摘要
目的探讨组蛋白去乙酰化酶2(histone deacetylase 2,HDAC2)在D-半乳糖(D-galactose)诱导的衰老心肌细胞自噬水平降低中的作用。方法使用D-galactose刺激大鼠心肌细胞H9c2衰老后,β-半乳糖苷酶染色验证模型是否成功;透射电镜观察未给予D-galactose对照(control)组与给予D-galactose组的自噬情况;Western Blot检测LC3B及p62的蛋白表达;分别采用qRT-PCR与Western Blot检测control组与D-galactose组HDAC2的mRNA与蛋白表达;同时,D-galactose组干扰HDAC2的表达后,Western Blot检测HDAC2的自身表达及LC3B与p62的蛋白表达,免疫荧光染色观察LC3B的定位及表达;最后,Western Blot检测control组与D-galactose组H3K14ac的蛋白表达;D-galactose组干扰HDAC2的表达后,Western Blot检测H3K14ac的蛋白表达。结果成功复制了衰老心肌细胞模型;经过D-galactose干预后,细胞内的自噬结构较未干预组明显减少;Western Blot结果显示,LC3BⅡ/Ⅰ水平降低,p62表达升高;qRT-PCR与Western Blot结果均证实D-galactose组与control组相比HDAC2的mRNA和蛋白水平明显降低;干扰HDAC2的表达后,Western Blot结果显示干扰HDAC2后的D-galactose组(Ad-shHDAC2)与未干扰HDAC2的D-galactose组(AdshNC)相比,LC3BⅡ/Ⅰ水平降低,p62表达升高,免疫荧光染色结果显示Ad-shHDAC2与Ad-shNC相比LC3B荧光强度要低;Western Blot结果显示,D-galactose组H3K14ac水平与control组相比明显升高;干扰HDAC2的表达后,Western Blot结果显示Ad-shHDAC2组与Ad-shNC组相比,H3K14ac表达进一步增加。结论 D-galactose能够诱导衰老心肌细胞自噬水平降低,且HDAC2的表达降低介导H3K14高乙酰化水平可能是衰老心肌细胞自噬水平降低的重要机制之一。
Objective To investigate the role of HDAC2 in the reduction of autophagy in D-galactose-in-duced aging cardiomyocyte.Methods D-galactose was used to stimulate the aging of rat cardiomyocyte H9c2 cellline,β-galactosidase staining was used to verify the success of the model. Autophagy level in no D-galactose group(control)and D-galactose group were detected by transmission electron microscopy. Protein expression of LC3B and p62 in control and D-galactose groups were detected by Western Blot. The m RNA and protein expression ofHDAC2 in control and D-galactose groups were detected by qRT-PCR and Western Blot. After being treated with D-galactose to interfere with the expression of HDAC2,the expression of HDAC2,LC3B and p62 were detected byWestern Blot,immunofluorescence staining was used to observe the localization and expression of LC3B. The pro-tein expression of H3K14ac was detected by Western Blot in control and D-galactose groups. After being treatedwith D-galactose to interfere with the expression of HDAC2,the expression of H3K14ac was detected by WesternBlot.Results The aging cardiomyocyte model was successfully replicated. After D-galactose intervention,theautophagy structure in the cells was significantly reduced compared with the non-intervention group. The results ofWestern Blot showed that the level of LC3BⅡ/Ⅰ was decreased and the expression of p62 were increased in the D-galactose group. Both qRT-PCR and Western Blot results confirmed that the m RNA and protein levels of HDAC2 were significantly decreased in D-galactose group compared with control group. After D-galactose intervention,Ad-sh HDAC2 compared with Ad-sh NC,Western Blot results showed decreased level of LC3B Ⅱ/Ⅰ,elevated p62 expression. The results of immunofluorescence staining showed that Ad-sh HDAC2 has lower LC3B compared withAd-sh NC. The results of Western Blot showed that the level of H3K14ac was increased in the D-galactose group.After D-galactose intervention,Ad-sh HDAC2 compared with Ad-sh NC,Western Blot results showed elevated H3K14ac expression.Conclusion D-galactose can decrease autophagy in aging cardiomyocyte,and the decreaseof HDAC2 expression may be one of the important mechanisms of autophagy in aging cardiomyocyte.
作者
王睿
王艳华
柴丽芬
吴琪瑞
吴凯
杨勇
杨晓玲
姜怡邓
李桂忠
WANG Rui;WANG Yanhua;CHAI Lifen;WU Qirui;WU Kai;YANG Yong;YANG Xiaoling;JIANG Yideng;LI Guizhong(School of Basic Medical Sciences,Ningxia Medical University,Yinchuan 750004,China;不详)
出处
《实用医学杂志》
CAS
北大核心
2020年第9期1157-1163,共7页
The Journal of Practical Medicine
基金
国家自然科学基金项目(编号:81560242,81860044)
宁夏自然科学基金项目(编号:2018A0151)
宁夏高等学校一流学科建设(宁夏医科大学西部一流建设学科基础医学)项目(编号:NXYLXK2017B07)
春晖计划项目(编号:Z2016043)。