The Modulation Of Orexin-A On Stroke-induced Immunodepression After Acute Ischemic Stroke In Mice

Part Ⅰ The Effect of Stroke induced immunodepression on susceptibility of pneumonia after cerebral ischemia-reperfusion in miceObjective:To investigate the effect of immunodepression on susceptibility of pneumonia in mice after cerebral ischemia-reperfusion.Methods:C57BL/6J male mice weighing 20-25 g were used in this experiment.A mouse model of cerebral ischemia-reperfusion(MCAO)was established by the suture-occluded method.And the injection of pathogenic bacteria into the trachea of the model animal to induce the pneumonia mouse model.The C57BL/6 mice were divided into control group(CON group),pneumonia model group(SP group),cerebral ischemia-reperfusion group(MCAO group),pneumonia group after cerebral ischemia-reperfusion(SAP group).24 h after the operation,the peripheral blood and BAL of each group were collected to detect the level of tumor necrosis factor-α(TNF-α),interferon-γ(IFN-γ),Interleukin-10(IL-10)by ELISA.BAL,peripheral blood and lung homogenate were used for bacterial culture by blood plate;The volume of cerebral infarction in MCAO group and SAP group was calculated by TTC staining after 72 h.And statistical analysis of the above indicators by SPSS 22.0 software.Results:The levels of TNF-α[(87.20 ± 4.37)ng/L]and IFN-γ[(86.71 ± 11.25)ng/L]in SAP group were lower than those in CON group[(112.96 ± 9.91)ng/L,(126.42 ± 14.61)ng/L,(t=5.32,4.815,P<0.05)],and IL-10 level[(192.36 ± 20.23)pg/ml]was higher in SAP group than that in control group[(148.85 ± 22.35)pg/ml,(t=-3.227,P<0.05)].There was no significant difference in the level of inflammatory factors among groups(P>0.05),except for the TNF-α between SAP and SP groups[(47.13± 3.84 ng/1)]vs.[(64.31±11.25)ng/l,t=-3.236,p<0.05]in BAL samples.A comparative study of bacterial load in peripheral blood[(6.77 ± 16.79)×104 CFU/ml vs.0 CFU/ml],BAL[(14.07 ± 7.59)×105 CFU/ml vs.(7.69 ±14.74)×104 CFU/ml]and lung homogenate[(5.03 ± 2.85)×106 CFU/ml vs.(9.76±9.24)×104 CFU/ml]revealed the mount of bacteria in SAP group increased when compared with SP group.The degree of pulmonary inflammatory reaction in SAP group was stronger than that in CON group and lighter than that in SP group,which was consistent with the pathological score of lung tissue;The total score of SAP group was 9.00 ± 2.27,higher than that of the control group(0.53±0.30),lower than that of SP group(15.20± 2.52),and the difference was statistically significant(all P<0.05).And the apoptosis of spleen cells in SAP group and MCAO group was observed by calculating spleen index and changing spleen histopathology.There was no significant difference in the volume of cerebral infarction between MCAO group and SAP group(t=-0.435,P=0.677).Conclusion:Stroke-induced immunodepression is a risk factor for pneumonia after stroke.Part Ⅱ Orexin-A alleviates stroke-induced immunodepression in miceObjective:To investigate the effect of Orexin-A on immunodepression induced by acute ischemic stroke(AIS).Methods:The C57/BL6 mice were randomly divided into control group(CON group),ischemic reperfusion group(MCAO group),Orexin-A intervention group after cerebral ischemia reperfusion(MCAO+OXA group),pulmonary infection simulation group(SP group),simulation of pulmonary infection group after Orexin-A intervention(SP+OXA group),ischemic reperfusion group with the pulmonary infection after simulation intervention group(SAP),Orexin-A intervention with pulmonary infection simulation following cerebral ischemia reperfusion group(SAP+OXA group).To identify the effect of Orexin-A on stroke-induced immunodepression,peripheral blood and alveolar lavage fluid(BAL)were collected,and the expression levels of tumor necrosis factor-α(TNF-α),interferon-γ(IFN-γ)and interleukin-10(IL-10)were detected by ELISA.The colony forming unit(CFU)of BAL,blood sample,and lung homogenate were calculated.72 h after ischemia-reperfusion injury,TTC staining was used to evaluate the effect of Orexin-A on cerebral infarction volume.Immunohistochemistry and immunofluorescence staining were used to evaluate whether Orexin-A could improve neuroinflammatory injury after ischemia-reperfusion.SPSS 22.0 statistical software was used for analysis,and t test was used for comparison between the two groups.Results:ELISA was used to detect inflammatory cytokines in serum and alveolar lavage fluid,respectively.(1)The expression of anti-inflammatory cytokine IL-10 in SP group was the highest in BAL,and the difference was significant between SP group and CON group(P>0.05).There was no significant statistical difference in the expression of other groups in BAL.(2)In blood sample,the level of IL-10 in MCAO group and SAP group were significantly higher than that of CON group(P<0.05).And the IL-10 level of MCAO+OXA group was significantly lower than that of MCAO group(P<0.05)after treatment with OXA.(3)There was no significant difference in the levels of TNF-α and IFN-γ in blood and BAL sample among groups.(4)The content of pro-inflammatory cytokine TNF-α in SAP+OXA group was significantly higher than that in SAP group.(5)After the intervention of OXA,the bacteria load in SAP+OXA group was significantly less than that in SAP group(P<0.01),in the streptococcus pneumoniae culture of lung homogenate,BAL and blood samples in each group.