清肾颗粒通过调控外泌体、miR-330-3p以及CREBBP表达抑制小鼠肾纤维化
目的 探讨清肾颗粒对腺嘌呤诱导的肾纤维化小鼠模型及尿酸刺激下NRK-49F细胞的作用及其调控外泌体、miR-330-3p和CREBBP的机制.方法 动物实验:构建腺嘌呤诱导的肾纤维化小鼠模型,分为正常组、模型组和清肾颗粒组(8.0 g·kg-1·d-1),6只/组,灌胃12周.采用尾静脉注射腺相关病毒载体,实验结束后收集双侧肾组织.观察小鼠肾组织外泌体标记蛋白CD9,Hsp70,TSG101蛋白水平变化;Western blotting及免疫荧光检测小鼠肾组织Col-Ⅲ、α-SMA、FN和E-cad表达变化;HE和Masson染色观察小鼠肾组织病理学变化.细胞实验:构建尿酸刺激下的NRK-49...
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| Published in | 南方医科大学学报 Vol. 44; no. 8; pp. 1431 - 1440 |
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| Main Authors | , , , , , , , , |
| Format | Journal Article |
| Language | Chinese |
| Published |
安徽中医药大学第一附属医院肾内科,安徽 合肥 230031%安徽中医药大学第一临床医学院,安徽 合肥 230038
01.08.2024
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| Subjects | |
| Online Access | Get full text |
| ISSN | 1673-4254 |
| DOI | 10.12122/j.issn.1673-4254.2024.08.01 |
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| Abstract | 目的 探讨清肾颗粒对腺嘌呤诱导的肾纤维化小鼠模型及尿酸刺激下NRK-49F细胞的作用及其调控外泌体、miR-330-3p和CREBBP的机制.方法 动物实验:构建腺嘌呤诱导的肾纤维化小鼠模型,分为正常组、模型组和清肾颗粒组(8.0 g·kg-1·d-1),6只/组,灌胃12周.采用尾静脉注射腺相关病毒载体,实验结束后收集双侧肾组织.观察小鼠肾组织外泌体标记蛋白CD9,Hsp70,TSG101蛋白水平变化;Western blotting及免疫荧光检测小鼠肾组织Col-Ⅲ、α-SMA、FN和E-cad表达变化;HE和Masson染色观察小鼠肾组织病理学变化.细胞实验:构建尿酸刺激下的NRK-49F细胞模型(尿酸400 μmol/L,SD大鼠灌胃制备清肾颗粒含药血清用于细胞实验,分为正常组、模型组和清肾颗粒含药血清组.观察NRK-49F细胞中外泌体标记蛋白CD9,Hsp70,TSG101蛋白水平变化;Western blotting及免疫荧光检测NRK-49F细胞中Col-Ⅲ、α-SMA、FN和E-cad表达变化;双荧光素酶报告基因检测miR-330-3p与CREBBP靶向关系;RT-qPCR和Western blotting检测NRK-49F细胞中miR-330-3p与CREBBP表达变化.结果 动物实验中,Western blotting结果显示,与正常组相比,模型组CD9、Hsp70、TSG101蛋白水平升高(P<0.05);与模型组相比,清肾颗粒组CD9、Hsp70、TSG101蛋白水平降低(P<0.05).Western blotting及免疫荧光结果显示,与正常组相比,模型组Col-Ⅲ、α-SMA、FN表达增加,E-cad表达减少(P<0.05);与模型组相比,清肾颗粒组Col-Ⅲ、α-SMA、FN表达减少,E-cad表达增加(P<0.05).肾脏病理检查显示,清肾颗粒可以明显减轻小鼠肾组织纤维化(P<0.05).RT-qPCR结果显示,尾静脉注射腺相关病毒病毒载体抑制miR-330-3p表达,增加CREBBP水平,减轻肾纤维化(P<0.05).双荧光素酶报告基因结果显示,CREBBP是miR-330-3p的靶点(P<0.05).RT-qPCR和Western blotting结果显示,与正常组相比,模型组miR-330-3p表达增加,CREBBP表达减少(P<0.05);与模型组相比,清肾颗粒组miR- |
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| AbstractList | 目的 探讨清肾颗粒对腺嘌呤诱导的肾纤维化小鼠模型及尿酸刺激下NRK-49F细胞的作用及其调控外泌体、miR-330-3p和CREBBP的机制.方法 动物实验:构建腺嘌呤诱导的肾纤维化小鼠模型,分为正常组、模型组和清肾颗粒组(8.0 g·kg-1·d-1),6只/组,灌胃12周.采用尾静脉注射腺相关病毒载体,实验结束后收集双侧肾组织.观察小鼠肾组织外泌体标记蛋白CD9,Hsp70,TSG101蛋白水平变化;Western blotting及免疫荧光检测小鼠肾组织Col-Ⅲ、α-SMA、FN和E-cad表达变化;HE和Masson染色观察小鼠肾组织病理学变化.细胞实验:构建尿酸刺激下的NRK-49F细胞模型(尿酸400 μmol/L,SD大鼠灌胃制备清肾颗粒含药血清用于细胞实验,分为正常组、模型组和清肾颗粒含药血清组.观察NRK-49F细胞中外泌体标记蛋白CD9,Hsp70,TSG101蛋白水平变化;Western blotting及免疫荧光检测NRK-49F细胞中Col-Ⅲ、α-SMA、FN和E-cad表达变化;双荧光素酶报告基因检测miR-330-3p与CREBBP靶向关系;RT-qPCR和Western blotting检测NRK-49F细胞中miR-330-3p与CREBBP表达变化.结果 动物实验中,Western blotting结果显示,与正常组相比,模型组CD9、Hsp70、TSG101蛋白水平升高(P<0.05);与模型组相比,清肾颗粒组CD9、Hsp70、TSG101蛋白水平降低(P<0.05).Western blotting及免疫荧光结果显示,与正常组相比,模型组Col-Ⅲ、α-SMA、FN表达增加,E-cad表达减少(P<0.05);与模型组相比,清肾颗粒组Col-Ⅲ、α-SMA、FN表达减少,E-cad表达增加(P<0.05).肾脏病理检查显示,清肾颗粒可以明显减轻小鼠肾组织纤维化(P<0.05).RT-qPCR结果显示,尾静脉注射腺相关病毒病毒载体抑制miR-330-3p表达,增加CREBBP水平,减轻肾纤维化(P<0.05).双荧光素酶报告基因结果显示,CREBBP是miR-330-3p的靶点(P<0.05).RT-qPCR和Western blotting结果显示,与正常组相比,模型组miR-330-3p表达增加,CREBBP表达减少(P<0.05);与模型组相比,清肾颗粒组miR- |
