Establishment And Application Of Myocardial Fibrosis Cell Model Based On Endothelial-Mesenchymal Transition

Objective:To established a novel cell model of myocardial fibrosis based on endothelial-mesenchymal transition (EndMT) used adult human oronary artery endothelial cells. Application of this model to observe the effect of Gualou Xeibai Decotion (GXD) on myocardial fibrosis. To clear the mechanisms and targets of GXD on myocardial firbosis. The results of the studies would show the experimental base for the clinic application of GXD.Methods:The studies included four parts.1. To bulid the novel cell model of myocardial fibrosis:Human oronary artery endothelial cells were used to establish the cell model and rhTGFβ1、rhTGFβ32、rhTGFβ3were candidate inducers. Endothelial cell marker protein CD31and mesenchymal cell specific proten FSP1and a-SMA were detected. The appropriate inducer was screened. The expression of indexes associated with fibrosis were used to identify the model.2. Application of myocardial fibrosis cell model-the effect of GXD on EndMT of myocardial fibrosis:The cell model of myocardial fibrosis were established. The expression of CD31, FSP1and a-SMA were detected to observe the effect of GXD on EndMT. The changes of the expression of Smadl, Smad2, Jaggedl, Jagged2, DLL1, DLL2, DLL3were detected to explain the target of GXD on EndMT of myocardial fibrosis.3. The validation of GXD on EndMT at the whole body level:To observe the effect of GXD on EndMT of myocardial fibrosis rats, myocardial fibrosis rats were induced by left coronary artery occlusion. The following indices of the rats were detected:cardiac index, hemodynamics, M-mode echo card iography parameters, myocardial enzymes, Ⅰ and Ⅲ collagen, histology examination, collagen volume fraction(CVF).The study above might lay a good foundation for the application of the new cell model. The expression of CD31, FSP1and α-SMA were determined by immunofluorescence and Westernblot.The indices above might decide whether the results in vitro are consistent with those in vivo.Results:1. RhTGFβ1、rhTGFβ、rhTGFβ3had the same inhibitory effect on cell viability in the dose range100ng/mL～0.01ng/mL (vs control group, P<0.05～0.01). RhTGFβ1、rhTGFβ2、 rhTGFβ3also had the same effect on endothelial cell phenotype change. The expression of CD31was reduced and the expression of FSP. and a-SMA were upregulated when coronary enaothelial cells incubated with candidate inducers. The content of TGFβ1was significantly higher than that of TGFβ2and TGFβ3in the organizational environment. So we choosed TGFβ1as inducing agent. The transformation curve of TGFβ1was builded. The optimal dose was10ng/mL and the optimal inducing time was7days. So this novel myocardial fibrosis cell model could be established with TGFβ1inducing7days at a final concentration10ng/mL.2.The was increase and the expression of FSP1and a-SMA were inhibited by GXD by flow cytometry and Westerblot in the novel cell model so as to achive the regulation of EndMT Further study found that the expression of Smadl, Smad2and North1,Jagged1, DLL1and DLL2were down-regulated in GXD groups These indices illustrated that GXD inhibited TGF-β pathway through the Notch pathway in endothelial cells.3. LVID, LVEDV, LVESV were significantly decreased in GXD groups (vs control, group, P<0.05～0.01)and IVS、LVPW、EF、FS were increased (vs control group, P<0.05-0.01). The hemodynamic level in GXD treated groups was improved with increased LVSP,±dp/dtmax, HR, SAP,DAP (vs control group, P<0.05～0.01)and decreased LVEDP. AST, LDH and CK-MB in serum were significantly inhibited by GXD (vs control group, P<0.05). LVM, HM, LVMI and HMI in high-dose group were decreased compared with MI group. HE stain of the heart from GXD roup found that muscle fibers arranged orderly, mild myocardial hypertrophy,interstitial infiltration of inflammatory cells, mild interstitial fibrous tissue hyperplasis and normal stain. Masson staining showed GXD could increase the myocardial cells, arrange the cells in orderly rules, reduce collagen fibers and significantly reduce CVF. The expression of Ⅰ and Ⅲ collagen in GXD groups were significantly decreased (vs control group, P<0.05～0.01).GXD had protective effect on myocardial fibrosis. It was found that the expression CD31was upregulated and TGF-β1,α-SUA and FSP1were downregulated in myocardial fibrosis rats compared with MI group (P<0.05～0.01). The result was consistent with that in vitro.The research data from the cell model of myocardial fibrosis was true, reliable and trustworthy.Conclusion:Novel cell model of myocardial fibrosis based on endothelial-mesenchymal transition (EndMT) was established:Incubation of adult human coronary endothelial cells with rhTGFβ1at a final concentration10ng/mL resulted in the differentiation of endothelial cells into mesenchymal cells expression of α-SMA/FSP1, and the expression of indexes associated with fibrosis were upregulated. The experimental method is simple and feasible. This model is successfully applied to the study of Gualou Xiebai Baijiu Decoction (GXD) on myocardial fibrosis. If was found that GXD substantially inhibited EndMT associated with myocardial fibrosis. Further research showed that GXD suppressed TGF-β pathway through Notch pathway to achieve the protection of endothelial cells and inhibition of EndMT.