Qingshen Granules inhibits renal fibrosis in mice by regulating glycolytic reprogramming and H3K18 lactylation

doi:10.12122/j.issn.1673-4254.2026.03.12

Abstract

Abstract:

Objective To investigate the effects of Qingshen Granules (QSG) on folic acid (FA)-induced renal fibrosis in mice and TGF-β1-stimulated HK-2 cells and the underlying mechanism. Methods A mouse model of FA-induced renal fibrosis were treated daily with QSG at 4.0 g·kg^-¹ via gavage for 4 weeks. Renal histopathological changes were evaluated using HE and Masson staining, and renal expressions of fibrosis markers, key glycolytic pathway proteins, and histone lactylation-related proteins (Pan Kla, and lactylation sites of H3/H4) were detected using Western blotting. Renal expressions of α-SMA, FN, E-cad, LDHA, and H3K18 lactylation (H3K18la) were also examined with immunofluorescence staining. Serum creatinine (SCR) and blood urea nitrogen (BUN) were measured by ELISA, and renal lactate content was determined with colorimetric assay. In HK-2 cells stimulated with TGF-β1, the effects of QSG-medicated serum from SD rats on cell viability, proliferation, protein expressions of α‑SMA, E-cad, LDHA, and H3K18la, extracellular oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and intracellular lactate levels were analyzed. Results The mouse models of renal fibrosis showed increased renal expressions of α‑SMA, FN, HIF-1α, HK2, PFKM, PKM2, LDHA, Pan Kla, and H3K18la, elevated serum SCR and BUN and renal lactate level, and decreased E-cad expression. QSG treatment effectively reversed these changes and alleviated renal fibrosis in the mouse models. Immunofluorescence staining showed that QSG treatment significantly reversed the elevation of renal α‑SMA, FN and LDHA expressions and reduction of LDHA/H3K18la co-localization, and enhanced E-cad expression. In HK-2 cells, TGF‑β1 treatment significantly reduced cell viability, proliferation and OCR, and increased ECAR, lactate, α‑SMA, LDHA, and H3K18la, which were all reversed by treatment with QSG-medicated serum. Exogenous lactate obviously reversed the inhibitory effects of QSG on these proteins. Conclusion QSG alleviates renal fibrosis in mice by modulating glycolytic reprogramming, reducing lactate accumulation, and inhibiting H3K18la.

Key words: Qingshen Granules, glycolytic reprogra-mming, histone lactylation, renal fibrosis

Yizhen CHEN, Weili WANG, Meng CHENG, Wei ZHANG, Yilin GAO, Xin HONG, Lei ZHANG, Rong DAI, Yiping WANG. Qingshen Granules inhibits renal fibrosis in mice by regulating glycolytic reprogramming and H3K18 lactylation[J]. Journal of Southern Medical University, 2026, 46(3): 582-591.

Figures/Tables 6

Fig.1 QSG inhibits FA-induced renal fibrosis in mice. A, B: HE and Masson staining (Scale bar=50 µm) and the quantitative data (n=6). C, D: Western blotting and quantitative analysis of expression levels of α-SMA, FN and E-cad in the renal tissues in each group (n=3). E, F: Immunofluorescence staining of α-SMA, FN and E-cad (Scale bar=50 µm) and the quantitative data (n=6). G, H: Serum creatinine (SCR) and blood urea nitrogen (BUN) levels of the mice (n=6). *P<0.05 vs Vehicle group; #P<0.05 vs FA group.

Fig.2 QSG regulates glycolytic reprogramming in renal tissues of the mice with FA-induced renal fibrosis. A, B: Western blotting and quantitative analysis of expression levels of HIF-1α, HK2, PFKM, PKM2 and LDHA in the renal tissues in each group (n=3). C, D: Immunofluorescence analysis of LDHA (Scale bar=50 µm) and the quantitative data (n=6). *P<0.05 vs Vehicle group; #P<0.05 vs FA group.

Fig.3 QSG inhibits lactate accumulation and histone lactylation in renal tissues of the mice. A: Renal tissue lactate levels in each group (n=6). B: Western blotting of Pan Kla in the renal tissues in each group (n=3). C, D:Western blotting and quantitative analysis of expression levels of H3K9la, H3K14la, H3K18la, H3K23la, H3K56la, H4K5la, H4K8la, H4K12la and H4K16la in the renal tissues in each group (n=3). E: Immunofluorescence co-staining of LDHA (green) and H3K18la (red) in renal tissues (Scale bar=20 µm). F: Quantitative analysis of Pearson colocalization coefficient for LDHA/H3K18la in the renal tissues (n=6). *P<0.05 vs Vehicle group; #P<0.05 vs FA group.

Fig.4 QSG-medicated serum inhibits TGF-β1-induced fibrosis in HK-2 cells. A: Cell viability of HK-2 cells treated with different concentrations of QSG (n=6). B: Cell viability of TGF‑β1-stimulated HK-2 cells treated with different concentrations of QSG (n=6). C, D: EdU cell proliferation assay (Scale bar=100 μm; n=3). E, F: Western blotting and quantitative analysis of expression levels of α-SMA and E-cad in the renal tissues in each group (n=3). *P<0.05 vs Control group; #P<0.05 vs TGF-β1 group.

Fig.5 QSG-medicated serum regulates TGF-β1-induced glycolytic reprogramming in HK-2 cells. A, B: Changes in extracellular oxygen consumption rate (OCR) in HK-2 cells in each group (n=3). C, D: Changes in extracellular acidification rate (ECAR) in HK-2 cells in each group (n=3). *P<0.05 vs Control group; #P<0.05 vs TGF-β1 group.

Fig.6 QSG-medicated serum inhibits TGF-β1-induced lactate accumulation and histone lactylation in HK-2 cells. A: Renal tissue lactate levels in each group (n=6). B, C: Western blotting and quantitative analysis of expression levels of LDHA, H3K18la and α‑SMA in the HK-2 cell in each group (n=3). *P<0.05 vs Control group; #P<0.05 vs TGF‑β1 group; †P<0.05 vs QSG-medicated serum group.

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