BHMT对高同型半胱氨酸环境下大鼠晶状体上皮细胞的保护作用  

Protective effects of betaine-homocysteine methyl transferase on rat lens epithelial cells under high homocysteine

作　　者：周海燕[1] 薛雨顺[1] 王新川 李迪[1] 高宁宁 ZHOU Hai-Yan;XUE Yu-Shun;WANG Xin-Chuan;LI Di;GAO Ning-Ning(Department of Ophthalmology,Shaanxi Provincial People's Hospital ,Xi'an 710068,Shaanxi Province,China;Basic Medical College,Air Force Military Medical University ,Xi'an 710038,Shaanxi Province,China)

机构地区：[1]陕西省人民医院眼科,陕西省西安市710068 [2]空军军医大学基础医学院,陕西省西安市710038

出　　处：《眼科新进展》2019年第9期801-804,共4页Recent Advances in Ophthalmology

基　　金：国家自然科学基金资助(编号:81600720);陕西省自然科学基金资助(编号:2017JQ8012)~~

摘　　要：目的观察甜菜碱高半胱酸甲基转移酶(betaine-homocysteine methyl transferase,BHMT)在高同型半胱氨酸(homocysteine,Hcy)环境下对大鼠晶状体上皮细胞的保护作用。方法 SPF级SD大鼠28只,颈椎脱臼法处死后取出晶状体,随机分为对照组(A组)、BHMT组(B组)、Hcy+BHMT组(C组)、Hcy组(D组),每组7只。每组孵育24 h后观察晶状体透明度的改变,晶状体研磨超声粉碎,提取各组总蛋白,Bio-Rad蛋白质定量后,Western blot检测以下蛋白质变化:内质网应激蛋白相关的葡萄糖调节蛋白78(glucose-regulated protein 78,GRP78),抗氧化损伤类蛋白质转录因子NF-E2相关因子2(nuclear factor-erythroid 2-related factor 2,Nrf2),谷胱甘肽还原酶(glutathione reductase,GR),凋亡相关酶半胱氨酸蛋白水解酶-12(cysteinyl aspartate specific proteinase-12,Caspase-12),凝胶成像分析系统显影。使用流式细胞术检测细胞内2’,7’-二氯双氢荧光素(2’,7’-Dichlorofluorescein,DCF)的荧光强度,间接检测细胞内活性氧(reactive oxygen species,ROS)水平。结果体外培养24 h后,A、B组晶状体透明,D组晶状体混浊,C组较D组晶状体混浊程度明显减轻;ROS定量检测结果显示:D组表达量明显高于A、B、C组(均为P<0.05),而C组与A、B组比较差异均无统计学意义(均为P>0.05);内质网应激蛋白GRP78:D组表达量均明显高于A、B、C组(均为P<0.05),而C组与A、B组比较差异均无统计学意义(均为P>0.05);Nrf2与GR的表达量:D组均明显低于A、B、C组(均为P<0.05),而C组与A、B组比较差异均无统计学意义(均为P>0.05);Caspase-12表达量:D组均明显高于A、B、C组(均为P<0.05),而C组与A、B组比较差异均无统计学意义(均为P>0.05)。结论 BHMT在体外可以有效防护高Hcy环境下大鼠晶状体氧化损伤,减轻内质网应激反应,清除ROS,阻止细胞凋亡,减轻晶状体混浊度,证明了其在防治与Hcy有关的疾病方面的积极作用。Objective To observe the protective effect of betaine-homocysteine methyl transferase(BHMT)on rat lens epithelial cells damaged under high homocysteine environment.Methods A total of 28 SD rats were sacrificed by cervical dislocation method.The lens was removed and randomly divided into 4 groups,control group(A group),BHMT group(B group),Hcy+BHMT group(C group) and Hcy group(D group),7 rats in each group.The changes of lens transparency were observed in each group after cultured for 24 hours.The change of the following proteins was measured by Western blot and the results were analyzed by gelimaging and analysis system,including endoplasmic reticulum stress-associated protein(glucose regulated proteins 78,GRP78),antioxidant-damaging protein transcription factors(nuclear factor-erythroid 2 related factor 2,Nrf2),glutathione reductase(GR),apoptosis-related enzyme(Cysteine proteolytic enzyme 12,Caspase 12).The intracellular reactive oxygen species(ROS) levels were estimated by staining with 2’,7’-dichlorofluorescein diacetate(DCF) by flow cytometry.Results After cultured 24 hours,the lens was transparent in group A,B and opacity in group D.The degree of lens opacification in group C was significantly reduced compare with group D.The expression level of ROS in D group was obviously higher than other three groups(all P<0.05),and there was no statistically significant difference between C group and A,B groups(both P>0.05).The expression of endoplasmic reticulum stress protein GRP78 in D group was obviously higher than other three groups(all P<0.05),and there was no statistically significant difference between C group and A,B groups(both P>0.05).The expression of Nrf2 and GR in D group were obviously lower than other three groups(all P<0.05),and there was no statistically significant difference between C group and A,B groups(both P>0.05).The expression of Caspase-12 in D group was obviously higher than other three groups(both P<0.05),and there was no statistically significant difference between C group and A,B group

关 键 词：甜菜碱高半胱氨酸甲基转移酶 高同型半胱氨酸 大鼠晶状体上皮细胞 保护作用 

分 类 号：R776.1[医药卫生—眼科]