| Traumatic brain injury(TBI)is defined as brain injury caused by external mechanical force,which leads to a large number of deaths and disabilities.Many victims have dysfunction,such as motor and sensory dysfunction,and even cognitive impairment.It is estimated that 50 million people worldwide are diagnosed with TBI every year.According to the different processes and stages of brain injury,it can be divided into primary and secondary brain injury,which are complex diseases caused by brain injury.In fact,tissue loss and cell death are the main injuries.After TBI,secondary brain injury caused by primary brain injury,activated microglia,recruited neutrophils and macrophages,and inflammatory reaction in blood metabolites can lead to secondary brain injury,and then lead to further functional and organic brain injury.Inflammatory response is considered to be an important mechanism of TBI secondary injury.Theoretically,reducing inflammation and improving energy and metabolism of damaged brain tissue can reduce the secondary brain injury caused by TBI.Calcitonin gene-related peptide(CGRP)is a peptide composed of 37 aminoacids,which is widely expressed in the central and peripheral nervous system.At present,CGRP is considered to be the most effective vasodilator.CGRP plays a protective role in brain tissue.CGRP is one of the most important protective substances produced by TBI when brain tissue is damaged and ischemic.Studies have shown that in the pathological process after TBI,the level of CGRP increased after TBI,and the number of neurons in brain tissue increased relatively.The purpose of the increase is to make the cerebral vessels dilate and provide blood supply for the brain tissue with ischemia and hypoxia as soon as possible.However,when the cerebral ischemia is serious,CGRP produced by brain tissue can not completely protect against this kind of injury.Therefore,serious secondary and permanent brain injury appears.Considering that CGRP has the potential to protect against brain injury,and autophagy and apoptosis play an important role in TBI induced secondary injury,we examined whether CGRP could inhibit the expression of autophagy and apoptosis in TBI mice.The results showed that CGRP can protect the brain by reducing brain edema,regulating neuronal autophagy and apoptosis,and reducing the neuroinflammatory response after TBI.Part one Relationship between serum CGRP level and prognosis inpatients with traumatic brain injuryObjective: To observe the changes of CGRP expression in patients with different level of TBI,judge the degree of injury,and further explore the relationship between CGRP and the degree of illness and prognosis of TBI patients,so as to help clinicians judge in time and give patients the most timely and effective treatment according to the changes of illness.And to explore whether CGRP can be used as a reliable index to judge the prognosis of TBI.Methods: A total of 138 TBI patients were divided into three groups:severe TBI group(n=73);There were 65 patients in mild TBI group and 54 healthy subjects(control group).Serum CGRP levels in patients with TBI were determined by enzyme-linked immunoassay(ELISA).The severity of TBI was defined using the Glasgow Coma Scale(GCS).CGRP comparison at different time was performed by ANOVA with repeated measurements.One-way ANOVA was used for comparison between groups at the same time,and LSD method was used for pair comparison.Result:1.Serum CGRP changes after TBIThe control group presented a stable state over time.while TBI(mild group)and TBI(severe group)showed a trend of decreasing first and then gradually increasing,and the serum CGRP levels in the two groups were significantly different.TBI in the severe group was lower than that in the mild group at admission,1,3,7 and 14 days,and the difference was statistically significant.TBI in the mild group was lower than that in the control group at admission,1 day and 3 days,and the difference was statistically significant.TBI in the mild group was lower than that in the control group at 7 and 14 days of admission,but the difference was not significant.2.The relationship between serum CGRP level and clinicopathological characteristics in patients with severe TBI at admissionCompared with patients with GCS score < 5,serum CGRP level was higher in patients with GCS score ?5(Z=-2.277,P=0.023).Serum CGRP levels in patients who died were lower than those in patients who survived(Z=-3.571,P<0.001).3.The value of serum CGRP level on admission in predicting death after injury in patients with TBIThe specificity and sensitivity of serum CGRP level for predicting death after injury in patients with TBI were 88.24% and 68.82%,and the optimal cut-off value of CGRP was 13.255pg/ml.4.Analysis of the association between serum CGRP levels and prognosis in patients with TBIThe study found that the survival of