| Objective:Traumatic brain injury(TBI)is an important disease with high mortality and disability.It significantly affects human health.According to the statistics,TBI is the main cause of death and disability among people under 45 years old.Because the affected population is mainly young and middle-aged people,it has caused greater economic burden to individuals,families and society.At present,TBI lacks effective treatment methods and it is very difficult to improve the prognosis.To find effective neuroprotective agents has become an urgent medical problem.As a new type of medical gas molecule,hydrogen has been used to treat many diseases and achieve satisfactory results.There are many obvious advantages in hydrogen for medical purposes:(1)Hydrogen selectively scavenges oxygen free radicals,and does not react with reactive oxygen species which have important signaling function.Hydrogen also does not destroy the endogenous antioxidant defense system.(2)Hydrogen can easily penetrate the blood-brain barrier and subcellular structure.(3)Hydrogen can combine with hydroxyl radicals to produce water,which is safe and non-toxic.Excess hydrogen can also be expelled from the body through breathing.miR-21 plays an important role in the development of TBI.Previous studies by our team found that miR-21 is the only microRNA that has more than twice increase in the expression level at 6h,24 h,48h and 72 h after trauma.Previous studies also confirmed that the expression of miR-21 increased after TBI in neurons,astrocytes,microglia and brain microvascular endothelial cells.Furthermore,up-regulation of miR-21 in brain tissue can improve learning,memory and motor function.In addition,miR-21 can inhibit cell apoptosis,reduce blood-brain barrier leakage and promote angiogenesis and repair.More importantly,hydrogen can affect the expression of miR-21 in brain tissue.The purpose of this study was to confirm the therapeutic effects of hydrogen on neurological impairment,vestibular dysfunction,volume of traumatic focus,blood-brain barrier leakage and brain edema after TBI.The study further explored the effects of hydrogen on neurite regeneration,neuronal apoptosis and oxidative stress after TBI Finally,the study tried to reveal whether miR-21 participates in and regulates the neuroprotective mechanism of hydrogen on TBI,and to provide new findings about therapeutic mechanism of hydrogen.Methods:The part one explored the neuroprotective effects of hydrogen in an animal model of TBI.TBI model was established by CCI.Some TBI rats were treated with hydrogen inhalation.Modified Neurological Defect Score(mNSS)was used to evaluate the effects of hydrogen therapy on neurological and behavioral deficits in TBI rats.Balance beam test was used to evaluate the effects of hydrogen therapy on balance ability and vestibular function in TBI rats.Cresol violet staining combined with image analysis technique was used to determine the volume percentage of brain injury,which was used to evaluate the effects of hydrogen therapy on TBI focus.The study detected the water content in brain tissue of TBI rats,which was used to evaluate the effect of hydrogen treatment on brain edema.EB exudation was measured to evaluate the effect of hydrogen treatment on blood-brain barrier leakage in TBI rats.The study also detected the activity of antioxidant enzymes SOD and CAT and the levels of oxidant products MDA and 8-iso-PGF2 alpha,which was used to evaluate the effect of hydrogen treatment on oxidative stress in TBI rats.The part two explored whether miR-21 is involved in the regulation of neuroprotective effects of hydrogen on TBI animal models.TBI rat models were established by CCI.Some TBI rats were treated with hydrogen inhalation,and some TBI rats were treated with miR-21 antagomir carried by liposome through lateral ventricle injection.The expression of miR-21 was detected by qRT-PCR from 0 to 3days after TBI.The methods mentioned above were used to evaluate the neurological impairment,balance ability,vestibular function,volume of brain injury,degree of brain edema,blood-brain barrier leakage and oxidative stress.These findings were adopted to evaluate whether miR-21 antagomir affects the protective effect of hydrogen on TBI.The part three explored the neuroprotective effect of hydrogen-rich solution in a cell model of TBI.TBI model of primary neuron cells was made by scratch cell method.Some neurons were intervened by hydrogen-rich