The Effect Of Spata2 Knockdown On The Brain Inflammation In Cerebral Ischemia/Reperfusion Rats

Objectives:1.To study the expression and distribution of Spata2 in the brain of normal rats.2.To study the temporal dynamics and spatial distribution of Spata2 in the cerebral cortex of rats subjected to transient middle cerebral artery occlusion followed by reperfusion(t MCAO/R).3.To study the effect of Spata2 knockdown on the outcomes of t MCAO/R rats.4.To study the effect of Spata2 knockdown on the brain inflammation induced by cerebral ischemia/reperfusion.Methods:1.The quantitative real-time reverse transcription-polymerase chain reaction(q RT-PCR)and western blot were used to explore the expression and distribution of Spata2 in different brain regions and testes of normal rats.The immunofluorescence was used to study the subcellular localization of SPATA2 and its co-localization with CYLD.2.Rats were randomly divided into the sham and t MCAO/R groups.The t MCAO/R animal model was established using a monofilament nylon suture with a rounded tip.The q RT-PCR and western blot were used to study the expression of SPATA2 in the peri-infarct cortex at different time points(6 h,12 h,24 h,48 h,and 72 h after reperfusion).The q RT-PCR was used to investigate the m RNA levels of TNF-α,IL-1β,and IL-18 in the peri-infarct cortex at different time points.The immunofluorescence was used to study the co-localization of SPATA2 with Neu N,GFAP,or Iba-1 in the peri-infarct cortex.3.The lentivirus-mediated sh RNA(LV-sh Spata2)was employed to inhibit Spata2 expression,and the scramble(LV-scramble)was served as the control.Rats were randomly divided into four groups: sham,t MCAO/R,t MCAO/R+LV-scramble,and t MCAO/R+LV-sh Spata2 group.At 24 h after reperfusion,the Garcia scores were used to assess neurological deficits.The TTC staining was used to determine the infarct volume.4.SD Rats were randomly divided into four groups: sham,t MCAO/R,t MCAO/R+LV-scramble,and t MCAO/R+LV-sh Spata2 group.At 24 h after reperfusion,the q RT-PCR was used to investigate the m RNA levels of TNF-α,IL-1β,and IL-18 in the peri-infarct cortex.The immunofluorescence was used to determine the Iba-1 positive cell counts.The western blot was used to determine the protein level of CYLD,p-IκBα,t-IκBα,p-P65,t-P65,p-JNK,t-JNK,p-P38,t-P38,and NLRP3.Results:1.Spata2 was expressed throughout the rat brain.SPATA2 was expressed in the nucleus and marginally expressed in the cytoplasm.SPATA2 was co-localized with CYLD.2.SPATA2 was mainly co-localized with Neu N,partially with Iba-1,and rarely with GFAP in the peri-infarct cortex.The m RNA level of Spata2 was reduced significantly(at 6,24,48,and 72 h,P < 0.0001,at 12 h,P <0.01),and the protein level of SPATA2 was reduced significantly at 24 h and 48 h(P < 0.01)after reperfusion.The m RNA level of TNF-α and IL-1β was gradually reduced with time and was significantly higher in the t MCAO/R group than in the sham group at 6 h to 48 h after reperfusion.(TNF-α at 6 h and 24 h,P < 0.01,at 12 h and 48 h,P < 0.05,IL-1β at 6 h,12 h,and 24 h,P < 0.01,at 48 h,P < 0.01).The m RNA level of IL-18 had no significant difference between sham and t MCAO/R group at any time points.3.The lentivirus-mediated sh RNA inhibited Spata2 expression effectively at both m RNA and protein levels(P < 0.01).At 24 h after reperfusion,the Garcia scores in the t MCAO/R+LV-sh Spata2 group were significantly lower than that in the other three groups(P < 0.05)while the infarct volumes in the t MCAO/R+LV-sh Spata2 group were significantly higher than that in the other three groups(P < 0.01).No significant differences in the Garcia scores and infarct volumes were found between t MCAO/R and t MCAO/R+LV-scramble groups(P > 0.05).4.At 24 h after reperfusion,there were more phosphorylated IκBα and P65 in the t MCAO/R group or the t MCAO/R+LV-scramble group than that in the sham group.The phosphorylated IκBα and P65 in the t MCAO/R+LV-sh Spata2 group were more than in the other three groups(P< 0.01).No significant difference in the phosphorylated JNK was found among the four groups(P > 0.05).There were more phosphorylated P38 in the t MCAO/R+LV-sh Spata2 group than in the other three groups(P <0.01),while no significant difference was found among the sham group,t MCAO/R,and t MCAO/R+LV-scramble groups(P > 0.05).The protein level of NLRP3 was higher in the t MCAO/R+LV-sh Spata2 group than in the t MCAO/R or t MCAO/R+LV-scramble groups(P < 0.01),while no significant difference was found among the other three groups(P > 0.05).Conclusions:Spata2 was expressed throughout the rat brain.SPATA2 knockdown led to stronger inflammatory response and poorer outcome in t MCAO/R rats,and one possible mechanism for that is the activation of P38MAPK/NF-κB and NLRP3 inflammasome.Therefore,SPATA2 plays a protective role against brain inflammation induced by ischemia/reperfusion injury so that SPATA2 could be a potential therapeutic target for treating ischemic stroke.