| Objective Preliminary exploration of the mechanism of action of bioactive peptides through the TGF-β1/Smads signaling pathway in regulating myocardial fibrosis.Methods(1)The first part:In vitro cell experiments: After 24 h induction of primary cardiac fibroblasts using different concentrations of TGF-β1,the best induction concentration of TGF-β1 modeling was selected by detecting the cell survival rate by CCK8 method.The cells were then divided into normal control group,group treated with bioactive peptide only,model group,and group treated with high,medium and low doses of bioactive peptide.The survivability of each group was detected by CCK8 method.The relative expression of α-SMA,collagen I and collagen III in each group of cells was measured by RT-q PCR.The Western blot method was used to detect the expression ofα-SMA,Smad2,Smad3 and their phosphorylated proteins.(2)The second part: In vivo studies.Thirty male C57BL/6J mice were randomly assigned into 3 groups of 10 mice each,normal control group,model group and bioactive peptide group.except for the normal control group,the other two groups were moulded by subcutaneous injection of 25 mg/kg isoproterenol,and after 5 days,the model was identified as successful by echocardiographic monitoring of cardiac function and other indicators,the bioactive peptide group was given bioactive peptide.The normal control group and the model group were flooded with saline at 360 μl per day for 14 days.At the end of treatment,the anesthetized animals were quickly removed from the heart,and a portion of the isolated heart was quickly fixed in 4% paraformaldehyde,followed by preparation of paraffin sections for subsequent HE staining,Masson staining and Sirius red staining as well as immunofluorescence staining to observe the myocardial histology and collagen deposition in each group;small pieces of the heart were immediately placed in 2.5% glutaraldehyde to prepare sections for transmission electron microscopy.The apical portion of the heart was put in liquid nitrogen and proteins were withdrawn for Western blot to determine the phosphorylated expression levels of collagen I,FN1,TGF-β(16)and Smad2/3 in all groups of myocardial tissue.Results Cell experiments:(1)The results of CCK8 assay showed that 10ng/ml of TGF-β1 effectively stimulated the proliferation of cardiac fibroblasts(P<0.01),and different concentrations of bioactive peptide significantly inhibited the proliferation of cardiac fibroblasts induced by TGF-β1(P<0.05).(2)RT-q PCR results revealed that the expression of α-SMA,collagen I and collagen III was decreased in the bioactive peptide group as compared to the model group(P<0.01,P<0.01,P<0.05).(3)Western blot results showed that the protein expression of α-SMA,Smad2,Smad3 and their phosphorylation levels were decreased in cells of the bioactive peptide group compared with the model group(P<0.05,P<0.05,P<0.01).Animal experiments:(1)In comparison to the model group,the bioactive peptide group demonstrated increased ejection fraction,shortening fraction(P>0.05)and decreased left ventricular systolic internal diameter and left ventricular diastolic internal diameter(P>0.05,P<0.05).(2)HE staining and transmission electron microscopy results showed that bioactive peptides improved ISOinduced myocardial histopathology as well as ultrastructural pathological changes.(3)Masson staining and Sirius red staining results were consistent with the bioactive peptide intervention significantly reducing the deposition of myocardial collagen fibres(P<0.05,P<0.05).(4)Protein expressed by α-SMA and collagen I were significantly downregulated following the action of the bioactive peptide(P<0.05),whereas the reduction of FN1 was not appreciably distinct.(5)Bioactive peptide intervention reduced ISO-induced elevated TGF-β1 and phosphorylated Smad2/3 expression,but only for the reduction of TGF-β1 expression was significantly different(P<0.05).Conclusion(1)The bioactive peptide significantly inhibited the proliferation and activation of TGF-β1 on cardiac fibroblasts and suppressed collagen secretion.(2)The bioactive peptide alleviated isoprenaline-induced fibrosis in a mouse model of myocardial fibrosis.(3)Preliminary studies showed that the inhibitory effect of bioactive peptide on myocardial fibrosis may be mediated and regulated by the TGF-β1/Smads signaling pathway. |
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