α7 nicotinic acetylcholine receptor agonist reduced myocardial fibrosis possibly through inhibiting p38 mitogen activated protein kinase signaling pathway

Abstract

Objective To investigate the role of α7 nicotinic acetylcholine receptor (α7nAchR) agonist in myocardial fibrosis and its possible mechanism. Methods The cardiac fibroblasts were isolated and cultured from neonatal rats. The cardiac fibroblasts were divided into the following four groups: blank control group (cardiac fibroblasts without any intervention); model group [cardiac fibroblasts treated with 10-6 mol/L angiotensin Ⅱ (Ang Ⅱ) only]; α7nAchR agonist group (cardiac fibroblasts treated with 5×10-6 mol/L PNU-282987, and 1 h later treated with 10-6 mol/L Ang Ⅱ); α7nAchR antagonist group (cardiac fibroblasts treated with 10-6 mol/L MLA, and 1 h later treated with 10-6 mol/L Ang Ⅱ). The proliferation abilities of cardiac fibroblasts in different treatment groups were measured using WST-1 method. The expression levels of α7nAchR genes in cardiac fibroblasts in different treatment groups were examined by real-time fluorescent quantitative polymerase chain reaction (FQ-PCR). The expression levels of α7nAchR, type Ⅰ and type Ⅲ collagen, α-smooth muscle actin (α-SMA), p38 mitogen activated protein kinase (p38MAPK) and phosphorylated p38MAPK (p-p38MAPK) proteins in cardiac fibroblasts were detected by Western blotting. Results (1) The final absorbance in the α7nAchR agonist group (0.50±0.13) was significantly lower than α7nAchR antagonist group and model group (0.75±0.10, 0.63±0.11, F=10.567, P=0.000). It suggested that activation of α7nAchR could inhibit the proliferation of cardiac fibroblasts. (2) The relative expression levels of α7nAchR mRNA and protein in α7nAchR agonist group (0.87±0.15, 0.76±0.08) were significantly higher than the α7nAchR antagonist group (0.45±0.09, 0.40±0.14), model group (0.62±0.11, 0.59±0.10) and blank control group (0.32±0.13, 0.30±0.07, F=25.402, P=0.000). It suggested that α7nAchR agonists can upregulate the expression of α7nAchR gene in cardiac fibroblasts. (3) The relative expression levels of type Ⅰ and type Ⅲ collagen proteins, α-SMA protein and p-p38MAPK protein in α7nAchR agonist group (0.53±0.09, 0.50±0.12, 0.38±0.08, 0.27±0.09) were lower than the model group (0.65±0.12, 0.62±0.10, 0.57±0.11, 0.45±0.11) and α7nAchR antagonist group (0.81±0.13, 0.71±0.11, 0.70±0.10, 0.68±0.08), while were higher than the blank control group (0.41±0.08, 0.35±0.06, 0.19±0.07, 0.16±0.07, F=29.647, 32.962, 30.549, 53.665, P=0.000, 0.000, 0.000, 0.000). The relative expression of p38MAPK protein in the α7nAchR agonist group (0.71±0.12) was higher than the model group (0.52±0.10) and α7nAchR antagonist group (0.35 ± 0.09), but was lower than the blank control group (0.85 ± 0.14, F=44.347, P=0.000). Conclusion α7nAchR agonists could inhibit the proliferation of cardiac fibroblasts and reduce the synthesis of collagen between cells. The mechanism might be related to inhibition of p38MAPK signaling pathway activation. Key words: Cardiac fibroblasts; α 7 nicotinic acetylcholine receptor agonist; Fibrosis; p38 mitogen-activated protein kinase signaling pathway