Xuefu Zhuyu Decoction Reduces Excessive Collagen Deposition and Crosslinking in Myocardial Fibrosis via CTR1–Copper–LOX Axis

Objective To explore the molecular mechanism of Xuefu Zhuyu Decoction (XFZYD) against myocardial fibrosis. Methods The active components of XFZYD were identified by high performance liquid chromatography (HPLC). Network pharmacology, molecular docking, and affinity dot-mass spectrometry (AD-MS) were used to predict and validate the binding interaction between XFZYD components and copper transporter 1 (CTR1). Sixty male Kunming mice were randomly divided into 6 groups using a random number table. Control, isoproterenol hydrochloride (ISO), captopril, low-, medium-, and high-dose XFZYD (5.69, 11.38, and 22.76 g/kg) groups, with 10 mice in each group. Except the control group, mice received daily subcutaneous injection of ISO (10 mg/kg) for 28 d to induce myocardial fibrosis, concurrently treated with respective drugs by gavage. Cardiac function was assessed by echocardiography (ECG) and electrocardiography (UCG). Serum myocardial injury markers and transforming growth factor beta 1 (TGF-β 1) were measured by a biochemical analyzer and enzyme-linked Immunosorbent assay (ELISA). Pathological changes and collagen deposition was observed using HE, Masson and sirius red staining methods. Copper content in cardiac tissue was determined by inductively coupled plasma mass spectrometry (ICP-MS), and protein expressions of collagen I, III, alpha-smooth muscle actin (α-SMA), lysyl oxidase (LOX), CTR1, copper chaperone for superoxide dismutase (CCS), and cytochrome c oxidase 17 (COX17) were detected by Western blot, immunohistochemistry, and immunofluorescence. In vitro, TGF-β 1 (5 ng/mL)-stimulated NIH-3T3 fibroblasts were treated with XFZYD (60 mg/L), with or without CuCl2 (20 µ mol/L) or SLC31A1 overexpression for 24 h, and the same indicators were evaluated. Results HPLC identified 7 major components in XFZYD. Network pharmacology revealed that XFZYD targets copper metabolism related genes. Molecular docking showed strong binding affinities between the 7 compounds and CTR1, and AD-MS confirmed direct binding of paeoniflorin and amygdalin to CTR1. In vivo, XFZYD could ameliorate the collagen deposition, cardiac dysfunction, and elevated levels of collagen I/III, α-SMA, LOX, and copper content exhibited in ISO-induced myocardial fibrosis mice (P<0.05 or P<0.01). In vitro, XFZYD had the effects of suppressing copper overload and the upregulation of LOX, collagen I/III, and α-SMA in TGF β 1 stimulated NIH-3T3 fibroblasts, whereas co-treatment with CuCl2 exacerbates these fibrotic responses and attenuates the protective effect of XFZYD (P<0.05 or P<0.01). XFZYD had the effect of partially reversing the enhanced copper accumulation, LOX activation, and collagen deposition induced by SLC31A1 (CTR1) overexpression in TGF β 1 stimulated fibroblasts, and SLC31A1 overexpression reduces its efficacy (P<0.05 or P<0.01). Conclusions XFZYD exerted a protective effect against ISO-induced myocardial fibrosis. The mechanism may be related to regulating the CTR1–copper–LOX axis, thereby reducing excessive collagen deposition and cross-linking in cardiac fibroblasts.