Effect Of OTULIN Expression Regulated By Electroacupuncture On Neuroinflammation And Its Mechanism In Focal Cerebral Ischemia/reperfusion Rats

PART 1: EFFECT OF OTULIN ON MICROGLIA ACTIVATION AND NEUROINFLAMMATION AND ITS MECHANISM IN FOCAL CEREBRAL ISCHEMIA/REPERFUSION RATSObjectives1.To study the time course of OTULIN expression in peri-ischemic cerebral cortex in focal cerebral ischemia/reperfusion rats.2.To explore whether OTULIN overexpression exerted neuroprotective effect in focal cerebral ischemia/reperfusion rats.3.To explore the effect of OTULIN on neuroinflammation and its mechanism in focal cerebral ischemia/reperfusion rats.4.To explore the effect of OTULIN on microglia-mediated neuroinflammation and its mechanism.Methods1.SD rats were randomly divided into Sham group and t MCAO group.The levels of OTULIN m RNA and protein in peri-ischemic cerebral cortex were detected by real-time quantitative polymerase chain reaction(RT-q PCR)and Western Blot following 6 h to 72 h after reperfusion.72 h after reperfusion,immunofluorescence was used to co-stain microglial marker Iba-1 with OTULIN protein in peri-ischemia cerebral cortex in rats of Sham and t MCAO group.2.OTULIN overexpression Lentivirus(LV-OTULIN)was injected into the lateral ventricle,and empty lentivirus(LV-Scramble)acted as a control.The rats were randomly divided into Sham,t MCAO,t MCAO+LV-Scramble and t MCAO+LV-OTULIN group.72 h after reperfusion,the neurological deficit of the rats in each group were evaluated by Longa score,Garcia score and Bederson score.The cerebral infarct volume was measured by TTC staining.Nue N+ and MAP2+ neurons were stained by immunofluorescence.3.Rats were randomly divided into Sham,t MCAO,t MCAO+LV-Scramble and t MCAO+LV-OTULIN group.Immunofluorescence was applied to stain Iba-1+ cells in the non-ischemic cerebral cortex and peri-ischemic cerebral cortex at 24 h,48 h and 72 h after reperfusion,respectively.4.Infected neuroinflammation model was established by LPS intraperitoneal injection.Rats were randomly divided into LPS(-),LPS(+),LPS(+)+LV-Scramble,and LPS(+)+LV-OTULIN group.Iba-1+ cells in the cerebral cortex in each group were observed by immunofluorescence at 24 h after LPS injection.5.Rats were randomly divided into four groups: Sham group,t MCAO group,t MCAO+LV-Scramble group and t MCAO+LV-OTULIN group.24 h after reperfusion,the content of TNF-α,IL-1β and IL-6 protein in peri-ischemic cerebral cortex was assessed by ELISA,and the content of p-IκBα,IκBα,cytoplasmic NF-κB p65 and nuclear NF-κB p65 protein was determined by Western Blot.6.Neuronal medium treated with oxygen glucose deprivation(OGD),namely conditioned medium(CM),was used to stimulate primary microglia(PM)and N9 microglial cell(N9).The cells were randomly divided into normal control(NC),OGD(-)CM and OGD(+)CM group,respectively.OTULIN m RNA and protein in each group were detected by RT-q PCR and Western Blot after 24 h culture.The microglia in NC,OGD(-)CM and OGD(+)CM group were transfected with LV-OTULIN or LV-Scramble,and the whole six groups were divided as following:NC+LV-Scramble,NC+LV-OTULIN,OGD(-)CM+LV-Scramble,OGD(-)CM+LV-OTULIN,OGD(+)CM+LV-Scramble and OGD(+)CM+LV-OTULIN group.ELISA was applied to detect the levels of TNF-α,IL-1β and IL-6 protein in each group.PM and N9 cells were randomly divided into OGD(+)CM+LV-Scramble and OGD(+)CM+LV-OTULIN group.Western Blot was used to detect the content of p-IκBα,IκBα,cytoplasmic p65 protein and nuclear p65 protein.Results1.OTULIN m RNA and protein expression in Sham group was very low from 6 h to 72 h after reperfusion.The OTULIN m RNA and protein in t MCAO group were significantly increased than those in Sham group at each time point except at 6 h after reperfusion,and peaked at 48 h.72 h after reperfusion,OTULIN in t MCAO group was enriched in activated microglia,while very few Iba-1+OTULIN+ cells were detected in Sham group.2.LV-OTULIN effectively increased OTULIN m RNA and protein expression in brain(P<0.001).Compared with t MCAO group,the Longa score(P<0.01)and Bederson score(P<0.01)in