Critical Role Of Inhibited Caspase-1 In Treatment Of Hypoxic-ischemic Brain Damage In Neonatal Rats

Objective:Hypoxic-ischemic encephalopathy（HIE）is the most common perinatal diseases of the central nervous system,however,there is no effective way to cure these hypoxic-ischemic（HI）-related diseases.Recent studies have found that NLRP3 inflammasome pathway plays an important role in adult brain damage induced by HI,it is unclear whether and how NLRP3 inflammasome pathway is involved in the development of HIE in newborns.Our studies were focused on exploring whether and how NLRP3 inflammasome pathway is involved in the development of HIE in newborns,which might provide a potential target on the prevention and treatment of HIE.Methods:（1）The expression of NLRP3 inflammasome-related proteins in HI models1)The expression of NLRP3 inflammasome-related proteins in HIBD rats7-day SD rats were used to establish hypoxic-ischemic brain damage（HIBD）models,the cerebral cortex and hippocampus were collected in 3h,6h,12h,24h,3d,7d after HIBD.Western blot was used to detect the expression of NLRP3,caspase-1,clv-caspase-1,pre-IL-1β,mature IL-1β.The intervention time window was decided to the expression peaks/valleys of each protein.2)The expression of NLRP3 inflammasome-related proteins in OGD neuronsThe primary neurons were used to establish oxygen-glucose deprivation（OGD）models,the neurons were collected in 3h,6h,9h,12h,24h after OGD.Western blot was used to detect the expression of NLRP3,caspase-1,clv-caspase-1,pre-IL-1β,mature IL-1β.The intervention time window was decided to the expression peaks/valleys of each protein.（2）Inhibition of caspase-1 on the behavior of HIBD models1)Pharmacological inhibition of caspase-1 in HIBD ratsUnsexed 7-day SD rats were randomly divided into four groups:sham,sham+vehicle,HIBD+vehicle,HIBD+AC-YVAD-CMK.HIBD+vehicle rats were pretreated with AC-YVAD-CMK（1mg/kg,i.p.）which is the specific inhibitor of caspase-1 1h before HIBD,daily AC-YVAD-CMK treatment was performed for 7 days and the first injection was carried out immediately after HIBD.Grasping test and rotarod test were performed 3weeks after HIBD,Morris water-maze test was performed 4 weeks after HIBD.2)Genetic ablation of NLRP3/caspase-1 in HIBD miceBoth NLRP3^-/-and caspase-1^-/-mice were provided by Professor Bo Pen（Fudan University,Shanghai,China）,animals were bred and maintained in the Laboratory Animal Center,Children’s Hospital of Chongqing Medical University.Unsexed 10-day WT/NLRP3^-/-/caspase-1^-/-mice were randomly divided into four groups:WT-sham,WT-HIBD,NLRP3^-/--sham/caspase-1^-/--sham,NLRP3^-/--HIBD/caspase-1^-/--HIBD.Grasping test and rotarod test were performed 3 weeks after HIBD,Morris water-maze test was performed 4 weeks after HIBD.（3）The mechanism of inhibited caspase-1 in treatment of HIBD7-day SD rats were used to establish HIBD models,the cerebral cortex and hippocampus were collected in 3h,6h,12h,24h,3d,7d after HIBD.The primary neurons were used to establish OGD models,the neurons were collected in 3h,6h,9h,12h,24h after OGD.Western blot was used to detect the expression of AMPARs and p97.The primary neurons were pretreated with AC-YVAD-CMK（5μM）1h before OGD,the neurons were collected in 6h after OGD.Western blot was used to detect the expression of caspase-1,clv-caspase-1,pre-IL-1β,mature IL-1β,AMPARs and p97.CO-IP was used to detect the interaction between AMPARs and p97.Results:（1）The expression of NLRP3 inflammasome-related proteins increased after HI1)The expression of NLRP3 inflammasome-related proteins increased after HIBDThe protein levels assessed by western blot,the expression of NLRP3,clv-caspase-1,mature IL-1βare significantly increased in HIBD rats compared with sham rats in 3-12h after HI.2)The expression of NLRP3 inflammasome-related proteins increased after OGDThe expression of NLRP3,caspase-1,clv-caspase-1,pre-IL-1β,mature IL-1βare significantly increased in neurons subjected to OGD compared with control.These results suggested that NLRP3 inflammasome is activated after HI.（2）Inhibition of caspase-1 improve motor and cognitive functions in HIBD models1)Pharmacological inhibition of caspase-1 improve motor and cognitive functions in HIBD ratsIn grasping test,AC-YVAD-CMK significantly recovered the grip strength of the right forelimb in HIBD rats.In rotarod test,HIBD+AC-YVAD-CMK rats spent more time on the rod compared with HIBD+vehicle rats.In Morris water maze test,compared with HIBD+vehicle rats,HIBD+AC-YVAD-CMK rats spent less time to the platform during the training period,spent more time on the quadrant where the platform was located and increased the number of entries into the platform zone during the test period.These results suggested that pharmacological inhibition of caspase-1 prevented the motor and cognitive impairment in HIBD model rats.2)Genetic inhibition of NLRP3/caspase-1 improve motor and cognitive functions in HIBD miceIn grasping test,genetic ablation of NLRP3/caspase-1 significantly recovered the grip strength of the right forelimb in HIBD mice.In rotarod test,NLRP3^-/--HIBD/caspase-1^-/--HIBD mice spent more time on the rod compared with WT-HIBD mice.In Morris water maze test,compared with WT-HIBD mice,NLRP3^-/--HIBD/caspase-1^-/--HIBD mice spent less time to the platform during the training period,spent more time on the quadrant where the platform was located and increased the number of entries into the platform zone during the test period.These results suggested that genetic inhibition of caspase-1 prevented the motor and cognitive impairment in HIBD model mice.（3）Inhibition of caspase-1 increases synaptic Glu A1 expression by reducing the interaction between p97 and Glu A1The protein levels assessed by western blot,the expression of syunaptic Glu A1 is notably decreased in HIBD rats compared with sham rats in 3-12h after HI.The expression of both cytoplasmic and membrane Glu A1 are notably decreased in neurons subjected to OGD compared with control.The expression of membrane Glu A2 is decreased in neurons subjected to OGD compared with control.The expression of Glu A1 but not Glu A2 is restored in the plasma membrane of primarily cultured neurons after AC-YVAD-CMK treatment.These results suggested that the expression of Glu A1 in membrane is decreased caused by the activation of NLRP3inflammasome.CO-IP showed that the interaction between p97 and Glu A1is increased after HI,AC-YVAD-CMK leads to the dissociation of p97 from Glu A1 compared with OGD groups in neurons.These results suggested that NLRP3 inflammasome is activated after HI,which induced the interaction between p97 and Glu A1 that leaded to the membrane Glu A1 decreased.Conclusion:（1）NLRP3 inflammasome pathway is activated under ischemia-like conditions,which enhances the interaction between p97 and Glu A1,thereby inducing a decrease in surface expression of Glu A1.（2）Inhibition of caspase-1 by pharmacological intervention or genetic ablation is able to improve the impairments of motor and cognitive functions following HIBD by increasing p97-mediated surface expression of Glu A1.