中华肾脏病杂志
中華腎髒病雜誌
중화신장병잡지
2014年
7期
530-534
,共5页
徐岩%沈学飞%宋年华%刘雪梅
徐巖%瀋學飛%宋年華%劉雪梅
서암%침학비%송년화%류설매
肾小管%上皮细胞%氧化性应激%NF-E2相关因子2%磷脂酰肌醇3-激酶
腎小管%上皮細胞%氧化性應激%NF-E2相關因子2%燐脂酰肌醇3-激酶
신소관%상피세포%양화성응격%NF-E2상관인자2%린지선기순3-격매
Kidney tubules%Epithelial cells%Oxidative stress%NF-E2-related factor 2%Phosphatidylinositol 3-kinase
目的 观察富含半胱氨酸蛋白61(Cyr61)对缺氧后人肾小管上皮细胞(HK-2)氧化应激的影响,探讨Cyr61对HK-2的保护机制.方法 将HK-2细胞分为5组:空白组、Cyr61处理组、MAPK抑制剂组(Cyr61+ PD98059预处理)、p38抑制剂组(Cyr61 +SB203580预处理)、PI3K抑制剂组[Cyr61+渥曼青霉素(Wortmannin)预处理].各组细胞预处理12h后进行缺氧培养.采用四甲基偶氮唑盐(MTT)法检测缺氧前后细胞存活率,流式细胞术检测细胞凋亡率,双氯荧光黄乙酸乙酯(DCFH-DA)染色检测细胞内活性氧簇(ROS)的生成量,比色法检测细胞中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性,Western印迹法检测细胞磷酸化(p)Akt及细胞核内Nrf2蛋白表达水平. 结果 缺氧培养后空白组HK-2细胞内ROS含量和Nrf2蛋白表达增加,SOD、CAT活性下降,同时细胞存活率下降,凋亡率增高(均P＜0.05).Cyr61处理组缺氧后存活率、细胞内SOD、CAT活性和p-Akt、Nrf2蛋白显著高于空白组,同时其凋亡率及ROS含量明显低于空白组(均P＜ 0.05).与Cyr61处理组相比,缺氧后PI3K抑制剂组HK-2细胞存活率、细胞内SOD、CAT活性和p-Akt、Nrf2蛋白显著下降,凋亡率及ROS含量显著增加(均P＜0.05);而MAPK抑制剂组和p38抑制剂组HK-2细胞的各项指标同Cyr61处理组相比差异无统计学意义(均P＞ 0.05).结论 Cyr61可通过PI3K途径促进Nrf2表达,增强抗氧化物质SOD、CAT产生,减少ROS生成,在缺氧诱导的HK-2细胞氧化应激损伤中发挥保护作用.
目的 觀察富含半胱氨痠蛋白61(Cyr61)對缺氧後人腎小管上皮細胞(HK-2)氧化應激的影響,探討Cyr61對HK-2的保護機製.方法 將HK-2細胞分為5組:空白組、Cyr61處理組、MAPK抑製劑組(Cyr61+ PD98059預處理)、p38抑製劑組(Cyr61 +SB203580預處理)、PI3K抑製劑組[Cyr61+渥曼青黴素(Wortmannin)預處理].各組細胞預處理12h後進行缺氧培養.採用四甲基偶氮唑鹽(MTT)法檢測缺氧前後細胞存活率,流式細胞術檢測細胞凋亡率,雙氯熒光黃乙痠乙酯(DCFH-DA)染色檢測細胞內活性氧簇(ROS)的生成量,比色法檢測細胞中超氧化物歧化酶(SOD)、過氧化氫酶(CAT)活性,Western印跡法檢測細胞燐痠化(p)Akt及細胞覈內Nrf2蛋白錶達水平. 結果 缺氧培養後空白組HK-2細胞內ROS含量和Nrf2蛋白錶達增加,SOD、CAT活性下降,同時細胞存活率下降,凋亡率增高(均P＜0.05).Cyr61處理組缺氧後存活率、細胞內SOD、CAT活性和p-Akt、Nrf2蛋白顯著高于空白組,同時其凋亡率及ROS含量明顯低于空白組(均P＜ 0.05).與Cyr61處理組相比,缺氧後PI3K抑製劑組HK-2細胞存活率、細胞內SOD、CAT活性和p-Akt、Nrf2蛋白顯著下降,凋亡率及ROS含量顯著增加(均P＜0.05);而MAPK抑製劑組和p38抑製劑組HK-2細胞的各項指標同Cyr61處理組相比差異無統計學意義(均P＞ 0.05).結論 Cyr61可通過PI3K途徑促進Nrf2錶達,增彊抗氧化物質SOD、CAT產生,減少ROS生成,在缺氧誘導的HK-2細胞氧化應激損傷中髮揮保護作用.