t MCAO+LV-OTULIN group significantly decreased,the Garcia score increased obviously(P<0.001),the cerebral infarct volume was significantly reduced(P<0.01),and the number of Nue N+ and MAP2+ cells located in peri-ischemic cerebral cortex was obviously increased(P<0.05).3.At each time point after reperfusion,Iba-1+ cells in Sham group were almost in resting form.Iba-1+ cells in t MCAO group were mainly in“half-activated” form at 24 h,mainly in amoeboid form at 72 h,and almost in amoeboid form at 7 d.Iba-1 mean immunofluorescence intensity in t MCAO+LV-OTULIN group was significantly lower than that in t MCAO and t MCAO+LV-Scramble group at each specific observation time point(P<0.001).4.Compared with LPS(-)group,Iba-1 mean immunofluorescence intensity in LPS(+)group was obviously increased.Iba-1 mean immunofluorescence intensity in LPS(+)+LV-OTULIN group was significantly lower than that in LPS(+)group(P<0.01).5.Compared with t MCAO,the content of TNF-α(P<0.01),IL-1β(P<0.05)and IL-6(P<0.01)protein in t MCAO+LV-OTULIN group was significantly decreased at 24 h after reperfusion,and NF-κB signaling pathway in brain was inhibited,as showed by enhanced IκBα protein degradation,IκBα protein phosphorylation and nuclear translocation of NF-κB p65 protein in t MCAO+LV-OTULIN group(P<0.01).6.The expression of OTULIN m RNA and protein was obviously enhanced in OGD(+)CM group than that in NC and OGD(-)CM group in both PM and N9 cells.In vitro,LV-OTULIN could significantly depress the secretion of TNF-α,IL-1β and IL-6 protein,and inhibit the activation of NF-κB signaling pathway in microglia activated by OGD(+)CM.Conclusions1.The focal cerebral ischemia/reperfusion injury induced enhanced OTULIN expression in peri-ischemic cerebral cortex of rats.2.OTULIN overexpression attenuated brain damage in focal cerebral ischemia/reperfusion rats.3.OTULIN overexpression attenuated microglia activation located in peri-ischemic cerebral cortex in focal cerebral ischemia/reperfusion rats.4.OTULIN overexpression alleviated the release of proinflammatory cytokines via inhibiting NF-κB signaling pathway in focal cerebral ischemia/reperfusion rats.5.OTULIN overexpression attenuated microglia-mediated neuroinflammation by inhibiting NF-κB signaling pathway.PART 2: THE EFFECT OF UP-REGULATING OTULIN EXPRESSION BY ELECTROACUPUNCTURE ON NEUROINFLAMMATION AND ITS MECHANISM IN FOCAL CEREBRAL ISCHEMIA/REPERFUSION RATSObjectives1.To investigate the effect of electroacupuncture on brain OTULIN expression in peri-ischemic cerebral cortex of focal cerebral ischemia/reperfusion rats.2.To investigate the cellular localization of OTULIN in peri-ischemic cerebral cortex of focal cerebral ischemia/reperfusion rats.3.To explore the possible role of OTULIN in electroacupuncture’s role of alleviating brain damage in focal cerebral ischemia/reperfusion rats.4.To explore the possible role of OTULIN in electroacupuncture’s role of alleviating neuroinflammation,and its mechanism in focal cerebral ischemia/reperfusion rats.5.To investigate the effect of enhanced neuronal OTULIN by electroacupuncture on neuronal injury,and its possible mechanism in focal cerebral ischemia/reperfusion rats.Methods1.SD rats were randomly divided into Sham,t MCAO and t MCAO+EA group.2 h-7 d after reperfusion were acted as observation time points.RT-q PCR and Western Blot were used to detect the content of OTULIN m RNA and protein in the peri-ischemic cerebral cortex in each group.The number of OTULIN+ cells was analyzed by immunofluorescence.2.Rats were randomly divided into Sham,t MCAO and t MCAO+EA group.Immunofluorescence was applied to stain OTULIN with neuronal marker Neu N,microglial marker Iba-1 and astrocyte marker GFAP in the peri-ischemic cerebral cortex in each group,respectively.3.Rats were randomly divided into Sham,t MCAO,t MCAO+EA,t MCAO+EA+LV-Scramble and t MCAO+EA+LV-sh OTULIN group.The degree of neurological deficits was evaluated by m NSS,inclined board test and rotarod test,and the cerebral infarct volume was determined by TTC staining.Immunofluorescence