Study On The Mechanism Of Recombinant Human Nerve Growth Factor’s Protective Effect On Brain Injury

Ischemic stroke is one of the main causes of human death and disability,and it causes a serious medical and social problem.Ischemic stroke usually occurs when the blood vessels supplying blood to the brain are blocked.Ischemia triggers an ischemic cascade reaction and induces a series of molecular events,including energy failure,ion imbalance,excitotoxicity,oxidative stress,cell death（apoptosis or necrosis）and neuroinflammation.This cascade reaction eventually leads to irreversible brain damage.At present,many strategies have been used for treatment of ischemic brain injury,mainly thrombolytic therapy and neuroprotective agent intervention.Thrombolytic therapy with tissue-type plasminogen activator（t-PA）is the most effective treatment for ischemic stroke,but its therapeutic window is narrow（≤4.5 h）and it is accompanied by serious adverse reactions such as bleeding risk and angioedema.Most neuroprotective agents also have some defects that can not be ignored,such as low solubility,short half-life and poor permeability of blood-brain barrier in vivo.Therefore,it is necessary to find safer and more effective new drugs to treat ischemic stroke.Nerve growth factor（NGF）is a kind of neurotrophic factor,and its efficacy in related parts of nerve injury has been widely and deeply studied and recognized.NGF has been proved to promote the survival,differentiation and maturation of neurons,and it also plays an important role in regulating the bidirectional signal network between nervous system and immune system to maintain the steady state of internal environment.Some clinical studies have focused on repairing nerve injuries in the Peripheral Nervous System（PNS）and Central Nervous System（CNS）,including peripheral neuropathy,optic nerve injury,ischemic stroke and neurodegenerative diseases.Recombinant human nerve growth factor（rhNGF）is the recombinant protein of NGF,whose biological function is closest to that of natural protein molecule,which can promote nerve regeneration and provide nutritional support for nerve repair after injury.The rhNGF expressed by CHO cells has high activity,low immune risk and broad market prospect.In the study of animal models of ischemic brain injury,it was found that NGF was delivered to the brain by intraventricular in situ injection,nasal drip or viral vector loading,which improved the neurological function to some extent.However,the neuroprotective effect of rhNGF in ischemic brain injury and its mechanism are not completely clear.In this study,rhNGF played protective efficacy on rat middle cerebral artery occlusion（MCAO）ischemia model,and the neuroprotective mechanism of rhNGF was further explored.The main contents and results are as follows:1.The therapeutic effect of rhNGF on ischemic stroke rat model.A rat model of ischemic stroke was established by MCAO for 2 h in male SD rats.Two groups（Vehicle and rhNGF）were set up,and 200μg·m L^-1 rhNGF was given by tail vein injection once a day for 3 days for rhNGF group.The neurological function was evaluated by behavioral tests,and the infarct volume was measured by TTC staining.The volume of cerebral infarction in MCAO rats in rhNGF group significantly decsrease,and the neurological function of some rats partly improved,suggesting that rhNGF can be used to treat ischemic stroke caused by MCAO by tail vein injection.2.Study on the regulatory effect of rhNGF on neuroinflammation.Firstly,the regulation of rhNGF on neuroinflammatory reaction was explored.The BV2 microglia was induced by LPS to establish a chronic inflammatory model.The cytotoxicity of LPS and the effect of rhNGF on cell viability were detected by CCK-8 assay.It was found that 1,10 and 100μg m L^-1 LPS reduced cell viability on a dose-dependent maner,with 1μg m L^-1 LPS could already cause cell damage,while rhNGF could significantly increase cell viability.Next,the relative gene expression level of inflammatory cytokines was detected by real-time fluorescence quantitative PCR,the protein expression of inflammatory cytokines was detected by CBA,the polarization state of BV2 microglia was detected by immunofluorescence,and the related pathways mediating the anti-inflammatory effect of rhNGF were detected by Western Blot.The results showed that in BV2 inflammation model,rhNGF significantly reduced the gene and protein expression of pro-inflammatory factors IL-1β,IL-6 and TNF-αin overactivated BV2 cells,and promoted the gene expression of anti-inflammatory factors IL-4 and IL-10.Therefore,rhNGF inhibited LPS-induced pro-inflammatory reaction of BV2 microglia and played an anti-inflammatory role.At the same time,based on the plasticity of microglia,we observed that rhNGF can induce BV2 microglia to change from pro-inflammatory M1 phenotype to anti-inflammatory M2 phenotype,thus regulating the inflammatory response.The results indicated that the regulation of rhNGF on microglia phenotypic transformation may be part of its anti-inflammatory mechanism.Therefore,rhNGF can play a neuroprotective role by regulating the polarization state of M1/M2,and inducing microglial inflammation to transform into a direction conducive to pathological development.In addition,NFκB was activated after LPS activating,while the phosphorylation level of NFκB was decreased after rhNGF intervention,suggesting that NFκB pathway may mediate the role of rhNGF in regulating inflammatory response.3.Study on the effect of rhNGF on neuronal apoptosis.In order to study the protective effect of rhNGF on neurons,a model of hypoxia injury induced by Na₂S₂O₄was established by mouse neuroblastoma N2a cell line.The viability of N2a cells was detected by CCK-8 assay,and it was found that 2.5 mmol·L^-1 Na₂S₂O₄could reduce the viability of N2a cells to 60%,which could be used to evaluate the neuroprotective effect in vitro.At the same time,N2a neurons were co-cultured with BV2 microglia and their conditioned medium to simulate the injury of neurons in inflammatory state.Tunel co-staining with immunofluorescence and flow cytometry were used to detect the apoptosis of neurons induced by inflammation or hypoxia.The results showed that both Na₂S₂O₄and microglial inflammation could cause the apoptosis of N2a neurons.Microglial cells activated by LPS and their cultured substances were more harmful to neurons than LPS,while rhNGF could significantly reduce the apoptosis rate of neurons induced by inflammation or hypoxia.Western Blot was used to detect whether PI3K signaling pathway mediated the anti-apoptosis effect of rhNGF.The results showed that rhNGF could activate PI3K protein and inhibit the activity of caspase-3,a pro-apoptotic protein.Therefore,rhNGF can inhibit neuronal apoptosis induced by hypoxia or inflammation,thus playing a neuroprotective role,and PI3K signaling pathway can mediate its apoptosis inhibition.Conclusion:rhNGF can reduce infarct volume and improve nerve functions in rat MCAO model.In BV2 cell inflammation model,rhNGF play an anti-inflammatory role by adjusting the polarization state of microglia,and inhibit the apoptosis of neurons mediated by microglia inflammation.At the same time,rhNGF can also inhibit the apoptosis of neurons during hypoxia.Rh NGF can regulate the inflammatory response by inhibiting NFκB pathway and then the expression of inflammatory factors downstream,and can also activate PI3K signaling pathway,inhibit Caspase-3 activity downstream,and play a neuroprotective role in inhibiting apoptosis.In a word,rhNGF plays a neuroprotective role by inhibiting neuroinflammation and apoptosis,which provides a new direction for the treatment of ischemic brain injury.