patients with high CGRP level was better than that of patients with low CGRP level,the difference was significant.Univariate Cox analysis showed that age,midline structure shift,GCS score,and CGRP level were correlated with prognosis of patients with TBI.Multivariate Cox analysis showed that midline structure shift and CGRP level were independent prognostic factors in patients with TBI.Summary:1.The change trend of serum CGRP in patients with TBI was firstly decreased and then gradually increased with the change of time after injury.It reaches its lowest point at about 24 hours and reaches its peak at about 14 days.2.Serum CGRP levels are highly predictive of poor prognosis and death in patients with TBI.Part two Protective effect of calcitonin gene-related peptide(CGRP)onneural function in mice after traumatic brain injuryObjective: Open Traumatic Brain Injury(TBI)animal model was established by Feeney's weight drop method,and the mice were treated with intracerebroventricular injection combined with caudal vein injection of extraneous calcitonin gene-related peptide(CGRP).The open field test,water maze test,barns maze and platform test were used to measure the behavioral scores of the experimental mice to verify the protective effect of CGRP on the neurological function of traumatic brain injury.To investigate the protective effect of CGRP on brain injury in TBI mice,the comparison of brain contusion area,brain edema and the damage degree of blood-brain barrier was measured before and after CGRP intervention.Methods: A total of 390 male BALB/C mice with clean grade(SPF)were randomly divided into Sham group,TBI group and TBI+CGRP group,TBI group and TBI+CGRP group.The mice were anesthetized with 1%pentobarbital sodium(40 mg/kg,I.P.I.)and the open craniocerebral trauma model was established according to Feeney's weight drop method.TBI+CGRP groups were injected with CGRP peptide(intracerebral ventricle injection +caudal vein injection),respectively.The degree of neurological impairment was measured by modified nerve severity score(m NSS)every day after surgery.Each group of mice is derived from post operative time points(1,3,5,and 7 days);Hematoxylin Eosin staining(HE staining)and(TTC)staining:The area of brain tissue damage was measured and compared between groups.The water content of brain tissue of different groups of mice was measured by dry and wet weight method.The extent of blood-brain barrier destruction after TBI was determined by measuring Evans Blue permeability.Western blot was used to measure the expression of CGRP protein in brain tissues.Through behavioral testing,the mouse neurological behavior was measured: 8th and9 th days after surgery the open field test was conducted;the water maze test was conducted within 10-15 days after surgery;the postoperative 16-22 days barnes maze test.The training and testing phase of the platform test was periodized 23,24 days after surgery.Result:1.Establishment of traumatic brain injury(TBI)animal modelThe average coma time of mice after operation was about 1.5-2 hours,the vital signs were stable,the body temperature recovered,the breathing was stable,gradually woke up and crawled,and they could eat and water.Anatomically,local contusion,local necrosis and congestion were found in all brain tissues,accompanied by depression and defect of some brain tissues;subarachnoid hemorrhage was found in the ipsilateral side of some brain tissues.2.The m NSS score of each group was differentOn the first day after surgery,both the TBI+CGRP group and the TBI group showed significant neurological impairment compared with the Sham group.The neurological function of the TBI+CGRP group was basically the same as that of the TBI group,and the difference was not statistically significant.At 2-7 days after TBI,the m NSS score in TBI+CGRP group was lower than that in TBI group,and the difference was statistically significant.The neurological function of the TBI+CGRP group and the TBI group gradually improved from day 2 to day 7,and the scores of the two groups gradually decreased and were gradually close to that of the Sham group.3.TBI mice neurological behavior resultsResults of open field test: total distance test: the total distance of Sham group was the longest,the total distance of TBI+CGRP group was in the middle level,and the total distance of TBI group was the shortest.The total distance of mice in the TBI+CGRP group was longer than that in the TBI group,and the difference was statistically significant.Mice in the TBI group had the largest number of immobility,while those in the Sham group and the TBI+CGRP group had lower immobility,and the difference was not statistically significant compared with those in the TBI group.Mice in the TBI group had the longest static time,while those in the Sham group and the TBI+CGRP group had the lowest static time,and the difference