| Abstract_FL | Objective To explore the effects of Qingshen Granules(QSG)on adenine-induced renal fibrosis in mice and in uric acid(UA)-stimulated NRK-49F cells and its mechanism for regulating exosomes,miR-330-3p and CREBBP.Methods A mouse model of adenine-induced renal fibrosis were treated daily with QSG at 8.0 g·kg-1·d-1 via gavage for 12 weeks.An adeno-associated virus vector was injected into the tail vein,and renal tissues of the mice were collected for analyzing exosomal marker proteins CD9,Hsp70,and TSG101 and expressions of Col-Ⅲ,α-SMA,FN,and E-cad using Western blotting and immunofluorescence and for observing pathological changes using HE and Masson staining.In the cell experiment,NRK-49F cells were stimulated with uric acid(400 μmol/L)followed by treatment with QSG-medicated serum from SD rats,and the changes in expressions of the exosomal markers and Col-Ⅲ,α-SMA,FN,and E-cad were analyzed.Dual luciferase reporter assay was employed to examine the targeting relationship between miR-330-3p and CREBBP,whose expressions were detected by RT-qPCR and Western blotting in treated NRK-49F cells.Results The mouse models of adenine-induced renal fibrosis showed significantly increased levels of CD9,Hsp70,and TSG101,which were decreased by treatment with QSG.The expressions of Col-Ⅲ,α-SMA,and FN increased and E-cad decreased in the mouse models but these changes were reversed by QSG treatment.QSG treatment obviously alleviated renal fibrosis in the mouse models.Intravenous injection of adeno-associated viral vector obviously inhibited miR-330-3p,increased CREBBP levels,and reduced fibrosis in the mouse models.Dual luciferase assay confirmed CREBBP as a target of miR-330-3p,which was consistent with the results of the cell experiments.Conclusion QSG inhibits renal fibrosis in mice by regulating the exosomes,reducing miR-330-3p levels,and increasing CREBBP expression. |
| Author | 刘传娇 张磊 王伟丽 陈义珍 戴荣 程梦 王亿平 葛永 曹泽平 |
| AuthorAffiliation | 安徽中医药大学第一附属医院肾内科,安徽 合肥 230031%安徽中医药大学第一临床医学院,安徽 合肥 230038 |
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| Author_FL | WANG Yiping GE Yong CHENG Meng ZHANG Lei CAO Zeping DAI Rong WANG Weili CHEN Yizhen LIU Chuanjiao |
| Author_FL_xml | – sequence: 1 fullname: DAI Rong – sequence: 2 fullname: CAO Zeping – sequence: 3 fullname: LIU Chuanjiao – sequence: 4 fullname: GE Yong – sequence: 5 fullname: CHENG Meng – sequence: 6 fullname: WANG Weili – sequence: 7 fullname: CHEN Yizhen – sequence: 8 fullname: ZHANG Lei – sequence: 9 fullname: WANG Yiping |
| Author_xml | – sequence: 1 fullname: 戴荣 – sequence: 2 fullname: 曹泽平 – sequence: 3 fullname: 刘传娇 – sequence: 4 fullname: 葛永 – sequence: 5 fullname: 程梦 – sequence: 6 fullname: 王伟丽 – sequence: 7 fullname: 陈义珍 – sequence: 8 fullname: 张磊 – sequence: 9 fullname: 王亿平 |
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| Keywords | 外泌体 exosomes Qingshen Granules 肾纤维化 miR-330-3p renal fibrosis 清肾颗粒 |
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| Publisher | 安徽中医药大学第一附属医院肾内科,安徽 合肥 230031%安徽中医药大学第一临床医学院,安徽 合肥 230038 |
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| Snippet | 目的 探讨清肾颗粒对腺嘌呤诱导的肾纤维化小鼠模型及尿酸刺激下NRK-49F细胞的作用及其调控外泌体、miR-330-3p和CREBBP的机制.方法 动物实验:构建腺嘌呤诱导的肾纤维化小鼠模型,分为正常组、模型组和清肾颗粒组(8.0... |
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| Title | 清肾颗粒通过调控外泌体、miR-330-3p以及CREBBP表达抑制小鼠肾纤维化 |
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