咖啡酸苯乙酯通过激活核因子E2相关因子2/血红素氧合酶1通路减轻氧化应激损伤改善腹膜透析相关性腹膜纤维化

Caffeic acid phenethyl ester improves peritoneal dialysis-associated peritoneal fibrosis by alleviating oxidative stress injury through activating nuclear factor erythroid-2-related factor 2/heme oxygenase-1 pathway

目的探讨咖啡酸苯乙酯(caffeic acid phenethyl ester,CAPE)是否通过激活核因子E2相关因子2(nuclear factor erythroid-2-related factor 2,Nrf2)/血红素氧合酶1(heme oxygenase-1,HO-1)信号通路减轻氧化应激损伤,从而改善腹膜透析(peritoneal dialysis,PD)相关性腹膜纤维化。方法按随机数字表法将32只雄性Sprague-Dawley大鼠随机分为4组:对照组(CON组,0.9%生理盐水20 ml/d腹腔注射)、单独CAPE组(CAPE组,0.9%生理盐水20 ml/d+CAPE 10 mg·kg-1·d-1腹腔注射)、模型组[PD组,4.25%葡萄糖腹膜透析液(peritoneal dialysis fluid,PDF)20 ml/d腹腔注射,并于第1、3、5及7天予脂多糖0.6 mg/kg腹腔注射]及CAPE干预组(PD+CAPE组,在PD组基础上予CAPE 10 mg·kg-1·d-1腹腔注射),每组8只。腹腔注射持续28 d,第28天行腹膜平衡试验检测腹膜转运功能后处死大鼠,收集壁层腹膜和网膜组织进行后续检测。为进一步探讨机制,分离培养原代大鼠腹膜间皮细胞(peritoneal mesothelial cells,PMCs),用2.5%葡萄糖PDF刺激PMCs,加5μmol/L CAPE干预,检测PMCs的形态和功能。应用Nrf2特异性抑制剂ML385阻断Nrf2探讨CAPE对PMCs保护作用与Nrf2/HO-1通路的关系。组织病理染色检测腹膜组织的结构变化,免疫组化分析Cleaved caspase-3、Bax、α平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、纤维连接蛋白(fibronectin,FN)及Ⅰ型胶原蛋白(typeⅠcollagen,Col-Ⅰ)的蛋白表达,Western印迹检测α-SMA、FN、转化生长因子β1(transforming growth factorβ1,TGF-β1)、HO-1及细胞核内Nrf2(N-Nrf2)的蛋白表达量,细胞凋亡检测试剂盒检测细胞凋亡情况,流式细胞术检测活性氧(reactive oxygen species,ROS)表达,丙二醛(malondialdehyde,MDA)、超氧化物歧化酶(superoxide dismutase,SOD)活性检测试剂盒分别检测MDA含量和SOD活性,细胞免疫荧光分析Nrf2蛋白的表达。结果与CON组相比,PD组大鼠腹膜较厚,凋亡相关蛋白Cleaved caspase-3、Bax、间皮下α-SMA、FN、Col-Ⅰ蛋白表达及MDA含量均较高,HO-1、N-Nrf2蛋白表达和SOD活性均较低(均P<0.05);与PD组相比,PD+CAPE组大鼠腹膜形态明显改善,Cleaved caspase-3、Bax、α-SMA、FN、Col-Ⅰ蛋白表达及MDA含量均较低,HO-1、N-Nrf2蛋白表达和SOD活性均较高(均P<0.05)。与CON组相比,PD组大鼠超滤量较低,腹膜通透性较高,CAPE干预后大鼠腹膜转运功能显著改善(均P<0.05)。在体外培养的PMCs中,PDF抑制Nrf2核转位和HO-1蛋白表达,上调细胞内ROS水平,诱导细胞凋亡增多和α-SMA、TGF-β1和FN蛋白表达增加(均P<0.05);CAPE可激活Nrf2核转位,增加HO-1蛋白表达,下调细胞内ROS水平,部分逆转PDF诱导的细胞凋亡及上皮-间充质转化(均P<0.05);CAPE对PMCs的保护作用可部分被ML385抵消(均P<0.05)。结论CAPE可减轻PD对腹膜的氧化应激损伤,减少PMCs凋亡和上皮-间充质转化,改善腹膜纤维化及超滤功能。CAPE对腹膜的保护作用与激活Nrf2/HO-1通路有关。

