Protective Role Of Exendin-4 And The Effect Of TLR4 And Endoplasmic Reticulum Stress On MC3T3-E1 Cell In High Glucose Conditions

Objective:(1) From proliferation, apoptosis and osteogenesis mineralization rate to studying whether a high glucose environment plays a role to damage the proliferation and differentiation of osteoblast, osteoblast glucose toxic damage model is established.(2) To observe whether new hypoglycemic drug endogenous glucagon like peptide 1(glp-1) analogue Extendin- 4 in vitro can mediate protecting role of osteoblast in high glucose conditions.(3) To investigate whether the protecting role of Exendin-4 on the osteoblast was mediated through the TLR4/NF-κB pathway and the endoplasmic reticulum stress.Method:(1) MC3T3- E1 subclone 14 as the research object. Complete medium which contains 33mmol/L glucose used to intervention osteoblast 24 hours. Glucose toxic injury osteoblast model was established. Different concentrations Exendin-4(0, 25, 50, 100nmol/L) at different times(0, 24, 48, 72 h) used to intervention osteoblast. Verify the correctness of the glucose toxic damage model. In vitro to observation the protective role of Exendin-4 and the effect of TLR4/ NF-kappa B and endoplasmic reticulum stress on MC3T3-E1 cell in high glucose conditions.(2) Relative growth rate was measured by MTT and apoptosis was detected by flow cytometric analysis and TUNEL. The formation of osteoblast mineralization nodules was detected by Alizarin red staining. The expression of m RNA and protein levels of TLR4/NF-KB, endoplasmic reticulum stress-related proteins(PERK-1/2, CHOP, Caspase 12) and the osteogenesis related genes(BMP- 2) were examined by RT-PCR and Western blot. The concentration of OCN and ALP in osteoblast was detected by ELISA.Results:(1) The osteoblast growth in the high glucose(33 mmol/L) environment that the growth rate dropped significantly(p < 0.05), the apoptosis rate increased significantly(p < 0.05). Extendin- 4 can improve the decline of osteoblast growth rate and the increase of apoptosis rate caused by glucose toxic injury in time and dose dependent(all p < 0.05).(2) The osteoblast growth in the high glucose(33mmol/L) environment that the mineralization rate dropped significantly(p < 0.05), the expression of osteoblasts secreted proteins OCN and ALP increased significantly(p < 0.05). Exendin-4 could improve the decline of osteoblast mineralization rate and the expression of OCN and ALP mediated by high glucose injury.(3) The osteoblast growth in the high glucose(33mmol/L) environment that the low-grade chronic inflammation genes TLR4 / NF-kappa B and endoplasmic reticulum stress genes(p ERK-1/2, CHOP, Caspase12) m RNA and protein expression were significantly increased(p < 0.05). Osteogenesis related genes BMP2 m RNA and protein expression decreased significantly(p < 0.05). Exendin-4 reduced the upregulated expression of TLR4/NF-KB and endoplasmic reticulum stress-related proteins(PERK-1/2, CHOP, Caspase12) caused by high glucose injury, and upregulated the high glucose-induced decline expression of osteogenesis genes(BMP- 2)(all p < 0.05).Conclusions:High glucose environment can interfere with osteoblast proliferation and differentiation, and activate osteoblast in a variety of inflammatory pathways, cause damage to the osteoblast. Exendin-4 can mediate protective role of osteoblast injured by high glucose through anti- endoplasmic reticulum stress, which may be related to the activation of the TLR4/NF-KB pathway.