QiShenYiQi Pill Ameliorates Cardiac Fibrosis After Pressure Overload-Induced Cardiac Hypertrophy by Regulating FHL2 and the Macrophage RP S19/TGF-β1 Signaling Pathway

Abstract

Purpose: Heart failure (HF) is a leading cause of morbidity and mortality worldwide, and it is characterized by cardiac hypertrophy and fibrosis. However, effective treatments are not available to block cardiac fibrosis after cardiac hypertrophy. The QiShenYiQi pill (QSYQ) is an effective treatment for chronic HF. However, the underlying mechanism remains unclear. Methods: In the present study, a pressure overload-induced cardiac hypertrophy model was established in rats by inducing ascending aortic stenosis for 4 weeks. QSYQ was administered for 6 weeks, and its effects on cardiac fibrosis, myocardial apoptosis, RP S19 release, macrophage polarization, TGF-β1 production, and TGF-β1/Smad signaling were analyzed. In vitro studies using H9C2, Raw264.7, and RDF cell models were performed to confirm the in vivo study findings and evaluate the contribution to the observed effects of the main ingredients of QSYQ, namely, astragaloside IV, notoginsenoside R1, 3,4-dihydroxyl-phenyl lactic acid, and Dalbergia odorifera T. C. Chen oil. The role of four-and-a-half LIM domains protein 2 (FHL2) in cardiac fibrosis and QSYQ's effects were assessed by small interfering RNAs (siRNAs). Results: QSYQ ameliorated cardiac fibrosis after pressure overload-induced cardiac hypertrophy and attenuated cardiomyocyte apoptosis, low FHL2 expression, and TGF-β1 release by the injured myocardium. QSYQ also inhibited the following: release of RP S19 from the injured myocardium, activation of C5a receptors in monocytes, polarization of macrophages, and release of TGF-β1. Moreover, QSYQ downregulated TGF-βR-II expression induced by TGF-β1 in fibroblasts and inhibited Smad protein activation and collagen release and deposition. Conclusion: The results showed that QSYQ inhibited myocardial fibrosis after pressure overload, which was mediated by RP S19-TGF-β1 signaling and decreased FHL2, thus providing support for QSYQ as a promising therapy for blocking myocardial fibrosis.

Keywords: FHL2; QiShenYiQi pill; RP S19; SMADs; TGF-β1; cardiac fibrosis; heart failure; pressure overload.

FIGURE 1

QSYQ post-treatment inhibited myocardial fiber rupture and cardiac fibrosis. (A) Effects of QSYQ post-treatment on the ultrastructure of myocardial tissues of rats in the Sham4W (a1), Sham10W (a2), Sham10W + TMZ (a3), Sham10W + QSYQ (a4), AAS4W (a5), AAS10W (a6), AAS10W + TMZ (a7), and AAS10W + TMZ (a8) groups. Scale bar = 1 μm. (B) Representative images of Masson’s trichrome staining in the Sham4W (b1 and c1), Sham10W (b2 and c2), Sham10W + TMZ (b3 and c3), Sham10W + QSYQ (b4 and c4), AAS4W (b5 and c5), AAS10W (b6 and c6), AAS10W + TMZ (b7 and c7), and AAS10W + TMZ (b8 and c8) groups. Scale bar = 200 μm. (C,D) Quantification results of interstitial fibrosis and perivascular fibrosis measured by Masson’s trichrome staining. Data are presented as the mean ± SEM, n = 3. ^* p < 0.05 vs. sham; ^# p < 0.05 vs. AAS. AAS, ascending aortic stenosis; C: cardiac myofibril; M: mitochondrial.

