Exogenous H2S mitigates myocardial fibrosis in diabetic rats through suppression of the canonical Wnt pathway

Abstract

Hydrogen sulfide (H2S) has antifibrotic activity in the kidneys, heart, lungs, and other organs. The present study investigated the protective activity of exogenous H2S against myocardial fibrosis in a rat model of diabetes. Animals were assigned to normal control, diabetes mellitus (DM), DM + sodium hydrosulfide (NaHS; DM + NaHS) and NaHS groups. Fasting blood glucose (FBG), cardiac function and hydroxyproline were monitored. Heart histomorphology and ultrastructure were additionally evaluated. Wnt1‑inducible signaling pathway protein (WISP)‑1 protein expression in the myocardium was determined by immunohistochemical staining. Matrix metalloprotease (MMP)‑2, tissue inhibitor of metalloproteinase (TIMP)‑2, collagens, and canonical Wnt and transforming growth factor (TGF)‑β1/SMAD family member 3 (Smad3) pathway‑related proteins were assessed by western blotting. Cardiac function was decreased, and myocardial injury, hypertrophy and fibrosis were increased in the diabetes model rats. MMP‑2 expression was decreased, and the expressions of WISP‑1, TIMP‑2, collagens, and canonical Wnt and TGF‑β1/Smad3 pathway‑related proteins were increased in the myocardia of the diabetes model rats. The present results indicated that the canonical Wnt pathway promoted diabetic myocardial fibrosis by upregulating the TGF‑β1/Smad3 pathway. Except for FBG, exogenous H2S ameliorated the changes in diabetes‑associated indices in rats in the DM + NaHS group. The results are consistent with H2S protection of streptozotocin‑induced myocardial fibrosis in the diabetes model rats by downregulation of the canonical Wnt and TGF‑β1/Smad3 pathway and decreased myocardial collagen deposition.

Figure 1

Effects of exogenous hydrogen sulfide on FBG and HW/BW. (A) FBG, (B) BW, (C) HW and (D) HW/BW in the different groups. Data are presented as the mean ± SD. n=8/group. ^**P<0.01 vs. NC group; ^##P<0.01 vs. DM group. FBG, fasting blood glucose; BW, body weight; HW, heart weight; HW/BW, heart weight/body weight; NC, normal control; DM, diabetes mellitus; NaHS, sodium hydrosulfide.

Figure 2

Effect of exogenous hydrogen sulfide on cardiac function in the different groups. (A) LVSP, (B) LVEDP, (C) +dp/dt_max, (D) -dp/dt_max in the different groups. Data are presented as the mean ± SD. n=8/group. ^**P<0.01 vs. NC group; ^##P<0.01 vs. DM group. LVSP, left ventricular systolic pressure; LVEDP, left ventricular end diastolic pressure; +dp/dt_max, maximal rise rate of left ventricular pressure; −dp/dt_max, maximal fall rate of left ventricular pressure; NC, normal control; DM, diabetes mellitus; NaHS, sodium hydrosulfide.

Figure 3

Effects of exogenous hydrogen sulfide on histological and ultrastructural alterations in myocardial tissues. (A) Representative H&E staining of myocardial tissues. (B) Representative transmission electron micrographs of myocardial tissues. Arrows indicate the swollen mitochondria. H&E, hematoxylin and eosin; NC, normal control; DM, diabetes mellitus; NaHS, sodium hydrosulfide; TEM, transmission electron microscope.

Figure 4

Effect of exogenous hydrogen sulfide on collagen deposition in myocardial tissues. (A) Representative Masson's trichrome staining of myocardial tissues. Levels of (B) CVF and (C) Hyp content in the different groups. Data are presented as the mean ± SD. n=8/group. ^**P<0.01 vs. NC group; ^##P<0.01 vs. DM group. CVF, collagen volume fraction; Hyp, hydroxyproline; NC, normal control; DM, diabetes mellitus; NaHS, sodium hydrosulfide.

Figure 5

Effect of exogenous hydrogen sulfide on extracellular matrix-associated proteins and the TGF-β1/Smad3 pathway in myocardial tissues. (A) Protein expression levels of MMP-2, TIMP-2, collagen-I and collagen-III in myocardium. (B) Expression levels of TGF-β1/Smad3 pathway-related proteins in myocardium. Data are presented as the mean ± SD. n=8/group. ^**P<0.01 vs. NC group; ^##P<0.01 vs. DM group. TGF-β1, transforming growth factor-β1; Smad3, SMAD family member 3; MMP-2, matrix metalloproteinase-2; TIMP-2, tissue inhibitor of metalloproteinase-2; NC, normal control; DM, diabetes mellitus; NaHS, sodium hydrosulfide; p-, phosphorylated.

Figure 6

Effect of exogenous hydrogen sulfide on the canonical Wnt pathway and WISP-1 expression in myocardial tissues. (A) Western blot analysis of the protein expression of β-catenin, GSK-3β and p-GSK-3β. (B) Immunohistochemical study of WISP-1 was performed in myocardial tissues. (C) Ratio of WISP-1-positive cells in each group. Data are presented as the mean ± SD. n=8/group. ^**P<0.01 vs. NC group; ^##P<0.01 vs. DM group. WISP-1, Wnt1-inducible signaling pathway protein-1; GSK-3β, glycogen synthase kinase-3β; p-, phosphorylated; NC, normal control; DM, diabetes mellitus; NaHS, sodium hydrosulfide.

Figure 7

Possible antifibrotic mechanism of exogenous hydrogen sulfide in diabetic cardiomyocytes. H₂S, hydrogen sulfide; TGF-β1, transforming growth factor-β1; R, receptor; GSK-3β, glycogen synthase kinase-3β; APC, adenomatous polyposis coli; p-, phosphorylated; Smad3, SMAD family member 3; P, phosphate; TCF/LEF, T-cell factor/lymphoid enhancer factor; WISP-1, Wnt1-inducible signaling pathway protein-1; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase.