The Role Of NLRP3 Inflammasome In Peripheral Nerve Injury In Mice

Objective Peripheral nerve injury is caused by various reasons and characterized by sensory,motor and nutritional disorders in the areas innervated by the nerve.NLRP3 can detect various microbial motifs and endogenous danger signals,which can lead to the formation of inflammasome.In this study,NLRP3 gene knockout mice were used to construct a peripheral nerve injury model,aiming to further explore whether NLRP3inflammasome is involved in peripheral nerve injury diseases and the role of NLRP3inflammasome in the repair of peripheral nerve injury,so as to lay an experimental foundation for better exploration of peripheral nerve injury and repair mechanisms.Methods Three types of C57BL/6 wild-type mice（WT）,NLRP3 knockout mice(Nlrp3^-/-)and TLR4 knockout mice(Tlr4^-/-)were used in this experiment.Follow the steps below:1.Construct an animal model of peripheral nerve injury.The WT mice were randomly divided into a control group（42）,a crush group（60）;Nlrp3^-/-mice were randomly divided into a control group（12）,a crush group（48）and Tlr4^-/-mice crush group（3）.After the mice were anesthetized,the mice sciatic nerves were exposed for 60 seconds（s）in the control group;the mice sciatic nerves were crushed with hemostatic forceps for 60 s in the crush group.2.Detect NLRP3 inflammasome.Hematoxylin and eosin（HE）staining and luxol fast blue（LFB）staining was used to detect the inflammation and myelin changes of the mice sciatic nerve.The RT-qPCR method was used to detect the m RNA expression levels of NLRP3,Caspase-1,ASC,IL-18 and IL-1βin the sciatic nerve.The Western blot method was used to detect the protein level changes of NLRP3,Caspase-1,ASC,IL-18 and IL-1β.The immunofluorescence method was used to detect the ASC oligomerization nucleation and the infiltration of macrophages.3.To detect the recovery of WT mice and Nlrp3^-/-mice after sciatic nerve injury.Western blot was used to detect the protein expression of p75NTR and GAP-43 at one week（w）after the successful establishment of the animal model.The immunohistochemical method was used to detect the protein expression of p75NTR and GAP-43 at 2 and 3 w after the successful establishment of the animal model.Sciatic nerve function index（SFI）was used as evaluate recovery of motor function of mice after sciatic nerve injury.4.The Western blot was used to detect the involvement of TLR4 in regulating the formation of NLRP3.Results The model of peripheral sciatic nerve injury in mice was successfully established.1.Results of WT mice:Compared with the control group,the LFB staining results showed that the myelin structure of the nerve injury in the crush group was incomplete and demyelination occurred.The results of HE staining showed that there were more inflammatory cells in the crush group.RT-qPCR results showed that the m RNA levels of NLRP3 and IL-1βincreased significantly in the crush group（P<0.0001）;Western blot results showed that at 12 h and 24 h after sciatic nerve injury,the protein levels of Caspase-1 and ASC increased significantly in the crush group（P<0.01）,while the protein levels of NLRP3 and IL-1βincreased significantly at 24 h after nerve injury（P<0.01）;immunofluorescence analysis showed that the number of ASC specks was significantly increased（P<0.01）.2.Results of Nlrp3^-/-mice:Compared with the control group,the results of LFB staining and HE staining were the same in the WT mice crush group.Nonetheless,higher infiltration of CD68⁺macrophages in the Nlrp3^-/-mice crush group were observed in the immunofluorescence assay.Besides,in compared with the crush group of WT mice,RT-qPCR results showed that the m RNA levels of NLRP3 and IL-1βwere significantly reduced in the crush group of Nlrp3^-/-mice at 6 h,12 h,24 h,and 48 h after the sciatic nerve injury（P<0.0001）.At 24 h and 48 h,the m RNA levels of ASC and Caspase-1 were significantly reduced in the crush group of Nlrp3^-/-mice（P<0.001）;3.Repair outcome after nerve injury:Western blot and immunohistochemical analysis showed that,p75NTR and GAP-43 protein level was higher in the crush group of Nlrp3^-/-mice when compared with the crush group of WT mice.Moreover,the SFI score was obviously higher in the crush group of Nlrp3^-/-mice in compared with the crush group of WT mice.4.The expression of NLRP3 regulated by TLR4:Western blot showed that the protein levels of NLRP3,NF-κB and TLR4 were reduced in the crush group of WT mice compared with the crush group of WT mice.Conclusion Three models of sciatic nerve injury in mice were successfully established.Sciatic nerve injury will induce the formation and activation of NLRP3 inflammasomes,the TLR4 gene positively regulates the formation of NLRP3 inflammasomes to a certain extent.Moreover,knocking out of NLRP3 gene can weaken the inflammatory response after nerve injury and accelerate the repair of motor function of sciatic nerve.Collectively,these results provide a theoretical basis for the inflammatory mechanism of peripheral nerve injury and new clues for the clinical treatment of peripheral nerve injury.