FIGURE 2

Effects of QSYQ and its main ingredients on cardiomyocyte apoptosis both in vivo and in vitro. (A) TUNEL staining of rat heart sections in the Sham4W (a1), Sham10W (a2), Sham10W + TMZ (a3), Sham10W + QSYQ (a4), AAS4W (a5), AAS10W (a6), AAS10W + TMZ (a7), and AAS10W + TMZ (a8) groups. Scale bar = 25 μm. (B) Quantification result of TUNEL-positive cardiomyocytes in rat hearts. Data are presented as the mean ± SEM, n = 3. (C) Serum lactate dehydrogenase (LDH) level detected by ELISA in different groups. (D) Serum malondialdehyde (MDA) level detected by ELISA in the different groups. (E) Western blot of apoptosis-related proteins: cleaved caspase-3/caspase-3, cleaved caspase-9/caspase-9, Bax, Bcl-2, and Bax/Bcl-2. (F–J) Quantification analyses of the Western blot results for cleaved caspase-3/caspase-3, cleaved caspase-9/caspase-9, Bax, Bcl-2, and Bax/Bcl-2. Data are presented as the mean ± SEM. n = 4. (K) TUNEL and phalloidin staining of H9C2 cells in the control (a1, b1, c1, and d1), Ang II (a2, b2, c2, and d2), Ang II + TMZ (a3, b3, c3, and d3), Ang II + QSYQ (a4, b4, c4, and d4), Ang II + ASIV (a5, b5, c5, and d5), Ang II + R1 (a6, b6, c6, and d6), Ang II + DLA (a7, b7, c7, and d7), and Ang II + DO (a8, b8, c8, and d8) groups. Bar = 25 μm. (L) Quantification result of TUNEL-positive cells. Data are presented as the mean ± SEM, n = 3. (M) Lactate dehydrogenase (LDH) level detected in supernatants of H9C2 cells by ELISA. (N) Malondialdehyde (MDA) level detected in H9C2 cell lysates by ELSA. Data are presented as the mean ± SEM, n = 4. (O) Western blot of apoptosis-related proteins in H9C2 cells from the different groups: cleaved caspase-3/caspase-3, cleaved caspase-9/caspase-9, Bax, and Bcl-2. (P–R) Quantification of the Western blot results for cleaved caspase 3/caspase 3, cleaved caspase-9/caspase 9, and Bax/Bcl-2 in H9C2 cells from the different groups. Data are presented as the mean ± SEM, n = 4. ^* p < 0.05 vs. control; ^# p < 0.05 vs. Ang II.

FIGURE 3

QSYQ reduced the release of chemokine RP S19 and inhibited its colocalization with the C5a receptor. (A,B) Rat serum RP S19 and MCP-1 levels detected by ELISA in rats from different groups. (C) Representative photomicrographs of immunohistochemistry staining for RP S19 in rat myocardium in the Sham4W (c1), Sham10W (c2), Sham10W + TMZ (c3), Sham10W + QSYQ (c4), AAS4W (c5), AAS10W (c6), AAS10W + TMZ (c7), and AAS10W + QSYQ (a8) groups. (D) Quantitative analysis results of RP S19 positive area. Arrows indicate the RP S19-positive area. Bar = 50 μm. Data are presented as the mean ± SEM, n = 3. (E,F) RP S19 and MCP-1 levels detected by ELISA in the H9C2 cell supernatants. Data are presented as the mean ± SEM, n = 4. (G) Immunofluorescence images of C5aR, RP S19 and Raw264.7 monocyte nucleus in the control (a1, b1, c1, and d1), RP S19 (a2, b2, c2, and d2), RP S19 + TMZ (a3, b3, c3, and d3), RP S19 + QSYQ (a4, b4, c4, and d4), RP S19 + ASIV (a5, b5, c5, and d5), RP S19 + R1 (a6, b6, c6, and d6), RP S19 + DLA (a7, b7, c7, and d7), and RP S19 + DO (a8, b8, c8, and d8) groups. Scale bar = 25 μm. Data are presented as the mean ± SEM, n = 3. (H) Western blotting analysis of Raw264.7 monocyte C5aR. Data are presented as the mean ± SEM, n = 4. ^* p < 0.05 vs. sham or control; ^# p < 0.05 vs. AAS or RP S19.

FIGURE 4

QSYQ inhibited monocyte infiltration, macrophage polarization and TGF-β1 expression. The four panels on the left show the immunohistochemistry staining results for CD68 (A,B), CD80 (C,D), CD163 (E,F), and TGF-β1 (G,H) in the Sham4W (a1, b1, c1, and d1), Sham10W (a2, b2, c2, and d2), Sham10W + TMZ (a3, b3, c3, and d3), Sham10W + QSYQ (a4, b4, c4, and d4), AAS4W (a5, b5, c5, and d5), AAS10W (a6, b6, c6, and d6), AAS10W + TMZ (a7, b7, c7, and d7), and AAS10W + QSYQ (a8, b8, c8, and d8) groups in myocardium and its quantification analysis results, respectively. Bar = 50 μm. Data are presented as the mean ± SEM, n = 6. (I) Serum TGF-β1 level detected by ELISA. (J) TGF-β1 level in rat myocardial tissue detected by ELISA. Data are presented as the mean ± SEM, n = 4. (K,L) Protein level of TGF-β1 in Raw264.7 cells detected using Western blot. Data are presented as the mean ± SEM, n = 4. ^* p < 0.05 vs. sham or control; ^# p < 0.05 vs. AAS or RP S19.

