[1]刘越峰,罗卫民,张勇,等.二十二碳六烯酸经Rac1/NAPDH氧化酶/ROS/p38通路诱导ARPE-19细胞表达HO-1的机制[J].眼科新进展,2016,36(3):223-226.[doi:10.13389/j.cnki.rao.2016.0060]
　LIU Yue-Feng,LUO Wei-Min,ZHANG Yong,et al.Molecular mechanism of HO-1 expression in ARPE-19 cells induced by DHA via Racl/NADPH oxidase/ROS/p38 pathway[J].Recent Advances in Ophthalmology,2016,36(3):223-226.[doi:10.13389/j.cnki.rao.2016.0060]
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二十二碳六烯酸经Rac1/NAPDH氧化酶/ROS/p38通路诱导ARPE-19细胞表达HO-1的机制
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
36卷
期数:
2016年3期
页码:
223-226
栏目:
实验研究
出版日期:
2016-03-05

文章信息/Info

Title:
Molecular mechanism of HO-1 expression in ARPE-19 cells induced by DHA via Racl/NADPH oxidase/ROS/p38 pathway
作者:
刘越峰; 罗卫民; 张勇; 钟晓东
442000　湖北省十堰市，湖北医药学院附属十堰市太和医院眼科中心
Author(s):
LIU Yue-Feng ;  LUO Wei-Min ; ZHANG Yong; ZHONG Xiao-Dong
Department of Ophthalmology , Taihe Hospital of Shiyan Affiliated to Hubei University of Medicine , Shiyan 442000 . Hubei Province . China
关键词:
二十二碳六烯酸; 视网膜色素上皮细胞; 血红素氧合酶-1
Keywords:
docosahexaenoic acid ;  retinal pigment epithelial cell;  heme oxygenase-l
DOI:
10.13389/j.cnki.rao.2016.0060
文献标志码:
A
摘要:
目的　观察二十二碳六烯酸（ｄｏｃｏｓａｈｅｘａｅｎｏｉｃａｃｉｄ，ＤＨＡ）诱导人视网膜色素上皮细胞表达血红素氧合酶（ｈｅｍｅｏｘｙｇｅｎａｓｅ，ＨＯ）-１的分子机制。方法　培养人视网膜色素上皮细胞系ＡＲＰＥ-１９，加入３０～１００μｍｏｌ·Ｌ－１ＤＨＡ作用４～２４ｈ。ＥＬＩＳＡ法检测Ｒａｃ１的活性，Ｗｅｓｔｅｒｎｂｌｏｔ检测ＨＯ-１的表达及ＮＡＤＰＨ氧化酶ｐ４７ｐｈｏｘ亚基和ｐ３８的磷酸化；荧光探针Ｈ２ＤＣＦＤＡ检测活性氧（ｒｅａｃｔｉｖｅｏｘｙｇｅｎｓｐｅｃｉｅｓ，ＲＯＳ）的产生；采用Ｒａｃ１抑制剂ＮＳＣ２３７６６、ＮＡＤＰＨ氧化酶抑制剂ＤＰＩ、ＲＯＳ清除剂ＮＡＣ预处理细胞，检测其对ＨＯ-１表达的影响。结果　３０μｍｏｌ·Ｌ－１、５０μｍｏｌ·Ｌ－１和１００μｍｏｌ·Ｌ－１ＤＨＡ作用ＡＲＰＥ-１９细胞１０ｍｉｎ后，Ｒａｃ１的活性分别为（１２６．４１±１１．２５）％、（１８５．０５±１５．４１）％和（２６０．５２±１７．８３）％，与对照组相比，差异均有统计学意义（均为Ｐ＜０．０５）。同时，１００μｍｏｌ·Ｌ－１ＤＨＡ也能诱导ｐ４７ｐｈｏｘ亚基磷酸化。３０μｍｏｌ·Ｌ－１、５０μｍｏｌ·Ｌ－１和１００μｍｏｌ·Ｌ－１ＤＨＡ处理４ｈ后，细胞内ＲＯＳ的含量分别为（１３２．５２±８．３３）％、（１７７．９４±１０．２４）％和（２１１．６２±７．１４）％，与对照组相比，差异也均有统计学意义（均为Ｐ＜０．０５）。对照组细胞中磷酸化ｐ３８含量为０．１６±０．０１；当给予３０μｍｏｌ· Ｌ－１、５０μｍｏｌ·Ｌ－１和１００μｍｏｌ·Ｌ－１ＤＨＡ处理３０ｍｉｎ后，磷酸化ｐ３８含量分别为０．２８±０．０３、０．３７±０．０２和０．４５±０．００，与对照组相比，差异也均有统计学意义（均为Ｐ＜０．０５）；采用Ｒａｃ１抑制剂ＮＳＣ２３７６６或ＮＡＤＰＨ氧化酶抑制剂ＤＰＩ处理后，ｐ４７ｐｈｏｘ亚基的磷酸化水平和细胞内ＲＯＳ水平显著降低；而采用ＮＡＣ处理后，可抑制ＤＨＡ诱导ＡＲＰＥ-１９细胞表达ＨＯ-１。结论　ＤＨＡ可能通过Ｒａｃ１／ＮＡＰＤＨ氧化酶／ＲＯＳ／ｐ３８通路途径诱导视网膜色素上皮细胞表达ＨＯ-１，从而发挥对细胞的保护作用。
Abstract:
