The Function Of PI3K/Akt Signal Pathway And ER Stress In Spinal Cord Injury

Spinal cord injury is induced by various external or internal reason,and the structure and/or function of the spinal cord is destroyed.The external causes are mainly car accidents,falling from the sky or violent fractures.These causes can cause direct damage to the spinal cord.It is manifested as dyskinesia,sensory disturbance,urinary and other autonomic dysfunction,accompanied by pathological reflexes.There are mainly primary diseases such as myelitis or spinal cord tumors that damage the spinal cord.According to the results of previous studies,the pathophysiological changes after spinal cord injury can be divided into primary injury and secondary injury Primary injury is an irreparable mechanical injury caused by external or internal factors directly acting on the spinal cord.It can cause tissue damage or congestion of the spinal cord.Pathophysiological changes are mainly caused by direct damage of neurons and glial cells.The change include metadentical necrosis,cell membrane rupture,barrier destruction between blood and spinal cord,tearing of axonal membrane,loss of myelin,damage to microtubule system,release of large amounts of inflammatory substances,and electrolyte imbalance.Secondary injury refers to a series of molecular-level damage caused by indirect causes,which seriously deteriorates the damage caused by primary injury,mainly manifested by vascular dysregulation caused by neurogenic shock after injury,electrolyte caused by serial biochemical reactions.The change include alterations,neurotransmitters and oxidized lipid accumulation,edema,and apoptosis,and the primary role of the change is apoptosis.Different from other tissues,the neurons are terminal cells,which do not have the regenerative function.When the spinal cord is damaged,the cells in the injured area undergo apoptosis or degeneration and necrosis,and the synaptic connections between the neurons are destroyed and transmitted.Under the action of self-repair and stress,a large number of glial cells,such as astrocytes proliferate,accumulate in the injured area and form glial scars which exerting neuroprotective effects.When the regenerated neurons touch the glial scar,they stop growing,and the glial scar can further hinder the extension of the regenerative neuron axons,resulting in limited recovery after spinal cord injury.Now,many medical assistance and treatment can prolong the life of patients,but there is still no effective treatment for promoting the regeneration of neurons.In recent years,some scholars have begun to pay attention to the series of changes caused by secondary spinal cord injury.They believe that limiting the apoptosis process and degeneration caused by secondary injury,improving the local microenvironment and reducing the release of various harmful factors can improve the recovery of injury.Rapid regeneration creates good conditions and is an effective way to improve the therapeutic effect after spinal cord injury.The PI3K/AKT signaling pathway controls a variety of cellular events such as cell proliferation,stress and apoptosis,and is currently widely used in ischemia-reperfusion injury,inflammatory response and tumor therapy research.The endoplasmic reticulum is an important organelle for neuronal and non-neuronal cells to maintain normal function and survival.Studies have shown that after spinal cord injury in mice,the mRNA level of mature XBP1,a characteristic molecule of endoplasmic reticulum stress,is significantly up-regulated.Reticulum stress may be involved in the pathological process of spinal cord injury.This study was to observe the expression changes of PI3K/Akt signaling pathway and endoplasmic reticulum stress-specific protein after spinal cord injury,and analyze the role of PI3K/Akt signaling pathway and endoplasmic reticulum stress in spinal cord injury.This study can provide experimental evidence for clinical prevention and reduction of secondary spinal cord injury.The Details are as following:Part 1 The expression and function of PI3K/Akt signaling pathway and ER stress in spinal cord injury[Objective]To observe the change tendency of PI3K/Akt signaling pathway and endoplasmic reticulum stress in rat spinal cord injury,and to analyze whether the two pathways are involved in spinal cord injury repair process and explore its possible role.