ROCK1 inhibitors(Medical University of Silesia)

The mechanisms underlying environmental endocrine disruptors (EEDs)-induced cardiovascular diseases (CVDs) are not well understood. Specifically, it is unclear whether nonylphenol (NP) exposure regulates myocardial fibrosis (MF) by activating myosin light chain (MLC) through the Ras homolog gene family member A (RhoA)/Rho-associated, coiled-coil containing protein kinase 1 (ROCK1) pathway. To confirm that NP induced MF and explore the underlying mechanism via the RhoA/ROCK1/MLC-signaling pathway. Male Sprague-Dawley (SD) rats were used in the in vitro experiments. Primary cardiac fibroblasts (CFs) were extracted and treated with recombinant human transforming growth factor-beta 1 (TGF-β1) for 24 h to establish a fibrosis model. The first phase of the experiment comprised three groups: control, NP-exposure (30 µmol/L NP), and model (10 ng/mL TGF-β1). The second phase involved five groups: control, ROCK1-inhibitor (50 µmol/L, Fas), NP+ROCK1-inhibitor (30 µmol/L NP+50 µmol/L Fas), NP-exposure (30 µmol/L NP), and model (10 ng/mL TGF-β1). For the in vivo experiment, the animal experiments included 60 male SD rats who were categorized into five groups of 12 rats each, as follows: control (corn oil), ROCK1-inhibitor (10 mg/kg/day Fas), NP+ROCK1-inhibitor (50 mg/kg/day NP+10 mg/kg/day Fas), NP-exposure (50 mg/kg/day NP), and model (isoprenaline hydrochloride). NP was administered for 90 days via gavage. Fas was injected intraperitoneally for 30 days, and isoprenaline hydrochloride (ISO) was injected subcutaneously on the back for 10 days. In vitro, when compared with the control group, the protein expression of MF markers (i.e., collagen I, collagen III, and α-SMA) and RhoA/ROCK1/MLC-signaling pathway markers (i.e., TGF-β1, RhoA, ROCK1, MLC, and p-MLC) increased in the NF-exposed CFs. Following intervention with the inhibitor, in comparison to the NP group, the protein and fluorescence expression of TGF-β1, RhoA, ROCK1, MLC, and p-MLC were alleviated in the NP+ROCK1-inhibitor group. In vivo, the NP group exhibited mitochondrial damage, disorganized myocardial fibers, abnormal collagen deposition, and increased collagen fibers, whereas the Fas+NP group exhibited an alleviation in these pathological changes. As opposed to the control group, both systolic and diastolic blood pressure levels were elevated in the NP exposure and model groups, reduced in the Fas group, and alleviated in the NP+Fas group. The protein expression of MF markers (i.e., collagen I, collagen III, and α-smooth muscle actin [α-SMA]) increased in the NP-exposure group, which was alleviated in the NP+inhibitor group. In comparison with the NP group, TGF-β1, RhoA, ROCK1, and MLC mRNA expression, whereas the TGF-β1, RhoA, ROCK1, MLC, and p-MLC protein expression were alleviated in the NP+ROCK1-inhibitor group. NP exposure may induce MF by activating the RhoA/ROCK1/MLC-signaling pathway.