| Objective: Skeletal muscle is a vital tissue that plays a key role in the movement,support,metabolism,and heat production.Its health is critical to maintaining a high quality of life and longevity.This study explores the Chinese herbal monomer Morroniside’s impact on the differentiation of C2C12 mouse myoblasts treated with TNFα.Additionally,we investigate the feasibility of using Morroniside to treat inflammation-related skeletal muscle atrophy and the underlying molecular mechanisms involved.Methods:The research subject of this study was the C2C12 mouse myoblast cell line.TNFα was used to induce the differentiation of cells and establish an in vitro model of skeletal muscle atrophy.The study observed the effects of different concentrations of TNFα on the formation of myotubes and investigated the role and mechanism of Morroniside in improving skeletal muscle atrophy.According to the experimental purpose,the experimental subjects were divided into four groups: Veh,TNFα,TNFα+Mor-L,and TNFα+MorH.The cell viability was assessed using the CCK-8 assay.Morphological parameters such as the number of myotubes,nucleus number,and relative diameter were measured and evaluated using the Image J software after immunofluorescence staining of the cells.The transcription levels of genes related to NF-k B classical and non-classical signaling pathways(Rel A,p50,Rel B,p52),pro-inflammatory cytokines(IL-6,IL-1b,CRP,TNFα),protein synthesis-related pathway genes(IGF-1,IGF-1R,PIK3R1,PIK3 CA,AKT1/2/3),and ubiquitination-related genes(Mu RF1,Atrogin1)were detected using RT-q PCR.The protein expression levels of Rel A,p50,Rel B,p52,TNFα,Mu RF1,Atrogin1,and My HC were detected using Western Blot.Results:(1)Compared with the Veh group,treatment with TNFα during differentiation significantly inhibited the formation of myotubes;the formation of myotubes in both TNFα+Mor-L and TNFα+Mor-H groups was better than that in the TNFα group.(2)The average number of myotubes in TNFα group C2C12 cells was significantly lower than that in the Veh group(p < 0.01);the average number of myotubes in the TNFα group was significantly lower than that in the TNFα+Mor-L group(p < 0.05)and TNFα+Mor-H group(p < 0.05).The average number of nuclei per myotube in the TNFα group was also lower than that in the Veh group(p < 0.01),TNFα+Mor-L group(p < 0.05),and TNFα+Mor-H group(p < 0.05).The relative diameter of myotubes in the TNFα group was significantly lower than that in the Veh group(p <0.01),as well as in the TNFα+Mor-L group(p < 0.05)and TNFα+Mor-H group(p < 0.05).(3)The m RNA levels of Rela in the TNFα group were significantly higher than those in the Veh group(p < 0.05);the m RNA levels of Rela in the TNFα+Mor group were significantly lower than those in the TNFαgroup(p < 0.05).The m RNA levels of p50 in both the TNFα and TNFα+Mor groups were significantly higher than those in the Veh group(p < 0.0001);the expression level of Rel A in the TNFα group was significantly higher than that in both the Veh group(p < 0.0001)and TNFα+Mor group(p < 0.0001).The protein expression levels of p50 in both the TNFα and TNFα+Mor groups were significantly higher than those in the Veh group(p < 0.0001);the m RNA levels of Relb in the TNFα and TNFα+Mor groups were significantly higher than those in the Veh group(p < 0.0001 and p < 0.001,respectively);the m RNA levels of Relb in the TNFα+Mor group were significantly lower than those in the TNFα group(p < 0.01).The m RNA levels of p52 in both the TNFα and TNFα+Mor groups were significantly higher than those in the Veh group(p < 0.0001);the m RNA levels of Relb in the TNFα+Mor group were significantly lower than those in the TNFα group(p < 0.01);the expression levels of Rel B in both the TNFα and TNFα+Mor groups were significantly higher than those in the Veh group(p < 0.0001 and p < 0.001,respectively).The expression level of Rel B in the TNFα+Mor group was significantly lower than that in the TNFα group(p < 0.01).Compared to the Veh group,the protein expression level of p52 was significantly increased in the TNFα group(p< 0.001);the protein expression level of p52 in the TNFα+Mor group was significantly higher than that in the Veh group(p < 0.05).However,the protein expression level of p52 in the TNFα+Mor group was significantly lower than that in the TNFα group(p < 0.05).