CREB Is Involved In The Hypothermia Protection Of SH-SY5Y Cells By Regulating The Expression Of RBM3

BackgroundMild hypothermia is an effective clinical therapy to reduce nerve damage.However,various complications may occur in the course of treatment due to factors such as individual patient differences.These complications affect the prognosis and life of patients,which greatly limits the wide application of mild hypothermia therapy in clinical practice.RNA-binding protein motif 3（RBM3）is a cold-shock protein with dramatically increased transcript level at mild temperature.Meanwhile,RBM3 was identified as a key protein mediating hypothermic neuroprotection,which provids a promising molecular therapeutic target for simulating hypothermia.Our previous study showed that the FAK/Src axis responds to mild hypothermia stress and regulates the transcription of gene RBM3 under mild hypothermia.However,the mechanism of the transcriptional regulation of RBM3 is currently unknown.And the key transcription factors remain to be elucidated.Objectives1.To analysis of the effect of mild hypothermia（32℃）on CREB signaling pathway in SH-SY5 Y cells.2.To clarify the transcriptional regulation of RBM3 by CREB and its relationship with the FAK/Src signaling axis.3.To explore the neuroprotective effect of CREB under mild hypothermia conditions.Methods1.Detecting the expression of CREB in SH-SY5 Y cells after mild hypothermia（32°C）treatment.SH-SY5 Y cells were cultured at 32°C for 0 h,0.5 h,1 h,2 h,4 h and 24 h,respectively.Cytoplasmic and nuclear proteins were isolated.And the phosphorylation expressions of RBM3,CREB and CREB were detected by Western blot.2.Analysis of the effect of CREB on RBM3 gene transcription.SH-SY5 Y cells were transfected with CREB-specific siRNA and CREB recombinant plasmid pc DNA3.1-Myc-CREB.And the cells were treated at room temperature（37°C）and mild hypothermia（32°C）for 24 h.And the phosphorylation of RBM3,CREB and CREB was detected by Western blot.RBM3 m RNA was detected by q PCR.3.Evaluation of the effects of FAK and Src signaling pathways on CREB expression under mild hypothermia.SH-SY5 Y cells were treated with FAK and Src-specific inhibitors for 1 h.And then treated at mild hypothermia for 24 h.SH-SY5 Y cells were transfected with FAK and Src kinase-specific siRNA.And cells were treated at mild hypothermia for 24 h.The expressions of FAK,Src,and FAK,Src,CREB phosphorylated proteins were detected by Western blot.4.Evaluating the effect of CREB on the neuroprotective effect of mild hypothermia.SH-SY5 Y cells were transfected with CREB-specific siRNA and incubated for 24 h,treated with mild hypothermia at 32°C for 24 h.And then induced to apoptosis by the drug rotenone（Rot）.Western blot was used to detect the expressions of cl.PARP and CREB proteins.Using CCK-8 detection of cell viability levels.Results1.Mild hypothermia stimulates activation of the transcription factor CREB.2.CREB-specific siRNA significantly inhibited the transcription of RBM3.Overexpression of CREB significantly promoted the transcription of RBM3.3.FAK/Src specific inhibitor and siRNA inhibited the expression of CBER protein.4.Inhibition of CREB expression can significantly eliminate the neuroprotective effect of rotenone（Rot）as a neuronal apoptosis cell model under mild hypothermia.ConclusionThe FAK/Src signaling axis and the transcription factor CREB together constitute a new mild temperature stress response signaling pathway,which are involved in the transcriptional regulation of the RBM3 gene by mild hypothermia.Meanwhile,CREB can mediate the neuroprotective effect of mild hypothermia.FAK/Src-CREB-RBM3 may play an important role in the treatment of neurodegenerative diseases with mild hypothermia,which is of great significance for elucidating the molecular mechanism of hypothermia neuroprotection.