Dai R, Cao Z, Liu C, Ge Y, Cheng M, Wang W, Chen Y, Zhang L, Wang Y
Department of Nephrology, First Affiliated Hospital of Anhui University of Traditional Chinese Medicine, Hefei 230031, China.
First Clinical College of Anhui University of Traditional Chinese Medicine, Hefei 230038, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2024 Aug 20;44(8):1431-1440. doi: 10.12122/j.issn.1673-4254.2024.08.01.
To explore the effects of 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.
A mouse model of adenine-induced renal fibrosis were treated daily with QSG at 8.0 g·kg·d gavage for 12 weeks. An adenoassociated 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, -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, -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.
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, -SMA, and FN increased and Ecad 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.
QSG inhibits renal fibrosis in mice by regulating the exosomes, reducing miR-330-3p levels, and increasing CREBBP expression.
探讨芪参颗粒（QSG）对腺嘌呤诱导的小鼠肾纤维化以及对尿酸（UA）刺激的NRK-49F细胞的影响，及其对外泌体、miR-330-3p和CREBBP的调控机制。
将腺嘌呤诱导的肾纤维化小鼠模型每日以8.0 g·kg⁻¹·d⁻¹的剂量经口灌胃给予QSG，持续12周。通过尾静脉注射腺相关病毒载体，收集小鼠肾组织，采用蛋白质免疫印迹法和免疫荧光法分析外泌体标志物蛋白CD9、Hsp70和TSG101以及Col-III、α-SMA、FN和E-cad的表达，并采用苏木精-伊红（HE）染色和Masson染色观察病理变化。在细胞实验中，用尿酸（400 μmol/L）刺激NRK-49F细胞，然后用SD大鼠含QSG的含药血清处理，分析外泌体标志物以及Col-III、α-SMA、FN和E-cad表达的变化。采用双荧光素酶报告基因检测法检测miR-330-3p与CREBBP之间的靶向关系，并用逆转录-定量聚合酶链反应（RT-qPCR）和蛋白质免疫印迹法检测经处理的NRK-49F细胞中它们的表达。
腺嘌呤诱导的肾纤维化小鼠模型中CD9、Hsp70和TSG101水平显著升高，QSG处理可使其降低。小鼠模型中Col-III、α-SMA和FN的表达增加，E-cad表达降低，但QSG处理可逆转这些变化。QSG处理明显减轻了小鼠模型中的肾纤维化。静脉注射腺相关病毒载体明显抑制了miR-330-3p，提高了CREBBP水平，并减轻了小鼠模型中的纤维化。双荧光素酶检测证实CREBBP是miR-330-3p的靶标，这与细胞实验结果一致。
QSG通过调节外泌体、降低miR-330-3p水平和增加CREBBP表达来抑制小鼠肾纤维化。
