Study On The Supressin Of Inflammatory Reatction And Its Mechanism After Traumatic Brain Injury By Useing Melatonin

Traumatic brain injury is the main cause of death among young and middle-aged people in developing countries,with high mortality and disability rates.Currently,there are no drugs and treatments that can significantly alleviate the symptom and it was always the key points of the recent research.The inflammasome-mediated inflammatory response has emerged as a prominent contributor pathophysiological process of traumatic brain injury.Recently,melatonin is an effective,small molecule NLRP3 inflammasome inhibitor that can penetrate the blood-brain barrier^[1].In this study,we investigated the effect of melatonin on inflammatory reaction,neurological functions and brain edema after traumatic brain injury in the mice model.We established the traumatic brain injury model by using the heavy object striking method,and used Western blotting and q PCR to determine the expression of NLRP3inflammasome and IL-1βand IL-18 after traumatic brain injury.We found that the expression level of NLRP3 inflammasome and IL-1βand IL-18 in the cerebral cortex after traumatic brain injury were significantly increased,and melatonin treatment could inhibit NLRP3 inflammasome and IL-1βand IL-18 expression level in cerebral cortex after traumatic brain injury,and we found that melatonin can inhibit the expression of NF-κB signal pathway to mediates NLRP3 inflammasome and IL-1βand IL-18 expression level.Then we analyzed the effect of melatonin on mice with traumatic brain injury using post-assessments including the neurological severity scores,balance beam test and brain edema content.We found that melatonin treatment can reduce brain edema,improve balance ability and reduce the neurological defect,so as to obtain better prognosis of neurological function after traumatic brain injury.These results suggest that melatonin may inhibit the activation and expression of NLRP3 inflammasome,which may be a promising therapeutic approach for patients with traumatic brain injury,and it may have a good application prospect in the treatment of traumatic brain injury.【Objective】（1）The model of traumatic brain injury in C57 mice was established by using the method of heavy object striking,and to explore the expression level of the NLRP3inflammasome and the related inflammatory factor IL-1βand IL-18 in the brain tissue at the acute and chronic stages of traumatic brain injury,and to determine the role of melatonin in traumatic brain injury.（2）To explore the effect of melatonin on the neurological function after traumatic brain injury,the balance ability after traumatic brain injury and the brain edema after traumatic brain injury.（3）To explore the effect of melatonin on the expression level of NLRP3inflammasome and related inflammatory factors in traumatic brain injury by regulating NF-κB signaling pathway.【Methods】（1）The healthy adult male SPF clean grade C57/B6 mice were randomly divided into three groups:sham operation group（Sham）（n=6）,traumatic brain injury operation（TBI）group（n=7）,melatonin treatment group（TBI+Mel）（n=7）.The traumatic brain injury model was established by using the heavy object striking method.The brain tissue of mice in each group was randomly selected at 1,2,3,7 and28 days after operation,and then Western blotting and qRT-PCR were performed to determination the expression of NLRP3 inflammasome and IL-1β.（2）The healthy adult male SPF clean grade C57/B6 mice were randomly divided into three groups:sham operation group（Sham）（n=6）,traumatic brain injury operation（TBI）group（n=7）,melatonin treatment group（TBI+Mel）（n=30）.The balance ability of mice was evaluated continuously at the first day before operation,the first day,the second day,the third day,the fourth day and the fifth day after operation,and the neurological function of mice was evaluated at the first day before operation,the first day,the 7th day,the 14th day,the 21st day and the 28th day after operation.（3）The healthy adult male SPF clean grade C57/B6 mice were randomly divided into three groups:sham operation group（Sham）（n=6）,traumatic brain injury operation（TBI）group（n=7）,melatonin treatment group（TBI+Mel）（n=7）.Mice were randomly selected from each group to take brain tissue for brain water content measurement at the 1st,2nd,3rd,7th and 28th day after operation.（4）The healthy adult male SPF clean grade C57/B6 mice were randomly divided into three groups:sham operation group（Sham）（n=6）,traumatic brain injury operation（TBI）group（n=7）,melatonin treatment group（TBI+Mel）（n=7）.Brain tissues of mice were randomly selected from each group at 1,2,3,7 and 28 days after operation,and then to determine the expression of NF-κB signal pathway protein and p-NF-κB by using Western blotting and RT-PCR.【Results】（1）Compared with the sham group,the expression of NLRP3 inflammasome and IL-1βin TBI group at 1,2,3,7 and 28 days after operation increased significantly;Compared with the TBI group,after melatonin treatment,The expression level of IL-1βand was significantly decreased at the 2nd,3rd,7th and 28th days after operation,and the expression level of NLRP3 inflammasome was significantly decreased at the1st,2nd,3rd and 7th days after operation,and the expression of NLRP3 inflammsome and related inflammatory factor IL-1βand IL-18 in brain tissue of mice with traumatic brain injury after administration of melatonin were decreased gradually with time elapsing.（2）Compared with the sham group,the expression of NF-κB and p-NF-κB in TBI group at 1,2,3,7 and 28 days after operation were significantly increased.Compared with the TBI group,after melatonin treatment,The expression level of NF-κB and p-NF-κB decreased significantly at the 1st,2nd,3rd,7th and 28th days after TBI,and theexpression of NF-κB signal pathway protein in brain tissue of mice with TBI after melatonin administration was decreasedgradually with time elapsing;Compared with sham,the transformation of NF-κB to p-NF-κB in TBI group at each time node after operation were significantly increased,and after melatonin treatment,NF-κB phosphorylation could be inhibited.（3）Compared with the sham group,the neurological severity score in TBI group were significantly rose.Compared with the TBI group,after melatonin treatment,at 7,14,21 and 28 daysΔNSS were decreased significantly.（4）Compared with the sham group,the Beam Test scores in TBI group were significantly increased at the 1st,2nd,3rd,4th and 5th day after operation,and significantly decreased at the 1st,2nd,3rd,4th and 5th day after melatonin treatment compared with the TBI group.（5）Compared with the sham group,the brain tissue water content in TBI group was significantly rose.Compared with the TBI group,the brain tissue water content after melatonin treatment was significantly reduced at the 1st,2nd,3rd,7th and 28th days,and the brain tissue water content of mice with traumatic brain injury after melatonin treatment gradually decreased with time elapsing.【Conclusions】（1）Mel administration inhibited the TBI-induced activation of NLRP3inflammasome and IL-1β.（2）Mel administration attenuates neurological deficits,balance ability and brain edema after TBI.（3）Mel administration attenuates the expression of NLRP3 inflammasome and IL-1βafter TBI by NF-κB signal pathway.