Role Of AMPK-SIRT1 Signaling Pathway In Regulating Neuronal Autophagy And Apoptosis Of Neural Cells After Spinal Cord Injury

Objective: Spinal cord injury(SCI)is a serious central nervous system injury,with a high morbidity and mortality,has become a worldwide medical problem.Although significant progresses treating this disease in animal models has been made,currently there is no effective measure to promote functional recovery after human spinal cord injury.The development of effective treatment strategies requires the support of relevant basic theories.Therefore,to clarify the complicated pathological regulation mechanism after SCI is the primary task of SCI treatment at present.Studies confirm that the AMP-activated protein kinase / silent information regulator of transcription 1(AMPK /SIRT1)signaling pathway is involved in the regulation of autophagy in a variety of disease models,such as diabetes,fatty liver,and cancer disease.However,there are few reports on this signaling pathway in CNS injury.Recently,the study found that AMPK and SIRT1 expressed abnormally after SCI in rodents,but how the AMPK / SIRT1 pathway regulates autophagy in SCI is not clear.The time course of AMPK and SIRT1 expression in SCI in vivo and in vitro were detected firstly,then further to pretreat the mechanical injury(MI)model of primary spinal cord neurons with AMPK-SIRT1 agonist resveratrol,and detected the effect of resveratrol on the autophagy and apoptosis of neuronal cells in MI model.So as to explore how AMPK / SIRT1 pathway is involved in the regulation of autophagy and apoptosis after SCI.Methods:1.Detection of AMPK-SIRT1 pathway,autophagy,and apoptosis after SCI in vivo:The rat model of SCI was established by modified Allen's method.The rats were randomly divided into sham operation group(sham group)and spinal cord injury group(SCI group).The spinal cord tissues were harvested at 1 d,7 d,14 d and 5 w after SCI respectively.Western blot and immunohistochemical staining were performed to detect the expression of AMPK,p-AMPK and SIRT1.Western blot was used to measure the expression of autophagy-related proteins(LC3?,LC3 II and p62)in the spinal cord after SCI.The changes of apoptosis in the spinal cord after SCI were observed by TUNEL-IF.2.Measurement of AMPK-SIRT1 pathway,autophagy,and apoptosis in primary spinal cord neural MI model in vitro:First,primary spinal cord neurons were isolatedfrom rat embryonic spinal cord,cultured for 7 days,and then these neurons were identified with NSE which is neuronal marker by immunofluorescence staining.These neurons were randomly divided into two groups: control group and mechanical injury group(MI group).Primary spinal cord neural MI models of were established and detected above related indicators in MI models at 6 h,24 h,48 h,72 h and 5 d.Western blot was used to detect the expression of AMPK,p-AMPK,SIRT1 and autophagy-related proteins(LC3?,LC3 II and p62)in MI model;Hoechst 33342 staining was used to observe the apoptosis in MI model.3.Effects of resveratrol at different concentrations on AMPK-SIRT1 pathway,autophagy and apoptosis in primary spinal cord neural MI model:They were randomly divided into two groups: control group and resveratrol pretreatment group(MI +Resveratrol group).Primary spinal cord neural MI model were pretreated with resveratrol(5,10 and 20?M)which is an AMPK-SIRT1 pathway agonist.Related indicators were detected at 24 h after MI.The changes of AMPK-SIRT1 pathway and autophagy-related proteins were detected by Western blot.The apoptosis of neurons in primary spinal cord neural MI model was observed by Hoechst 33342 staining.Results:1.In vivo SCI model Western blot results showed that p-AMPK at 1 d,7d,14 d and 5 w in SCI group(P <0.05,P <0.001,P <0.05 and P <0.05,respectively)and SIRT1(both P <0.001)increased significantly compared with Sham group.All of which peaked on the 7th day after SCI.However,compared with Sham group,there was no significant change in AMPK expression in SCI group.Compared with Sham group,the ratio of LC3 II / LC3 I(P <0.001)and p62 level(P <0.001)increased at first and then decreased in SCI group.Compared with Sham group,the level of p62 in SCI group was significantly increased(P <0.001)at 7 d,14 d and 5 w after SCI.The ratio of LC3 II /LC3 I was significantly increased(P <0.001)at 1d after SCI.It is suggested that the autophagic flow was blocked at this time.The results of immunohistochemistry showed that compared with Sham group,the expression of p-AMPK and SIRT1 in spinal cord increased significantly on the 7 th day after SCI.The results of TUNEL-IF staining showed that the number of apoptotic cells in spinal cord increased significantly at 1 d,7 d,14 d and 5 w after SCI compared with Sham group.2.In vitro primary spinal cord neurons MI model Cells from the spinal cord wereisolated and cultured them,then labeled with NSE fluorescent antibody.All the cells were labeled red indicating that these cells were primary spinal cord neurons.Western blot results showed that p-AMPK levels(P <0.01,P <0.01,P <0.01)and SIRT1(P <0.01,P <0.001,P <0.001 and P <0.001)of MI group at 24 h,48 h,72 h and 5 d were significantly increased with time compared with control group,consistent with experimental results in vivo.Compared with Control group,LC3 II / LC3 I ratio and p62 level in primary neurons of MI group increased firstly and then decreased,and all reached peak at 24 h(P <0.01).It is suggested that the autophagic flow was blocked at this time.Hoechst 33342 staining results showed that nuclei were more concentrated or split in primary neurons MI group than control group.3.Further to pretreat primary spinal cord neurons MI model with AMPK-SIRT1 activator resveratrol(5?M,10?M ? 20?M)Compared with the Control group,p-AMPK(P <0.01,P <0.001 and P <0.001)and SIRT1(all P <0.001)increased significantly,but there was no significant change in AMPK in the MI+Resveratrol group.Compared with MI group,the levels of p-AMPK(P <0.05 and P <0.01)and SIRT1(all P<0.05)in the MI+Resveratrol group(10?M and 20?M)were significantly up-regulated,while the levels of AMPK did not change significantly.Compared with MI group,the expression of AMPK,p-AMPK and SIRT1 in the MI+Resveratrol group(5?M)had no significant change.Compared with MI group,the ratio of LC3 II / LC3 I in the MI+Resveratrol group(10?M and 20?M)significantly increased.Compared with MI group,p62 levels in the MI+Resveratrol group(5?M,10?M ? 20?M)were significantly decreased(all P <0.001).It is showed that the autophagic flow was improved.The results of Hoechst 33342 staining showed that the concentration or fragmentation of nuclear condensation in the MI+Resveratrol group was significantly decreased compared with MI group,but still higher than that of control group.Conclusions:After SCI,the AMPK-SIRT1 pathway in the spinal cord neurons is activated,and further activation of the AMPK-SIRT1 pathway can promote neuronal autophagy and decrease neuronal apoptosis,finally reduce spinal cord injury.This study provides important clues for the target therapy of SCI.