Protective Role Of Metformin On MC3T3-E1 Cell In High Glucose Conditions And Its Relationship With PERK/ATF4/CHOP Signaling Pathway From Endoplasmic Reticulum Stress

Objective:(1)To explore the effect of metformin on osteoblasts in high glucose conditions from the aspects of the cell morphology,bone mineralization,cell secretion proteins and apoptosis.(2)To explore whether the protective role of metformin has a relationship with PERK/ATF4/CHOP proteins from endoplasmic reticulum stress on osteoblasts in high glucose conditions.(3)The effect of metformin on glucose toxic osteoblasts after inhibition of PERK.Methods:(1)object of research: MC3T3-E1 subclone 14.(2)Groups of experiment:1.Intervention experiment of metformin: MC3T3-E1 cells were divided into six groups:NC(treated with 5.5mmol/l of glucose for 24h),HG(treated with33mmol/l of glucose for 24h),TG(treated with 100nmol/l of thapsigargin for24h),MET(treated with 100umol/l of metformin for 24h),HG+MET(treated with 33mmol/l glucose for 24 h and then treated with 100umol/l of metformin for 24h),TG+MET(treated with 100nmol/l of thapsigargin for 24 h and then treated with 100umol/l of metformin for 24h).2.Intervention experiment of Inhibition of PERK activity:MC3T3-E1 cells were divided into 6 groups:NC(treated with 5.5mmol/l of glucose for24h),HG(treated with 33mmol/l of glucose for 24h),HG+MET(treated with33mmol/l glucose for 24 h and then treated with 100umol/l of metformin for24h),GSK(treated with 0.3umol/l of GSK2606414 for24h),GSK+HG(pretreated with 0.3umol/l of GSK2606414 for 4h,and thentreated with the combination of 33 mmol/l of glucose for24h),GSK+HG+MET(pretreated with 0.3umol/l of GSK2606414 for 4h,and then treated with the combination of 33 mmol/l of glucose and 100 umol/l of metformin for 24h).(3)Testing indexes: the formation of osteoblast mineralization nodules was detected by Alizarin red staining.The concentration of ALP and OCN in osteoblasts was detected by ELISA.The apoptosis was detected by flow cytometric analysis.And the expression of m RNA and protein levels of apotosis genes(BAX,Bcl2,Caspase3)and endoplasmic reticulum stress-related proteins(PERK,ATF4,CHOP)were examined by RT-PCR and Western blot.Results:(1)Metformin can reverse the damage to osteoblasts in high glucose condition during the intervention time.It can improve cell morphology and increase connections between cells,enhance the capacity of adherent,increase number of mineralized nodules.The cell secretory protein ALP/OCN increased(p<0.05).The rate of apoptosis is reduced(p<0.05)and the expression of m RNA and protein levels of BAX,Caspase3 were reduced,the expression of m RNA and protein levels of Bcl2 was increased(p<0.05).And it can reduce the expression of m RNA and protein levels of the endoplasmic reticulum stress-related proteins PERK,ATF4,CHOP(p<0.05).(2)After inhibiting the activity of PERK,it can reduce the activation of PERK/ATF4/CHOP signaling pathway from endoplasmic reticulum and then cooperate with metformin to work on glucose toxic osteoblasts.It had significantly change in cell number of mineralizaed nodules and the cell secretory protein ALP/OCN increased more(p<0.05).Further more the effect of anti-apotosis enhanced further(p<0.05).The expression of m RNA and protein levels of BAX,Caspase3 were reduced more and the expression of m RNA and protein levels of Bcl2 increased more(p<0.05).The expression of m RNA and protein levels of thedownstream proteins of endoplasmic reticulum stress ATF4,CHOP were reduced more(p<0.05).Conclusions:(1)Metformin plays a protective role of glucose toxic damage on osteoblasts from the aspects of the cell morphology,bone mineralization,cell secretion proteins and apoptosis.(2)The protective effect of metformin on osteoblasts in high glucose conditions may be mediated by inhibition of PERK/ATF4/CHOP signaling pathway from the endoplasmic reticulum stress.Inhibition of PERK can enhance the effect.