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

doi:10.12122/j.issn.1673-4254.2024.08.01

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (8): 1431-1440.doi: 10.12122/j.issn.1673-4254.2024.08.01

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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

戴荣¹(), 曹泽平², 刘传娇², 葛永², 程梦¹, 王伟丽², 陈义珍², 张磊¹(), 王亿平¹()

^1.安徽中医药大学第一附属医院肾内科，安徽合肥 230031
^2.安徽中医药大学第一临床医学院，安徽合肥 230038

收稿日期:2024-04-02 出版日期:2024-08-20 发布日期:2024-09-06
通讯作者: 张磊,王亿平 E-mail:azydairong@163.com;zhang0551lei@163.com;wypwyp54@aliyun.com
作者简介:戴荣，博士，医师，E-mail: azydairong@163.com
基金资助:
国家自然科学基金(82274307);合肥市科技局生命健康专项(GJ2022SM03);2022临床医学研究转化专项(202104j07020014);安徽省自然科学基金面上项目(2308085MH292);安徽省高等学校科学研究重点项目(2023AH050749);2023年度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科研项目(2023CXMMTCM018)

Qingshen Granules alleviates renal fibrosis in mice by regulating exosomes, miR-330-3p, and CREBBP expression

Rong DAI¹(), Zeping CAO², Chuanjiao LIU², Yong GE², Meng CHENG¹, Weili WANG², Yizhen CHEN², Lei ZHANG¹(), Yiping WANG¹()

^1.Department of Nephrology, First Affiliated Hospital of Anhui University of Traditional Chinese Medicine, Hefei 230031, China
^2.First Clinical College of Anhui University of Traditional Chinese Medicine, Hefei 230038, China

Received:2024-04-02 Online:2024-08-20 Published:2024-09-06
Contact: Lei ZHANG, Yiping WANG E-mail:azydairong@163.com;zhang0551lei@163.com;wypwyp54@aliyun.com
Supported by:
National Natural Science Foundation of China(82274307)

摘要/Abstract

摘要：

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-330-3p表达减少，CREBBP表达增加（P<0.05）。细胞实验结果与动物实验结果趋势一致。结论清肾颗粒通过干预外泌体，降低miR-330-3p水平，增加CREBBP表达抑制肾纤维化。

关键词: 清肾颗粒, 外泌体, miR-330-3p, 肾纤维化

Abstract:

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^-1via 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.

Key words: Qingshen Granules, exosomes, miR-330-3p, renal fibrosis

戴荣, 曹泽平, 刘传娇, 葛永, 程梦, 王伟丽, 陈义珍, 张磊, 王亿平. 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[J]. 南方医科大学学报, 2024, 44(8): 1431-1440.

Rong DAI, Zeping CAO, Chuanjiao LIU, Yong GE, Meng CHENG, Weili WANG, Yizhen CHEN, Lei ZHANG, Yiping WANG. Qingshen Granules alleviates renal fibrosis in mice by regulating exosomes, miR-330-3p, and CREBBP expression[J]. Journal of Southern Medical University, 2024, 44(8): 1431-1440.

图/表 9

图1 清肾颗粒可以减轻肾纤维化小鼠肾组织外泌体分泌

Fig.1 Qingshen Granules (QSG) inhibits renal exosome secretion in a mouse model of renal fibrosis. A, B: Western blotting and quantitative analysis of exosome marker proteins CD63, Hsp70 and TSG101 in the renal tissues of each group (n=6). *P<0.05 vs Sham group; #P<0.05 vs Model group.

图2 清肾颗粒对小鼠肾纤维化的影响

Fig.2 Effect of QSG on renal fibrosis in mice. A, B: Western blotting and quantitative analysis of expressions of Col-III, α-SMA, FN and E-cad in the renal tissues in each group (n=6). C, D: Immunofluorescence analysis of Col-III, α-SMA, FN and E-cad and the quantitative data (n=6). Scale bar=50 µm. E, F: HE and Masson staining and the quantitative data (n=6). Scale bar = 50 µm. *P<0.05 vs Sham group; #P<0.05 vs Model group.

图3 抑制miR-330-3p表达,减轻肾纤维化

Fig.3 Inhibition of miR-330-3p expression attenuates renal fibrosis in mice. A-E: Western blotting and quantitative data of Col-III, α-SMA, FN and E-cad expressions in mouse renal tissues in each group (n=6). F, G: Immunofluorescence analysis of Col-III and FN and the quantitative data (n=6). Scale bar=50 µm. *P<0.05 vs Sham group; #P<0.05 vs Model group.

图4 抑制miR-330-3p,CREBBP表达增加

Fig.4 Inhibition of miR-330-3p increases CREBBP expression in the renal tissues of the mouse models. A, B: Western blotting and quantitative data of CREBBP in mouse kidneys (n=6). C: RT-qPCR of CREBBP mRNA (n=6). *P<0.05 vs Sham group, #P<0.05 vs Model group.

图5 CREBBP是miR-330-3p的靶点

Fig.5 CREBBP is a target of miR-330-3p. A: miR-330-3p binding sites predicted in the 3'-UTR of CREBBP mRNA. B: Luciferase activity of NRK-49F cells after transfection with NC or miR-330-3p mimics and reporter vectors containing the CREBBP mutant binding sequence. #P<0.05.

图6 清肾颗粒抑制小鼠肾组织miR-330-3p表达, 增加CREBBP水平减轻肾纤维化

Fig.6 QSG inhibits miR-330-3p expression in mouse kidney tissue and increases CREBBP levels to attenuate renal fibrosis. A: Renal miR-330-3p levels in mice in each group. B: Renal CREBBP mRNA levels in mice in each group. C, D: Western blotting of protein expression and quantitative data of CREBBP in the renal tissues of the mice. *P<0.05 vs Sham group; #P<0.05 vs Model group.

图7 清肾颗粒含药血清可以减轻尿酸刺激下NRK-49F细胞外泌体分泌

Fig.7 QSG-medicated rat serum attenuates uric acid-stimulated exosome secretion from NRK-49F cells. A, B: Western blotting and quantitative data of exosome marker proteins CD63, Hsp70 and TSG101 in each group. *P<0.05 vs Sham group; #P<0.05 vs Model group.

图8 清肾颗粒含药血清对NRK-49F细胞活化的影响

Fig.8 Effect of QSG-medicated rat serum on activation of NRK-49F cells. A, B: Western blotting of protein expression levels of Col-III, α-SMA, FN and E-cad in NRK-49F cells treated with QSG-medicated serum. C, D: Immunofluorescence micrographs showing Col-III and FN expressions in NRK-49F cells treated with QSG-medicated serum. Scale bar=50 µm. *P<0.05 vs Sham group; #P<0.05 vs Model group.

图9 清肾颗粒含药血清降低miR-330-3p表达, 增加CREBBP水平抑制NRK-49F细胞活化

Fig.9 QSG-medicated rat serum decreases miR-330-3p expression and increases CREBBP level to inhibit NRK-49F cell activation. A: miR-330-3p levels in NRK-49F cells treated with QSG-medicated serum. B: CREBBP mRNA levels in NRK-49F cells treated with QSG-medicated serum. C, D: Western blotting of protein expression of CREBBP in NRK-49F cells treated with QSG-medicated serum. *P<0.05 vs Sham group; #P<0.05 vs Model group.

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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

Qingshen Granules alleviates renal fibrosis in mice by regulating exosomes, miR-330-3p, and CREBBP expression

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