Effects Of Notch Signal Pathway On Transdifferentiation Of Cardiac Fibroblasts To Myofibroblasts

【Background】The excessive proliferation and transdifferentiation of cardiac fibroblasts (CFs) into myofibroblasts, which expressedα-smooth muscle actin and led to excessive collagen synthesis and secretion, are essential pathology of cardiac fibrosis. Transforming growth factor -β1 (TGF-β1), which can promote the differentiation of CFs to the MFB and up-regulate the expression ofα-SMA and collagen synthesis, is an important profibrotic cytokine. AngiotensinⅡ(angiotensinⅡ, AngⅡ), main product of cardiac renin-angiotensin system (rennin-angiotensin system, RAS) can promote myocardial fibrosis either. Notch signaling pathway, which controls the cell differentiation by interaction of ligands and receptors, plays an key role in the development and pathology of cardiovascular system. Although it has been proved that Notch signaling regulated the differentiation of fibroblasts into myofibroblasts, its effects in the differentiation of CFs into MFB remains unclear.【Purposes】1. To detect the expression level of Notch receptors in the CFs in basic state, investigate the effects of DAPT on the expression level ofα-SMA and HYP. To clarify if Notch signaling pathway participated in the pathology of myocardial fibrosis preliminarily.2. To investigate the change of Notch receptors expression in the transdifferentiation of cardiac fibroblasts (CFs) into myofibroblasts (MFB) induced by TGF-β1.3. To investigate the change of Notch receptors expression in the transdifferentiation of CFs into MFB induced by AngⅡ.【Contents】1. Detect the expression level of Notch receptors in the CFs in basic state and the expression level ofα-SMA and HYP stimulated by DAPT at different concentration and for different time length. Real-time quantitative PCR and Western blot were used to detect the expression level of Notch receptors mRNA and protein. Cytochemical immunofluorescence and Western blot were used to detect the expression ofα-SMA. Digestion method was used to detect the content of HYP.2. The effects stimulation by TGF-β1 at different concentration and time length on the expression levelα-SMA and HYP in the CFs and the change of Notch receptors expression during the transdifferentiation of CFs into MFB. Cytochemical immunofluorescence and Western blot were used to detect the expression ofα-SMA. Digestion method was used to detect the content of HYP. Real-time quantitative PCR and Western blot were used to detect the expression level of Notch receptors mRNA and protein. 3. The effects stimulation by AngⅡat different concentration and time length on the expression levelα-SMA and HYP in the CFs and the change of Notch receptors expression during the transdifferentiation of CFs into MFB. Cytochemical immunofluorescence and Western blot were used to detect the expression ofα-SMA. Digestion method was used to detect the content of HYP. Real-time quantitative PCR and Western blot were used to detect the expression level of Notch receptors mRNA and protein.【Results】1. In basic state, the mRNA and protein of Notch receptors were expressed in CFs. The expression levels ofα-SMA and HYP showed uptrend as the concentration and the time length of DAPT increased.2. TGF-β1 could up-regulate the expression ofα-SMA and HYP in CFs. The expression levels ofα-SMA and HYP showed increasing trend as the concentration and the time length of TGF-β1 stimulation increased. However, the expression levels of Notch1, Notch3 and Notch4 decreased with the increase of TGF-β1 concentration and extension of time. The expression of Notch2 did not change significantly.3. AngⅡcould up-regulate the expression ofα-SMA and HYP in CFs. The expression levels ofα-SMA and HYP was up-regulated with the increase of AngⅡconcentration and extension of time. However, the expression levels of Notch1, Notch3 and Notch4 decreased with the increase of AngⅡconcentration and extension of time. The expression of Notch2 did not change significantly.【Conclusion】1. In basic state, the mRNA and protein of Notch receptors were expressed in CFs. Blockade of Notch signaling by DAPT up-regulated the expression ofα-SMA and HYP and resulted in the trandifferentiation of CFs into MFB.2. TGF-β1 could up-regulate the expression ofα-SMA and HYP significantly in a time- and dose-dependent manner and induce the trandifferentiation of CFs into MFB. During the trandifferentiation, the expression levels of Notch1, Notch3 and Notch4 decreased gradually, while the expression level of Notch2 did not change significantly.3. AngⅡ, which could up-regulate the expression ofα-SMA and HYP significantly in a time- and dose-dependent manner, could induce the trandifferentiation of CFs into MFB. During the trandifferentiation, the expression levels of Notch1, Notch3 and Notch4 decreased gradually, while the expression level of Notch2 did not change significantly.