[Methods]The spinal cord injury model of rat was established,and the effect of model was determined by histopathological staining and BBB scoring method.Then the expression of PI3K,Akt,p-Akt,GSK-3?,Bcl-2,caspase12,CHOP,Grp78,Eif-2 a,p-Eif-2 a,XBP1,Bad and p-Bad were detected by immunohistochemistry and western-blotting.The trends of various factors at different time points after spinal cord injury were analyzed[Results](1)The results of HE staining showed that the morphology of spinal cord tissue in normal and sham-operated rats showed no change at different time points after operation,which showed that the texture of spinal cord tissue was uniform,and the boundary between gray matter and white matter was clear.After amplification,the number of neurons was observed,the cell body was larger,the nucleolus was obvious,and the cells did not show bleeding,edema or necrosis.In the 1st,3rd,and 7th days after spinal cord injury,the morphological structure of the spinal cord injury area was incomplete,the cavities of different sizes appeared,the nerve tissue had different degrees of defects,and the boundary between gray matter and white matter disappeared or unclear.It is observed that there are a large number of foam cells in the center of the damaged area,accompanied by inflammatory cell infiltration,the number of neurons in the gray matter area is reduced,irregular shape,nuclear pyknosis,Nissl reduction,dissolution further aggravation;the fibers in the white matter region are reduced,the distribution is disordered,the morphological structure is incomplete,the gap between the myelin sheaths is enlarged,and the arrangement is disordered.Compared with 1 day,3 days,and 7 days,the morphology of neurons in the 14-day and 28-day groups after spinal cord injury recovered some extent,the nucleolus was visible,and some Nissl bodies recovered,but the distribution was uneven.(2)BBB scores showed that the normal and sham-operated rats had continuous ground movement,gait coordination,strong toe grip,and the position of the limbs remained parallel with the body during the exercise,the trunk was stable,and the tail was stable sustained lifting,the BBB score is 21 points.One day after spinal cord injury,the rats were still in complete paralysis,the hind limbs did not have any exercise ability,the tail was pulled downwards,and there was no spontaneous movement,the BBB score was recorded as 0 points;in the 3 days after injury,the rats showed slight activity,however,the BBB score did not reach the standard of 1 point;7 days after injury,one or two joints of the hind limbs of the rats showed slight activity,and the BBB score was 1 point;14 days after the injury,the functional activities of the hind limbs of the rats partial recovery,one joint has a large movement or one joint has a large movement and another joint has a slight activity,but the hind limb can not bear weight,the BBB score is 2 points;28 days after the injury,the two joints of the rat's hind limbs can have significant activities,however,the hind limbs were stiff and the BBB score was 3 points.(3)Immunohistochemical staining results showed that brown-yellow,granular positive staining areas were found in the cytoplasm of neurons under the microscope.These particles were the positive response of PI3K,Akt,CHOP,Grp78,Eif-2a,XBP1 and Bad.There were fewer positive granules in the spinal cord tissue of normal and sham-operated rats After spinal cord injury,there were more positive granules in the spinal cord tissue of each group.It was found that the expression levels of PI3K and Akt were gradually increased from 1 day after injury,the expression level was the highest at 14 days,then decreased gradually,but the absolute expression was higher than the normal group.The expression of CHOP and Grp78 was the highest at 1 day after injury,and then gradually decreased,but absolute expression.The expression of XBP1 and Bad increased in 1 day after injury,and was the highest at 3 days after injury,then decreased gradually,but the absolute expression was higher than the normal group;The expression level of Eif-2? factor increased gradually from 1 day after injury,and reached the highest expression level at 7 days,and then gradually decreased,but the absolute expression was higher than the normal group.(4)Western-blot results showed that the expression of PI3K,Akt,GSK-3?,caspasel2,Bcl-2,Grp78,CHOP.Eif-2a,p-Eif-2a,XBP1,Bad and p-Bad in the normal group and sham group was lower,and the expression was significantly increased after spinal cord injury,and the absolute expression was higher than that of the normal group(P<0.05).However,the peak expression time of different factors was inconsistent.The expression of PI3K factor was increased gradually from 1 day after the injury,and the expression level was the highest in 3 days,and then gradually decreased.The expression levels of Akt,GSK-3?,caspase12 and Bcl-2 gradually increased from 1 day after injury to 7 days,subsequently,the trend of gradual decrease was observed;the expression levels of CHOP,XBP1 and Eif-2a were highest at 1 day after injury,and then gradually decreased;the expression levels of Grp78,p-Eif-2a and Bad were from 1 after injury,and the expression level was the highest at 7 days,and then gradually decreased;the expression level of p-Bad gradually increased from 1 day after injury,and reached the highest level at 3 days,and then gradually decreased.