Hydrochlorothiazide ameliorates cardiac remodeling in rats with heart failure by inhibiting sodium hydrogen exchanger 1

Abstract

Abstract Background and Aim Our previous study showed that hydrochlorothiazide could improve cardiac function and myocardial fibrosis in rats with heart failure, reduce the expression of plasma inflammatory factors, and inhibit the myocardial TGF-β/Smad signaling pathway. Furosemide has no similar beneficial effect, but its mechanism must be further elucidated. Methods The rat model of heart failure was established by ligating the left anterior descending branch of the coronary artery, and hydrochlorothiazide was administered by gavage for 6 weeks. The cardiac function was evaluated using echocardiography and hemodynamics. The effects of hydrochlorothiazide on myocardial fibrosis, inflammation, oxidative stress, and apoptosis were evaluated using Masson staining, immunohistochemistry, immunofluorescence, enzyme-linked immunosorbent assay, quantitative polymerase chain reaction, and Western blot. H9C2 cells were cultured to further verify the effects of hydrochlorothiazide on inflammation, oxidative stress, and apoptosis, and explore its molecular mechanism. The molecular docking method was used to verify whether hydrochlorothiazide directly bound carbonic anhydrase (CA) Ⅱ and sodium hydrogen exchanger 1. Results Cardiac ultrasound and hemodynamics showed that hydrochlorothiazide could improve cardiac function in rats with heart failure. Masson staining showed that hydrochlorothiazide could improve the degree of interstitial fibrosis in noninfarcted myocardium. Immunohistochemistry showed that hydrochlorothiazide could reduce the deposition of collagens Ⅰ and Ⅲ in the cardiac interstitium. Further, hydrochlorothiazide could reduce mRNA and protein expression of myocardial collagen Ⅰ. In terms of inflammation, hydrochlorothiazide could reduce the levels of plasma endothelin-1 and C-reactive protein. Immunohistochemistry showed that hydrochlorothiazide could reduce the expression of MCP-1 in the myocardium. Moreover, hydrochlorothiazide could inhibit mRNA and protein expression of NF-кB p65. In terms of oxidative stress, hydrochlorothiazide could reduce the level of plasma MDA and increase the level of SOD, total antioxidant capacity, and mitochondrial respiratory chain complexes Ⅰ and Ⅳ. Immunohistochemistry showed that hydrochlorothiazide could reduce the expression of p47phox in the myocardium. Further, hydrochlorothiazide could inhibit mRNA and protein expression of p47phox and p67phox and increase SOD expression. In terms of apoptosis, terminal deoxynucleotidyl transferase–mediated dUTP nick-end labelingstaining, caspase3 immunofluorescence, and cytochrome c immunohistochemistry showed that hydrochlorothiazide could reduce cardiomyocyte apoptosis. Moreover, hydrochlorothiazide could inhibit mRNA and protein expression of Bax and caspase-3 in the myocardium. In H9C2 cells, hydrochlorothiazide had similar effects in terms of improving inflammation, oxidative stress, and apoptosis. In vivo and in vitro, hydrochlorothiazide could inhibit CAII, sodium/hydrogen exchanger 1 (NHE1), and p38 mitogen–activated protein kinase (p38 MAPK)/c-Jun N-terminal kinase (JNK) signaling pathways. The gene silencing of CAⅡ and NHE1 by siRNA resulted in the suppression of NCX1, NF-кB p65, p47phox, p67phox, and Bax protein expression. H9C2 cells were transfected with NHE1 overexpression plasmids by lentiviral transduction, and the results revealed that hydrochlorothiazide could inhibit the protein expression of NCX1, NF-кB p65, Bax, p38 MAPK, and JNK. Molecular docking simulation showed that hydrochlorothiazide had a strong binding effect on CAⅡ and NHE1 of myocardial cells. Conclusions Hydrochlorothiazide reduced intracellular calcium overload by inhibiting CAⅡ and NHE1, thereby reducing oxidative stress. It further inhibited the p38 MAPK/JNK signaling pathway, inflammation, and apoptosis, finally improving cardiac function and cardiac fibrosis remodeling in rats with heart failure.