Objective To observe the molecular mechanism of docosahexaenoic acid ( DHA) on the expression of heme oxygenase ( HO-I ) in human retinal pigment epithelium cells. Methods Human retinal pigment epithelium cell line ARPE-19 was cultured in vitro and was treated with 30 - 100 ymol . L -l DHA for 4 - 24 hours. Activity of Racl was measured by ELISA. HO-I expression and phosphorylation of NADPH oxidase p47phox subunit and p38 were detected by Western blot. Production of reactive oxygen species ( ROS) was detected by fluorescent probe H2DCFDA. NSC23766 .an inhibitor of Racl,and DPI,an inhibitor of NADPH oxidase or NAC ( ROS inhibitor) were used to test their effects on HO-I expression. Results Treatment of ARPE-19 cells by 30 ymol . 1’1 ,50 ymol . L-l and 100 ymol . L-1 0f DHA could up-regulate the Racl activity to ( 126. 41 + 11. 25 ) % . ( 185. 05 + 15. 41 ) % and ( 260. 52 + 17. 83 ) % , respectively, there were statistical differences between DHA groups and control group ( all P < 0. 05). 100 Vmol . L-l of DHA could increase the level of phosphorylated p47Phox. 30 ymol . 1’1 ,50 ymol . L-l and 100 ymol . L-l of DHA treatment could also increase the ROS to ( 132. 52 + 8. 33) % . ( 177. 94 + 10. 24 ) % and (211. 62 + 7. 14 ) % , respectively, there were statistical differences as compared with the control group ( all P < 0. 05 ) . The phosphorylated p38 level in control group was 0. 16 + 0. 01. After treated by 30 ymol . 1’1 ,50 ymol . L-l and 100 ymol . L-I DHA for 30 nunutes,phosphorylated p38 reached t0 0. 28 + 0. 03 , 0. 37 + 0. 02 and 0. 45 + 0. 005 , respectively , there were statistical differences as compared with the control group ( all P < 0. 05 ) . Pretreatment of the Racl inhibotor NSC23766 0r NADPH oxidase inhibitor DPI could inhibit p47Phox phosphorylation and ROS production, respectively. While NAC could significantly abrogate DHA induced HO-I expression. Conclusion DHA protects retinal pigment epithelial cells against oxidative stress by induction of HO-I via Racl/NADPH oxidase/ROS/p38 pathway.

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备注/Memo

备注/Memo:
十堰市科学技术研究与开发项目计划（编号：１４Ｙ４０）
更新日期/Last Update: 2016-03-08