Objective To investigate whether caffeic acid phenethyl ester(CAPE)would improve peritoneal dialysis(PD)-associated peritoneal fibrosis by alleviating oxidative stress through activating nuclear factor erythroid-2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)pathway.Methods Thirty-two male Sprague-Dawley rats were randomly divided into four groups by the random number table:control(CON)group(0.9%normal saline 20 ml/d intraperitoneal injection),CAPE group(0.9%normal saline 20 ml/d+CAPE 10 mg·kg-1·d-1 intraperitoneal injection),PD group[4.25%glucose peritoneal dialysis fluid(PDF)20 ml/d intraperitoneal injection with lipopolysaccharide 0.6 mg/kg intraperitoneal injection at day 1,3,5 and 7],and PD+CAPE group(CAPE 10 mg·kg-1·d-1 intraperitoneal injection in addition to PD group),with 8 rats per group.On day 28,rats were euthanized after peritoneal equilibration test,and then the parietal peritoneum and omentum were collected for follow-up tests.To further investigate the mechanism,primary peritoneal mesothelial cells(PMCs)of rats were isolated and cultured.The PMCs were stimulated with 2.5%glucose PDF and added with 5μmol/L CAPE intervention.The Nrf2 inhibitor(ML385)was used to identify whether CAPE protected PMCs from PDF by activating the Nrf2/HO-1 pathway.Histopathological staining was used to detect structural changes of the peritoneum,and immunohistochemical analysis was performed on cleaved caspase-3,Bax,α-smooth muscle actin(α-SMA),fibronectin(FN),and typeⅠcollagen(Col-Ⅰ)protein.Western blotting was used to detect the protein expression ofα-SMA,FN,transforming growth factor-β1(TGF-β1),HO-1 and nuclear Nrf2(N-Nrf2).The apoptosis detection kit was used to detect apoptosis and flow cytometry was used to detect reactive oxygen species(ROS)in PMCs.The malondialdehyde(MDA)and superoxide dismutase(SOD)activity detection kit were used to detect MDA content and SOD activity.Cell immunofluorescence was used to analyze the protein expression of Nrf2 in PMCs.Results Compared with the CON group,the PD group had thicker peritoneum,and the expression levels of cleaved caspase-3,Bax,α-SMA,FN,Col-Ⅰand MDA in peritoneum were significantly higher,while HO-1,N-Nrf2 protein expression and SOD activity were lower(all P<0.05).Compared with the PD group,the parietal peritoneum morphology of CAPE+PD group was improved,accompanied by reduced cleaved caspase-3,Bax,α-SMA,FN,Col-Ⅰprotein expression,and MDA content,while N-Nrf2,HO-1 protein expression,and SOD activity were higher(all P<0.05).Compared with the CON group,the PD group had significantly lower ultrafiltration volume and higher peritoneal permeability(both P<0.05).After CAPE intervention,the peritoneal transport function of the rats was significantly improved(P<0.05).In cultured PMCs,PDF inhibited nuclear translocation of Nrf2 and protein expression of HO-1,and upregulated intracellular ROS level.In addition,PDF increased cell apoptosis and the protein expression levels ofα-SMA,TGF-β1 and FN(all P<0.05).CAPE activated nuclear translocation of Nrf2,increased HO-1 protein expression,downregulated intracellular ROS level,and partially reversed PDF-induced cell apoptosis and epithelial-mesenchymal transition(all P<0.05).The protective effects of CAPE on PMCs were partially abolished by ML385(all P<0.05).Conclusions CAPE can reduce PD-induced PMCs apoptosis and epithelial-mesenchymal transition by attenuating oxidative stress,and significantly improve peritoneal fibrosis and ultrafiltration function.The beneficial effects of CAPE on peritoneum are related to activation of Nrf2/HO-1 pathway.