was not statistically significant compared with those in the TBI group.Water maze test: positioning sea trial stage;The incubation period of the water maze test mice found the platform.The incubation period of mice in the TBI+CGRP group was shorter than that in the TBI group,and the difference was statistically significant.The incubation period of mice in the TBI group was longer than that in the Sham group,and the difference was statistically significant.Space exploration test stage: On the fifth day of the test,the number of mice crossing the platform was the most in the sham group and the least in the TBI group,and the time in the TBI+CGRP group was between the two groups.The number of crossing the platform in the TBI group was less than that in the sham group,and the difference was statistically significant.The number of crossing platforms in TBI+CGRP group was more than that in TBI group,and the difference was statistically significant.Barnes maze test: Training Stage: The incubation period of mice in the TBI group was longer than that in the TBI+CGRP group,and the difference was significant.In the sixth day of the experiment,the number of error detection before the first exploration of the target hole(without black box)was compared with that of the TBI+CGRP group,and the difference was statistically significant.Platform test score: In the platform test stage,the incubation period of mice in the TBI+CGRP group was longer than that in the TBI group,and the difference was statistically significant.Error times: The error times of mice in the TBI+CGRP group were less than those in the TBI group,and the difference was statistically significant.4.Measurement and comparison of trauma area of mice brain tissue of different groups:Comparison of brain tissue sections of the three groups of mice:compared with the TBI+CGRP group and the TBI group,there was a certain degree of brain tissue damage and focal loss of nerve cell tissue,and the difference in the scope of early defect was small.Over time,brain tissue sections of TBI mice showed more severe brain damage.The volume of brain injury was quantitatively calculated.Compared with the brain tissue sections of TBI+CGRP group,the area of brain injury was slightly smaller,but the difference was not statistically significant.5.Measurement of brain edema:Exogenous CGRP intervention brought forward the time point of regression of brain edema after TBI,reduced the duration of TBI-induced brain edema after TBI,accelerated the absorption of TBI-induced brain tissue edema,and thereby alleviated brain injury;On the other hand,the degree of brain edema in TBI+CGRP group was significantly lower than that in TBI group at the four time points of 1 day,3 days,5 days and 7 days,the difference was statistically significant.6.The degree of blood brain barrier damage:On the first day after TBI,no obvious Evans Blue(EB)permeability was observed in the Sham group,and the permeability of EB in the TBI+CGRP group was not significantly different from that in the TBI group.On day 3,5and 7 after TBI,Evans Blue(EB)permeability was still higher in TBI+CGRP group and TBI group,but lower in TBI+CGRP group than in TBI group,and the difference was statistically significant.7.The difference of CGRP protein expression level in each group after TBI: The expression level of CGRP in the brain tissue of mice in TBI group was significantly decreased,and gradually increased with the passing of time;The expression level of CGRP protein in brain tissue of TBI+CGRP group was significantly higher than that of TBI group,and the difference was statistically significant.Summary:1.According to Feeney's weight dropping method,a standard and reliable open animal model of brain contusion was established.Normal feeding and behavioral changes were observed after surgery to confirm the establishment of the TBI model.2.The intervention of CGRP reduced the nerve function injury and the area of contusion of traumatic brain injury in mice.It reduced the degree of brain edema after traumatic head injury and the duration of brain edema after TBI.The damage degree of the blood-brain barrier after TBI was reduced.3.Western blotting showed that the expression level of CGRP in the brain tissues of mice after TBI decreased significantly,reached the lowest level at24 h,and gradually increased with time.The expression level of CGRP protein in brain tissue of TBI+CGRP group was significantly higher than that of TBI group,suggesting that the intervention approach of intracerebroventricular injection + caudal vein injection in this study was effective and reliable.Part three Effects of calcitonin gene-related peptide(CGRP)on Autop-hagy and apoptosis of neuronal cells after TBIObjective: By sampling the contusion brain tissue of mice,the autophagy and apoptotic-related signature proteins were detected,and the changes of