The Effects And Mechanisms Of 6-gingerol On Hypoxic Ischemic Brain Injury In Neonatal Mice

BackgroundHypoxic ischemic encephalopathy(HIE),a form of brain damage caused by hypoxia and ischemia,which is a major cause of neonatal death and disability.Surviving infants usually suffer from neurological sequelae such as mental retardation and motor dysfunction.At present,there is no specific effective treatment besides mild hypothermia therapy.The chief dealing in clinic are symptomatic relief and supportive treatment,which can block hypoxia ischemia primary injury and avoid secondary brain injury.Treatment includes maintaining proper ventilation and oxygenation,maintaining adequate cerebral blood perfusion,maintaining appropriate blood sugar levels and limiting the fluid intake to prevent cerebral edema.These nonspecific treatments produce poor effects.Therefore,it is very important to explore the new effective treatment of HIE.HIE can lead to a series of pathophysiological changes including neural cell apoptosis and necrosis,microglia activation,excitability toxicity,oxidative stress,mitochondrial dysfunction and so on.As the resident immune cell in brain,microglia was activated after HIE and released pro-inflammatory cytokines,causing the secondary death of nerve cells and myelin damage.Myelin damage is the main pathological changes of white matter lesions and white matter lesions are closely associated with long-term exercise and cognitive impairment.Therefore,it is of great significance to alleviate the inflammatory response after HIE,promote the survival of nerve cells and attenuate myelin sheath injury for the recovery of brain injury after HIE.At present,traditional Chinese medicine is widely applied in diseases.As a traditional Chinese medicine,ginger has been found to have valuable functions such as relieving the surface cold,warming the stomach to stop vomiting and warming the lung and relieving cough,which is widely used in clinic.A variety of studies have shown that it has certain therapeutic effects on cardiovascular system diseases,digestive system diseases and central nervous system diseases.6-gingerol is a ginger extract obtained in large quantities in fresh ginger roots and stems and it is one of the most important components of ginger which plays the pharmacological activity.Studies have proved that the toxicity of 6-gingerol is low and it can pass through the blood-brain barrier.It has a wide range of pharmacological effects including anti-tumor,anti-oxidative stress,anti-inflammation,anti-apoptosis and other effects.However,the role of 6-gingerol in neonatal HIE and its molecular mechanism need to be further investigated.ObjectiveTo investigate whether 6-gingerol can rescue the HIE-induced brain damage and improve the neurobehavioral disorder after HIE.Moreover,to explore its mechanisms.Furthermore,we hope that we can provide a new effective method for clinical treatment of HIE.Methods1.Establishment of HIE model7-day-old Kunming mice were selected as the experimental subject and the Rice-Vannucci model was applied as described in the literature.The mice in the experimental group(HIE group)were fixed in supine position after anesthesia and the right common carotid artery was removed for vascular ligation after skin cutting.After ligation,the mice returned to the nest and rested for 1 hour.Then the mice were placed in a 37℃ hypoxia incubator containing 5%carbon dioxide,86%nitrogen and 9%oxygen for 90 minutes for hypoxia treatment.For Sham group,the right common carotid artery was dissected without ligation or hypoxia treatment.2.Experimental grouping7-day-old Kunming mice were randomly divided into three groups:Sham group,HIE group and HIE+6-gingerol group(HIE+6G)group.6-gingerol was intraperitoneally injected 24 hours before HIE operation and was injected 1,2 and 3 days after HIE operation respectively.The Sham group and HIE group were injected with the corresponding volume of sterile normal saline.3.Detection methods3.1 Detection of brain tissue morphologyTTC(2,3,5-triphenyltetrazolium chloride)staining was used to detect the area of cerebral ischemia injury in mice.The effects of 6-gingerol on the brain injury,neurocyte apoptosis,microglia activation,myelin basic protein(MBP)expression,oligodendrocyte differentiation and apoptosis and myelin sheath injury were detected by HE,Nissl and TUNEL staining,IL-1β Iba-1,TNF-α Iba-1,MBP,PDGFR-α Olig2,APC Olig2,active Caspase3 Olig2 immunofluorescence and LFB staining after HIE respectively.Long-term brain atrophy of mice was detected by measuring brain weight.3.2 Detection of molecular level of brain tissueThe expressions of apoptosis-related proteins Bcl-2,Bax and Cleaved PARP1,inflammatory cytokines IL-1β and TNF-α,MBP,histones,and inflammation related signaling pathway proteins were detected by Western Blot.The expressions of inflammatory cytokines IL-1β and TNF-α were detected by qPCR.3.3 Behavioral testsShort-term neural reflexes and motor ability were measured by hind limb suspension,negative geotaxis reflex and righting reflex.The long-term motor function was evaluated by rotarod test and open field test.Results1.6-gingerol alleviated cerebral infarction and brain histomorphological damage after HIEDifferent concentrations of 6-gingerol(1mg/kg,2mg/kg and 4mg/kg)were selected for the experiment.TTC staining results showed that 2mg/kg and 4mg/kg 6-gingerol could reduce cerebral ischemia area to the same extent(P<0.05).Therefore,2mg/kg 6-gingerol was selected as