Effect Of Calcitonin Gene Related Peptide (CGRP) On MC3T3-E1 Osteoblast Apoptosis And Autophagy Induced By Serum Starvation

BackgroundOsteoblasts are one of the most important cells in the bone repair and remodeling during the process of fracture healing.Their normal physiological function relies mainly on the continuous supply of nutrients from the microenvironment and the pro-survival signal.However,when the supply of nutritional factors is limited by the lesion or damage of the bone tissue,the apoptosis rate of osteoblasts will rise to a higher level than that under normal conditions,leading to a serious impact on the bone remodeling.Studies have found that the nervous system can regulate the physiological functions of bone tissue.Calcitonin gene-related peptide(CGRP)is a kind of neurotransmitter that widely distributed in the nervous system,and it plays a crucial role in bone formation and bone resorption.In our early studies,CGRP has been shown to play an important role in the healing process of jaw fractures.CGRP receptors are found on the surface of osteoblasts,and CGRP regulates the activity of osteoblast by binding to the receptors directly.The molecular mechanism of CGRP acting on osteoblasts has been studied extensively.Recent studies have proved that CGRP can reduce the loss of the bone by inhibiting the apoptosis of osteoblasts,and CGRP may has a potential protective effect on osteoblasts,but the specific mechanism is not completely uncovered yet.Autophagy is a self-protecting mechanism for cells to cope with unfavorable environments.During the process of autophagy,damaged or abnormal organelles and proteins are tansported to the lysosome through the bi-layer membrane and then degraded and recycled.Apoptosis is an initiative and orderly way of death controlled by genes.Fracture healing process is accompanied by both apoptosis and autophagy;however,the specific mechanism of their interaction in promoting fracture healing is still not clear.The aim of this study is to verify the anti-apoptotic effect of CGRP on osteoblasts and the role of CGRP on autophagy activity of osteoblasts under conditions of starvation,and then preliminarily explore the relationship between the apoptosis and autophagy in the process.For the first time,we try to further reveal the the protective mechanism of CGRP on osteoblasts from the perspective of the autophagy.MethodsOsteoblasts were cultured in the serum starvation condition(?-MEM medium,0%FBS,100U/ml penicillin-streptomycin)for 12 h or 24 h,then the apoptosis rate was detected by the flow cytometry,and the expresion of LC3,an autophagy-related protein,was detected by western blotting.3-MA,an autophagy inhibitor,was used to treat osteoblasts for 1h.And then osteoblasts were cultured in serum starvation condition for 24 h,after which the apoptosis rate was detected by the flow cytometry.Osteoblasts were cultured in the serum starvation condition added with a series of concentrations of the CGRP for at least 24 h.The preponderant concentration of CGRP for autophagy was selected by detecting the expression of LC3 and P62 by western blotting,and then the preponderant time was selected by detecting the expression of LC3 by western blotting.Osteoblasts were cultured in the serum starvation condition added with CGRP for 24 h,the apoptosis rate was detected by the flow cytometry,and the autophagosomes were detected by the MDC staining and the immunofluorescence staining of LC3 using confocal laser microscopy.Under the condition of serum starvation,the osteoblasts were pretreated with 3-MA and then the CGRP was added to the medium,after which the apoptosis rate was detected by the flow cytometry.Results1.The apoptosis rate of osteoblasts was rised under the condition of serum starvation;and the LC3 ? / ? ratio was also elevated.2.Pretreted with 3-MA,the osteoblasts' apoptosis rate was increased under serum starvation.3.CGRP can inhibit the apoptosis of osteoblasts and induce the autophagosome formation ?Added with CGRP in osteoblasts culturing under serum starvation for 24 h,the LC3 ? / ? ratio increased gradually and the expression of P62 decresed as the concentration of CGRP elevated.The most obvious difference was reached at the concentration of 10-8mol/L.While comparing the effect of different period time of CGRP treatment,the change of LC3? / ? ratio was most obvious in 24 hours' treatment.CGRP can induce the autophagosome formation and the LC3 expression in osteoblasts under serum starvation4.3-MA pretreatment can increase the apoptosis rate of osteoblasts under serum starvation,while CGRP can partially reverse the result.Conclusion1.This study demonstrated that serum starvation can induce osteoblast apoptosis and increase autophagy activity,and inhibition of autophagy in osteoblasts under serum starvation leads to an increased apoptosis rate.2.This study confirmed that CGRP can inhibit the apoptosis of osteoblasts induced by serum starvation and induce autophagic activity under serum starvation.And CGRP could partially reverse the apoptosis of osteoblasts induced by autophagy inhibitor under serum starvation by enhence the autophagy activity in osteoblasts.