solution.MTT method was used to analyze the effect of hydrogen-rich solution on the viability of TBI cell model.LDH method was used to analyze the effect of hydrogen-rich solution on the damage level of TBI cell model.The neurite branches and neurite length were quantitatively analyzed using immunofluorescence staining of neuron-specific marker beta-III-Tubulin,which was used to evaluate the effect of hydrogen-rich solution on TBI cell model.The expression of apoptosis-related proteins was measured using Western blot,and apoptotic rate of neurons was measured using TUNEL staining,which was used to evaluate the inhibitory effect of hydrogen-rich solution on the apoptosis of TBI cells.The activities of antioxidant enzymes SOD and CAT and the levels of oxidant products MDA were detected to evaluate the inhibitory effect of hydrogen-rich solution on oxidative stress of TBI cells.The part three explored whether miR-21 participates in the regulation of neuroprotective effect of hydrogen-rich solution on TBI cell model.TBI models of PC12 neurons were made by scratch cell method.Some TBI cells were interfered by hydrogen-rich solution,and some TBI cell were interfered by miR-21 antagomir.QRT-PCR was used to detect the expression of miR-21.The cell viability,cell damage level,neurite repair,apoptosis and oxidative stress of PC12 neurons were evaluated using the methods mentioned above.These findings were adopted to evaluate whether antagomir could affect the protective effect of hydrogen-rich solution on TBI.Results:In animal experiments,(1)hydrogen can improve the neurological deficits after TBI;(2)hydrogen can improve the balance ability and vestibular function of TBI rats;(3)hydrogen can significantly reduce the volume percentage of brain injury;(4)hydrogen can reduce the brain edema and promote the blood-brain barrier leakage after TBI;(5)hydrogen can inhibit the oxidative stress after TBI.In animal experiments,(1)hydrogen can up-regulate the expression of miR-21 inTBI rats,and miR-21 antagomir can inhibit the expression of miR-21;(2)antagomir can attenuate the protective effect of hydrogen on neurological deficits.(3)antagomir can attenuate the protective effect of hydrogen on balance and vestibular function.(4)antagomir can attenuate the protective effect of hydrogen on the volume of brain injury.(5)antagomir can attenuate the protective effect of hydrogen on brain edema and blood-brain barrier leakage.(6)antagomir can attenuate the protective effect of hydrogen on oxidative stress.In cell experiments,(1)hydrogen-enriched solution can improve the decrease of primary neuronal cell viability;(2)hydrogen-enriched solution can improve the level of cell damage;(3)hydrogen-enriched solution can increase the number of neuritis and Lengthen the length of neurites;(4)hydrogen-enriched solution can inhibit the neuronal apoptosis;(5)hydrogen-enriched solution can inhibit the oxidative stress.In cell experiments,(1)hydrogen-rich solution can increase the expression of miR-21 in PC12 cell model,which can be antagonized by miR-21 antagomir;(2)miR-21 antagomir can attenuate the protective effect of hydrogen-rich solution on the viability of PC12 neurons;(3)miR-21 antagomir can attenuate the protective effect of hydrogen-rich solution on PC12 neurons;(4)miR-21 antagomir can attenuate the protective effect of hydrogen-rich solution on the regeneration of neuronal processes in PC12 neurons;(5)miR-21 antagomir can attenuate the protective effect of hydrogen-rich solution on apoptosis of PC12 neurons;(6)miR-21 antagomir can attenuate the protective effect of hydrogen-rich solution on oxidative stress in PC12 neurons.Conclusion:Hydrogen has significant neuroprotective effects on TBI model at both cell and animal levels.It can significantly improve nerve defect and balance function,reduce the volume of traumatic focus,inhibit brain edema and blood-brain barrier leakage,improve the viability and damage level of neurons,promote the regeneration of injured neurites,inhibit oxidative stress and cell apoptosis.MiR-21 participates in the regulation of the protective effect of hydrogen on TBI.Hydrogen can protect TBI by regulating the expression level of miR-21 in cell andanimal experiments.Hydrogen can be used as a potential therapy for secondary brain injury of TBI.This study lays a foundation for further research on the mechanism of hydrogen molecular therapy. |
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