Effect Of Niacinamide Mononucleotide(NMN) On Myoblast Proliferation And Differentiation

Nicotinamide Mononucleotide(NMN)is synthesized from water-soluble vitamin B3and 5-phosphoribo-1 pyrophosphate under the catalysis of nicotinamide phosphoribotransferase.NMN is not only found in a variety of foods,a natural substance that exists in the body itself.Exogenous ingested NMN exerts its important physiological functions mainly by increasing the level of NAD~+after entering the body.Current studies have found that systematic use of NMN can effectively improve the biosynthesis of NAD~+in various peripheral tissues such as pancreas,liver,adipose tissue,heart,skeletal muscle,testis,eyes and blood vessels,and play an important regulatory role in biological processes such as cell proliferation,anti-aging and improvement of cell state.However,there are few studies on the effect of NMN on myoblasts and its mechanism of action.In this study,by exploring the influence of NMN on myoblast proliferation and differentiation,and exploring the key factors and related mechanisms of NMN’s action on myoblast proliferation and differentiation,this study provides theoretical basis for further revealing the regulatory role of NMN on skeletal muscle development.In this study,CCK-8 and flow cytometry were used to screen the concentration and time of action of NMN on myoblast proliferation,and the optimal concentration and time of action were selected.In addition,real-time quantitative PCR,EdU immunofluorescence staining and western blotting were used to verify the effect of NMN on myoblast proliferation at the optimal concentration and time,and real-time quantitative PCR was used to screen out members of the Sirtfamily that play a major role in the regulation of proliferation by NMN,so as to determine the main regulatory pathway.Then,real-time fluorescence quantitative PCR was used to determine the concentration of NMN-induced myoblast differentiation,and the optimal concentration of NMN-induced myoblast differentiation was screened to determine its main regulatory pathway.The main findings are as follows:(1)CCK-8 and flow cytometry were used to preliminarily screen the proliferative concentration of myoblasts treated with NMN,and the experimental results showed that NMN promoted the proliferation of myoblasts at a concentration of 2 m M.EdU immunofluorescence staining,real-time fluorescence quantitative PCR(RT-PCR)and western blotting were used to detect the effect of 2 m M NMN on myoblast proliferation.The experimental results showed that the EdU positive cell rate of myoblasts in the treatment group was significantly higher than that in the experimental group when the concentration of 2 m M NMN was used(p<0.05).The mRNA expression levels of proliferation-related genes PCNA,CCND1,CDK4,CCNE1 and CDK2 were not significantly changed,but the protein expression levels were significantly up-regulated(p<0.05).(2)RT-PCR was used to detect the gene expression of Sirtfamily members in myoblasts treated with 2 m M NMN,and the results showed that the expressions of Sirt4,Sirt5and Sirt6 of Sirtfamily members were significantly increased(p<0.001).Sirt4,Sirt5 and Sirt6 gene overexpression vectors were constructed,and cell transfection experiments were performed on myoblasts.The experimental results showed that the expressions of Sirt4(p<0.001),Sirt5(p<0.01)and Sirt6(p<0.001)genes were significantly up-regulated after cell transfection.The successful transfection of myoblast with overexpressed vector was determined.The cells successfully transfected with overexpressed genes were tested for proliferation-related genes.RT-PCR results showed that after overexpression of Sirt5 gene,The expressions of proliferation-related genes PCNA(p<0.001),CCN D1(p<0.001),CDK4(p<0.01),CCN E1(p<0.001)and CDK2(p<0.001)were significantly up-regulated.Overexpression of Sirt4 gene and Sirt6 gene showed no significant differences in proliferation-related genes.After overexpression of Sirt5 gene,The number of cells in S phase of cell cycle was significantly increased(p<0.001),the rate of EdU positive cells was significantly increased(p<0.05),the proliferation-related protein PCNA(p<0.001),CCN D1(p<0.001),CDK4(p<0.05),The expressions of CCN E1(p<0.05)and CDK2(p<0.05)were significantly increased.In myoblasts treated with 2 m M NMN,P-ERk1/2 protein expression was significantly up-regulated(p<0.001).After overexpression of Sirt5 gene,p-ERK1/2 protein expression was significantly up-regulated(p<0.001),and there were no significant differences in the rate of EdU positive cells and P-ERK1/2 protein expression after the use of pathway inhibitor U0126.(3)The concentration of NMN-induced myoblast differentiation was preliminarily screened by RT-PCR.The experimental results showed that the expressions of Myod and Myog genes related to differentiation were significantly up-regulated at 2 d,4 d and 6 d,respectively,after the treatment with 1 m M NMN concentration(p<0.001).MyHC immunofluorescence staining was performed on muscle tubes at 2 d,4 d and 6 d after myoblast differentiation.The experimental results showed that the fusion length of muscle tubes and nuclear fusion index were significantly increased.The expressions of differentiation-related proteins Myod,Myog and MyHC were significantly up-regulated on 2 d,4 d and 6 d after induction of differentiation,respectively.After myoblast differentiation was induced by 1 m M NMN,the expression of p-ERK1/2 protein was significantly decreased at 2d,4d and 6d after myoblast differentiation(p<0.05).After the use of pathway inhibitor U0126,MyHC immunofluorescence staining showed no significant differences in muscle tube fusion length and nuclear fusion index,and no significant differences in p-ERK1/2 protein expression.In conclusion,the treatment of myoblasts with 2 m M NMN can up-regulate Sirt5 and promote myoblast proliferation by acting on ERK1/2 pathway.When treated with 1 m M NMN,it promotes myoblast differentiation and acts by inhibiting ERK1/2 pathway phosphorylation.