The Role And Mechanism Of Gentianella Acuta And Its Active Component(Bellidifolin) On Myocardial Fibrosis

Myocardial fibrosis(MF),which is a morphological feature of ventricular remodeling.Acute myocardial infarction(MI)is one of the most common diseases,which lead to ventricular remodeling and myocardial fibrosis.Inflammation is contributed to the whole process of myocardial fibrosis after infarction.Transforming growth factor(TGF)-β is one of the most important pro-inflammatory factors and pro-fibrosis factors,which mediates the fibrosis through the canonical and non-canonical signaling pathway.Inhibition of TGF-β signaling may be an effective strategy to alleviate myocardial fibrosis.According to the clinical characteristics of coronary heart disease and myocardial infarction,it is assigned to chest stuffiness and angina pectoris in traditional Chinese medicine.Heat-toxin is considered one of the key pathogenesis of chest stuffiness and angina pectoris,and the method of clearing the heat-toxin is effective in the clinical treatment of myocardial infarction.Gentianella acuta(G.acuta)belongs to the Gentianaceae,which has the effect of clearing the heat-toxin,and it is effective in treating angina pectoris in Inner Mongolia of China.Studies have shown that G.acuta and its active components have anti-inflammatory,antioxidant,antibacterial and a protective effect on the heart.However,it is not clear whether G.acuta and its active components can prevent myocardial fibrosis by repressing TGF-β signaling pathway.Therefore,the aim of this study was to elucidate the role and mechanism of G.acuta and bellidifolin,one of active components of G.acuta,on myocardial fibrosis by suppressing TGF-β signaling pathway in vivo and in vitro.Part one Gentianella acuta improves isoproterenol(ISO)-induced rat myocardial fibrosis by inhibiting the TGF-β1/Smads signaling pathwayObjective: To investigate the effects of G.acuta on rat myocardial fibrosis,and explore the underlying mechanism of G.acuta anti-myocardial fibrosis via repressing TGF-β/Smads signaling pathway.Methods: The model of myocardial fibrosis was established as described previously.Briefly,ISO(5 mg/kg/day)was injected subcutaneous into rats for7 days.A total of 50 rats were divided into the following 5 groups at random:the control group(Control),the model group(ISO),the low-dose drug intervention group(ISO + G.acuta 0.3 g/kg),the middle-dose drug intervention group(ISO + G.acuta 0.6 g/kg)and the high-dose drug intervention group(ISO+ G.acuta 1.2 g/kg).Histopathological changes in the heart were observed using HE,Masson and WGA(Wheat germ agglutinin)staining.The expression of Collagen Ⅰ,Ⅲ,α-SMA,TGF-β1,Smad2,3 and 4 were investigated by immunohistochemical staining in heart tissue.The expression of Collagen Ⅰ,Ⅲ,α-SMA,TGF-β1,TβRI,Ⅱ,Smad2,3,4 and the levels of TβRI,Ⅱ,Smad2,3phosphorylation were analyzed by Western blotting.Results:1.G.acuta ameliorates ISO-induced rat myocardial fibrosis and cardiac remodelingCompared with the control group,heart tissues from ISO-treated rats exhibited widespread myocardial structural disorder,necrotic degeneration,and inflammatory cell infiltration.Compared with the model group,treatment with G.acuta produced a marked improvement in ISO-induced myocardial necrosis,structural disorder and inflammatory cell infiltration.Masson’s trichrome staining revealed that a mass of collagen was deposited in the hearts of ISOtreated rats compared with the control group.However,a significant reduction in collagen deposition was observed in the G.acuta treatment group compared to the model group.Compared with the control group,the LVMI of the model group was significantly increased(P < 0.01);however,treatment with G.acuta at the indicated doses significantly attenuated this ISO-induced change in LVMI(P <0.05).WGA staining indicated that ISO could induce cardiomyocytes injury and hypertrophy,and treatment with G.acuta could inhibit myocardial hypertrophy(P < 0.01).2.G.acuta inhibits the expression of Collagen I,III and a-SMA induced by ISOImmunohistochemical staining revealed that the expression of myocardial Collagen I and III were markedly enhanced in the model group versus the control group.G.acuta treatment decreased ISO-induced Collagen I and III expression enhancement.Western blotting results indicated that G.acuta treatment inhibited the Collagen I and III expression.These findings demonstrate that G.acuta treatment caused a decrease in Collagen I and III expression in the process of ISO-induced myocardial fibrosis(P < 0.01).In the control group,α-SMA-positive staining was observed in the blood vessels.In comparison with the control group(without ISO),an abundance of ISO-induced brown particles was present in the