autophagy and apoptotic signature proteins before and after CGRP intervention were observed and compared,so as to explore the regulatory effects of CGRP on autophagy and apoptosis under TBI environment.Methods:LC3,Neun and GFAP proteins were localized and analyzed by immunofluorescence double staining.The expression levels of LC3,P62,Beclin-1,cleaved caspase-3 were quantitatively analyzed by protein coprecipitation method.Tunel and Neun double staining were used to analyze the expression changes of neuronal apoptosis after TBI.Result:1.Effect of CGRP on the autophagy of neuronal cells of TBI mice:Immunofluorescence dual staining: LC3(+)protein was localized in the cytoplasm of neurons at the contusion site,and a small amount of positive expression was observed in the Sham group,and the degree of positive expression remained unchanged over time.LC3(+)cells were significantly increased in TBI group on the first day after injury,and the expression degree of LC3(+)cells was gradually enhanced over time,which was higher than that in Sham group,and the difference was statistically significant.The number of LC3(+)cells in TBI+CGRP group was less than that in TBI group,and the expression of LC3(+)cells was significantly decreased.On the 3rd,5th and 7th day after TBI,the difference between the two groups gradually increased and the difference was statistically significant.Western blotting showed that the expression of partial autophagy markers LC3,P62 and Beclin-1 was consistent with the results of double staining.The LC3II/LC3 I ratio and Beclin-1 level in the TBI+CGRP group were lower than those in the TBI group significantly,and the difference was statistically significant.Compared with the Sham group,the expression of P62 was decreased by TBI,and the expression of P62 protein was gradually enhanced and maintained at a relatively high stage after the intervention of CGRP.P62 in the TBI+ CGRP group was significantly higher than that in the TBI group,and the difference was statistically significant.2.Effects of CGRP on neuronal apoptosis in TBI mice:TUNEL and Neun double staining showed that TBI induced the increase of neuronal apoptosis,and the TUNEL(+)cells around the contusion were significantly increased.The TUNEL(+)in TBI+CGRP group was significantly lower than that in TBI group,and the difference was statistically significant;Western blot: TBI induced neuronal apoptosis,increased expression of cleaved-caspase-3 protein in brain tissue of contusion foci,and increased apoptosis in cortical tissue.Cleaved caspase-3 was significantly lower in the TBI+ CGRP group than in the TBI group,and the difference was statistically significant.3.The effects of CGRP on glial fibrillary acidic protein(GFAP)of neurons in TBI miceImmunofluorescence double staining:The expression of glial fibrillary acidic protein(GFAP)in the contusion area of cerebral cortex increased after TBI,and a small amount of GFAP was found in the Sham group.The degree of positive expression remained unchanged over time.GFAP(+)was significantly increased in TBI group at 1 day after injury,which was higher than that in Sham group,and the difference was statistically significant.The number of GFAP(+)cells in TBI+CGRP group was lower than that in TBI group.On the first day after TBI,the difference was not significant.On the3 rd,5th and 7th day after TBI,the difference between the two groups gradually increased and the difference was statistically significant.Neu N(+)expression in the contusion area of cerebral cortex: A moderate amount of Neu N(+)expression was observed in the Sham group.Neu N(+)cells decreased significantly in TBI group and TBI+CGRP group at 1,3,5 and7 days after injury.The Neu N(+)count rate in TBI group was lower than that in Sham group,and the difference was statistically significant.The Neu N(+)count rate in TBI+CGRP group was higher than that in TBI group,and the differences were statistically significant on day 1,3,5 and 7.Summary:1.After TBI,the levels of autophagy and apoptosis of neuronal cells in mice were increased,and the intervention of exogenous CGRP effectively inhibited the increase of autophagy and apoptosis of neuronal cells in mice induced by TBI.2.After TBI,glial fibrillary acidic protein(GFAP)was significantly increased in mice.After CGRP intervention,glial fibrillary acidic protein(GFAP)was reduced in the craniocerebral injury site of TBI mice and the function of nerve cells was protected.Part four Akt /m TOR signaling pathway is involved in theregulationof autophagy and apoptosis of neuronal cellsby exogenous CGRPObjective: By using CGRP to intervene TBI mouse model,the regulation of CGRP on autophagy and apoptosis related pathway proteins was explored at the signaling pathway level,and the regulatory mechanism of CGRP on autophagy and apoptosis was further explored.Method: Western blot was used to determine