组蛋白去乙酰化酶抑制剂Chidamide抑制C2C12骨骼肌细胞分化及其机制

目的:探讨组蛋白去乙酰化酶抑制剂西达苯胺（Chidamide）对C2C12小鼠骨骼肌细胞分化作用及其分子机制。方法:CCK-8（cellcountingKit-8）法检测0、0.5、1、2、4μmol/LChidamide处理后C2C12细胞的活力；将C2C12骨骼肌细胞分为对照组（control）和Chidamide组，Western印迹检测Chidamide处理后组蛋白H3乙酰化（Pan-acetylH3）水平，细胞免疫荧光染色肌管细胞分化成熟标志蛋白肌球蛋白重链（MyHC），检测肌细胞分化，Western印迹和qPCR检测MyHC、成肌分化抗原（MyoD）、肌细胞生成素（MyoG）的表达及肌原纤维Ⅰ型（Myh7）、Ⅱa型（Myh2）、Ⅱb型（Myh4）和Ⅱx型（Myh1）的表达。结果:0、0.5、1、2、4μmol/L的Chidamide均不影响细胞活力。Chidamide升高Pan-acetylH3蛋白水平（t=3.22，P<0.05）。细胞免疫荧光结果显示，与对照组相比，Chidamide处理后肌细胞融合受损，分化程度降低。Western印迹和qPCR结果显示，与对照组相比，Chidamide下调MyHC、MyoD和MyoG蛋白水平（t=5.71、6.05、7.37，均P<0.01）和mRNA水平（t=26.87、5.81、4.86，均P<0.01）；降低Myh7蛋白（t=3.93，P<0.01）和mRNA水平（t=4.85，P<0.01）；降低Myh1、Myh2蛋白（t=14.13、5.05，均P<0.01）和mRNA水平（t=15.48、16.82，均P<0.001），但不影响Myh4的蛋白（t=2.19，P>0.05）和mRNA水平（t=1.50，P>0.05）。结论:Chidamide可能通过下调MyoD和MyoG的表达抑制C2C12骨骼肌细胞分化，且主要抑制Ⅰ型、Ⅱa和Ⅱx型肌原纤维，不影响Ⅱb型肌原纤维。

The inhibitive effect of histone deacetylase inhibitor Chidamide on the differentiation of C2C12 skeletal muscle cells and its mechanism

Objective:To investigate the effect of histone deacetylase inhibitor Chidamide on the differentiation of C2C12 skeletal muscle cells and its potential molecular mechanism. Methods: The cell counting Kit-8（CCK-8）method was used to detect the viability of C2C12 cells after 0，0.5，1，2，4 μmol/L Chidamide treatment；C2C12 skeletal muscle cells were divided into control group and Chidamide group. Western blotting was used to detect the acetylation of histone H3（Pan-acetyl H3）level after Chidamide treatment. Immunofluorescence staining of differentiation and maturation marker proteins myosin heavy chain（MyHC）was used to detect muscle cell differentiation. The protein and mRNA expressions of MyHC，myogenic differentiation antigen（MyoD），myogenin （MyoG），and the typeⅠ（Myh7），typeⅡa（Myh2），typeⅡb（Myh4）and typeⅡx（Myh1）of myofibrils were detected byWestern blotting and qPCR， respectively. Results: 0，0.5，1，2，4 μmol/L Chidamide did not affect cell viability. Chidamide increased the protein level of Pan-acetyl H3（t=3.22，P<0.05）. Immunofluorescence results showed that compared with the control group，myocyte fusion was impaired and th e degree of differentiation decreased after Chidamide treatment. Western blotting and qPCR results showed that compared with the control group，Chidamide decreased the protein levels（t=5.71，6.05，7.37，all P<0.01）and mRNA levels（t=26.87，5.81，4.86，all P<0.01） of MyHC，MyoD and MyoG. Chidamide also decreased protein and mRNA levels of Myh7（t=3.93，4.85，both P<0.01），Myh1（t=14.13， 15.48，both P<0.001）and Myh2（t=5.05，16.82，both P<0.01）. However，Chidamide did not affect the protein（t=2.19，P>0.05）and mRNA levels （t=1.50，P>0.05）of Myh4. Conclusion :Chidamide may inhibit the differentiation of C2C12 skeletal muscle cells by down-regulating the expressions of MyoD and MyoG，and mainly inhibits typeⅠ，Ⅱa andⅡx myofibrils，but does not affect typeⅡb myofibrils.