Protective Effect And Mechanism Of Orexin-A On Secondary Brain Injury After Intracerebral Hemorrhage

Part one Analysis of related risk factors for poor prognosis in patients with intracerebral hemorrhageObjective:To investigate the short-term prognosis and influencing factors of patients with intracerebral hemorrhage（ICH）.Methods:The subjects of this study were ICH patients admitted to our department from September 2018 to March 2022.At 6 months after discharge,they were divided into good prognosis group and poor prognosis group according to the Glasgow Outcome Scale（GOS）score.The general data and clinical indicators of the two groups were compared,and Logistic binary regression analysis was used to explore the risk factors of poor prognosis in patients with ICH.Results:There were significant differences in education level（P<0.001）,bleeding site（P<0.05）,Glasgow coma score（GCS）（P<0.001）,hematoma volume（P<0.001）and intracranial infection（P<0.01）between ICH patients with good prognosis and ICH patients with poor prognosis.Logistic binary regression analysis showed that bleeding site,GCS score,hematoma volume and intracranial infection were independent risk factors for poor prognosis of ICH.Conclusions:The prognosis of ICH is related to age,gender,education level,history of hypertension,time from onset of symptoms to admission,bleeding site,GCS score,hematoma volume,treatment,intracranial infection and pulmonary infection.The bleeding site,GCS score,hematoma volume and intracranial infection were independent risk factors for poor prognosis of ICH.Part two Effect of exogenous administration of orexin-A on poor prognosis in rats with intracerebral hemorrhageObjective:This part aims to explore whether Orexin-A（OXA）can reduce the secondary brain injury（SBI）after experimental intracerebral hemorrhage（ICH）in rats and play a neuroprotective role.Methods:A total of40 healthy adult male Sprague-Dawley（SD）rats（250-300 g,10-12 weeks）were selected,and were randomly and evenly allotted to 4 groups of 10 rats each,namely,Sham group,ICH group,ICH+Vehicle group and ICH+OXA group.ICH model of rats was established by tail artery autologous blood infusion,and treatment drugs of OXA（30μg/kg）was administered by intracerebroventricular injection 1 h after successful modeling,and the Vehicle group was given an equal volume of dimethyl sulfoxide（DMSO）at the same time point.The neurological function was evaluated by corner turn test and modified neurological severity score（m NSS）at 48 h after ICH.The brain water content was detected by dry-wet weight method.The neuronal damage was observed by hematoxylin-eosin（H&E）staining and Nissl staining.Results:Compared with Sham group,the tissue space of ICH group and ICH+Vehicle group was larger,the number of neurons decreased,the brain water content increased,and obvious sensory and motor dysfunction were occurred（P<0.05）.However,after treatment with OXA,the neuronal damage and brain edema were significantly alleviated,and the neurological dysfunction was significantly improved（P<0.05）.Conclusions:OXA treatment can significantly reduce brain edema and neurological damage after ICH in rats,and has a neuroprotective effect on SBI after ICH.Part three Study on the molecular mechanism of exogenous administration of orexin-A in improving poor prognosis after intracerebral hemorrhageObjective:To determine the activity of autophagy after ICH and whether OXA exerts neuroprotective effects by inhibiting over-activated autophagy in vivo.To further explore the protective effect of OXA and its molecular mechanism at the cellular level.Methods:According to the experimental design,27 SD rats were randomly divided into 9 groups:Sham group,ICH 12 h group,ICH 24 h group,ICH 48 h group,ICH 3 d group,ICH 5 d group,ICH 7 d group,ICH 14 d group and ICH 28 d group.The ICH model was established by tail arterial autologous blood infusion,and the expression of autophagy-related proteins（including LC3B and p62）after ICH was observed by Western blot.Then another 36 SD rats were randomly divided into 4 groups:Sham group,ICH group,ICH+Vehicle group and ICH+OXA group.Western blot,immunohistochemistry and transmission electron microscopy（TEM）were used to observe the effect of OXA on autophagy activity at 48 h after ICH.Subsequently,hemin-induced PC12 cell injury was used as ICH model in vitro.After determining the optimal concentration and time of Hemin-induced injury and the optimal concentration of OXA to protect cells from injury,PC12 cells were divided into 7 groups:Control group,Hemin group,Hemin+Vehicle group,Hemin+OXA group,Hemin+OXA+Rapamycin group,Hemin+OXA+PD98059 group and Hemin+OXA+SB334867 group.Cell counting kit-8（CCK-8）was used to detect cell viability.The apoptosis of PC12 cells was detected by flow cytometry.The protein expressions of LC3B,Beclin-1,p62,Bax,Bcl-2,p-ERK_1/2,t-ERK_1/2,p-m TOR,and t-m TOR were detected by Western blot analysis.Results:1.Compared with the Sham group,the ratio of LC3B-II/LC3B-I increased significantly at 12 h after ICH,peaked at 2 d,and decreased significantly at 14 d,while the protein expression level of p62showed an opposite trend at the same time point（P<0.05）.2.Immunohistochemistry results showed that compared with Sham group,the expression of LC3B in perihematomal brain tissue of ICH group was significantly increased,while the expression of LC3B in perihematomal brain tissue of OXA group was significantly decreased;a similar trend was observed by Western blot（P<0.05）.Transmission electron microscopy showed that there were autophagosomes and autolysosomes in the area around the hematoma.After OXA treatment,the double membrane structure observed by electron microscopy was significantly reduced.3.OXA can significantly increase cell viability and inhibit Hemin-induced autophagic cell death（P<0.05）.4.In addition,compared with the Control group,the LC3B-II/LC3B-I ratio and Beclin-1/Bcl-2 ratio,as well as the expression of Bax and p-ERK_1/2were significantly increased and the expression of p62 and p-m TOR were significantly decreased in Hemin group（P<0.05）.However,after OXA treatment,the LC3B-II/LC3B-I ratio,the protein level of Bax,the Beclin-1/Bcl-2 ratio and the expression of p-ERK_1/2were significantly decreased,and the expression of p62 and p-m TOR protein levels were significantly increased（P<0.05）.5.Rapamycin,an autophagy activator,partially eliminated the inhibitory effect of OXA on autophagy（P<0.05）.6.PD98059,an inhibitor of ERK/m TOR pathway,or SB334867,an inhibitor of orexin 1 receptor,partially blocked the protective effect of OXA（P<0.05）.Conclusions:Autophagy is activated after ICH,and OXA plays a protective role in SBI after ICH by inhibiting autophagy.OXA could significantly reduce Hemin-induced PC12 cell damage by inhibiting over-activated autophagy.The molecular mechanism of the protective effect of OXA may be related to OXR1-mediated ERK/m TOR signaling pathway.