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,Beclin-1,LC3Ⅱand LC3Ⅱ/Ⅰratio in Qingshen granule group and miR-23b-mimic group were significantly higher than those in TGF-β1 group,and the expressions of P62 andα-SMA were significantly lower than those in TGF-β1 group(P<0.05).The performance of miR-23b-mimic+Qingshen granule group was better than that of miR-23b-mimic group(P<0.05).Conclusions Qingshen granules can up-regulate the expression of miR-23b-5p in NRK-52E cellsand inhibit the transdifferentiation process of NRK-52E cells by enhancing the mitochondrial autophagy activity mediated by PINK1/Parkin pathway.
作者
金华
张叶青
呼琴
张磊
陈诺
韩燕全
王亿平
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)
出处
《中国药理学通报》
CAS
CSCD
北大核心
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)。
