| Objective To establish models of tunicamycin-induced endoplasmic reticulum stress (ERS) and to explore the effect of ghrelin on osteoblasts of tunicamycin-induced ERS. Methods MC3T3-E1 cells were chosen for the study. (1) MC3T3-E1 osteoblasts cultured in vitro were treated with different concentrations of tunicamycin (0, 0.5, 1.0, 1.5μg/mL) for 24h. CCK-8 assay was used to determine cell viability. DCFH-DA probe was used to detect intracellular ROS level. Real-time quantitative PCR was used to measure the expression of BIP，CHOP，caspase-12 mRNA in each group. Finally, the most sensitive concentration of tunicamycin (1.5μg/mL) was chosen to establish the ERS model. (2) The effect of ghrelin on osteoblasts under ERS was observed. MC3T3-E1 osteoblasts cultured in vitro were pretreated with different concentrations of ghrelin (0, 10-11, 10-9, 10-7mmol/L) for 4h. Then the cells were treated with 1.5ug/mL tunicamycin to establish ERS model. After the completion of the culture, the cell proliferation activity, the content of intracellular ROS, and the expression of ERS-related marker genes were detected using the above methods. Results Compared with the control group, the osteoblast viability and proliferation decreased in a dose-dependent manner after treated with different concentrations of tunicamycin for 24h. The statistical significance appeared in cells treated with 1.0ug/mL and 1.5ug/mL of tunicamycin (P<0.05), but not in those with 0.5μg/mL of tunicamycin (P>0.05). Compared with the control group, the content of intracellular ROS increased in a dose-dependent manner (P<0.05). qRT-PCR results showed that the expressions of CHOP mRNA in all concentration of tunicamycin-additioned cells were higher than those in the control group (P<0.05). The expressions of BIP and caspase-12 mRNA were statistically significant only in cells with 1.0ug/mL and 1.5ug/mL of tunicamycin (P<0.05), but not in cells with 0.5μg/mL of tunicamycin (P>0.05). (2) Compared with cells with addition of 1.5ug/mL tunicamycin alone, cells pretreated 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. The statistical significance appeared in 10-9 and 10-7 mmol/L of ghrelin pretreatment (P<0.05), but not in 10-11mmol/L ghrelin pretreatment (P>0.05). Also, the content of intracellular ROS reduced significantly in cells pretreated with different concentrations of ghrelin for 4h (P<0.05). qRT-PCR results showed that after pretreatment with 10-9 and 10-7mmol/L of ghrelin, the decrease of CHOP mRNA expression was statistically significant (P<0.05), but the difference was not statistically significant after 10-11 mmol/L ghrelin pretreatment (P>0.05). The mRNA expressions of BIP and caspase-12 were statistically significant after pretreatment with different concentrations of ghrelin (P<0.05). Conclusion Tunicamycin can induce ERS in osteoblasts. Ghrelin can inhibit ERS in osteoblasts to some extent. |