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

doi:10.12122/j.issn.1673-4254.2024.08.01

Abstract

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

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.

Figures/Tables 9

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.

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.

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.

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.

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.

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.

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.

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.

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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