Kanglexin protects against cardiac fibrosis and dysfunction in mice by TGF-β1/ERK1/2 noncanonical pathway

Figures

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  FIGURE 1. The chemical structure of KLX.

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  FIGURE 2. KLX alleviates cardiac dysfunction in mice of TAC. Cardiac function including LVEF% (A) and LVFS% (B) was investigated by echocardiographic analysis (n = 5 in each group). (C) Cumulative mouse mortality data of control (n = 35), vehicle (n = 41), or KLX administration at dose of 20 mg/kg/d (n = 38) were recorded at each time point after TAC operation. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with control group and # p < 0.05 compared with TAC + vehicle group.

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  FIGURE 3. Role of KLX on TAC-induced cardiac hypertrophy. (A) Hearts from control, TAC + vehicle, and TAC + KLX (20, 10, and 5 mg/kg/d) groups are shown. (B) Heart tissues stained with HE, magnification, ×200 and scale bar = 100 µm. The black arrows point to hypertrophic cardiomyocytes. (C) The ratio of HW/BW revealed that KLX ameliorates cardiac index changes in TAC-operated mice (n = 9 in each group). (D) Quantitative data revealed that long-term administration of KLX dose-dependently decreased the cell surface area (CSA) during pressure overload (n = 5 in each group). (E) Pressure overload increased the transcription of ANP, BNP, and β-MHC compared with controls, which was dramatically suppressed by long-term administration of KLX (n = 5 in each group). *p < 0.05, **p < 0.01, and ***p < 0.001 compared with control group and # p < 0.05, ## p < 0.01, and ### p < 0.001 compared with TAC + vehicle group.

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  FIGURE 4. Role of KLX on cardiac fibrosis. (A) Heart tissues stained with Masson’s trichrome. Blue areas indicate collagen deposition, magnification, ×200 and scale bar = 100 µm. (B) Quantitative data showed that collagen synthesis and deposition increased markedly in TAC-operated groups compared with controls in both perivascular area and intramyocardial area; KLX administration at dose of 20 mg/kg/d strongly prevented collagen accumulation (n = 5 in each group). Pressure overload significantly upregulated collagen Ⅰ and collagen Ⅲ transcription (C; n = 5 in each group) and cardiac hydroxyproline content compared with controls (D; n = 9 in each group), which was significantly suppressed by long-term administration of KLX in a dose-dependent manner. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with control group and # p < 0.05, ## p < 0.01, and ### p < 0.001 compared with TAC + vehicle group.

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  FIGURE 5. KLX relieves fibroblast activation and function induced by TGF-β1 in primary cultured adult mouse CFs. (A) KLX showed no significant effect on cell viability of CFs. TGF-β1 significantly upregulated α-SMA expression (B), proliferation (C), and collagen secretion (D) in adult mouse CFs compared with untreated cells, which was concentration-dependently suppressed by KLX cotreatment (n = 3–4 in independent experiments). **p < 0.01 and ***p < 0.001 compared with control group and # p < 0.05 and ## p < 0.01 compared with TAC + vehicle group.

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  FIGURE 6. KLX alleviates EndMT in TAC-pressured heart. (A) Western blot analysis of CD31, VE-cadherin, vimentin, and α-SMA in mouse hearts. The relative protein levels of CD31 (B), VE-cadherin (C), vimentin (D), and α-SMA (E) in mouse hearts (n = 4 in each group). *p < 0.05, **p < 0.01, and ***p < 0.001 compared with control group and # p < 0.05 and ### p < 0.001 compared with TAC + vehicle group.

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  FIGURE 7. KLX suppresses EndMT stimulated by TGF-β1 in HUVECs. (A) Representative immunofluorescence images showing staining of endothelial marker CD31 and mesenchymal marker vimentin in different treatment of HUVECs, magnification, ×200 and scale bar = 100 µm. (B) KLX showed no significant effect on cell viability of HUVECs. TGF-β1 significantly increased cell proliferation (C) and collagen secretion (D) in HUVECs compared with untreated cells, which was concentration-dependently suppressed by KLX cotreatment (n = 3–4 in independent experiments). *p < 0.05 and ***p < 0.001 compared with control group and # p < 0.05 and ## p < 0.01 compared with TAC + vehicle group.

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  FIGURE 8. The effect of KLX on canonical and noncanonical pathways of TGF-β1 signaling. (A) ELISA results showed that cardiac TGF-β1 protein expression was upregulated in long-term pressure overload heart compared with the control cohort, which was reversed by KLX administration (n = 7 in each group). (B) The expression levels of p-Smad2/3, t-Smad2/3, and Smad4 in mouse hearts were measured by Western blot. TAC-operated hearts showed no significant P-Smad2/3 protein expression in total, cytoplasmic, and nuclear fractions. TAC-operated hearts exhibited no significant nuclear expression of Smad4. TGF-β1-stimulated HUVECs, as a positive control, showed a highlighted protein band of p-Samd2/3, indicating activation of Smad2/3. (C) The relative expression levels of p-ERK1/2 and t-ERK1/2 in mouse hearts were measured by Western blot. TAC operation significantly increased ERK phosphorylation compared with controls, which was suppressed by KLX administration (n = 5 in each group). **p < 0.01 and ***p < 0.001 compared with control group and ## p < 0.01 compared with TAC + vehicle group.

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  FIGURE 9. Schematic diagram of the underlying mechanisms by which KLX exerts a regulatory role in cardiac fibrosis.