fibrotic areas,while G.acuta treatment ameliorated the ISO-induced positive brown particles.Western blotting results indicated that the expression level of α-SMA was downregulated by G.acuta(P < 0.05).3.G.acuta treatment inhibits TGF-β1 expression and the phosphorylation of TβR induced by ISOImmunohistochemical analysis revealed that the expression levels of TGF-β1 were increased in the model group compared with the control group.However,compared with the model group,G.acuta treatment caused a significant decrease in the ISO-induced TGF-β1 expression levels.Western blotting results showed that TGF-β1 expression were significantly decreased in G.acuta treatment group(P < 0.01).The results indicated that TβRI expression and TβRI and TβRII phosphorylation levels were significantly upregulated in the model group comparing with the control group(P < 0.01).Moreover,G.acuta treatment abolished upregulation of these ISO-induced proteins(P < 0.01).4.G.acuta inhibits ISO-induced TGF-β1/Smads signalingIn comparison with the control group,the immunoreactivity of Smad2,3,and 4 were significantly higher in the model group.Moreover,G.acuta treatment significantly reduced the increase in ISO-induced Smad2,3,and 4expression.Western blot analysis showed that G.acuta treatment caused a decrease in the Smad2 and 4 expression(P < 0.01).In addition,compared with the control group,Smad2 and 3 phosphorylation levels were markedly increased in the model group(P < 0.01).G.acuta treatment caused a significant reduction in Smad2 and 3 phosphorylation levels,compared with the model group(P <0.05 or P < 0.01).Part two Bellidifolin inhibits the proliferation and activation of cardiac fibroblasts induced by TGF-β1Objective: To demonstrate the effect of bellidifolin,one of component of G.acuta,on the proliferation and the phenotypic transformation of CFs induced by TGF-β1.Method: Primary cardiac fibroblasts were extracted from neonatal rat,and cardiac fibroblasts were cultured and passaged.The identification of cardiac fibroblasts was investigated by morphological observation and vimentin detection.The cell viability of cardiac fibroblasts treated with bellidifolin,the TGF-β1-induced proliferation of cardiac fibroblasts and the effect of bellidifolin on the TGF-β1-induced proliferation were detected by CCK-8,respectively.The expression of Collagen Ⅰ,Ⅲ,α-SMA,Smad3 and the level of phosphorylated Smad3 were analyzed by Western blotting at different times of TGF-β1 stimulation.The expression of Collagen Ⅰ,Ⅲ and α-SMA were analyzed by Western blotting and immunofluorescence staining to investigate the phenotypic transformation of cardiac fibroblasts induced by TGF-β1.The expression of Collagen Ⅰ,Ⅲ and α-SMA were detected by immunofluorescence staining to determine the effect of bellidifolin on phenotypic transformation of cardiac fibroblasts induced by TGF-β1.Results:1.Bellidifolin inhibits the cardiac fibroblasts proliferation induced by TGF-β1The cardiac fibroblasts were thin and triangular under inverted light microscopy,and vimentin expression was positive.The cell viability of cardiac fibroblasts was detected by CCK-8.The results showed that there were no effects of bellidifolin on the viability of cardiac fibroblasts without TGF-β1treatment(P > 0.05),the proliferation of cardiac fibroblasts induced by TGF-β1(5,10,20 ng/ml)was significantly increased comparing with the control group(P < 0.01),while the administration of bellidifolin markedly inhibited the cardiac fibroblasts proliferation induced by TGF-β1(P < 0.01).2.TGF-β1 induces transition of cardiac fibroblasts to myofibroblastsThe expression of α-SMA,Collagen Ⅰ,Ⅲ,Smad3 and the level of phosphorylated Smad3 were analyzed at different times of TGF-β1 stimulation by Western blotting,and the results showed that the most obvious stimulated effects occurred at 24 h.The morphology of cardiac fibroblasts changed significantly at different doses of TGF-β1(5,10,20 ng/ml)stimulation.Stimulating with TGF-β1(10 ng/ml)for 24 h induced the cell phenotypic transformation.Immunofluorescence staining results showed that TGF-β1induced the significant increasing in the expression of α-SMA,Collagen Ⅰ andⅢ comparing with the control group.Consistent with the immunofluorescence staining,Western blotting results showed that TGF-β1 induced high expression of α-SMA,collagen Ⅰ and Ⅲ comparing with the control group(P < 0.01).3.Bellidifolin inhibits the phenotypic transformation of cardiac fibroblasts induced by TGF-β1Immunofluorescence staining results showed that TGF-β1 induced the significant enhancements in the expression of α-SMA,Collagen Ⅰ and Ⅲcompared with the control group,and the expression of α-SMA,Collagen Ⅰ andⅢ were markedly reduced by the bellidifolin administration.Part