The Effect Of Nesfatin-1 On Activity Of Human Gastrointestinal Smooth Muscle Cells And Interrelated Signaling Pathway

Object:This study was aimed to explore the relationship between Nesfatin-1 and HGSMC.Firstly,we determined the optimal concentration of Nesfatin-1.Then,we studied the effect of Nesfatin-1 on cell viability,apoptosis and adhesion.Finally,the potential cell signaling pathway was detected.Methods:Firstly,HGSMC were treated with different concentrations of Nesfatin-1,including0 nM,10 nM,50 n M,100 nM and 200 nM.In addition,we assayed the mRNA and protein expressions of eNOS by Quantitative Real-time PCR(qRT-PCR)and Western Blot.The optimal concentration of Nesfatin-1 was confirmed.Then,the HGSMC were divided into three groups: control group(C),Nesfatin-1 group(N),Nesfatin-1 and NGNitro-L-arginine Methyl Ester(L-NAME)group(NL).Thereafter,the cell viability,apoptosis and adhesion of HGSMC were assessed by Methyl Thiazolyl Tetrazolium(MTT)colorimetric assay,flow cytometry and related kits,respectively.Besides,the m RNA and protein expression of apoptosis factor p53 and Fas were assessed.Finally,in order to further investigate the effects of Nesfatin-1 on the ERK/MAPK/mTOR signaling pathway,we assayed the mRNA and protein expressions of ERK1/2,p38 MAPK and m TOR by qRT-PCR and Western blot in the control(C)group,Nesfatin-1(N)group,Nesfatin-1 and NG-Nitro-L-arginine Methyl Ester(L-NAME)(NL)group.Results:To determine the concentration of Nesfatin-1 on HGSMC: the results showed that no statistical difference was witnessed on mRNA expressions of e NOS among Nesfatin-1concentration of 0 nM,10 nM and 50 nM,respectively(P > 0.05).At concentration of100 nM and 200 nM,the mRNA expressions of e NOS raised observably compared to that at concentration of 0 nM(P < 0.05).The Western blot result declared as concentration of Nesfatin-1 increased,the protein expression of eNOS were higher.The protein expression of eNOS was at the highest at the concentration of 100 nM.The effect of Nesfatin-1 on cell viability,apoptosis and adhesion: we divided HGSMC into three groups: control group(C),100 nM Nesfatin-1 group(N),100 nM Nesfatin-1 and NG-Nitro-L-arginine Methyl Ester(L-NAME)group(NL).The result displayed that the cell viability in the N group was statistically lower than that in the C group(P < 0.05)at day 4.The addition of eNOS inhibitor L-NAME increased cell viability compared to the level of C group and there was no difference between C and NL group(P > 0.05);Apoptosis cells went up significantly by Nesfatin-1 compared to the C group(P < 0.01).The addition of eNOS inhibitor L-NAME made apoptosis cells reduced and there was no difference between C and NL group(P > 0.05).Both pro-apoptotic factors p53 and Fas were markedly upregulated in the N group compared to those in the C group(P < 0.05);The cell adhesion experiment showed that the percentage of relative adhesion in the N group was statistically lower than that in the C group(P < 0.05).In addition,there was no difference between C and NL group(P > 0.05).The effect of Nesfatin-1 on theERK/MAPK/mTOR signaling pathway in HGSMC:The results showed that,as compared with the C group,the mRNA expressions of ERK1/2,p38 MAPK and mTOR were significantly down-regulated in the N group(P <0.05).Nevertheless,the mRNA expressions of ERK1/2,p38 MAPK and mTOR in the NL group were significantly increased(P < 0.05 or P < 0.01).Western blot showed that the protein expressions of p-ERK1/2,p-p38 MAPK and p-mTOR were significantly lower than that in the C group.However,there were no significant differences in the proteins expressions of p-ERK1/2,p-p38 MAPK and p-mTOR between NL group and C group.Conclusions:This study reveals that the Nesfatin-1 can regulate the expression of eNOS.With the increase of concentration of Nesfatin-1,e NOS mRNA and protein expression levels have increased and reached the highest when the concentration is 100 nM.The Nesfatin-1statistically suppresses the HGSMC viability,adhesion and expedites apoptosis.Accordingly,it is concluded that Nesfatin-1 serves as an inhibitory factor on HGSMC.Besides,addition of eNOS inhibitor L-NAME could relieve effects of Nesfatin-1 in HGSMC;Nesfatin-1 also reduces the mRNA and protein expressions level of ERK1/2,p38 MAPK and mTOR.In addition,the activities of ERK1/2,p38 MAPK and mTOR signal pathways are inhibited by the Nesfatin-1.Interestingly,these effects can be relieved by the e NOS inhibitor L-NAME.In summary,Nesfatin-1 can inhibit the activities of ERK/MAPK/mTOR signaling pathway by upregulating the expression of e NOS in HGSMC.The Nesfatin-1 also results in the decline of HGSMC viability and adhesion.Nesfatin-1 remarkably expedites apoptosis.The addition of e NOS inhibitor L-NAME could relieve these effects in HGSMC.It reveals that eNOS inhibitor is praised to be a novel therapeutic strategy and target for DM induced gastrointestinal complications.