Ershen Zhenwu Decoction suppresses myocardial fibrosis of chronic heart failure with heart–kidney Yang deficiency by down-regulating the Ras Homolog Gene Family Member A/Rho-Associated Coiled-Coil Kinases signaling pathway

The therapeutic efficacy of Ershen Zhenwu Decoction (ESZWD)—a famous formulation from Xin'an for patients with chronic heart failure heart–kidney Yang deficiency (CHF-HKYD)—is well established. Still, the underlying molecular mechanism is not clear. This study investigated mechanisms by which ESZWD suppresses cardiac pathology, including myocardial fibrosis, in CHF-HKYD model rats and Ang II-stimulated cardiac fibroblasts (CFs). The components in ESZWD were analyzed by ultra-high-performance liquid chromatography coupled with Quadrupole Time-Of-Flight mass spectrometry (UHPLC-Q-TOF-MS). CHF-HKYD model was established in the male Sprague–Dawley rats through bilateral thyroidectomy and intraperitoneal administration of 0.02% doxorubicin (DOX), twice weekly for 3 weeks. Subsequently, the CHF-HKYD model rats were randomly categorized into the Model, ESZWD-L (3.96 g/kg/d ESZWD), ESZWD-M (7.92 g/kg/d ESZWD), ESZWD-H (15.84 g/kg/d ESZWD), and Sac/Val (68 mg/kg/d sacubitril/valsartan) groups and treated daily for 4 weeks. As a control, the sham surgery group (Sham) was used. Primary cardiac fibroblasts (CFs) were categorized into Control, Model, ESZWD, and Sac/Val groups. Then, the CFs were stimulated with Ang-II. The ESZWD and Sac/Val groups were incubated with different concentrations of drug-containing sera and their effects on CF viability were assessed via the CCK-8 assay. The ESZWD and Sac/Val groups received drug-containing serum concentrations determined by CCK-8 assay results. The cardioprotective effects of ESZWD were determined using echocardiography, Hematoxylin & Eosin (H&E) staining, Masson staining, and Sirius red staining, and the Enzyme Linked Immunosorbent Assay (ELISA). ESZWD's effects on the Ras Homolog Gene Family Member A (RhoA)/Rho-Associated Coiled-Coil Kinases (ROCKs) signaling pathway and myocardial fibrosis were assessed by Western blotting and Quantitative Real-Time PCR (qRT-PCR) analyses. Immunofluorescence was used to observe fibrotic markers in CFs. ESZWD treatment improved cardiac function in the CHF-HKYD rats by significantly reducing myocardial fibrosis and ventricular remodeling. ESZWD treatment increased the rats' body temperature (Tb) and 24-h urine volume, left ventricular ejection fraction (LVEF) and LV fractional shortening (LVFS), and decreased LV internal systolic diameter (LVIDs), LV internal diastolic diameter (LVIDd), and heart weight/body weight (HW/BW) compared to the Model group. In comparison to the model rats, ESZW