HO-1/CO/PPAR-γ信号通路在小鼠动静脉内瘘内膜增生过程中的变化

HO-1/CO/PPAR-γ signaling pathway alterations in the course of intimal hyperplasia of arteriovenous fistula in mice

目的观察HO-1/CO/PPAR-γ信号通路在小鼠动静脉内瘘内膜增生病理损害中的变化规律。方法选取雄性野生型小鼠随机分为模型组及对照组,每组18只。应用显微外科技术建立小鼠颈动静脉内瘘模型(模型组),对照组仅做手术切口缝合。术后21天使用过量戊巴比妥钠处死小鼠,获取血液及病理标本。应用全自动生化分析仪检测血清中总胆固醇、甘油三酯、高密度脂蛋白、低密度脂蛋白、脂蛋白α、总胆红素水平;HE染色检测血管壁变化;应用分子染色方法对标记的活性氧(reactive oxygen species,ROS)进行检测;应用反转录聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)测定目标基因血红素加氧酶-1(heme oxygenase,HO-1)、过氧化物酶增殖物激活受体-γ(peroxisome proliferators-activated receptors-γ,PPAR-γ)及基质金属蛋白酶-9(matrix metalloprotein-9,MMP-9)的mRNA水平;应用Western blot法测定目标基因HO-1、PPAR-γ及MMP-9的蛋白表达;应用酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)法测定HO-1/CO/PPAR-γ信号通路下游因子转化生长因子-β(transforming growth factor-β,TGF-β)、白细胞介素(interleukin,IL)-1β、IL-8表达水平。结果模型组小鼠血清中总胆固醇、甘油三酯、高密度脂蛋白、低密度脂蛋白和总胆红素较对照组明显降低,差异具有统计学意义[mmol/L:(3.45±0.32)比(4.03±0.16),(1.62±0.42)比(2.06±0.35),(3.87±0.30)比(5.11±0.26),(0.45±0.04)比(0.65±0.09),(11.28±1.73)比(13.47±0.87),t值分别为-6.88、-3.41、-13.25、-8.62和-4.80,P值均<0.05]。HE染色提示,模型组小鼠静脉壁内膜厚度较对照组明显增厚,血管壁中活性氧ROS水平较对照组明显升高,差异具有统计学意义[EU/mg:(99.11±6.34)比(89.71±8.41),t=3.79,P<0.05]。与对照组相比,模型组小鼠HO-1/CO/PPAR-γ信号通路目标基因及蛋白HO-1、PPAR-γ、MMP-9 mRNA表达明显升高,差异具有统计学意义[目标基因:(1.02±0.69)比(7.17±2.16),(0.96±0.04)比(6.75±0.24),(0.61±0.35)比(1.02±0.03),t值分别为11.51、100.96和4.95,P值均<0.05;目标蛋白:(0.07±0.02)比(0.41±0.01),(0.27±0.04)比(0.35±0.05),(0.12±0.02)比(0.45±0.08),t值分别为81.43、5.19和17.39,P值均<0.05]。与对照组相比,模型组小鼠HO-1/CO/PPAR-γ信号通路下游炎性因子TGF-β、IL-1β、IL-8分泌明显升高,差异有统计学意义[pg/mL:(3.52±2.92)比(27.46±2.70),(16.78±0.60)比(38.54±2.53),(35.20±1.95)比(101.71±7.75),t值分别为25.53、35.55和35.31,P值均<0.05]。结论HO-1/CO/PPAR信号通路参与了小鼠动静脉内瘘血管内膜增生发生发展过程,并通过提高目标基因、目标蛋白的表达水平,增强相关炎性因子的释放等途径保护内膜功能,减轻血管的损伤。

Objective To observe the changes of HO-1/CO/PPAR-γsignaling pathway in the course of intimal hyperplasia of arteriovenous fistula in mice.Methods Male wild-type mice were randomly divided into model group and control group,with 18 mice in each group.The model group was equipped with microsurgical techniques to establish mice carotid arteriovenous fistula model,while the control group was only sutured with surgical incision.On day 21,mice were sacrificed with pentobarbital sodium,blood and pathological specimens were obtained.The serum levels of total cholesterol,triglyceride,high density lipoprotein,low density lipoprotein,lipoprotein alpha and total bilirubin were detected by automatic biochemical analyzer.Pathological staining was used to detect the change of vascular wall.The labeled reactive oxygen species(ROS)were detected by molecular staining.The mRNA levels of heme oxygenase(HO-1),peroxisome proliferators-activated receptors-γ(PPAR-γ)and matrix metalloprotein-9(MMP-9)were determined by reverse transcription-polymerase chain reaction(RT-PCR).The protein expression of HO-1,PPAR-γand MMP-9 was determined by Western blot,and the levels of transforming growth factor-β(TGF-β),interleukin(IL)-1βand IL-8 were determined by enzyme linked immunosorbent assay(ELISA).Results Compared with control group,serum levels of total cholesterol,triglyceride,high density lipoprotein,low density lipoprotein and total bilirubin in model group were significantly decreased[mmol/L:(3.45±0.32)vs(4.03±0.16),(1.62±0.42)vs(2.06±0.35),(3.87±0.30)vs(5.11±0.26),(0.45±0.04)vs(0.65±0.09),(11.28±1.73)vs(13.47±0.87),t values were-6.88,-3.41,-13.25,-8.62 and-4.80 respectively,all P values<0.05].HE staining showed that the thickness of the vein wall in the model group was significantly thicker than that in the control group,and the level of ROS in the vascular wall was significantly higher than that in the control group[EU/mg:(99.11±6.34)vs(89.71±8.41),t=3.79,P<0.05].Compared with the control group,the expression of HO-1,PPAR-γand MMP-9 in the HO-1/CO/PPAR-γsignaling pathway was significantly increased in the model group[Target gene:(1.02±0.69)vs(7.17±2.16),(0.96±0.04)vs(6.75±0.24),(0.61±0.35)vs(1.02±0.03),t values were 11.51,100.96 and 4.95 respectively,all P values<0.05;target protein:(0.07±0.02)vs(0.41±0.01),(0.27±0.04)vs(0.35±0.05),(0.12±0.02)vs(0.45±0.08),t values were 81.43,5.19 and 17.39 respectively,all P values<0.05].Compared with the control group,the secretion of inflammatory factors TGF-β,IL-1βand IL-8 in the HO-1/CO/PPAR-γsignaling pathway was significantly increased in model group[pg/mL:(3.52±2.92)vs(27.46±2.70),(16.78±0.60)vs(38.54±2.53),(35.20±1.95)vs(101.71±7.75),t values were 25.53,35.55 and 35.31 respectively,all P values<0.05].Conclusions The HO-1/CO/PPAR signaling pathway is involved in the development of intimal hyperplasia of arteriovenous fistula in mice.It protects endothelial function and mitigates vascular injury by increasing the expression levels of target genes and proteins,and enhancing the release of related inflammatory factors.