(4)The transcription levels of IL-6 were significantly higher in both the TNFα group(p < 0.0001)and the TNFα+Mor group(p < 0.0001)than in the Veh group.However,the transcription level of IL-6 in the TNFα+Mor group was significantly lower than that in the TNFα group(p< 0.01).The transcription level of IL-1b was significantly higher in the TNFα group than in the Veh group(p < 0.05),while the transcription level of IL-1b in the TNFα+Mor group was significantly lower than that in the TNFα group(p < 0.01).The transcription level of Crp was significantly higher in the TNFα group than in the Veh group(p < 0.01),while the transcription level of Crp in the TNFα+Mor group was significantly lower than that in the TNFα group(p < 0.01).The transcription level of Tnfα was significantly higher in the TNFα group than in the Veh group(p < 0.0001),while the transcription level of Tnfα in the TNFα+Mor group was significantly lower than that in the TNFα group(p < 0.001)but higher than that in the Veh group(p < 0.05).The TNFα expression levels in both the TNFα group and the TNFα+Mor group were significantly higher than those in the Veh group(p < 0.0001).The protein expression level of TNFα in the TNFα group was significantly more significant than that in the TNFα+Mor group(p < 0.001).(5)Compared with the Veh group,the transcription levels of Igf-1were significantly reduced in the TNFα group(p < 0.001)and TNFα+Mor group(p < 0.01);however,the degree of reduction in Igf-1 transcription levels was higher in the TNFα group than in the TNFα+Mor group(p <0.05).Compared with the Veh group,the transcription level of Igf-1r was significantly reduced in the TNFα group(p < 0.01),and the reduction in the transcription level of Igf-1r was higher in the TNFα group than in the TNFα+Mor group(p < 0.05).Compared with the Veh group,the transcription level of Irs-1 was significantly reduced in the TNFα group(p< 0.01),and the reduction in the transcription level of Irs-1 was lower in the TNFα+Mor group than in the TNFα group(p < 0.05).Compared with the Veh group,the transcription level of Pik3r1 was significantly reduced in the TNFα group(p < 0.01),and the transcription level of Pik3r1 in the TNFα+Mor group was significantly increased relative to that in the TNFαgroup(p < 0.05).Compared with the Veh group,the transcription level of Pik3 ca was significantly reduced in the TNFα group(p < 0.001),and the transcription level of Pik3 ca in the TNFα+Mor group was significantly increased relative to that in the TNFα group(p < 0.05).Compared with the Veh group,the transcription level of Akt1 was significantly reduced in the TNFα group(p < 0.001),and the transcription level of Akt1 in the TNFα+Mor group was significantly increased relative to that in the TNFαgroup(p < 0.05);the transcription level of Akt1 was also reduced in the TNFα+Mor group compared with the Veh group(p < 0.05).The transcription levels of Akt2 and Akt3 were significantly reduced in the TNFα group compared with the Veh group(p < 0.01 and p < 0.05,respectively).(6)Compared with the Veh group,the transcription levels of Atrogin1 were significantly increased in the TNFα and TNFα+Mor groups(p <0.0001).Compared with the TNFα group,the transcription levels of Atrogin1 were significantly decreased in the TNFα+Mor group(p < 0.001).Compared with the Veh group,the protein expression level of Atrogin1 was significantly increased in the TNFα group(p < 0.01),while it was significantly lower in the TNFα+Mor group than in both the Veh group(p< 0.0001)and the TNFα group(p < 0.0001).Compared with the Veh group,the transcription levels of Mu RF1 were significantly increased in the TNFαgroup(p < 0.001)and the TNFα+Mor group(p < 0.0001).Compared with the TNFα group,the transcription levels of Mu RF1 were significantly decreased in the TNFα+Mor group(p < 0.05).Compared with the Veh group(p < 0.01)and the TNFα+Mor group(p < 0.01),the protein expression level of Mu RF1 was significantly increased in the TNFα group.The protein expression level of Mu RF1 in the TNFα+Mor group tended to decrease compared with the Veh group but was not statistically different.My HC expression was significantly induced after 48 hours,and different expression levels of My HC were observed among the treatment groups.Compared with the Veh group,the expression levels of My HC were significantly decreased in the TNFα group(p < 0.05)and the TNFα+MorH group(p < 0.01)after induction for 48 hours.At 72 hours,compared with the Veh group,the expression levels of My HC were significantly decreased in the TNFα group(p < 0.0001),the TNFα+Mor-L group(p <0.001),and the TNFα+Mor-H group(p < 0.00001).The expression level of My HC in the TNFα+Mor-L group was significantly higher than that in the TNFα group(p < 0.001)and the TNFα+Mor-H group(p < 0.001).Conclusion:Morroniside inhibits the activation of NF-κB canonical and non-canonical signaling pathways,downregulates the transcription and expression of relevant pro-inflammatory factors,and promotes protein synthesis while inhibiting protein degradation,thus treating skeletal muscle atrophy. |
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