Folic acid treatment regulates C2C12 myoblast diferentiation via JNK/p38 MAPK signaling pathway / 中国病理生理杂志

AIM:To observe the effect of folic acid(FA)on C2C12 myoblast proliferation and differentia-tion,and to explore its mechanism.METHODS:During the proliferation stage,C2C12 myoblasts were treated with vari-ous concentrations of FA(0,2.5,5,10 and 20 μmol/L).The cell status was observed under a microscope,cell viability was detected using the MTT method,and cell proliferation was assessed using the EdU method.In the differentiation stage,C2C12 cells were divided into control(Ctrl)group(0 μmol/L FA)and FA group(10 μmol/L FA).On day 2 or 4 of differentiation,immunofluorescence staining and Western blot were employed to detect the expression of myoblast differen-tiation-related proteins,myoblast determination protein 1(MyoD),myogenin(MyoG)and myosin heavy chain(MyHC).The myotubule formation in each group was analyzed.On day 4 of differentiation,C2C12 cells were treated with FA for 0,1,3 and 6 h,and the protein levels of p-JNK,JNK,p-p38 MAPK and p38 MAPK at each time point were detected by Western blot.Additionally,C2C12 cells after 4-day differentiation were divided into Ctrl group,FA group,FA+ SP600125(specific inhibitor of JNK)group,and FA+SB203580(specific inhibitor of p38)group.The cells in FA+ SP600125 and FA+SB203580 groups were treated with 10 μmol/L SP600125 or SB203580 for 1 h,followed by treatment with 10 μmol/L FA for 24 h.The cells in FA group were treated with 10 μmol/L FA for 24 h,while the cells in Ctrl group were left untreated.The protein levels of p-JNK,JNK,p-p38 MAPK,p38 MAPK and MyHC were detected by Western blot.RESULTS:(1)Compared with 0 μmol/L FA group,the number of the cells in other concentration groups in-creased,cell viability was raised(P＜0.05 or P＜0.01),and the rate of EdU positive cells increased(P＜0.05).(2)Com-pared with Ctrl group,the expression levels of MyoD,MyoG and MyHC in FA group were increased(P＜0.05),and the myotube fusion index was raised(P＜0.05 or P＜0.01).(3)Compared with 0 h group,the ratios of p-JNK/JNK and p-p38 MAPK/p38 MAPK were elevated after FA treatment for 1,3 and 6 h(P＜0.05 or P＜0.01),and showed a trend of gradual increase with the extension of treatment time.(4)After FA treatment,the ratios of p-JNK/JNK and p-p38 MAPK/p38 MAPK,and the expression of MyHC were elevated(P＜0.01).Treatment with SP600125 decreased the ratio of p-JNK/JNK and the expression of MyHC(P＜0.05),while SB203580 intervention cut down the ratio of p-p38 MAPK/p38 MAPK and the expression of MyHC(P＜0.05 or P＜0.01).CONCLUSION:Folic acid can promote the differentiation of C2C12 myoblasts by activating the JNK/p38 MAPK signaling pathway.

Protective effects of Qigu Capsule-medicated serum on C2C12 cells atrophy induced by dexamethasone / 中成药

AIM To observe the protective effects of Qigu Capsule-medicated serum on C2C12 myotube cell atrophy induced by dexamethasone.METHODS The Qigu Capsule-medicated serum was prepared.C2C12 cells cultured in vitro were divided into the normal control group,the dexamethasone group,the 10%blank serum group and the 10%Qigu Capsule-medicated serum group for 48 h,corresponding drug treatment,followed by 24 h culture with 10 μmol/L dexamethasone except the control group,and had cell viability detection by CCK-8 method.With their myotube cells intervened accordingly by the aforementioned regime following 6-7 days differentiation with 2%horse serum induction,the C2C12 cells had their myotube cells diameter measured;and the expressions of proteins related to MyHC,MuRF-1,Atrogin-1 and PI3K/Akt signaling pathway detected by Western blot.The impact of PI3K inhibitor LY294002 on the diameter of myotube cells and the expression of MuRF-1,Atorgin-1 and PI3K/Akt signaling pathway related proteins were detected as well.RESULTS Compared with the normal control group,the dexamethasone group and the blank serum group were observed with decreased viability of C2C12 cells(P<0.01),decreased diameter of myotube cells(P<0.01),increased protein expressions of MuRF-1 and Atrogin-1(P<0.01),and decreased expression of MyHC protein and phosphorylation levels of PI3K,Akt,mTOR and FoxO3a(P<0.01).Compared with the dexamethasone group,the Qigu Capsule-medicated serum group showed increased viability of C2C12 cells(P<0.01);increased diameter of myotube cells(P<0.01);decreased protein expressions of MuRF-1 and Atrogin-1(P<0.01);and increased expression of MyHC protein and phosphorylation levels of PI3K,Akt,mTOR and FoxO3a(P<0.05,P<0.01).The intervention of LY294002 upon the Qigu Capsule-medicated serum group offset the anti-muscular tube atrophy effect(P<0.01),increased the protein expressions of MuRF-1 and Atorgin-1(P<0.01),and decreased the phosphorylation levels of PI3K,Akt,mTOR and FoxO3a(P<0.05,P<0.01).CONCLUSION The Qigu Capsule-medicated serum can alleviate the atrophy of C2C12 myotubes induced by dexamethasone,and its mechanism may be related to the activation of PI3K/Akt signaling pathway.

