Dept of Pharmacy,the First Affiliated Hospital,Anhui University of Chinese Medicine,Hefei 230031,China)

机构地区：[1]安徽中医药大学第一附属医院肾病科,安徽合肥230031 [2]合肥综合性国家科学中心大健康研究院新安医学与中医药现代化研究所,安徽合肥230012 [3]安徽中医药大学研究生院,安徽合肥230012 [4]安徽中医药大学第一附属医院药学部,安徽合肥230031

出　　处：《中国药理学通报》2024年第1期162-170,共9页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 82274307,82004165);安徽省高等学校自然科学重点研究项目(No KJ2021A0546)。

摘　　要：目的 探讨TGF-β1诱导大鼠NRK-52E细胞转分化模型中miR-23b对PINK1/Parkin通路的靶向调节机制,并阐明清肾颗粒含药血清对NRK-52E细胞转分化的干预机理。方法 采用超高效液相色谱(UPLC)指纹图谱法对清肾颗粒进行全指纹图谱分析。构建TGF-β1诱导大鼠NRK-52E细胞转分化模型,转染siRNA后分为模拟物空载对照组、miR-23b-5p模拟物组、抑制剂空载对照组、miR-23b-5p抑制剂组,观察miR-23b-5p对PINK1表达量的影响。再将NRK-52E细胞分组为正常组、TGF-β1组、清肾颗粒组、miR-23b-mimic-NC组、miR-23b-mimic组、miR-23b-mimic+清肾颗粒组,Western blot法检测NRK-52E细胞中Pink1、Parkin、LC3Ⅱ、Beclin-1、P62、α-SMA蛋白表达,RT-qPCR法检测NRK-52E细胞中miR-23b-5p、Pink1、Parkin、Beclin-1、α-SMA mRNA的表达,双荧光素酶报告基因实验检测miR-23b-5p与PINK1的靶向关系。结果 UPLC指纹图谱法鉴定出清肾颗粒中11个活性成分。miR-23b-5p过表达后,PINK1 mRNA表达量也显著增加(P<0.05);而miR-23b-5p表达沉默后,PINK1 mRNA表达量也显著减少(P<0.05)。双荧光素酶报告显示,Rno-miR-23b-5p能显著下调Rno-PINK1-WT荧光素酶活性(P<0.05),但未能下调突变Rno-PINK1-mut荧光素酶活性(P>0.05)。清肾颗粒含药血清干预实验发现,TGF-β1组的miR-23b-5p、Pink1、Parkin、Beclin-1、LC3Ⅱ表达及LC3Ⅱ/Ⅰ比值均明显低于正常组,P62和α-SMA表达明显高于正常组(P<0.05)。清肾颗粒组和miR-23b-mimic组的miR-23b-5p、Pink1、Parkin、Beclin-1、LC3Ⅱ表达及LC3Ⅱ/Ⅰ比值均明显高于TGF-β1组,P62和α-SMA表达明显低于TGF-β1组(P<0.05)。miR-23b-mimic+清肾颗粒组的表现更优于miR-23b-mimic组(P<0.05)。结论 清肾颗粒能够上调NRK-52E细胞内miR-23b-5p表达,并通过增强PINK1/Parkin通路介导的线粒体自噬活性,抑制NRK-52E细胞转分化进程。Aim To investigate the targeting mechanism of miR-23b on PINK1/Parkin pathway in transdifferentiation of NRK-52E cellsinduced by TGF-β1,and to elucidate the intervention mechanism of Qingshen granules drug-containing serum on NRK-52E cell transdifferentiation.Methods Ultra-high performance liquid chromatography(UPLC)fingerprinting method was used to analyze Qingshen granules.The NRK-52E transdifferentiation model induced by TGF-β1 was constructed.The NRK-52E cells were divided into simulated no-load control group,miR-23b-5p simulated group,inhibitor no-load control group,and miR-23b-5p inhibitor group,after transfection with siRNA,and the effect of miR-23b-5p on PINK1 expression was observed.The NRK-52E cells were then divided into normal group,TGF-β1 group,Qingshen granule group,miR-23b-mimic group,miR-23b-mimic group,and miR-23b-mimic+Qingshen granule group.Western blot was used to detect the expression of Pink1,Parkin,LC3Ⅱ,Beclin-1,P62 andα-SMA proteins,and RT-PCR was used to detect the expression of miR-23b-5p,Pink1,Parkin,Beclin-1 andα-SMA mRNA in NRK-52E cells.Dual-Luciferase Reporter gene experiment was used to detect the targeting relationship between miR-23b-5p and PINK1.Results UPLC fingerprinting method found 11 active components in Qingshen granules.After overexpression of miR-23b-5p,the expression of PINk1 mRNA significantly increased(P<0.05).And after silencing of miR-23b-5p expression,the expression of PINk1 mRNA also significantly decreased(P<0.05).Dual-Luciferase Reporter Assay showed that Rno-miR-23b-5p could significantly down-regulate the luciferase activity of Rno-PINK1-WT(P<0.05),but could not down-regulate the luciferase activity of mutant Rno-PINK1-mut(P>0.05).The experimental results showed that the expressions of miR-23b-5p,Pink1,Parkin,Beclin-1,LC3Ⅱand LC3Ⅱ/Ⅰratio in TGF-β1 group were significantly lower than those in normal group,but the expressions of P62 andα-SMA were significantly higher than those in normal group(P<0.05).The expressions of miR-23b-5p,Pink1,Parkin,Bec

关 键 词：miR-23b-5p PINK1/Parkin信号通路 线粒体自噬 NRK-52E细胞 清肾颗粒 上皮细胞转分化 

分 类 号：R-332[医药卫生] R285.5R322.61R329.24R342.22