(6)Meanwhile,the number of microglia infiltration in the ischemic penumbra decreased,and the difference was significant(P<0.01)after OXA treatment.(7)Compared to the CON group,the percentage of cerebral infarct volume was decreased in MCAO+OXA group,while the neurological function was improved(P<0.05).Conclusion:The results showed that the area of cerebral infarction was reduced,the neuroinflammatory injury was alleviated,and the degree of peripheral immunosuppression was also alleviated in mice with the treatment of OXA after cerebral ischemia-reperfusion.Part Ⅲ Orexin-A inhibits cerebral ischaemic inflammatory injury in microglia through nuclear factor-KB signalling pathwayObjective:To investigate the mechanism of Orexin-A in regulating neuroinflammatory injury after ischemia-reperfusion.Methods:C57/BL6 mice were randomly divided into control group(CON group),cerebral ischemia-reperfusion group(MCAO group),and Orexin-A treatment group after cerebral ischemia-reperfusion(MCAO+OXA group).Western blotting were used to detect the expression of p65,p-p65,IKBα and p-IKBα in the ischemic penumbra region of mice.The transcriptional levels of inflammatory cytokines IL-1β(mRNA)and TNF-α(mRNA)were detected by PCR.Immunofluorescence staining was used to label Iba1 positive cells and p65 positive cells,respectively,and the nuclear translocations of p65 were assessed.SPSS 22.0 statistical software was used for analysis.T test was used for comparison between the two groups.Results:(1)After 72 h of cerebral ischemia reperfusion,the expression of IKBαwas significantly lower than that of CON group(P<0.01).However,the expression of IKBα was significantly increased compared with MCAO group after the treatment of Orexin-A,with statistically significant difference(P<0.01).(2)The expression of p65,p-IKBα and p-p65 protein in cytoplasm was increased in MCAO group,which was significantly higher than that of CON group(P<0.05).But the increase of each index in MCAO+OXA group was inhibited to different degrees,and the expression levels were significantly lower than those in MCAO group(P<0.05).(3)Immunofluorescence co-localization results showed that in MCAO group,the number of microglia infiltration and the expression of p65 protein were significantly increased(P<0.05).(4)In the MCAO+OXA group,the number of microglia infiltration and the expression of p65 protein in the Iba1 positive cells were higher than those in the CON group,but lower than those in the MCAO group.Moreover,p65 protein was rarely found in the nucleus of Iba1 positive microglia in MCAO+OXA group.(5)Transcription levels of TNF-α and IL-1β in pro-inflammatory cells were also decreased after OXA treatment(P<0.05).Conclusion:OXA can regulate the activation of microglia through NF-κB signaling pathway,and playing a neuroprotective role in regulating neuroinflammatory injury after cerebral ischemia reperfusion.Part Ⅳ The changes of serum Orexin-A level in patients with Acute Ischemic Stroke and correlation to pulmonary infectionObjective:To explore the correlation between the stroke-associated pneumonia and serum Orexin-A levels in patients with acute ischemic stroke(AIS).Methods:A total of 78 patients with acute ischemic Stroke(Stroke group)admitted to our hospital from July 2019 to January 2020,were divided into the pulmonary infection group and the non-pulmonary infection group according to diagnostic criteria of stroke-associated pneumonia(SAP).Ten patients who were hospitalized with contemporaneous and diagnosed with Transient Ischemic Attacks(TIA)were selected as the control group.Medical records and blood samples of each group were collected and collated.SPSS 22.0 statistical software was used to analyze the baseline and clinical data of patients to explore the risk factors of SAP.The level of serum Orexin-A in each group was detected by ELISA kit to explore the correlation between the level of Orexin-A and the occurrence of SAP.Results:Of the 78 patients with AIS,31 patients were grouped into stroke-associated pneumonia(pulmonary infection group),and 47 patients were grouped into non-pulmonary infection group.There were significant difference in Orexin-A content between the stroke group and the control group(P<0.05),but no significant difference was found in other clinical data and baseline data(P>0.05).The comparison of baseline data,clinical data and Orexin-A level between pulmonary infection group and non-pulmonary infection group showed that there were significant differences in age,gender,atrial fibrillation,NIHSS score,swallowing dysfunction,infarct size,white blood cell count(WBC),neutrophil count,platelet count and ICU length of stay(P<0.05).Binary Logistic regression analysis was performed on the above-mentioned indicators,and found that NIHSS score,WBC count,atrial fibrillation and infarct size were the independent risk factors for SAP after adjusting for confounding factors.What’s more,the level of Orexin-A was negatively correlated with NIHSS(r=-0.241,P<0.05).Conclusion:Compared with the control group,the level of serum Orexin-A decreased in patients with AIS and were even lower in patients with SAP.The changes of Orexin-A was correlated with the severity of brain tissue injury after AIS,which might be a potential prediction and therapeutic target,but further large sample studies are needed.