Liraglutide increases FNDC5 expression in C2C12 myotubes via activa-tion of CAMKK2/AMPK signaling pathways / 中国病理生理杂志

AIM:To investigate the effect of liraglutide ( LG) on the expression of fibronectin type Ⅲdomain-containing protein 5 (FNDC5) in the C2C12 myotubes.METHODS:The C2C12 mouse myoblast cell line was induced to differentiation.Differentiated cells were stimulated with gradient concentrations (1 ~1000 nmol/L) of LG for different time (0 ~24 h).The effects of LG on the expression of FNDC5 and the activation of adenosine 5'-monophosphate ( AMP)-activated protein kinase ( AMPK) signaling pathway were determined .After pretreated with glucagon-like peptide-1 ( GLP-1 ) receptor antagonist exendin 9-39 , the inhibitor of Ca 2+/calmodulin-dependent protein kinase kinase 2 (CAMKK2), STO609, or the inhibitor of AMPK, Compound C, the LG-induced FNDC5 expression in C2C12 myotubes was examined.The expression of FNDC5 and the activation of AMPK were determined by Western blot .RESULTS: In C2C12 myotubes, LG promoted the expression of FNDC5 in a dose-and time-dependent manner .LG also activated AMPK signaling pathway .These effects of LG were partly abolished by exendin 9-39 , STO609 and Compound C .CONCLUSION:LG promotes the expression of FNDC5 via GLP-1 receptor in the C2C12 myotubes possibly through activation of the CAMKK2/AMPK signaling pathways .

Effect of different concentrations of calpain inhibitor ALLN on proliferation and apoptosis of C2C12 myoblasts / 中华临床营养杂志

Objective To explore the effect of different concentrations of ALLN on proliferation and apoptosis of C2C12 myoblasts.Methods After intervention with Ca2+ and ALLN,methyl thiazolyl tetrazolium and flow cytometry were used to determine the effect of Ca2+ and ALLN on the proliferation and apoptosis of C2C12 cells,respectively.The morphological changes of C2C12 myoblasts were observed using Giemsa staining.Results The absorbance of Ca2 + group was significantly lower than that of the control group (P ＜0.05).After 6,12,24,36 hours of intervention,the absorbance in ALLN groups 1 to 7 (cultured in serum-free media containing 16 mmol/L Ca2+ and ALLN at final concentrations of 3.125,6.25,12.5,25,50,100,200 μmol/L) were all significantly higher than that in the 16 mmol/L Ca2+ group (after 6 hours:0.449±0.024,0.472±0.022,0.513 ±0.008,0.540±0.014,0.588±0.016,0.607±0.030,0.700±0.020 vs.0.355 ±0.012,all P =0.000; after 12 hours:0.407 ±0.007,0.414 ±0.006,0.434 ±0.004,0.441 ±0.003,0.460 ±0.010,0.484 ± 0.006,0.525 ± 0.006 vs.0.368 ± 0.027,all P =0.000; after 24 hours:0.436±0.005,0.431 ±0.015,0.441 ±0.006,0.459 ±0.013,0.527 ±0.009,0.581 ±0.005,0.599 ±0.011 vs.0.386 ± 0.007,all P =0.000 ; after 36 hours:0.464 ± 0.022,0.460 ± 0.018,0.461 ± 0.007,0.434 ± 0.020,0.454 ± 0.028,0.479 ± 0.006,0.524 ± 0.011 vs.0.379 ± 0.011,all P =0.000),while no significant differences were observed after 48-72 hours of intervention.After treatment for 36 hours,the apoptosis rate in ALLN 10,50,100,and 200 μmol/L groups were (6.00 ± 1.20) ％,(5.02 ± 1.13) ％,(4.89±1.11)％,and (2.71 ± 1.15)％,all significantly lower than that in the Ca2+ group [(13.70 ±2.30)％] (all P =0.000).Giemsa staining showed apoptotic morphological changes in the Ca2+ group,which were obviously alleviated in the ALLN group.Conclusions Ca2+ at a concentration of 16mmol/L can induce apoptosis of C2C12 cells.In contrast,ALLN can inhibit cell apoptosis and promote proliferation in a time-and dose-dependent manner.

Cigarette smoke extract promotes senescence by reducing expression of HDAC2 in C2C12 myoblasts / 中国病理生理杂志

AIM:To investigate whether the cigarette smoke extract (CSE) causes senescence of C2C12 myo-blasts and the relationship between senescence and histone deacetylase 2(HDAC2).METHODS: Murine C2C12 cells were induced to differentiate into myoblasts.The HDAC2 activator and inhibitor were used to investigate the effects of CSE in the myoblasts on cell senescence and the expression of HDAC2.The expression of HDAC2 at mRNA and protein levels was determined by real-time PCR and Western blot, respectively, and the positive cell rate of β-galactosidase staining for cell senescence was also detected.RESULTS:The optimal concentration of CSE was 60 mL/L and the intervention time was 24 h.After the intervention of CSE, the positive cell rate ofβ-galactosidase staining was increased, accompanied with the reduction of HDAC2 expression at mRNA and protein levels.The expression of HDAC2 at mRNA and protein levels was increased by 4, 5, 6, 7-tetrabromobenzotriazole (TBB), accompanied with the reduction of positive cell rate ofβ-galacto-sidase staining.Furthermore, when HDAC2 expression at mRNA and protein levels was reduced by HDAC2 inhibitor valp-roic acid, the positive cell rate of β-galactosidase staining was increased.CONCLUSION: CSE promotes the senescence by reducing the expression of HDAC2 in C2C12 myoblasts.