The Effect Of Tanshinone â…¡A On Endothelial-mesenchymal Transition Of Cardiac Microvascular Endothelial Cells Cultured In The Condition Of High Glucose

Background and Objective: Studies have shown that the activation of cardiacfibroblasts originated from heart-resident cardiac fibroblasts in several pathologicalconditions.In addition to these mechanisms of recruiting activated fibroblasts,recentlyresearch demonstrated that endothelial-to-mesenchymal transition (EndMT) plays asignificant role in cardiac fibrosis.The process of EndMT can be stimulated byinflammatory factor, high glucose, and hypoxia,which makes the Endothelial cellsobtain mesenchymal marker and change from Pebble-like into the spindle-shapedwith the functions and simple shape of fibroblasts. The cardiac microvascularendothelial cells were isolated from the heart of SD rats, the EndMT model of thecardiac microvascular endothelial cells were constructed in high glucosecondition．To observe the effect of tanshinone ⅡA on Endothelial-mesenchymaltransition of cardiac microvascular endothelial cells cultured in the condition of highglucose and the secretion of type I collagen and type Ⅲ collage.Methods: The cardiac microvascular endothelial cells were separated from theSD rats of5-7days old by the enzyme digestion method. The two times of differentialadhesion method was used to isolate and purify the cells.The cardiac microvascularendothelial cells were identified through the immunofluorescence CD31and theexpression of factor Ⅷ.The second generation of endothelial cells were used to dothe experiments in groups.The experiment was divided into five groups.A: normalcontrol group;B:mannitol permeation pressure control group; C: high glucose(30mmol/L)group;D: high glucose+low dose tanshinone ⅡA(10-5mol/L TSNⅡA)group;E:high glucose+high dose tanshinone ⅡA(10-4mol/L TSNⅡA) group.Allgroups were cultured to the third day,expression of CD31and a-SMA byimmunofluorescence combined with the change of the cell morphological were usedto identify the EndMT in cardiac microvascular endothelial cells.The expression ofmRNA of a-SMA, TGF-β1,type I collagen and type Ⅲ collage of each cell weredetected by semi-quantitative PCR. Results: The cells were in line with the characteristics of myocardialmicrovascular endothelial cells by identifying the expression of CD31and factorⅧ,the immunofluorescence displayed that part of the cells showed double-stainingpositive of CD31and a-SMA in high glucose group; the semi-quantitative PCRresults showed that the mRNA expression of a-SMA,TGF-β1,type I collagen and typeⅢ collagen in high glucose group were significantly up-regulated compared with thenormal control group(p<0.05),the expression between mannitol group and normalgroup had no difference;compared with the high glucose group, the mRNAexpression of a-SMA, TGF-β1, type I collagen and type Ⅲ collagen in high dosetanshinoneⅡA group was reduced(p<0.05).Conclusion: The high glucose can induce the EndMT of cardiac microvascularendothelial cells and can also increase the mRNA expression of type I collagen andtype Ⅲ collagen,while tanshinone ⅡA can inhibit the process at a certain extent,which may be related to the inhibition on the expression of TGF-β1.