生肌玉红膏对血栓性脉管炎大鼠血管平滑肌细胞凋亡、炎性因子表达的影响及机制

Effects of Shengji Yuhong ointment on apoptosis of vascular smooth muscle cells and expression of inflammatory factors in rats with thromboangiitis

目的探究生肌玉红膏对血栓性脉管炎大鼠血管平滑肌细胞凋亡、炎性因子表达的影响及机制。方法取40只大鼠,随机分为正常组、模型组、血管内皮祖细胞(EPC)干预组、生肌玉红膏组,每组10只,除正常组外,均建立血栓性脉管炎模型,正常组与模型组尾静脉每天1次输注1 mL生理盐水,EPC干预组输注数量为5×105个EPC细胞后创面外敷生肌玉红膏,生肌玉红膏组创面外敷生肌玉红膏。将EPC细胞培养后观察细胞形态,检测IL-6、IL-8、TNF-α、TGF-β_(1)表达,采用免疫荧光法检测CD34、CD133,TUNEL法检测平滑肌细胞凋亡率,免疫印迹法检测内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)蛋白表达。结果模型组大鼠血清中IL-6、IL-8、TNF-α、TGF-β_(1)水平及平滑肌细胞数高于正常组(P均<0.05),EPC干预组以上指标均低于模型组(P均<0.05),生肌玉红膏组以上指标均高于EPC干预组(P均<0.05)。模型组大鼠血管组织中CD34、CD133及血管组织中VEGF、bFGF蛋白表达低于正常组(P均<0.05),EPC干预组以上指标高于模型组(P均<0.05),生肌玉红膏组以上指标低于EPC干预组(P均<0.05)。正常组大鼠生长良好;模型组大鼠血栓显著,可见大量炎性细胞浸润;EPC干预组血栓变化缓解,伴随少量炎性细胞,血管平滑肌细胞排列趋于整齐;生肌玉红膏组少量血栓,血管平滑肌细胞排列略显紊乱。模型组大鼠血管组织中CD34、CD133表达低于正常组(P均<0.05),EPC干预组以上指标高于模型组(P均<0.05),生肌玉红膏组CD34、CD133指标低于EPC干预组(P均<0.05)。与正常组比较,模型组血管组织中平滑肌细胞凋亡率高(P<0.05);与模型组比较,EPC干预组血管组织中平滑肌细胞凋亡率高(P均<0.05);与EPC干预组比较,生肌玉红膏组平滑肌细胞凋亡率较低(P均<0.05)。模型组大鼠血管组织中VEGF、bFGF蛋白表达低于正常组(P均<0.05),EPC干预组VEGF、bFGF蛋白表达高于模型组,生肌玉红膏组VEGF、bFGF蛋白表达低于EPC干预组(P均P<0.05)。结论生肌玉红膏可促进血栓性脉管炎大鼠平滑肌细胞凋亡,降低炎性因子表达,激活VEGF与bF⁃GF蛋白表达,发挥血管保护作用;其机制可能与介导EPC分化相关。

Objective To explore the effects of Shengji Yuhong ointment on apoptosis of vascular smooth muscle cells and expression of inflammatory factors in rats with thromboangiitis and their mechanisms.Methods Forty rats were randomly divided into the normal group,model group,endothelial progenitor cells(EPCs)intervention group,and Shengji Yuhong ointment group,with 10 rats in each group.Except the normal group,thromboangiitis models were established.The tail vein normal saline(1 mL)was injected once a day in the normal group and model group;the rats were injected with 5×105 EPCs in the EPCs intervention group,and the wound surface was treated with Shengji Yuhong ointment;Shengji Yuhong ointment was applied on the wound surface in the Shengji Yuhong ointment group.We observed the morphology of EPCs after culture,and detected the interleukin-6(IL-6),IL-8,tumor necrosis factor-α(TNF-α),transforming growth factor-β1(TGF-β1).The expression of inflammatory factors,CD34 and CD133 were detected by immunofluorescence;the apoptosis rate of smooth muscle cells was detected by TUNEL;and the expression levels of vascular endothelial growth fac⁃tor(VEGF)and basic fibroblast growth factor(bFGF)protein were detected by immunoblotting.Results IL-6,IL-8,and TNF-αand the TGF-β1 in serum,and the number of smooth muscle cells was higher in the model group than in the nor⁃mal group(all P<0.05),the above indexes in the EPCs intervention group were lower than those in the model group(all P<0.05),and the above indexes in the Shengji Yuhong ointment group were higher than those in the EPCs intervention group(all P<0.05).The expression levels of CD34,CD133 and VEGF,bFGF protein in the vascular tissues of rats in the model group were lower than those in the normal group,the above indexes in the EPCs intervention group were higher than those in the model group(all P<0.05),and the above indexes in the Shengji Yuhong ointment group were lower than those in the EPCs intervention group(all P<0.05).The normal rats grew well;in the model group,the thrombus was significant,and a large number of inflammatory cells infiltrated;in the EPCs intervention group,the changes of thrombus were alleviated,ac⁃companied by a small number of inflammatory cells,and vascular smooth muscle cells were arranged neatly;in Shengji Yuhong ointment group,there was a small amount of thrombosis,and the arrangement of vascular smooth muscle cells was slightly disordered.The expression levels of CD34 and CD133 in the vascular tissues of rats in the model group were lower than those in the normal group,the above indexes in the EPCs intervention group were higher than those in the model group(all P<0.05),and the indexes of CD34 and CD133 in the Shengji Yuhong ointment group were lower than those in the EPCs intervention group(all P<0.05).Compared with the normal group,the apoptosis rate of smooth muscle cells in the vascular tissues of the model group increased.Compared with the model group,the apoptosis rate of smooth muscle cells in the vas⁃cular tissues of the EPCs intervention group was higher(P<0.05);compared with the EPCs intervention group,the apopto⁃sis rate of smooth muscle cells in the Shengji Yuhong ointment group was lower(P<0.05).The expression of VEGF and bF⁃GF protein in vascular tissues of rats in the model group was lower than that in normal group(P<0.05),the expression lev⁃els of VEGF and bFGF protein in the smooth muscle of rats in the EPCs intervention group were higher than those in the mod⁃el group,and the expression levels of VEGF and bFGF protein in the Shengji Yuhong ointment group were lower than those in the EPCs intervention group(all P<0.05).Conclusions Shengji Yuhong ointment can promote the apoptosis of smooth muscle cells in rats with thromboangiitis,reduce the expression of inflammatory factors,and activate VEGF and bF⁃GF proteins for vascular protection.The mechanism may be related to the differentiation of EPCs.