Neuroprotective Protective Effect Of Vitamin K2 On Rats With Traumatic Brain Injury By Reducing Pyroptosis

Background and ObjectiveTraumatic brain injury(TBI)can cause severe neuroinflammatory reaction and eventually lead to neuronal cell death.Pyrolysis is the most common type of inflammatory cell death after brain injury.Vitamin K2(VK2)is an anti-inflammatory antioxidant which can cross the blood-brain barrier,it can inhibit the inflammatory response and oxidative stress of damaged cells,suggesting that VK2 can play a neuroprotective effect by reducing the neuroinflammatory response after brain injury.In this study,a rat model of traumatic brain injury(TBI)was constructed to observe and analyze the neuroprotective effect of VK2 on TBI rats and its effect on pyrolysis-related protein,NOD-like receptor protein 3(nucleotide-binding protein 3).oligomerization domain-like receptor protein 3,NLRP3),Caspase-1(cysteinyl aspartate specific proteinase),inflammatory factors interleukin-1β(IL-1β)and interleukin-18(IL-18)expression,to explore whether VK2 has a neuroprotective effect on traumatic brain injury rats by inhibiting nerve cell pyrolysis.MethodsThirty six healthy SPF grade male SD rats(purchased fromLiaoning Changsheng Biotechnology Co.,Ltd.)weighing 280-300 g were randomly divided into three groups(n= 12 / group),including sham+ vehicle group,TBI + vehicle group and VK2 treatment group.The VK2 treatment group was given intraperitoneal injection of VK2 at a dose of400 mg / kg 0.5hours after modeling.The modified neurological deficit score(mNSS)and open field test were used to evaluate the changes of neurological function 24 hours after operation.After anesthesia,the brain was decapitated and the brain edema was assessed by brain water content.TTC staining was used to detect the degree of brain injury.Western blot was used to detect the protein expression of NLRP3,Caspase-1 and IL18 involved in the process of cell pyrolysis,immunofluorescence staining was used to detect the expression intensity of NLRP3,and Elisa was used to detect the changes in the contents of pyroptosis-related inflammatory factors IL1β and IL18.Result24 hours after TBI modeling,mNSS evaluation systemwas used to analyze the neurological function of experimental rats.Compared with the Sham+vehicle group,the mNSS scores of the TBI+vehicle group(P < 0.001)was significantly increased after brain injury;however,compared with TBI + vehicle group,the mNSS scores of TBI +VK2 group were significantly decreased(P < 0.01);then the spontaneous activity and anxiety degree of rats were evaluated by open field experiment.The analysis results are as follows:After brain injury,the total distance of the TBI+vehicle group(P < 0.001)was significantly less than the Sham+vehicle group.Compared with the TBI+vehicle group,the total distance of the TBI+VK2 group was significant increased(P < 0.05);compared with the Sham+vehicle group,the residence time in the central area of the TBI+vehicle group(P < 0.01)had a significant reduction;after VK2 was given,compared with the TBI+vehicle group,the residence time in the central area of the TBI+VK2 group was significantly increased(P < 0.01).These results suggest that 24 hours after TBI modeling,the rats in the TBI model group have neurological dysfunction,decreased spontaneous activity and increased anxiety,while VK2 can alleviate the neurological dysfunction,decreased spontaneous activity and increased anxiety of TBI rats.24h after TBI modeling,the degree of cerebral edema in rats was judged by the determination of brain water content.The results were as follows: compared with the Sham+vehicle group,the brain water content(%)of rats in the TBI+vehicle group(P <0.01)increased significantly after brain injury;After VK2 treatment,the water content of brain tissue in the TBI+VK2 group was significantly lower than that in the TBI+vehicle group(P < 0.05).The brain tissue damage volume of rats 24 h after TBI modeling was detected by brain TTC staining.The results were as follows: Compared with the Sham+vehicle group,the injury volume of rat brain tissue of the TBI+vehicle group(P< 0.001)was significantly increased;after administration of VK2,the damage volume of the TBI+VK2 group was significantly lower than that of the TBI+vehicle group(P <0.05).These results suggest that VK2 can significantly reduce the volume of brain injury and brain edema in TBI rats.Western blot was used to detect the expression of pyroptosis related target proteins,including NLRP3,caspase-1 and IL-18 in the area around the cortical injury of TBI rats.The results are the gray scale ratio of the target protein expression to the corresponding gray scale of the internal reference protein GAPDH.The results were as follows:compared with the Sham+vehicle group,NLRP3(P < 0.05),Caspase-1(P < 0.05)and IL-18(P < 0.01),the protein contents in the brain tissue around the injured area of the TBI+vehicle group were significantly up-regulated;after administration of VK2,compared with TBI + vehicle group,NLRP3(P < 0.01),caspase-1(P < 0.05),IL-18(P< 0.05)in TBI + VK2 group were significantly higher than those in TBI + vehicle group.Elisa method was used to detect the content of pyrolysis-related target molecules Caspase-1,IL-18,and IL-1β in the area around the cortex injury.Results were as follows:compared with the Sham+vehicle group,the expression of Caspase-1(P < 0.001),IL-18(P < 0.01)and IL1β(P < 0.05)in the TBI+vehicle group increased significantly;After administration of VK2,the expressions of Caspase-1(P < 0.05),IL-18(P < 0.05)and IL-1β(P < 0.05)in the TBI+VK2 group were significantly lower than those in the TBI+vehicle group.Immunofluorescence staining was used to detect the immunoreactivity of the target molecule NLRP3 related to pyrolysis in the area around cortical injury of TBI rats.The results are as follows: compared with the Sham+vehicle group,the NLRP3 immunoreactive positive signals of the TBI+vehicle group(P < 0.05)increased significantly;after administration of VK2,the positive signal of NLRP3 immunoreaction of rats in the TBI+VK2 group was significantly lower than that in the TBI+vehicle group(P < 0.05).These results suggest that 24 hours after TBI,the expression and content of pyrolysis-related target molecule proteins in cortex injury area of TBI model group were significantly increased,while VK2 can significantly reduce the brain damage of TBI rats,down-regulate the expression of pyroptosis-related target proteins NLRP3,Caspase-1,IL-18 and IL-1β,and exert neuroprotective effects on TBI rats by inhibiting nerve cell pyroptosis.Conclusion1.VK2 can alleviate the neurological dysfunction,decrease the spontaneous activity and increase the degree of anxiety in TBI rats;2.VK2 can reduce the volume of brain injury and alleviate brain edema in TBI rats;3.VK2 can reduce the protein expression of NLRP3 and caspase-1 and the immunoreactivity of NLRP3,which are significantly increased in the area around cortical injury in TBI rats.reduce the content of IL-18 and IL-1 β in TBI rats.VK2 can play a neuroprotective role in TBI rats by reducing pyroptosis.