JiangyaTongluo decoction ameliorates tubulointerstitial fibrosis via regulating the SIRT1/PGC-1α/mitophagy axis in hypertensive nephropathy

Abstract

Introduction: With the increasing prevalence of hypertension, the incidence of kidney diseases is also increasing, resulting in a serious public burden. Jiangya Tongluo decoction (JYTL), a recognized prescription in traditional Chinese medicine (TCM), is commonly used to calm an overactive liver and reduce excess yang, while also promoting blood flow to alleviate obstructions in the meridians. Previous research has indicated that JYTL may help mitigate kidney damage caused by hypertension; however, the underlying mechanisms have not been thoroughly assessed.

Methods: First, an amalgamation of UPLC-QE/MS and network pharmacology techniques was employed to pinpoint potential active components, primary targets, and crucial action mechanisms of JYTL in treating hypertensive nephropathy (HN). Then, we used spontaneous hypertensive rats (SHRs) and Wistar-Kyoto rats (WKYs) to evaluate the efficacy of JYTL on HN with valsartan as a positive reference. We also conducted DCFH-DA fluorescence staining in rat renal tissues to detect the level of ROS. Western blotting and immunohistochemistry were performed to investigate further the effect of JYTL decoction on key targets and signaling pathways.

Results: Through UPLC-QE/MS and network analysis, 189 active ingredients and 5 hub targets were identified from JYTL. GSEA in the MitoCarta3.0 database and PPI network analysis revealed that JYTL predominantly engages in the Sirt1-mitophagy signaling pathway. Tanshinone iia, quercetin, and adenosine in JYTL are the main active ingredients for treating HN. In vivo validation showed that JYTL decoction could improve kidney function, ameliorate tubulointerstitial fibrosis (TIF), and improve mitochondrial function by inhibiting ROS production and regulating mitochondrial dynamics in SHRs. JYTL treatment could also increase the expression of SIRT1, PGC-1α, Nrf1, and TFAM, and activate PINK1/Parkin-mediated mitophagy.

Conclusion: JYTL decoction may exert renal function protective and anti-fibrosis effects in HN by ameliorating mitochondrial function and regulating the SIRT1/PGC-1α-mitophagy pathway.

Keywords: Chinese medicine; SIRT1; hypertensive nephropathy; mitochondrial dysfunction; mitophagy; tubulointerstitial fibrosis.

FIGURE 1

Chemical ingredients analysis of JYTL decoction. Total ion chromatography in positive (A) and negative (B) ion modes for JYTL samples and blank serum and dosed serum samples in positive (C) and negative (D) modes are shown. Among them, red represents administered serum, and green represents blank serum.

FIGURE 2

Target genes of active ingredients in JYTL and HN-associated microarray data analysis. (A) Network analysis depicting the relationship between JYTL components and their target genes. (B) Batch effect before and after correction. (C) Volcano plot of differentially expressed genes, with red indicating upregulation of differential expression and blue indicating downregulation of differential expression. (D–F) Results of GSEA of gene ontology (GO) for biological processes (BP), cellular components (CC), and molecular functions (MF) of differentially expressed genes. (G) KEGG pathway enrichment analysis of differentially expressed genes. (H) Annotation of significant MitoPathways by GSEA analyses.

FIGURE 3

Prediction of disease targets of JYTL in HN through network pharmacology. (A) The common target genes of JYTL and HN. (B, C) PPI network for common targets. The darker the color, the greater the degree value, indicating the gene was more likely to be the core gene. (D) Enrichment analysis of GO for BP, CC, and MF of common genes. (E) KEGG pathway enrichment analysis of common genes.

FIGURE 4

Effect of JYTL Decoction on blood pressure and renal function in SHRs. Changes of Systolic blood pressure (A) and Diastolic blood pressure (B) in different groups since the start of the gavage intervention; The detection of Scr (C), BUN (D), UA (E), and 24-h urinary protein quantification (F) levels in circulation (n = 8); (G). Comparisons of renal index among different groups (n = 8), renal index = kidney weight (G)/body weight (G). Data were presented as means ± SEM. Compared to the WKY rats group, **p < 0.01, compared to the model group, #p < 0.05, ##p < 0.01. The * and ^# in the figure below represent the same comparisons.

FIGURE 5

JYTL decoction treatment attenuated renal injury in SHRs. (A) H&E and Masson’s trichrome were performed to evaluate the general morphological changes of kidneys. The magnification of the images is ×400, scale bar = 50 μm. (B) A semi-quantitative analysis of the collagen areas according to Masson’s trichrome staining. (C–F) Expression levels of TGF-β1, a-SMA, and COLⅢ in the kidney were detected by immunohistochemistry, and the optical intensity of the abovementioned proteins was measured (n = 6). The magnification of the images is ×400, scale bar = 75 μm. Data were presented as means ± SEM (n = 6).

FIGURE 6

JYTL improves mitochondrial function and morphology. (A) DCFH-DA fluorescent staining for detecting reactive oxygen species (ROS). (B) Image-J-based quantification of DCFH-DA fluorescent intensity. Data are shown as the means ± SD from three independent experiments.^△△ denote statistical significance at the P < 0.01 level compared to the JYTL group. (C) ATP content in rat renal tissues. The data is represented by Mean ± SD (n = 6). **P < 0.01 versus the WKY rats group; ^## P < 0.01 versus the model group.^△ P < 0.05, ^△△ P < 0.01 versus the JYTL group. (D) Transmission electron microscopy (TEM) images of mitochondrial morphology in the renal cortex following pharmacological intervention (n = 3). The red triangles indicate mitophagosomes. Green-boxed areas are enlarged and presented in the bottom panel. Red arrows indicate damaged mitochondria. Scale bars: 2 μm (top panel); 500 nm (bottom panel).

FIGURE 7

JYTL decoction modulated the expression of proteins involved in mitochondrial dynamics. (A) Expression levels of mitochondrial fusion protein Mfn1, Mfn2, and OPA1 and mitochondrial fission protein Drp1, Mff, and Fis in the kidney were detected by immunohistochemistry. (B–G) The analytical result of the abovementioned proteins was measured. The magnification of the images is ×400, scale bar = 75 μm. Data were presented as means ± SEM (n = 6). **P < 0.01 versus the WKY rats group; ^## P < 0.01 versus the model group.^△ P < 0.05, ^△△ P < 0.01 versus the JYTL group.

FIGURE 8

JYTL decoction promotes mitochondrial biogenesis in SHRs. (A–E) The expression of SIRT1, PGC-1α, Nrf1, and TFAM by Western blot (n = 3) and analyzed semi-quantitatively. Data are presented as mean ± standard deviation and analyzed using one-way ANOVA, followed by Tukey’s multiple comparison test for the post hoc test. (F–I) Levels of SIRT1 (F) and Nrf1 (G) in the renal cortex of rats were detected using immunohistochemistry. The boxed areas are enlarged and presented in the bottom panel. The magnification of the images is ×5 (top panel) and ×200 (bottom panel). Scale bars: 500 μm and 100 μm respectively. Data are expressed as means ± SEM; **P < 0.01 versus the WKY rats group; ^# P < 0.05, ^## P < 0.01 versus the model group.^△△ P < 0.01 versus the JYTL group.

FIGURE 9

JYTL decoction promotes PINK1/Parkin-Mediated Mitophagy by Activating the SIRT1 signaling. (A–E) Representative Western blot analysis of PINK1, Parkin, Beclin1, and P62 in kidney tissue (n = 3). (F) Representative TEM images of mitophagy in rats (n = 3). Red quadrilateral indicated autophagosomes (scale bar: 2 μm).