[Conclusion]In the spinal cord injury model of rat,the expression levels of PI3K,Akt,p-Akt,GSK-3? and other key molecules of PI3K/Akt signaling pathway,apoptosis-related factors Bcl-2,caspase 12 and endoplasmic reticulum stress key molecule such as CHOP,Grp78,Eif-2a,p-Eif-2a,XBP1,Bad and p-Bad showed a gradual increase trend,and after reaching the peak,the decrease occurred.This result is consistent with the "window period" change after spinal cord injury,and the treatment given during this time can obtained good effect.Part 2 The expression and function of PI3K/Akt signaling pathway and ER stress after primary neuronal injury[Objective 1 To analyze the expression levels of PI3K/Akt signal pathways and endoplasmic reticulum stress in primary neuronal injury and to analyze the role of PI3K/Akt signaling pathway and endoplasmic reticulum stress in vitro.[Methods]The primary neurons of rats were isolated and cultured,and the neuron injury model was made by scratch method.The apoptosis of cells was detected by flow cytometry at different time after injury.Then,the distribution density and expression level of some factors,such as PI3K,Akt,p-Akt,CHOP,Grp78,Eif-2a,p-Eif-2a,XBP1,Bad and p-Bad were tested by immunocytochemistry and western-blotting.The trends of various factors at different time points after spinal cord injury were analyzed.[Results](1)The results of flow cytometry of AnnexinV-FITC/P1 double staining showed that the non-apoptotic cells in the normal group were as high as 90.5%,and the apoptotic cells were 4.2%;4h after the neuron injury,the non-apoptotic cells were 75.1%,the apoptotic cells were 18.5%;the non-apoptotic cells decreased by 61%and the apoptotic cells were 34.4%at 12 h after neuronal injury;24 h after neuronal injury,non-apoptotic cells were 66%,and apoptotic cells were 24.7%.At the same time,with the continuous change of time after injury,the proportion of apoptotic rate increased gradually after neuron injury,and the apoptosis rate was the highest at 12h,and decreased slightly after 24h injury.(2)Immunocytochemical staining results showed that after primary cultured neuron injury,endoplasmic reticulum stress-related proteins such as CHOP,Grp78,XBP1,Eif-2a,Bad and p-Bad were mainly expressed in the cell.In the quality,it is granular and scattered.(3)Western-blot results showed that the expression of PI3K,Akt and endoplasmic reticulum stress-induced apoptosis proteins Grp78,CHOP,Eif-2a,p-Eif-2a,XBP1,Bad and p-Bad were almost absent in normal neurons,then was significantly increased after the scratch injury,and the absolute expression was higher than that of the normal neurons(P<0.05).However,the peaks of the expression of different factors were inconsistent.The expression of PI3K and Akt were the highest at 24 after the injury;the expression of CHOP and XBP1 was the highest at 4 hours after injury,and the expression of Grp78 was higher at 12,24,and 48 hours after injury.The expression of Eif-2a,p-Eif-2a,Bad and p-Bad were the highest expression at 12 hours after injury.[Conclusion]In the primary neuron injury model,the trend of various factors after spinal cord injury was consistent with the in vivo experiment,suggesting that PI3K/Akt signal pathway and endoplasmic reticulum stress may be involved in the process of apoptosis after neuronal injury.Part 3 Mechanism and function of PI3K/Akt signaling pathway and endoplasmic reticulum stress in spinal cord injury[Objective]To explore the mechanism of PI3K/Akt signaling pathway and endoplasmic reticulum stress in spinal cord injury,and to analyze the intersection and association between PI3K/Akt signaling pathway and endoplasmic reticulum stress.[Methods]A rat model of spinal cord injury was made.After the injury,PI3K and Akt specific inhibitors were intraperitoneally injected daily.BBB scoring method was used to evaluate the recovery of behavior,immunostining and western-blotting was used to observe the expression of PI3K,Akt,GSK-3?,p-GSK-3? and apoptosis relevant factors such as Bcl-2,bax,caspase 3,caspase 9 and caspase12.[Results]The results of BBB showed that the recovery of behavior was higher after adding inhibitor;the results of Western Blot showed that the expression of PI3K and Akt at different time points after spinal cord injury was significantly lower than that that of the inhibited group(P<0.05),and the expression level of GSK-3?,p-GSK-3?and apoptosis-related factors such as Bcl-2,bax,caspase 3,caspase 9,caspase12 were significantly increased,and the absolute expression was higher than the normal group(P<0.05).[Conclusion]After adding specific inhibitors of PI3K/Akt signaling pathway,the expression level of GSK-3?,p-GSK-3? and apoptotic proteins were increased.The pathophysiological changes after spinal cord injury were complex processes involving multiple factors.We hypothesize that there is a crossover of key factors between the PI3K/Akt signaling pathway which inhibiting apoptosis and the endoplasmic reticulum stress which promoting apoptosis during this process.In the future,we can select some key downstream factors of PI3K/Akt signaling pathway to provide a new target for effective treatment of secondary spinal cord injury.