侯氏黑散含药血清对氧糖剥夺损伤血管内皮细胞PLCγ2/ERK及TGF-β/Smad2/3表达的影响

Influence of medicated serum of Houshi Hei San on expressions of PLCγ2/ERK and TGF-β/Smad2/3 in vascular endothelial cells after injury of oxygen-glucose deprivation

目的探讨侯氏黑散含药血清对氧糖剥夺损伤人脐静脉内皮细胞(HUVEC)中磷脂酶Cγ2(PLCγ2)、细胞外调节蛋白激酶(ERK)、转化生长因子β(TGF-β)及膜受体调控蛋白Smad2/3表达的影响。方法将HUVEC随机分为对照组,模型组,2.5%、5.0%、10.0%、20.0%侯氏黑散含药血清组。将模型组及各含药血清组细胞更换为无糖培养液后放入低氧培养箱连续培养6 h构建氧糖剥夺细胞模型,对照组正常培养相同时间。然后,对照组、模型组更换为含10.0%空白血清的完全培养基,各给药组用含对应质量分数的侯氏黑散含药血清正常培养24 h后提取细胞总蛋白。Western blot法检测细胞中PLCγ2、ERK、p-ERK、TGF-β及Smad2/3蛋白的表达。结果与对照组相比,模型组细胞PLCγ2、Smad2/3蛋白表达上调(P<0.01),TGF-β蛋白表达下调(P<0.01);与模型组比较,5.0%侯氏黑散含药血清上调TGF-β蛋白表达,2.5%、5.0%、10.0%侯氏黑散含药血清下调PLCγ2蛋白表达,各质量分数含药血清均下调Smad2/3的表达(P<0.01或P<0.05)。结论侯氏黑散含药血清可调节PLCγ2/ERK及TGF-β/Smad2/3信号通路,促进氧糖剥夺损伤的HUVEC增殖。

Objective To investigate the influence of medicated serum of Houshi Hei San(Hou’s Black Powder,HHS-MS)on expressions of phospholipase Cγ2(PLCγ2),extracellular regulated kinase(ERK),transforming growth factor-β(TGF-β)and Smad2/3 in human umbilical vein endothelial cells(HUVEC)after injury of oxygen-glucose deprivation(OGD).Methods HUVEC were randomly divided into control group,model group,2.5%HHS-MS group,5.0%HHS-MS group,10.0%HHS-MS group and 20.0%HHS-MS group.The OGD model was established through putting HUVEC in a hypoxic incubator for continuous cultivation for 6 h after changed to sugar-free nutrient solution in model group and all HHS-MS groups.HUVEC were cultured normally in the same duration.After modeling,the nutrient solution was changed to complete nutrient solution with 10%blank serum in control group and model group,and MS with corresponding mass fraction of HHS-MS was used in all HHS-MS groups.The HUVEC total protein was extracted after normal cultivation for 24 h.The protein expressions of PLCγ2,ERK,p-ERK,TGF-βand Smad2/3 in HUVEC were detected by using Western blotting assay.Results Compared with control group,the expression of PLCγ2 and Smad2/3 were up-regulated(P<0.01)and expression of TGF-βwas down-regulated(P<0.01)in model group.Compared with model group,the expression of TGF-βwas up-regulated in 5.0%HHS-MS group and expressions of PLCγ2 and Smad2/3 were down-regulated in 2.5%HHS-MS group,5.0%HHS-MS group and 10.0%HHS-MS group(P<0.01 or P<0.05).Conclusion HHS-MS can regulate PLCγ2/ERK signaling pathway and TGF-β/Smad2/3 signaling pathway,and improve the proliferation of HUVEC after OGD injury.