Ghrelin Inhibits Tunicamycin-induced Endoplasmic Reticulum Stress In Osteoblasts

Objective To establish models of tunicamycin-induced endoplasmic reticulum stress（ERS）and explore the effect of ghrelin on osteoblasts of tunicamycin-induced ERS.Methods Select MC3T3-E1 cell to study,（1）Different concentrations of tunicamycin（0,0.5,1,1.5μg/ml）medium were prepared,and different concentrations of osteoblasts were incubated for 24 h;CCK-8 assay was used to determine cell viability.DCFH-DA probe was used detect intracellular ROS level.Real-time quantitative PCR was used to measure each group the expression of CHOP,BIP,caspase-12 mRNA.Finially,then the cells were treated with 1.5μg/ml tunicamyin to establish ERS models.（2）Observe the impacts of ghrelin on osteoblastes of ERS.MC3T3-E1 osteoblastes cultured in vitro were pretreated by different concentrations(0,10^-11,10^-9,10^-77 mmol/L)of ghrelin for 4h,then the cells were treated with1.5μg/ml tunicamyin to establish ERS model.After the completion of the culture,the cell proliferation activity,the content of intracellular ROS,and the expression of endoplasmic reticulum stress-related marker genes were detected by the above methods.（3）Statistical method.The analysis was performed using statistical analysis software SPSS 22.0,and the experimental data were expressed as mean±standard deviation（`X±S）.The independent sample t test was used for comparison between the two groups.One-way ANOVA was used for comparison between groups.P<0.05 was considered statistically significant.Results Compared with the control group,the osteoblasts viability and proliferation of different concentrations of tunicamyin treated osteoblasts cell for 24 h that were decreased in a dose-dependent way.The cells were treated 1.0μg/ml and 1.5μg/ml tuniacmyin were statistically significant（P<0.05）,but there was no statistical significance after the intervention of 0.5μg/ml tunicamycin（P>0.05）;Compared with the control group,the content of intracellular ROS were increased in a dose-dependent way（P<0.05）;qRT-PCR results showed that the expressions of CHOP mRNA in different concentrations were higher than those in the control group and all had statistically significant（P<0.05）,but the expressions of BIP and caspase-12 mRNA in 1.0ug/ml and 1.5μg/ml tuniacmyin had statistically significant（P<0.05）,0.5μg/ml tuniacmyin had no statistically significant（P>0.05）;（2）Compared with the addition of 1.5μg/ml tunicamycin,pretreatment of cell with different concentrations of ghrelin for 4h followed by addition of 1.5μg/ml tunicamycin revealed that cell proliferation and survival increased with increasing ghrelin concentration,10^-9、10^-77 mmol/L ghrelin pretreatment was statistically significant（P<0.05）,10^-1111 mmol/L ghrelin pretreatment was not statistically significant（P>0.05）;Compared with the addition of 1.5μg/ml tunicamycin,pretreat of cells to differern concentrations ghrelin for4 h significantly reduced the content of intracellular ROS（P<0.05）;qRT-PCR results showed that after pretreatment with cells in 10^-9、10^-77 mmol/L ghrelin,the expressions of CHOP mRNA was statistically significant（P<0.05）,but there was no statistically significant after 10^-1111 mmol/L ghrelin pretreatment（P>0.05）.For the expression of BIP and caspase-12mRNA,pretreatment with cells in different concentrations ghrelin,the expressions were statistically significant（P<0.05）.Conclusion Tunicamycin can induce the occurrence of endoplasmic reticulum stress in osteoblasts and is aggravated with increasing concentration.Ghrelin can inhibit the endoplasmic reticulum stress of osteoblasts to a certain extent.