| Objective To explore the effects of Ghrelin on endoplasmic reticulum stress in osteoblast,and its possible mechanism.Methods（1）Various concentrations tunicamycin were exposed to MC3T3E1 osteoblast line for inducing and establishing endoplasmic reticulum stress model system.On the basis of references and previous studies,after being treated with different concentrations of tunicamycin（0-8.0μg/m L）for 20 hours,and then the appropriate experimental concentration of tunicamycin was determined by CCK8;（2）Different concentrations of Ghrelin pretreatment groups(10-11,10-9,10-7mol/L)were set up to compare the effects of pretreatment with different concentrations for 4 hours on endoplasmic reticulum stress apoptosis and reactive oxygen species production induced by tunicamycin,and the appropriate concentration of Ghrelin was selected for subsequent experiments.（3）Set up control group and GSK621（AMPK agonist）and/or Ghrelin with treatment group,the incidence of apoptosis and the production of reactive oxygen species were compared among those groups after being pretreated with AMPK agonist GSK621 for 3 h and treated with Ghrelin for 4 h and then tunicamycin for 20 h.（4）q RT-PCR technique was utilized to detect the m RNA expression of endoplasmic reticulum stress related marker genes（Bip,Chop,IRE1α）in different treatment groups,and to explore the effects of Ghrelin on endoplasmic reticulum stress induced by TM and its possible mechanism.（5）Western blotting was used to analyze the levels of Bip and p-AMPK in each group.Cells were pretreated with GSK621to explore the relationship among Ghrelin,AMPK pathway and endoplasmic reticulum stress.The experiments of each group were repeated three times independently.Results（1）CCK8 test showed that the concentration of TM with cell survival rate of 50%was between 1.0 and 2.0μg/m L,and so 1.5μg/m L was selected as the model concentration of endoplasmic reticulum stress,and the apoptosis rate and the level of reactive oxygen species were significantly increased in 1.5μg/m L TM group（C vs TM,P=0.000）.（2）Compared to the group treated with TM alone,the apoptosis rate(TM vs G10-11M+TM,P=0.003)and the level of reactive oxygen species(TM vs G10-11M+TM,P=0.002)decreased significantly after pretreatment with 10-11mol/L Ghrelin for 4 h.In addition,the effect of Ghrelin was obviously concentration-dependent and enhanced with the increase of concentration（P<0.05）.（3）The apoptosis rate and the production of reactive oxygen species in group GSK621+TM pretreated with Ghrelin were significantly lower than those in group GSK621+TM(GSK621+TM vs GSK621+G10-7M+TM,P<0.001).（4）The m RNA expression of endoplasmic reticulum stress related marker gene（Bip,Chop and IRE1α）in TM treatment group,by comparing with the Control group,were increased obviously（C vs TM,P<0.01）;and the difference between Ghrelin and Control group exists no statistically significant(C vs G10-7M,P>0.05).The expression levels of related genes in Ghrelin and TM co-treatment group decreased compared with TM group(TM vs G10-7M+TM,P<0.05).Those gene expression were also declined significantly in GSK621+TM group pretreated with Ghrelin compared to the GSK621+TM group(GSK621+TM vs GSK621+G10-7M+TM,P<0.001).（5）The results of Western blot indicated that Bip and p-AMPK level in TM group increased obviously,while Bip and AMPK phosphorylation levels decreased after Ghrelin pretreatment(TM vs G10-7M+TM,P<0.05).Compared with GSK621+TM,Ghrelin pretreatment in GSK621+TM group can obviously downregulation the expression of p-AMPK and Bip(GSK621+TM vs GSK621+G10-7M+TM,P<0.05).Conclusion Ghrelin can alleviate the endoplasmic reticulum stress induced by tunicamycin in a dose-dependent manner,which may improve the apoptosis of endoplasmic reticulum stress induced by tunicamycin through inhibiting AMPK phosphorylation. |
