Inhibitory Effect Of Stevioside On Myocardial Fibrosis And Its Potential Mechanisms

Objective:To investigate the inhibitory effect of stevioside on myocardial fibrosis and its potential mechanisms.Methods:The mouse cardiac fibroblasts M6300 cells were used in the vitro study and divided into control,transforming growth factor-β1(TGF-β1)stimulation,and stevioside 100,200,and 400 μM groups.After stimulation of M6300 cells with 10 μM TGF-β1 for 2 h,the cells were treated with different concentrations of stevioside for 24 h.The intracellular total mRNA and proteins were then extracted according to manufacturer’s instructions,and the expressions of α-SMA,collagen Ⅰ,collagen Ⅲ,Smad2/3,P-Smad2/3,Smad4,and Smad7 mRNA or proteins were measured by real-time PCR and western blot methods,respectively.To verify the anti-fibrotic effect of stevioside,the model animals of myocardial fibrosis were used here.Male Kunming mice were randomly divided into the control group,model group,stevioside 75,150,and 300 mg/kg groups,and positive drug captopril 25 mg/kg group.After pretreatment of the medicine-treated mice with stevioside or captopril by gavage for 3 days in the morning,these mice,like model mice,were simultaneously given isoproterenol(ISO)5 mg/kg by subcutaneous injection once in the afternoon,subsequently,the dose of ISO was reduced to 2.5 mg/kg/d for 30 days.After stopping the injection of ISO,the medicine-treated mice were continuously given stevioside or captopril by gavage for 7 days.At the end of the experiment,the cardiac weight index(CWI),glutathione peroxidase(GSH-PX)and superoxide dismutase(SOD)levels in serum and myocardial tissues,and myocardial hydroxyproline(HYP)content and cardiac morphological changes were examined.The expressions of myocardial peroxisome proliferator-activated receptor gamma(PPARy),nuclear factor κB p65(NF-κB p65),TGF-β1,α-SMA,collagen Ⅰ,collagen Ⅲ,Smad2/3,P-Smad2/3,and Smad7 proteins were determined by Western blot method.To verify the activation of PPARy by stevioside,a PPARy inhibitor GW9662 was used.After pretreatment of ISO-stimulated M6300 cells with GW9662 for 2 h,the effects of stevioside on PPARγ downstream gene NF-κB and TGF-β1/Smad signaling pathway related protein expressions were examined.Results:In vitro,the results showed that after treatment with 200-400 μM stevioside,the expressions of a-SMA,collagen Ⅰ,collagen Ⅲ,Smad2/3,P-Smad2/3,and Smad4 mRNA or proteins in M6300 cells were significantly decreased(P<0.05 or P<0.01),while these of Smad7 mRNA and protein were significantly increased(P<0.05).In vivo,after oral administration of stevioside for 40 days,the CWI and myocardial HYP content were decreased,the collagen area’s ratio of myocardial tissues was also reduced,especially in the stevioside 300 mg/kg group(P<0.01).Stevioside treatment could increase the levels of GSH-PX and SOD in serum and myocardial tissues(P<0.05 or P<0.01).Western blot analysis showed that stevioside could decrease the expressions of myocardial a-SMA,collagen Ⅰ,collagen Ⅲ,NF-κB p65,TGF-β1,Smad2/3,and P-Smad2/3 proteins(P<0.05 or P<0.01),and increase the expressions of myocardial PPARy and Smad7 proteins(P<0.05).After pretreatement of ISO-stimulated M6300 cells with PPARy inhibitor GW9662,stevioside-reduced these protein expressions were attenuated(P<0.05),but stevioside-induced PPARγ and Smad7 protein expressions were not affected.Conclusion:Stevioside possessed an inhibitory effect on myocardial fibrosis,and its main mechanisms might be related to the increments of myocardial antioxidant capacity,PPARy activation,and Smad7 expression,which might cause a synergic inhibition of the NF-κB/TGF-β1/Smad signaling pathway.