组蛋白去乙酰化酶2在衰老心肌细胞自噬水平降低中的作用

Role of HDAC2 in decrease of autophagy level in aging cardiomyocytes

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.