环氧化酶2抑制剂parecoxib对C2C12骨骼肌细胞分化的影响及机制研究

Study of effect and mechanism of cyclooxygenase-2 inhibitor parecoxib on the differentiation of C2C12 skeletal muscle cells

目的:探讨环氧化酶2抑制剂帕瑞昔布(parecoxib)对C2C12小鼠骨骼肌细胞分化的影响及其分子机制。方法:CCK-8法检测不同浓度的parecoxib处理后C2C12细胞的活力;分化培养基诱导C2C12小鼠骨骼肌细胞48 h后,将细胞分为对照组(control组)和parecoxib组继续分化48 h。免疫荧光检测肌管细胞分化成熟的标志蛋白肌球蛋白重链(MyHC),qPCR和Western印迹分别检测My HC、肌原纤维Ⅰ型(Myh7)、Ⅱa型(Myh2)、Ⅱb型(Myh4)、Ⅱx型(Myh1)、肌生成决定因子(MyoD)、肌生成因子5(Myf5)、肌生成素(myogenin)的基因和蛋白表达。结果:0、100、150、200、250、300μmol/L的parecoxib均不影响细胞活力。与对照组相比,parecoxib组MyHC阳性肌纤维数量减少,肌管融合受损,分化程度降低,MyHC、MyHCⅡa、MyHCⅡb、MyHCⅡx、MyoG、MyoD和Myf5的mRNA水平降低(t=19.04、53.93、72.38、33.72、15.32、3.061、18,均P<0.05),MyHCⅠ的mRNA水平升高(t=17.12,P<0.01)。Western印迹结果显示,与对照组相比,parecoxib组MyHC、MyHCⅡa、MyHCⅡb、MyHCⅡx、MyoG和MyoD蛋白水平降低(t=7.297、7.852、11.43、227、80.14、11.76,均P<0.01),MyHCⅠ蛋白水平升高(t=5.891,P<0.01)。结论:Parecoxib可能通过下调MyoG、MyoD和Myf5的表达,抑制C2C12骨骼肌细胞的分化。

Objective:To investigate the effect of parecoxib,a cyclooxygenase-2 inhibitor,on the differentiation of C2C12 skeletal muscle cells and its potential molecular mechanism.Methods:The Cell Counting Kit-8(CCK-8)was used to detect the viability of C2C12 cells treated with different concentrations of parecoxib.C2C12 mouse skeletal muscle cells were incubated with differentiation medium for 48 hours.Then the cells were divided into control group and parecoxib group for further differentiation for 48 hours.Immunofluorescence was used to detect myosin heavy chain(MyHC),a marker of differentiation and maturation of myotubule cells.The mRNA and protein expression of MyHC,MyHCI(Myh7),Ⅱa(Myh2),Ⅱb(Myh4),Ⅱx(Myh1),myogenic determinant(MyoD),myogenic factor 5(Myf5)and Myogenin were detected by qPCR and Western blotting,respectively.Results:0,100,150,200,250 and 300μmol/L parecoxib did not affect cell viability.Immunofluorescence data showed that compared with the control group,the number of MyHC-positive muscle fibers in the parecoxib group was reduced,muscle tube fusion was impaired,and the degree of differentiation was reduced.The results of qPCR showed that compared with the control group,the mRNA levels of MyHC,MyHCⅡa,MyHCⅡb,MyHCⅡx,MyoG,MyoD and Myf5 in the parecoxib group were decreased(t=19.04,53.93,72.38,33.72,5.32,3.061,18,all P<0.05),and the mRNA level of MyHCⅠwas increased(t=17.12,P<0.01).Western blotting results showed that compared with the control group,the protein levels of MyHC,MyHCⅡa,MyHCⅡb,MyHCⅡx,MyoG and MyoD in the parecoxib group were decreased(t=7.297,7.852,11.43,227,80.14,11.76,all P<0.01),and the MYHCⅠprotein level was increased(t=5.891,P<0.01).Conclusion:Parecoxib may inhibit the differentiation of C2C12 skeletal muscle cells by down-regulating the expression of MyoG,MyoD and Myf5.