Hirudin Protects Ang II-Induced Myocardial Fibroblasts Fibrosis by Inhibiting the Extracellular Signal-Regulated Kinase1/2 (ERK1/2) Pathway

Abstract

BACKGROUND Myocardial fibrosis is closely related to all types of cardiovascular diseases. Hirudin is widely used in the prevention and treatment of cardiovascular diseases and cancers. In this study, we examined the potential role(s) and mechanism of hirudin in angiotensin II (Ang II)-induced myocardial fibrosis. MATERIAL AND METHODS The viability of myocardial fibroblasts, and reactive oxygen species (ROS) rates were measured respectively using cell counting kit-8 (CCK-8) and flow cytometry. Malondialdehyde (MDA) content, the activities of lactate dehydrogenase (LDH), and superoxide dismutase (SOD) were detected by the respective kits. The mRNA and protein levels of fibrosis-related factors were separately assessed by qRT-PCR and western blot. RESULTS Our data revealed that hirudin suppressed the viability of myocardial fibroblasts, and that it relieved the proliferation induced by Ang II in a dose-dependent manner. We also found that hirudin reduced ROS production, LDH activity, and MDA content; however, it enhanced SOD activity. Moreover, while hirudin significantly downregulated the levels of matrix metalloproteinase-2 (MMP-2), MMP-9, fibronectin (FN), transforming growth factor beta 1 (TGF-β1), collagen-I (COL-I), and COL-III, it upregulated the expression level of tissue inhibitor of metalloproteinases-2 (TIMP-2). Furthermore, phosphorylated extracellular signal-regulated kinase1/2 (p-ERK1/2) was decreased by hirudin, compared to the Ang-II group. CONCLUSIONS Hirudin depressed Ang II-induced myocardial fibroblasts via inhibiting oxidative stress, regulating fibrosis-related factors, and repressing the ERK1/2 pathway.

Figure 1

Morphological characteristic of myocardial fibroblasts. (A, B) Cells were cultured in DMED containing 10% FBS and 1% penicillin-streptomycin in a humidified incubator with 5% CO₂ at 37°C for 24 hours. Next day, the cells were observed and photographed under a microscope (100× and 200×).

Figure 2

Hirudin repressed the viability of Ang-II induced myocardial fibroblasts. (A) Cells were treated respectively with PBS (control), hirudin (10, 20 40, 80, 160, and 320 μg/mL) for 12, 24, and 48 hours. CCK-8 was used to detect cell viability. * P<0.05, ** P<0.01 vs. control. (B) Cells were treated respectively with PBS (control), Ang II 10⁻⁶ mol/L, hirudin 20 μg/mL+Ang II 10⁻⁶ mol/L (Hirudin1+Ang II), hirudin 40 μg/mL+Ang II 10⁻⁶ mol/L (Hirudin2+Ang II) and Hirudin 80 μg/mL+Ang II 10⁻⁶ mol/L (Hirudin3+Ang II) for 12, 24 and 48 hours. The viability of cells was measured by CCK-8. * P<0.05, ** P<0.01 vs. control. ^# P<0.05, ^## P<0.01 vs. Ang-II. Ang II – angiotensin II; PBS – phosphate buffered saline; CCK-8 – cell counting kit-8.

Figure 3

Hirudin decreased the oxidative stress of Ang II-induced myocardial fibroblasts. (A) The rate of ROS was analyzed using ROS detection kit. (B) The activity of LDH was measured by LDH assay kit. (C) The content of MDA was assessed by lipid peroxidation MDA assay kit. (D) The activity of SOD was detected by SOD assay kit. * P<0.05, ** P<0.01, *** P<0.001 vs. control. ^# P<0.05, ^## P<0.01, ^### P<0.001 vs. Ang II. ROS – reactive oxygen species; LDH – lactate dehydrogenase; MDA – malondialdehyde; SOD – superoxide dismutase, Ang II – angiotensin II.

Figure 4

Hirudin regulated fibrosis-related factors in Ang II-induced myocardial fibroblasts. (A) The relative mRNA levels of MMP-2, MMP-9, TIMP-2 was evaluated by qRT-PCR. (B) The relative protein levels of MMP-2, MMP-9, TIMP-2 was examined by western blot. (C) qRT-PCR was performed to determine the mRNA levels of FN, TGF-β1, COL-I and COL-III. (D) Western blot was carried out to detect the protein levels of FN, TGF-β1, COL-I and COL-III, and normalized to β-actin expression. Gray value was detected and counted by quality one. * P<0.05, ** P<0.01, *** P<0.001 vs. control. ^# P<0.05, ^## P<0.01, ^### P<0.001, vs. Ang II.

Figure 5

Hirudin downregulated ERK1/2 pathway in Ang II-induced myocardial fibroblasts. (A) The proteins expression levels of ERK1/2 and p-ERK1/2 were examined by western blot. (B) Quantification of protein expression was carried out with GraphPad prism 7.0. β-actin was used as internal control. The gray value was evaluated and calculated by quality one. * P<0.05, ** P<0.01, *** P<0.001 vs. control. ^# P<0.05, ^## P<0.01, ^### P<0.001, vs. Ang II.