Explore The Effect Of β3-AR On Fibrosis In Cardiac Fibroblasts Induced By AngⅡ And Its Potential Mechanism

Objective: We use Ang II induce fibrosis of cardiac fibroblasts and intervene cardiac fibroblasts with lentivirus which containes β3-AR, β3-AR agonist and antagonist to observe the influence on fibrosis of β3-AR and to discuss β3-AR and the TGF-β/Smad signaling pathway in fibroblasts on myocardial fibrosis. We want to prove that the activation of β3-AR may mediate fibrosis of cardiac fibroblasts through the TGF-β/Smad signaling pathway and may aggravate myocardial remodeling.Methods: Differential adhesion was used to collect primary cardiac fibroblasts. We used the methods of lentivirus which containes β3-AR, β3-AR agonist and antagonist to intervene myocardial fibrosis.The best infection rate that lentivirus infection in cardiac fibroblasts was detected by the method of Annexin V-FITC/PI double staining. We want to selecte the most suitable MOI that lentivirus infection in cardiac fibroblasts. Extract kit method were used to extract cardiac fibroblasts β3-AR membrane proteins and total proteins. The BCA method was applied for protein quantification. Relative content of β3-AR membrane protein, fibrosis related proteins and proteins in TGF-β/Smad signaling pathway in each sample were detected by western blot to observe the difference between each group.We use the method of q RT-PCR to observe the m RNA expression of these factors in each group. The method of WST-1 to detect the proliferation rate of cardiac fibroblasts in each group.The first grouping method:normal control group: cardiac fibroblasts that without any intervention; lentivirus infection group: cardiac fibroblasts was intervened one hours with lentivirus which containes β3-AR then we cultivate it 72 hours with Ang II; empty lentivirus control group: cardiac fibroblasts was intervened one hours with lentivirus that without β3-AR then we cultivate it 72 hours with Ang II; Ang II group: cardiac fibroblasts was cultivated 72 hours with Ang II. The second grouping method: normal control group: cardiac fibroblasts that without any intervention;β3-AR agonist group: cardiac fibroblasts was intervened one hours with β3-AR agonist then we cultivate it 72 hours with Ang II; β3-AR antagonist group: cardiac fibroblasts was intervened one hours with β3-AR antagonist then we cultivate it 72 hours with Ang II; Ang II group: cardiac fibroblasts was cultivated 72 hours with AngII.Results: 1. Lentivirus infected cardiac fibroblasts successfully, the most suitable MOI is100 and 72 hours is the best intervention time. 2. The western blot result showed that: the expression of β3-AR protein in groups with lentivirus and β3-AR agonist increased more than Ang II group and normal control group(P<0.05). The group of lentivirus is a little higher thanβ3-AR agonist group(P>0.05). The expression of β3-AR protein in β3-AR antagonist group is lower than that in β3-AR agonist group and Ang II group, but higher than normal control group(P<0.05). 3. The western blot result also showed that: the proteins expression of TGF-β/Smad signaling pathway and fibrosis related proteins such as TGF-β1-R, TGF-β1,p-Smad-2, COL-I and COL-III in the group of lentivirus increased more than empty lentivirus control group, Ang II group and normal control group(P<0.05). There is no significant difference between empty lentivirus control group and Ang II group(P>0.05), but they all higher than that in normal control group(P<0.05). The expression of those proteins in β3-AR agonist group is higher than that in Ang II group, β3-AR antagonist group and normal control group(P<0.05). The expression of those proteins in β3-AR antagonist group is lower than that in Ang II group, but higher than that in normal control group(P<0.05). 4. The method of q RT-PCR result showed that: the m RNA expression of TGF-β1、COL-I、COL-III in the group of lentivirus is increased more than empty lentivirus control group, Ang II group and normal control group(P<0.05). There is no significant difference between empty lentivirus control group and Ang II group(P>0.05), but they all higher than that in normal control group(P<0.05). The expression of those m RNA in β3-AR agonist group is higher than that in Ang II group, β3-AR antagonist group and normal control group(P<0.05). The expression of those m RNA in β3-AR antagonist group is lower than that in Ang II group, but higher than that in normal control group(P<0.05). 5. The method of WST-1 result showed that: The proliferation level of cardiac fibroblasts in lentivirus group is higher than empty lentivirus control group,Ang II group and normal control group(P<0.05). There is no significant difference between empty lentivirus control group and Ang II group(P>0.05), but they all higher than that in normal control group(P<0.05). The proliferation level of cardiac fibroblasts in β3-AR agonist group is higher than that in Ang II group, β3-AR antagonist group and normal control group(P<0.05). The proliferation level of cardiac fibroblasts in β3-AR antagonist group is lower than that in Ang II group, but higher than that in normal control group(P<0.05).Conclusion: 1. Methods of β3-AR agonist and lentivirus which containes β3-AR can induceβ3-AR of cardiac fibroblasts overexpression successfully. 2. Overexpression of β3-AR in cardiac fibroblasts can promote proliferation and fibrosis. 3. Overexpression of β3-AR may mediate the fibrosis of cardiac fibroblasts through the signaling pathway of TGF-β/Smad,promote myocardial remodeling and contribute to the progression of heart failure.