목적 관찰부함반광안산단백61(Cyr61)대결양후인신소관상피세포(HK-2)양화응격적영향,탐토Cyr61대HK-2적보호궤제.방법 장HK-2세포분위5조:공백조、Cyr61처리조、MAPK억제제조(Cyr61+ PD98059예처리)、p38억제제조(Cyr61 +SB203580예처리)、PI3K억제제조[Cyr61+악만청매소(Wortmannin)예처리].각조세포예처리12h후진행결양배양.채용사갑기우담서염(MTT)법검측결양전후세포존활솔,류식세포술검측세포조망솔,쌍록형광황을산을지(DCFH-DA)염색검측세포내활성양족(ROS)적생성량,비색법검측세포중초양화물기화매(SOD)、과양화경매(CAT)활성,Western인적법검측세포린산화(p)Akt급세포핵내Nrf2단백표체수평. 결과 결양배양후공백조HK-2세포내ROS함량화Nrf2단백표체증가,SOD、CAT활성하강,동시세포존활솔하강,조망솔증고(균P＜0.05).Cyr61처리조결양후존활솔、세포내SOD、CAT활성화p-Akt、Nrf2단백현저고우공백조,동시기조망솔급ROS함량명현저우공백조(균P＜ 0.05).여Cyr61처리조상비,결양후PI3K억제제조HK-2세포존활솔、세포내SOD、CAT활성화p-Akt、Nrf2단백현저하강,조망솔급ROS함량현저증가(균P＜0.05);이MAPK억제제조화p38억제제조HK-2세포적각항지표동Cyr61처리조상비차이무통계학의의(균P＞ 0.05).결론 Cyr61가통과PI3K도경촉진Nrf2표체,증강항양화물질SOD、CAT산생,감소ROS생성,재결양유도적HK-2세포양화응격손상중발휘보호작용.
Objective To investigate the effect and mechanism of cysteine-rich protein 61 (Cyr61) on oxidative stress in human kidney tubular epithelial cell line after anoxia.Methods Human kidney tubular epithelial cell line (HK-2 cells) were divided into 5 groups:control group,Cyr61 group,MAPK inhibitor group (Cyr61 +PD98059),p38 inhibitor group (Cyr61 +SB203580) and PI3K inhibitor group (Cyr61+Wortmannin).Each group was pretreated for 12 h and then injured by anoxia.The cell viability was determined by MTT assay and the apoptosis rate of HK-2 cells was determined by flow-cytometry.The cellular ROS level was measured by spectro-fluorometry.The cellular superoxide dismutase (SOD) and catalase (CAT) were measured by nephelometry test.The expression of Nrf2 in HK-2 cells was detected by Western blotting.Results Anoxia enhanced the expression of ROS and Nrf2,decreased the expression of SOD and CAT significantly,meanwhile decreased HK-2 viability and increased HK-2 apoptosis (all P ＜ 0.05).Cyr61 increased the expression of p-Akt,Nrf2,SOD and CAT in HK-2,and decreased the expression of ROS,at the same time increased HK-2 viability and decreased HK-2 apoptosis (all P ＜ 0.05).Wortmannin inhibited the expression of p-Akt,Nrf2,SOD and CAT,meanwhile decreased HK-2 viability and increased HK-2 apoptosis (P ＜ 0.05).PD98059 and SB203580 had no affect on HK-2 compared to Cyr61 group (P＞0.05).Conclusions Cyr61 promotes the expression of Nrf2 through PI3K pathway in HK-2,which enhances the expression of SOD and CAT,and decreases the expression of ROS.Cyr61 exhibits protective effects on HK-2 cells injured by oxidative stress after anoxia.