was used to detect brain Iba-1 mean immunofluorescence intensity and different shapes of Iba-1+ cells in all Iba-1+ cells,TUNEL+ cell ratio,TUNEL+Neu N+ cell ratio,and NF-κB p65 protein distribution in Neu N+ cells located in peri-ischemic cerebral cortex.ELISA was used to detect TNF-α,IL-1β and IL-6 protein content.Western Blot was used to assay the content of OTULIN protein,p-IκBα/IκBα,nucleus/ cytoplasm NF-κB p65 protein.Injured neurons in peri-ischemic cerebral cortex were detected by Nissl staining.Results1.The OTULIN m RNA and protein in t MCAO group decreased sharply at 2 h after reperfusion(P<0.05),and increased with the prolongation of reperfusion time.The content of OTULIN m RNA and protein in t MCAO group was obviously increased from 12 h to 7 d.48 h after reperfusion,OTULIN+ cells in t MCAO group increased obviously than Sham group(P<0.01),and OTULIN+ cells in t MCAO+EA group further increased than t MCAO group(P<0.01).The OTULIN protein in each group was mainly in located cytoplasm in each group.2.OTULIN protein in Sham group was mainly located in neurons.In t MCAO group,OTULIN protein was mainly enriched in Iba-1+ cells,and lower OTULIN was presented in Neu N+ cells.OTULIN protein in t MCAO+EA group was mainly located in Neu N+ cells and Iba-1+ cells.Moreover,OTULIN protein was not found in GFAP+ cells in three groups.3.72 h after reperfusion,the neurological function of t MCAO+EA group was significantly improved than t MCAO group,which showed by a decrease in m NSS score(P<0.05),an increase in holding angle(P<0.05)and prolonged relative latency time(P<0.05),and smaller cerebral infarct volume(P<0.01).After silencing OTULIN,the neuroprotective effect of electroacupuncture was significantly weakened.4.24 h and 72 h after reperfusion,Iba-1 mean immunofluorescence intensity in t MCAO+EA+LV-sh OTULIN group was significantly higher than that in t MCAO+EA and t MCAO+EA+LV-Scramble group.24 h after reperfusion,compared with t MCAO group,the proportion of“half-activated” microglia was significantly decreased(P<0.001),and the proportion of “resting” microglia was obviously increased in t MCAO+EA group(P<0.001).After silencing OTULIN,the proportion of“half-activated” microglia was increased(P<0.001),and the proportion of“resting” microglia was obviously decreased(P<0.001)in t MCAO+EA+LV-sh OTULIN group.72 h after reperfusion,compared with t MCAO group,the proportion of “Ameoboid” microglia in t MCAO+EA group decreased(P<0.001),and the proportion of “half-activated” microglia increased obviously(P<0.001).After silencing OTULIN,this effect of electroacupuncture was significantly suppressed(P<0.001).Meanwhile,compared with t MCAO+EA group,the content of TNF-α,IL-1β and IL-6protein in t MCAO+EA+LV-sh OTULIN group was increased obviously,the phosphorylation of IκBα and nuclear translocation of NF-κB p65 protein were significantly enhanced(P<0.001).5.After inhibiting OTULIN expression,the effect of electroacupuncture on improving pathological damage of neurons in periischemic cerebral cortex following ischemic stroke was attenuated.Moreover,compared with t MCAO group,the proportion of TUNEL+ and TUNEL+Neu N+ cells in t MCAO+EA+LV-sh OTULIN group decreased(P<0.001),and the nuclear NF-κB p65 protein in the Neu N+ cells increased significantly,while the cytoplasmic NF-κB p65 protein decreased obviously.Conclusions1.Electroacupuncture enhanced OTULIN expression in peri-ischemic cerebral cortex in focal cerebral ischemia/reperfusion rats.2.OTULIN protein was mainly expressed in microglia and neurons located in peri-ischemic cerebral cortex of focal cerebral ischemia/reperfusion rats.3.Electroacupuncture alleviated brain damage by enhancing OTULIN expression in focal cerebral ischemia/reperfusion rats.4.Electroacupuncture up-regulated OTULIN expression to alleviate neuroinflammation via inhibiting NF-κB signaling pathway activation in focal cerebral ischemia/reperfusion rats.5.Electroacupuncture may alleviate neuronal damage by up-regulating neuronal OTULIN expression in focal cerebral ischemia/reperfusion rats.