Investigation Of Involvment Of Aldehyde Dehydrogenase-2 And Wnt/β-Catenin Pathway In The Inhibitory Effects Of Magnolol On The Proliferation And Collagen Synthesis Of Cardiac Fibroblasts

Myocardial fibrosis is a pathological change which is characterized by the proliferation of cardiac fibroblasts(CFs)as well as the extreme accumulation of extracellular matrix.There pathologic changes closely related to varieties of cardiovascular diseases,such as hypertension,hypertrophic cardiomyopathy,dilated cardiomyopathy,viral myocarditis,affecting the systolic and diastolic function of the heart,and eventually leading to heart failure.ALDH2 and Wnt/β-catenin pathway play an important role in myocardial fibrosis.In our previous experiments,we butt the ALDH2 molecular protein with a number of Chinese herbs through molecular docking.The results showed that Mag could dock with the active site of ALDH2 and affect the activity of ALDH2.Many modern studies demonstrated that Mag exerted protective effects on cardiovascular and attenuated the progression of myocardial fibrosis,particularly the myocardial apoptosis caused by ischemia/reperfusion injury.However,the effects and mechanism of Mag on the myocardial fibrosis remains unclear.Here,the involvement of aldehyde dehydrogenase-2(ALDH2)on the inhibitory functions of Mag on proliferation,collagen synthesis and Wnt/β-catenin activation in cardiac fibroblasts(CFs)was investigated.Effect of Mag on proliferation of CFs was assessed by MTT and EdU assays,followed by detection of the expression of proliferation-associated proteins,Cyclin D1 and CDK4.The interaction between Mag and ALDH2 was measured using molecular docking as well as molecular dynamics(MD)simulations.A commercial kit was purchased to assess ALDH2 enzymatic activity.Daidzin,an ALDH2 inhibitor,was utilized to verify that ALDH2 was involved in the suppressive functions of proliferation,collagen synthesis.Effects of Mag on the collagen synthesis and activation of Wnt/β-catenin pathway were evaluated by detecting the expression levels of type I and III collagen,and the phosphorylated-GSK-3β(p-GSK-3β)and β-catenin,respectively.Treatment with Mag significantly decreased cell viability of CFs in a dose-and time-dependent pattern.Accordingly,the EDU positive cell ratio and expression levels of proliferation-related proteins,including Cyclin D1 and CDK4,were significantly reduced by Mag in a dose-dependent manner.Additionally,administration with Mag significantly inhibited the expressions of type I and III collagen.Molecular docking(MD)simulation demonstrated that Mag could dock into the agonist binding site of ALDH2.The binding energy between ALDH2 and Mag was-7.91 kcal/mol.MD simulations showed the stability of the binding of ALDH2-Mag complex.Treatment with Mag dose-dependently enhanced the ALDH2 activity without alteration of ALDH2 protein expression.Moreover,inhibition of ALDH2 by ALDH2 inhibitor(Daidzin)raised the cell viability,EdU positive cell ratio as well as the expressions of type Ⅰ,Ⅲ collagen,p-GSK-3β and-catenin in CFs.Co-treatment with Daidzin significantly negated the inhibitory actions of Mag on CFs proliferation and collagen synthesis.In addition,the expressions of p-GSK-3 β and β-catenin were significantly reduced by Mag treatment in a dose-dependent manner.Co-treatment with Daidzin partially abolished the inhibitory actions of Mag on p-GSK-3(3 and β-catenin in CFs.In summary,Mag might acts as a natural ALDH2 agonist,and exerts the inhibitory actions on proliferation,collagen synthesis and activation of Wnt/β-catenin signaling in CFs.