the changes of Akt /m TOR and Fox O3 a pathway related marker proteins in local brain tissue of contusion lesions before and after CGRP intervention,and to compare the changes of protein expression before and after CGRP intervention.Result:1.The effects of CGRP on Akt/m TOR pathway were as followsAfter TBI,the P-Akt/Akt and P-m TOR/m TOR levels were reduced in mice brain neuronal cells,and the TBI+CGRP group and the TBI group were lower than the sham group,and the difference was statistically significant.CGRP increases both ratios.Among them,CGRP activation of P-Akt/Akt is more obvious.At four time points of 1,3,5,and 7 days after surgery,the P-Akt/Akt of the TBI+CGRP group has increased significantly compared to the expression of TBI groups;CGRP is weak to P-m TOR/m TOR activation.The p-m TOR/m TOR of the TBI+CGRP group was significantly different from that of the TBI group on the 1st and 3rd postoperative days.On the 5th and 7th day after surgery,p-m TOR/m TOR in TBI+CGRP group was not statistically significantly different from that in TBI group.At the same time,the activation of the AKT/m TOR signal is also gradually enhanced after TBI.2.The effects of CGRP on Fox O3 a expression were as followsAfter TBI,the level of Fox O3 a protein in the nucleus was significantly increased,while that in the cytoplasm was significantly decreased,which indicated that TBI caused the change of Fox O3 a protein distribution in the brain neurons of mice.The increase of Fox O3 a in the nucleus was gradually weakened by CGRP,while the increase of Fox O3 a in the cytoplasm was gradual.The expression of Fox O3 a protein in the nucleus of TBI+CGRP group was lower than that of TBI group,and the difference was statistically significant.As can be seen from the expression of Fox O3 a in the cytoplasm,the expression of Fox O3 a protein in the TBI+CGRP group was higher than that in the TBI group at four time points 1,3,5 and 7 days after TBI operation,and the difference was statistically significant.Summary:1.TBI inhibited the activation of the Akt/m TOR signaling pathway and had a damaging effect on neurons.Exogenous CGRP intervention promoted the activation of Akt/m TOR signaling pathway,led to the decrease of autophagy and apoptosis of neuronal cells,and played a role in brain protection.2.TBI promoted the transfer of Fox O3 A protein into the nucleus of neurons,which had a damaging effect on neurons.Exogenous CGRP intervention can alleviate the increase of Fox O3 a in the nucleus and protect the neurons.Conclusion:1.The change trend of CGRP in serum samples of clinical TBI patients was firstly rapidly decreased and then gradually increased,reaching the lowest point in about 24 hours and reaching the peak around 14 days,which was the same as the trend of change in animal models.2.Serum CGRP levels can predict poor prognosis and death in patients with TBI,and this prediction is highly effective.The long-term prognosis and survival rate of high level CGRP group were better than those of low level CGRP group.Serum CGRP levels in patients who survived were higher than those who died of TBI.CGRP can be used as a reference index for poor prognosis and mortality evaluation of TBI.3.According to Feeney's weight dropping method,a standard and reliable animal model of brain contusion can be established.After the operation,normal feeding and behavioral changes can be observed to confirm the TBI model establishment.4.CGRP intervention reduced the nerve function injury and the area of contusion of traumatic brain injury in rats;It reduced the degree of brain edema after traumatic head injury and the duration of brain edema after TBI.The damage degree of blood-brain barrier after TBI was reduced.Western blot showed that the expression level of CGRP protein in the brain tissue of the TBI+CGRP group was significantly higher than that of the TBI group,suggesting that the exogenous intervention approach of intracerebroventricular injection combined with caudal vein injection was effective and reliable.5.After TBI,the levels of autophagy and apoptosis were increased,and the glial fibrillary acidic protein(GFAP)was increased.The intervention of CGRP effectively inhibited the autophagy and apoptosis of neuronal cells induced by TBI,and reduced the expression of GFAP in the brain injury site of TBI mice.6.TBI inhibited the activation of Akt/m TOR signaling pathway,led to the increase of autophagy and apoptosis levels of neuronal cells,and promoted the transfer of Fox O3 a protein into the nucleus,which had a damaging effect on neuronal cells.On the contrary,the intervention of exogenous CGRP can promote the activation of Akt/m TOR signaling pathway,inhibit the autophagy and apoptosis of neuronal cells,alleviate the increase of Fox O3 a in the nucleus,and play a role in brain protection. |
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