Zinc Inhibits NLRP3 Inflammasome And Promotes Recovery From Spinal Cord Injury Through Autophagy And Ubiquitination

ObjectiveAim to explore the molecular mechanism of the regulation of NLRP3 inflammasome and the protective effect on the neuromotor function of mice after treatment with zinc gluconate after spinal cord injury.MethodsThe mice were kept in an SPF animal room for one week and randomly divided into three groups: sham operation group,control group,and zinc treatment group.A moderate contusion model of C57BL/6J mice was established by Allen’s percussion method.The success was marked by spastic paralysis of the lower limbs,the tail was upturned,and the hind limbs twitched.In the sham operation group,only the lamina was lifted and no blow was performed;the zinc treatment group was given intraperitoneal injection of zinc gluconate(30 mg/kg.d)after successful model building;the control group was given the same amount of control solvent glucose after successful model building The solution is injected intraperitoneally.The first injection was given 2hours after the operation,and then once a day until the end of the experiment.Use BMS scoring standards to evaluate the recovery of mice’s motor function on day 1,3,7,14,21,28.On the day 3 after spinal cord injury,use western blot,q RT-PCR,and Elisa to detect Inflammatory body,autophagy-related protein expression;use projection electron microscope to observe the substructure of tissue cells to clarify the expression level of autophagosome;use immunofluorescence technique to detect the expression of Neu N at 28 days and Nissl staining technique to detect spinal cord The number of motor neurons in the ventral anterior horn of the transverse section.The autophagy inhibitor 3-MA and Bafilomycin A1 were used to clarify the regulatory effect of autophagy on NLRP3 inflammasome.In in vitro experiments,LPS and ATP were used to polarize the BV2 microglia cell line into a pro-inflammatory phenotype;and co-immunoprecipitation(co-IP)was used to detect the ubiquitination of NLRP3 protein.ResultsOur data shows that zinc can promote the recovery of motor function after spinal cord injury.In vivo,zinc treatment can significantly inhibit the protein expression level of NLRP3 and increase the level of autophagy.These effects have been fully verified by in vitro experiments,after polarizing the microglial cell line BV2 cells to a pro-inflammatory phenotype.At the same time,when3-MA and Baf A1 were applied,the promotion of autophagy by zinc was blocked,and the inhibitory effect of zinc on NLRP3 was reversed.In addition,co-IP confirmed that zinc promotes autophagy also activates ubiquitin protein expression and inhibits high levels of NLRP3 expression.ConclusionsResearch results show that after spinal cord injury,zinc administration can increase autophagy and ubiquitination to regulate NLRP3 inflammasomes and provide neuroprotection.