回阳生肌膏对人来源微血管内皮细胞的干预作用

Intervention effects of Huiyang Shengji cream on human microvascular endothelial cells

目的研究回阳生肌膏对人来源微血管内皮细胞体外凋亡和迁移的影响及其可能存在的机制。方法细胞实验分为空白组、模型组、实验组和阳性对照组,空白组与模型组不予干预,实验组予回阳生肌膏,阳性对照组予龙珠软膏,均作用48小时。采用MTT法筛选药物无毒浓度;流式细胞术观察细胞凋亡情况;酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测细胞上清液中基质金属蛋白酶1(matrix metallopeptidase 1,MMP1)、基质金属蛋白酶9(matrix metallopeptidase 9,MMP9)、基质金属蛋白酶抑制剂1(tissue inhibitors of matrix metalloproteinase 1,TIMP1)、基质金属蛋白酶抑制剂2(TIMP2)、血管内皮生长因子(vascular endothelial growth factor,VEGF)含量;细胞迁移实验检测细胞迁移能力;蛋白质印迹法检测血管内皮细胞生长因子受体2(VEGF receptor 2,VEGFR2)蛋白的表达。结果确定了实验组予50μg/mL回阳生肌膏,对照组予25μg/mL龙珠软膏为后续试验浓度,作用48小时后,与模型组相比,回阳生肌膏可有效抑制内皮细胞的凋亡(P<0.05),而龙珠软膏能显著抑制内皮细胞凋亡(P<0.01);与模型组相比,回阳生肌膏、龙珠软膏在体外均能显著下调糖尿病足内皮细胞MMP9、TIMP1的分泌(P<0.01),显著提高VEGF的含量(P<0.01),对MMP1、TIMP2的表达也有下调趋势;细胞迁移试验结果显示,回阳生肌膏可增多人来源微血管内皮细胞的迁移数(P<0.05),龙珠软膏能显著增多细胞迁移数(P<0.01);蛋白质印迹法结果显示,回阳生肌膏、龙珠软膏均促进了糖尿病足创面内皮细胞VEGFR2蛋白的表达(P<0.05)。结论回阳生肌膏可抑制糖尿病足创面内皮细胞凋亡,促进糖尿病足创面内皮细胞迁移增殖,其机制可能与其调控基质金属蛋白酶类及抑制酶类,促进VEGF、VEGFR2的表达有关。

Objective To study the effects of Huiyang Shengji cream on apoptosis and migration of human microvascular endothelial cells in vitro and its possible mechanism.Methods The experiment cells were divided into blank group,model group,experimental group and positive control group.No intervention was performed in the blank group and the model group,the experimental group was given Huiyang Shengji cream,and the positive control group was given Longzhu ointment,which both acted for 48 hours.The non-toxic concentration of the drug was screened by MTT method.Cell apoptosis was observed by flow cytometry.The contents of matrix metallopeptidase 1(MMP1),matrix metallopeptidase 9(MMP9)and tissue inhibitors of matrix metalloproteinase 1(TIMP1),tissue inhibitors of matrix metalloproteinase 2(TIMP2)and vascular endothelial growth factor(VEGF)were detected by enzyme linked immunosorbent assay(ELISA).Cell migration ability was detected by cell migration experiment.The protein expression of vascular endothelial growth factor receptor 2(VEGFR2)was detected by Western blot.Results The experimental group was given 50 g/mL of Huiyang Shengji cream,and the positive control group was given 25 g/mL of Longzhu ointment.After 48 hours of action,compared with the model group,Huiyang Shengji cream could effectively inhibit the apoptosis of endothelial cells(P<0.05),while Longzhu ointment could significantly inhibit the apoptosis of endothelial cells(P<0.01).Compared with the model group,both Huiyang Shengji cream and Longzhu ointment significantly down-regulated the secretion of MMP9 and TIMP1 of diabetic foot endothelial cells in vitro(P<0.01),increasing the content of VEGF significantly(P<0.01),and having a down-regulated trend on the expression of MMP1 and TIMP2.The results of cell migration test showed that Huiyang Shengji cream could increase the migration number of human microvascular endothelial cells(P<0.05),while Longzhu ointment could increased it significantly(P<0.01).Western blot results showed that both Huiyang Shengji cream and Longzhu ointment could promoted the protein expression of VEGFR2 in endothelial cells of diabetic foot wounds(P<0.05).Conclusion Huiyang Shengji cream can inhibit the apoptosis of endothelial cells in diabetic foot wounds,and promote the migration and proliferation of endothelial cells in diabetic foot wounds.The mechanism may be related to the regulation of matrix metalloproteinases and inhibitory enzymes,and the promotion of the expression of VEGF and VEGFR2.