three Bellidifolin suppresses cardiac fibroblasts activation by repressing the TGF-β1/Smads and p38 signaling pathwayObjective: To illustrate the underlying inhibitory mechanism of bellidifolin on the activation of cardiac fibroblasts induced by TGF-β1stimulation.Method: Cardiac fibroblasts were divided into the following 6 groups: the control group(Control),the model group(TGF-β1),the model and DMSO group(TGF-β1 + DMSO),the low-dose bellidifolin intervention group(TGF-β1 + B15),the middle-dose bellidifolin intervention group(TGF-β1 + B30)and the high-dose bellidifolin intervention group(TGF-β1 + B60).The model group was administrated with TGF-β1(10 ng/ml)for 24 h.The bellidifolin intervention groups were pretreated with bellidifolin(15,30,60 μM)for 0.5 h,then administrated with TGF-β1 for 24 h.The expression of Collagen Ⅰ,Ⅲ andα-SMA were detected by immunofluorescence staining and Western blotting to determine the effects of different doses of bellidifolin on the cardiac fibroblasts phenotypic transformation induced by TGF-β1.The expression of Smad2,3,4and the levels of Smad2,3 phosphorylation were detected by immunofluorescence staining and Western blotting to determine the effects of bellidifolin on TGF-β1/Smads canonical signaling.The protein expression and phosphorylation of non-canonical signaling pathway were analyzed by Western blotting to determine the effects of bellidifolin on non-canonical signaling pathway.Further,cardiac fibroblasts were treated with SB203580(the inhibitor of p38),or LY2157299(the inhibitor of TβRⅠ),or combination SB203580 and LY2157299.The expression of Smad3 and the level of phosphorylated Smad3 were analyzed by Western blotting to investigate the target of bellidifolin on TGF-β1 signaling.Results:1.Bellidifolin inhibits the TGF-β1-induced cardiac fibroblasts activation at different dosesImmunofluorescence staining results showed that TGF-β1 induced a significant increasing in the expression of α-SMA,Collagen Ⅰ and Ⅲ compared with the control group,while the increased expression of α-SMA,Collagen Ⅰand Ⅲ were remarkably reduced by different doses of bellidifolin administration.Consistent with the immunofluorescence staining,Western blotting results showed that TGF-β1 induced the up-regulated expression of α-SMA,Collagen Ⅰ and Ⅲ comparing with the control group(P < 0.01),while the expression of α-SMA,Collagen Ⅰ and Ⅲ were significantly down-regulated in cardiac fibroblasts by bellidifolin treatment(15,30,60 μM)(P < 0.01).2.Bellidifolin inhibits the activation of TGF-β1/Smads signaling in cardiac fibroblastsImmunofluorescence staining results showed that TGF-β1 induced the enhancements in the expression of Smad2,4 and the phosphorylation of Smad2,3 comparing with the control group,and bellidifolin inhibited the enhancing in the expression of Smad2,4 and the phosphorylation of Smad2,3.Western blotting results showed that bellidifolin reduced the increases in the expression of Smad2,4 and the phosphorylation of Smad2,3 induced by TGF-β1(P < 0.05 or P < 0.01).Smads proteins are mediated by the upstream molecules of TβRⅠ and TβRⅡ.Western blotting results showed that TGF-β1induced the phosphorylation elevating of TβRⅠ and TβRⅡ comparing with the control group(P < 0.01),while bellidifolin decreased the phosphorylation of TβR(P < 0.01).3.Bellidifolin inhibits the phosphorylation of p38 induced by TGF-β1 in cardiac fibroblastsWestern blotting results showed that TGF-β1 induced the increase in the phosphorylation of p38(P < 0.01),but not in the expression of p38,ERK and the level of phosphorylated ERK,comparing with the control group,while bellidifolin treatment inhibited the phosphorylation of p38(P < 0.05).4.Bellidifolin inhibits the phosphorylation of Smad3 independent of p38 signaling in cardiac fibroblastsWestern blotting results showed that TGF-β1 stimulation induced the Smad3 phosphorylation(P < 0.01),and bellidifolin administration significantly inhibited the p38 and Smad3 phosphorylation(P < 0.05).SB203580(the inhibitor of p38)could not inhibit the phosphorylation of Smad3(P > 0.05),while LY2157299(the inhibitor of TβRⅠ)alone or combination with SB203580 significantly inhibited the phosphorylation of Smad3(P < 0.01).Based on these results,we preliminarily speculate that the inhibitory effect of bellidifolin on Smad3 may be not related to the inhibition of p38.Conclusion:1.The inhibitory role of G.acuta on rat myocardial fibrosis is based on the inhibition of TGF-β1/Smads signaling pathway.2.Bellidifolin can inhibit the proliferation and activation of cardiac fibroblasts induced by TGF-β1,and the underlying role and mechanism of bellidifolin is by suppressing the Smads canonical signaling pathway and the p38 non-canonical signaling pathway.