FIGURE 5

QSYQ inhibited AAS-induced increases of TGF-β1 and its receptor TGFβR II as well as activation of Smads. (A) Immunofluorescence images of TGFβR II, TGFβR I, and nucleus in RDF cells in the control (a1, b1, c1, and d1), TGF-β1 (a2, b2, c2, and d2), TGF-β1+TMZ (a3, b3, c3, and d3), TGF-β1+QSYQ (a4, b4, c4, and d4), TGF-β1+ASIV (a5, b5, c5, and d5), TGF-β1+R1 (a6, b6, c6, and d6), TGF-β1+DLA (a7, b7, c7, and d7), and TGF-β1+DO (a8, b8, c8, and d8) groups. (B) Expression of TGFβR II, p-Smad3, and Smad3 in RDF cells examined by Western blot. (C,D) Quantification of the Western blot results for TGFβR II and p-Smad3/Smad3. Data are presented as the mean ± SEM, n = 4. (E) Immunofluorescence images of p-Smad3 and nuclei in RDF cells. Scale bar = 25 μm. (F) Expression of TGF-β1, TGFβR II, p-Smad3, Smad4, Smad6, and Smad7 in rat myocardium examined by Western blot. (G–L) Quantification of the Western blot results for TGF-β1, TGFβR II, p-Smad3/Smad3, Smad4, Smad6, and Smad7 in Panel 5E. Data are presented as the mean ± SEM, n = 4. ^* p < 0.05 vs. sham or control; ^# p < 0.05 vs. AAS or TGF-β1.

FIGURE 6

QSYQ regulated abnormal expression of MMP/TIMP. (A) Expression of MMP2, MMP9, TIMP1, TIMP2, and cathepsin B in rat myocardium examined by Western blotting. (B–F) Quantification of Western blot results of MMP2, MMP9, TIMP1, TIMP2, and cathepsin (B). (G) Expression of MMP2, MMP9, TIMP1, and TIMP2 in RDF cells examined by Western blot. (H–K) Quantification of Western blot results for MMP2, MMP9, TIMP1, and TIMP2. Data are presented as the mean ± SEM, n = 4. ^* p < 0.05 vs. sham or control; ^# p < 0.05 vs. AAS or TGF-β1.

FIGURE 7

QSYQ ameliorated collagen production both in vivo and in vitro. (A) Expression of α-SMA, collagen I and collagen III in rat myocardium examined by Western blot. (B–D) Quantification of the Western blot results for α-SMA, collagen I and collagen III. Data are presented as the mean ± SEM, n = 4. (E,F) Serum collagen I and collagen III levels detected by ELISA. (G,H) Collagen I and collagen III levels in TGF-β1-stimulated RDF cell supernatants examined by ELISA. Data are presented as the mean ± SEM, n = 6. ^* p < 0.05 vs. sham or control; ^# p < 0.05 vs. AAS or TGF-β1.

FIGURE 8

QSYQ inhibited cardiac myocyte release of TGF-β1 in a FHL2 dependent manner. (A) Immunofluorescence images of FHL2, phalloidin, and nuclei in rat myocardium in the Sham4W (a1, b1, c1, and d1), Sham10W (a2, b2, c2, and d2), Sham10W + TMZ (a3, b3, c3, and d3), Sham10W + QSYQ (a4, b4, c4, and d4), AAS4W (a5, b5, c5, and d5), AAS10W (a6, b6, c6, and d6), AAS10W + TMZ (a7, b7, c7, and d7), and AAS10W + QSYQ (a8, b8, c8, and d8) groups. Bar = 25 μm. (B) Expression of FHL2 in rat myocardium examined by Western blot with quantification shown below. (C) Expression of FHL2 in 1 µM Ang II stimulated H9C2 cells examined by Western blot, with quantification shown below. (D) Expression of FHL2 in FHL2 knock-downed H9C2 cells using siRNA examined by Western blot, with quantification shown below. (E) Expression of FHL2 and TGF-β1 in FHL2 knock-down H9C2 cells examined by Western blot. (F) Quantification of the results presented previously. Data are presented as the mean ± SEM, n = 4. ^* p < 0.05 vs. sham or control; ^# p < 0.05 vs. AAS or Ang II. (G) Immunofluorescence images of FHL2, TGF-β1, and nucleus in H9C2 cells in NC-siRNA: NC (a1, b1, c1, and d1), Ang II (a2, b2, c2, and d2), Ang II + QSYQ (a3, b3, c3, and d3) and FHL2-siRNA: NC (a’1, b’1, c’1, and d’1), Ang II (a’2, b’2, c’2, and d’2), and Ang II + QSYQ (a’3, b’3, c’3, and d’3). Scale bar = 25 μm.