Effect And Mechanism Of Orexin-a Combined With Transplanted Neural Stem Cells In Promoting Brain Injury Repair In Rats

Background and Purpose:Traumatic brain injury(TBI)is organic damage to the brain tissue that is caused by violent action on the head.The incidence of TBI is increasing year by year,and the mortality and disability rates are high.It is one of the major public health problems in the world.With the advancement of medical technology,especially the development of emergency medicine and critical care medicine,The overall survival rate of craniocerebral injury is also increasing,but there are also more and more patients with neurological deficits and cognitive impairment caused by craniocerebral injury,which brings a heavy burden to society and families.Traumatic brain injury is divided into primary injury and secondary injury according to the time of injury.Primary injury is the direct action of external forces,Secondary injury is a secondary tissue injury caused by a series of biochemical changes triggered by the primary injury,and its injury mechanism is very complex,including excitotoxicity,increased free radicals,intracellular calcium overload,inflammatory response,apoptosis,etc.Primary damage is irreversible,while secondary damage is salvageable.The current treatment methods are still limited to surgical decompression and supportive treatment,among which the supportive treatment for central nervous system repair is mainly neuroprotective drugs,neuromodulation,and rehabilitation training.Neuroprotective drugs are mainly aimed at improving the intracranial microenvironment;Neural regulation is mainly to promote neuron excitation or inhibition,change axonal output,etc.;Rehabilitation training can promote the expression of various nerve growth-related factors in the central environment,enhance the intrinsic growth ability of nerves,and induce a nerve regeneration environment.The treatment effect is difficult to achieve the expected value of the patient,Such as drugs,neuromodulation,and rehabilitation training,due to the very limited ability of nerve regeneration in the central nervous system.Neurons are the basic unit of structure and function of the nervous system.Some studies have shown that neural stem cells(NSCs)are cells that have differentiation potential and self-renewal ability.It can not only generate new neural stem cells but also differentiate into neurons and glial cells to repair damaged brain tissue.In addition,it can also secrete neurotrophic factors to regulate the neural microenvironment.Therefore,neural stem cell transplantation therapy has become a research hotspot in the field of regenerative medicine.However,how to regulate neural stem cells to differentiate in a specific direction without excessive proliferation,How to ensure long-term integration of neural stem cells with host tissue at the site of injury.None of these problems have reliable solutions.In recent years,there have been reports of adjuvant drugs combined with stem cell transplantation.However,more focus on the comparison of treatment effects,there are few studies on its specific mechanism.The previous study of the research group found that Orexin-A has neuroprotective and wake-promoting effects in brain-injured coma rats.In addition,the research group found that electrical stimulation can increase the secretion of Orexin-A in the lateral hypothalamus,and reduce the degree of brain damage by inhibiting oxidative stress,inflammation,and apoptosis.However,whether Orexin-A affects the neural regeneration of neural stem cells after brain injury is unclear.The purpose of this study was to investigate the effect of Orexin-A on the biological behavior of neural stem cells in the inflammatory microenvironment of traumatic brain injury in vitro,and the effect of Orexin-A combined with neural stem cell transplantation on the repair of brain injury in rats in vivo.Method:At first,our study used an in vitro model that lipopolysaccharide induces inflammatory damage in traumatic brain injury,CCK-8 was used to test the level of cell proliferation;Transwell test were used to observe the migration ability of neural stem cells;The damage of neural stem cells was observed by HE staining of cell climbing slides;Griess kit is used to detect the release level of NO;Western blot was used to analyze the expression levels of pathway-related proteins p-Ik Bα,Ik Bα,p-P65,P65,P38,p-P38,Erk,p-Erk,JNK,p-JNK;q RT-PCR was used to evaluate the m RNA expression levels of downstream inflammatory factors IL-1β,IL-6,TNF-α,In order to explore the effect and possible mechanism of Orexin-A on the inflammatory response of neural stem cells induced by LPS.Subsequently,EDU was used to detect cell proliferation under normal physiological conditions and in an LPS-induced inflammatory microenvironment,respectively;Western blot and immunofluorescence were used to analyze the expression levels of neural stem cell differentiation markers Tuj1,GFAP,and Olig2;Western blot was used to evaluate the expression levels of pathway-related proteinsβ-catenin and TCF-1;q RT-PCR was used to analyze the m RNA expression levels of downstream target genes C-Myc,Cyclin D1,and MMP-9 in the pathway.Thus,its effects and possible mechanisms were explored that the effect of Orexin-A on neural stem cell differentiation in the inflammatory microenvironment.Finally,an in vitro Feeney fall-body brain injury model was constructed,Tissue HE staining was used to observe the morphological changes of rat brain;Nissl staining was used to observe neuronal changes after brain injury;q RT-PCR was used to evaluate the m RNA expression levels of neurotrophic factors BDNF;Evaluation of neurological deficits in rats with m NSS score;Positioning navigation experiment and space exploration experiment was used to observe the cognitive impairment of rats.Therefore,the effect of Orexin-A combined with neural stem cell transplantation on the repair of brain injury in rats was explored.Result:For LPS-induced inflammatory injury model studies in vitro,The results suggest that Orexin-A can promote the proliferation of neural stem cells in vitro,and the optimal concentration is 10-1μM;Orexin-A can reduce the m RNA expression levels of inflammatory factors IL-1β,IL-6,and TNF-α,thereby reducing LPS-induced inflammatory damage;Orexin-A can up-regulate the expression level of IKBα and down-regulate the expression levels of p-P65 and p-IKBα,which may be involved in inhibiting the activation of NF-κB signaling pathway induced by LPS;Orexin-A can down-regulate the expression levels of p-P38 and p-Erk,which may be involved in reducing the phosphorylation of MAPK/P38/Erk(1/2)pathway induced by LPS.Under normal physiological conditions,Orexin-A had no significant effect on the expression levels of neural stem cell differentiation markers Tuj1,GFAP,and Olig2;However,in the inflammatory microenvironment,Orexin-A upregulates the expression level of Tuj1 to promote the differentiation of neural stem cells into neurons.In the inflammatory microenvironment,Orexin-A can up-regulate the expression of β-catenin and TCF-1 in the nucleus,and down-regulate the expression of β-catenin in the cytoplasm.The results suggest that Orexin-A may be involved in the regulation of Wnt/β-catenin in the inflammatory microenvironment Signaling pathways promote neural stem cell differentiation.The SD rat brain injury model was constructed to explore the effect of Orexin-A combined with neural stem cell transplantation on the repair of brain injury in rats.The results showed that: Orexin-A combined with neural stem cell transplantation can reduce the infiltration of inflammatory cells,edema,and vacuolar degeneration in brain tissue;reduce Nissl body disintegration;decreased m NSS score in rats with brain injury;shorten the escape latency of the positioning navigation experiment;increase the time proportion and the number of crossing the water platform in the first quadrant of the space exploration experiment.Conclusion:(1)Orexin-A may reduce the inflammatory response of lipopolysaccharide-induced neural stem cells by regulating the phosphorylation of NF-κB and MAPK/P38/Erk pathways.(2)In the inflammatory microenvironment,Orexin-A may promote the differentiation of neural stem cells into neurons by activating the Wnt/β-catenin pathway.(3)The effect of Orexin-A combined with neural stem cell transplantation in improving neurological and cognitive function in rats with brain injury is better than that of single NSC transplantation and Orexin-A treatment.