The Role Of Rev-erbα And Agonist SR9009 In Myogenic Differentiation Of C2C12 Myoblasts During Inflammation Induced By Lipopolysaccharide

Objective:Chronic inflammation or excessive accumulation of inflammatory cytokines can cause muscle atrophy and ultimately lead to impaired skeletal muscle function.The interaction between circadian clock and inflammatory response is involved in maintaining skeletal muscle homeostasis and muscle regeneration and repair.Rev-erbα,an important member of the circadian clock,is a key connection point between the core clock network and inflammatory pathways and is involved in regulating the balance of pro-inflammatory and anti-inflammatory.The mechanism of targeted activation of Rev-erbα in regulating inflammation-related skeletal muscle atrophy in inflammatory environment in vitro needs to be further elucidated.In this study,C2C12 myoblasts were stimulated with lipopolysaccharide(LPS)to establish an in vitro inflammatory model.The specific objectives of the study were as follows:(1)In order to observe the effects of 0.1μg/m L and1μg/m L LPS on the differentiation of C2C12 myoblasts and the expression of inflammatory cytokines;(2)Rev-erbα was activated by SR9009,a small molecule agonist,to investigate the regulatory effect of SR9009 on NF-κB signaling pathway by LPS stimulation;(3)To elucidate the regulatory effects of SR9009 on myogenic differentiation process and muscular atrophy genes in inflammatory environments in vitro.Methods:1.The differentiation process of C2C12 myoblasts with LPS was observed and collected at 0h,12 h,24h,72 h and 168 h.The mRNA levels of inflammatory cytokines IL-6and TNF-α and Clock genes Bmal1,Clock and Rev-erbα during the differentiation of C2C12 myoblasts stimulated by LPS were detected by RT-PCR.Furthermore,10μm SR9009 was added to myoblasts to activate Rev-erbα receptor.The expression of clock genes was analyzed by RT-PCR under the co-action of LPS and SR9009.The protein levels of REV-ERBα,BMAL1 and CLOCK were detected by Western blot.2.Rev-erbα was activated by SR9009.ELISA and RT-PCR were used to detect the release of IL-6 and TNF-α in myoblasts under the action of SR9009,and the mRNA level of CD14 and TLR4 on cell surface was further verified.3.Western blot detection NF-κB and p-NF-κB p65 protein levels,according to the results in the 72 h and 168 h SR9009+LPS group p-NF-κB p65/NF-κB p65 ratio with the LPS group.The activation of REV-ERBα inhibits the NF-κB signaling pathway.4.The expression trends of Clock genes Rev-erbα,Bmal1 and Clock under the combined action of LPS and SR9009 were analyzed by RT-q PCR,and the protein levels of REV-ERBα,BMAL1 and clock were detected by Western blot.5.Flow cytometry was used to determine the effect of SR9009 and LPS co-treatment on cell cycle and the proliferation of myoblasts by CCK8.6.RT-PCR was used to detect the mRNA levels of myogenic regulators Myf5,Myod,Myogenin and Myh3 and atrophies related genes Atrogin,Foxo1 and Foxo3.Results:1.Under LPS stimulation,the mRNA level of IL-6 increased in a time-dependent manner and reached its peak at 168 h.The mRNA level of TNF-α increased in a time-and concentration-dependent manner before 72 h and then decreased slightly.It proved that the in vitro inflammatory model was successfully constructed.2.Rev-erbα agonist SR9009 reduced the mRNA levels of inflammatory factors IL-6,TNF-α,Cd14 and Tlr4 during myoblast differentiation,especially after 72 h.3.The expressions of Bmal1 and Clock were relatively stable and fluctuated at a small and low level during the experimental period.Rev-erbα showed an up-regulated trend,and its fluctuation increased with the induction differentiation time.After the addition of the agonist SR9009,the self-transcription level of Rev-erbα was down-regulated and the protein level continued to be highly expressed during myoblast differentiation.5.In SR9009+LPS group,the number of cells tending to divide was increased,and the proliferation ability of cells was enhanced.The mRNA expression levels of myoregulatory factor Myf5 were up-regulated,while the mRNA expression levels of MyoD,Myogenin and Myh3 were down-regulated.6.Atrophy related gene,Atrogin-1,was significantly up-regulated and maintained a relatively high level of fluctuation by LPS,and the expressions of upstream transcription factors Foxo1 and Foxo3 a were up-regulated simultaneously.Under the treatment of SR9009,the expression of genes related to muscular atrophy up-regulated by LPS stimulation tended to recover to the control level.Conclusion:1.LPS inhibited the differentiation process of C2C12 myoblasts and interfered with the expression of clock genes to a certain extent,in which Rev-erbα was more sensitive to LPS stimulation.2.During the differentiation of C2C12 myoblasts stimulated by LPS,the clock gene Rev-erbα is more sensitive to inflammatory stimulation.3.Rev-erbα agonist SR9009 can alleviate LPS-stimulated inflammatory response in C2C12 myoblasts by inhibiting NF-κB p65 phosphorylation.4.Rev-erbα agonist SR9009 can partially reverse LPS-induced up-regulation catabolism related genes Atrogin,Foxo1 and Foxo3 a.5.The agonist SR9009 could effectively maintain the proliferation ability of C2C12 myoblasts under LPS stimulation.It suggests that Rev-erbα may be the key clock gene coordinating C2C12 myoblasts to respond to external stimuli during myogenic differentiation.