吡非尼酮抑制转化生长因子β1/转化生长因子活化激酶1通路改善大鼠腹膜纤维化  

Pirfenidone inhibits transforming growth factorβ/transforming growth factor-activated kinase 1 pathway to improve peritoneal fibrosis in rats

作　　者：邹臻寰[1] 张小红[1] 李镇洲[1] 林佳群 万建新[1] ZOU Zhen-huan;ZHANG Xiao-hong;LI Zhen-zhou;LIN Jia-qun;WAN Jian-xin(Department of Ne phrology,First Affiliated Hospital of Fujian Medical University,Fuzhou.Fujian 350005,China)

机构地区：[1]福建医科大学附属第一医院肾内科,福建福州350005

出　　处：《中华高血压杂志》2021年第5期460-469,共10页Chinese Journal of Hypertension

基　　金：福建省卫生计生委青年科研课题(2017-1-48)。

摘　　要：目的观察吡非尼酮(PFD)对高糖诱导大鼠的腹膜纤维化(PF)的影响,并探讨其分子作用机制。方法雄性SD大鼠腹腔注射4.25%腹膜透析液100 mL/(kg·d),连续4周,建立PF大鼠模型;随机分为4组:空白对照组、PF组、PF+PFD 250 mg/(kg·d)(PF-PFD250组)、PF+PFD 750 mg/(kg·d)(PF-PFD750组)。检测超滤量(UFV)及最大葡萄糖转运量(MTG),代表腹膜转运功能;Masson染色观察腹膜胶原沉积;免疫组化法测壁层腹膜中转化生长因子β1(TGF-β1)、α平滑肌肌动蛋白(α-SMA)及E-钙黏蛋白表达;免疫印迹试验(Western-blot)检测腹膜组织TGF-β1、α-SMA、E-钙黏蛋白、Ⅰ型胶原、转化生长因子活化激酶1(TAK1)/磷酸化TAK1(P-TAK1)、P38/磷酸化P38(P-P38)蛋白的表达。体外培养腹膜间皮细胞(PMCs),并分别使用TGF-β1、TGF-β1+PFD、TGF-β1+P-P38抑制剂(SB20358020 nmol/L)进行干预。细胞计数试剂(CCK8)评估细胞活力;Western-blot测细胞α-SMA、E-钙黏蛋白、Ⅰ型胶原、TAK1/P-TAK1、P38/P-P38蛋白表达。结果与空白对照组相比,PF组超滤量减少[(1.588±0.955)比(9.675±2.626)mL,P<0.01],MTG升高[(18.625±3.042)比(8.880±1.940)mmol/kg,P<0.01],腹膜厚度明显增加(P<0.01);与PF组相比,PF-PFD250组及PF-PFD750组超滤量增加,MTG降低,腹膜厚度降低(均P<0.01)。与空白对照组相比,PF组腹膜组织中TGF-β1、Ⅰ型胶原、P-TAK1、P-P38、α-SMA表达水平升高,E-钙黏蛋白表达水平降低(均P<0.01);PFD可抑制PF大鼠腹膜组织TGF-β1、α-SMA、Ⅰ型胶原、P-TAK1、P-P38蛋白表达,促进E-钙黏蛋白的表达(P<0.01)。与空白对照组相比,TGF-β1(5μg/L)组细胞活力明显降低(P<0.01),P-TAK1、P-P38、α-SMA、Ⅰ型胶原蛋白水平增加,E-钙黏蛋白表达减少(均P<0.01);与TGF-β1组相比,TGF-β1+PFD(10-3 mol/L)组和TGF-β1+P-P38抑制剂组细胞活力增加,α-SMA和Ⅰ型胶原蛋白水平降低,E-钙黏蛋白表达升高(均P<0.01);TGF-β1+PFD组中P-TAK1和P-P38表达均减少;而TGF-β1+P-P38抑制剂组中仅P-PObjective To observe the effect of pirfenidone(PFD)on high glucose-induced peritoneal fibrosis(PF)and explore its molecular mechanism in rats.Methods Male Sprague-Dawley rats were infused with 100 mL/(kg·d)of 4.25%glucose-based standard peritoneal dialysis fluid for 4 weeks.The rats were divided into four groups:control group(Ctr group),peritoneal fibrosis group(PF group),PF rats with PFD 250 mg/(kg·d)(PF-PFD250 group)and PF rats with PFD 750 mg/(kg·d)(PF-PFD750 group).Ultrafiltration volume(UFV)and mass transfer of glucose(MTG)were used to assess peritoneal transport function.Masson staining was performed to evaluate the deposition of collagen in peritoneal.Immunohistochemistry was performed to measure the expression levels of transforming growth factorβ1(TGF-β1),αsmooth muscle actin(α-SMA)and E-cadherin.Western-blot was performed to measure the expression levels of TGF-β1,α-SMA,E-cadherin,collagen-Ⅰ,transforming growth factor-activated kinase 1(TAK1)/posphorylated TAK1(P-TAK1)and P38/posphorylated P38(P-P38).In vitro,peritoneal mesothelial cells(PMCs)were cultured,which were divided into blank group,TGF-β1 group,TGF-β1+PFD(10-3 mol/L)group and TGF-β1+P38 inhibitor(SB20358020 nmol/L)group.Cell counting kit-8(CCK-8)assay was used to measure cell viability.Western-blot was performed to measure the expression levels ofα-SMA,E-cadherin,collagen-Ⅰ,TAK1/P-TAK1 and P38/P-P38.Results Compared with control group,PF group showed decreased UFV[(1.588±0.955)vs(9.675±2.626)mL,P<0.01]and increased MTG[(18.625±3.042)vs(8.880±1.940)mmol/kg,P<0.01]as well as markedly increased peritoneal thickness and higher expression of TGF-β1,α-SMA,collagen-Ⅰ,P-TAK1 and P-P38,lower expression of E-cadherin(all P<0.01).Compared with PF group,PF-PFD250 group and PF-PFD750 group showed increased UFV and decreased MTG as well as markedly decreased peritoneal thickness(all P<0.01).The elevated TGF-β1,α-SMA,collagen-Ⅰ,P-TAK1 and P-P38 in PF rats were significantly decreased and the declined E-cadherin in PF rats were si

关 键 词：吡非尼酮 腹膜纤维化 转化生长因子Β1 转化生长因子活化激酶1 Α平滑肌肌动蛋白 E-钙黏蛋白 大鼠 

分 类 号：R965[医药卫生—药理学]