the standard concentration.The results of HE staining showed that there was cell edema and necrosis in the cerebral cortex and hippocampus of mice after HIE.The degree of edema and necrosis was significantly reduced after drug treatment.2.6-gingerol improved the short-term neural reflex after HIEBehavioral test results showed that the time of negative geotaxis reflex and righting reflex were significantly increased in HIE group(P<0.001,P<0.0001,respectively).The reflex time of mice in the HIE+6G group was shorter than that in the HIE group(P<0.05,P<0.01,respectively).In hind limb suspension experiment,the suspension time of HIE group was slightly shorter than that of Sham group(P<0.0001),while the suspension time of 6-gingerol treatment group was improved compared with HIE group(P<0.01).3.6-gingerol alleviated neurocyte apoptosis and regulated the expression of apoptosis-related proteinsNissl staining showed that the cell edema and necrosis in the cortex and hippocampus were severe and decreased Nissl body can be observed after HIE.6-gingerol treatment could reduce the degree of cell injury.Meanwhile,TUNEL immunofluorescence staining was used to evaluate cell apoptosis.Results showed that the number of TUNEL+cells in the cortex of the affected side of mice increased after HIE(P<0.0001),while 6-gingerol decreased the number of TUNEL+cells(P<0.01).Western Blot results showed that after 6-gingerol treatment,the ratio of Bcl-2/Bax increased(P<0.01)and the protein level of Cleaved PARP Ⅰ was decreased(P<0.01),suggesting that 6-gingerol could play a neuroprotective role by anti-apoptosis.4.6-gingerol inhibited the expression of proinflammatory genes and microglia activationqPCR and Western Blot results showed that the mRNA and protein expression levels of pro-inflammatory cytokines TNF-α and IL-1β were significantly decreased after 6-gingerol treatment(P<0.001,P<0.0001,P<0.01,P<0.05,respectively).Immunofluorescence staining results showed that the number of activated microglia(IL-1β Iba-1 and TNF-α Iba-1 positive cells)was significantly reduced after drug treatment(P<0.01,P<0.0001,respectively),indicating that 6-gingerol could significantly inhibit the expression of pro-inflammatory factors and the activation of microglia after HIE.5.6-gingerol attenuated HIE-induced brain atrophyThe brain was weighed 4 weeks after HIE.Results showed that the brain weight of mice in HIE group decreased significantly(P<0.0001),and severe brain atrophy appeared on the injured side.After 6-gingerol treatment,the brain weight increased(P<0.05)and the degree of brain atrophy on the injured side was reduced.HE staining results also showed that 6-gingerol alleviated brain atrophy after HIE.6.6-gingerol improved long-term motor dysfunction after HIEWe evaluated the motor ability of mice by rotarod test and open field test.HIE can significantly reduce the movement distance of mice(P<0.0001),while medication can improve the motor ability after HIE(P<0.01,P<0.001,respectively).7.6-gingerol rescued myelin injury induced by HIELFB staining and MBP immunofluorescence staining showed that 6-gingerol could reduce myelin loss after HIE.Western Blot results showed the protein level of MBP was increased after 6-gingerol treatment(P<0.05).8.6-gingerol stimulated oligodendrocytes maturation and reduced oligodendrocyte death after HIEImmunofluorescence staining showed that the number of oligodendrocyte progenitor cell(PDGFR-α+Olig2+)in the affected side of the corpus callosum was increased(P<0.001)and the number of mature oligodendrocyte(APC+Olig2+)decreased after HIE(P<0.0001).Treatment with 6-gingerol can reduce the number of oligodendrocyte progenitor cell and increase the number of mature oligodendrocyte(P<0.001,P<0.05,respectively),indicating that 6-gingerol improved the maturation of oligodendrocyte after HIE.Compared with the Sham group,the number of active Caspase3+Oligo 2+cells in the affected side of corpus callosum was significantly increased in the HIE group(P<0.0001),while the number of double positive cells in the 6-gingerol group was significantly decreased(P<0.001).9.6-gingerol regulated Akt,ERK and NF-κB signaling pathwaysWestern Blot results showed that the expression of p-Akt in the cerebral cortex of the affected side was decreased in the HIE group(P<0.01),while the phosphorylation level of Akt was increased after 6-gingerol treatment(P<0.05).In addition,the levels of ERK and NF-κB phosphorylation were increased after HIE(P<0.01),which were reversed by 6-gingerol treatment(P<0.01,P<0.05,respectively).10.6-gingerol regulated epigenetic modifications of histonesWestern Blot results showed that histone acetylation in the cerebral cortex of the affected side was decreased at 72h after HIE(P<0.01).However,6-gingerol had no significant effect on histone acetylation.Moreover,results showed that the trimethylation levels of H3K4 and H3K9 did not change significantly at 72h after HIE.Notably,the level of H3K27 trimethylation was increased at 72h after HIE(P<0.05)and 6-gingerol decreased the level of H3K27 trimethylation after HIE(P<0.05).Conclusion6-gingerol exerted protective effects on HIE by reversing the nerve cells apoptosis,inhibiting the release of inflammatory cytokines and inhibit the activation of microglia cells.It can improve the short-term behavioral reflex and long-term brain atrophy of mice after HIE.Moreover,it can promote the maturation of oligodendrocytes,reduce the apoptosis of oligodendrocytes and alleviate myelin damage,through which it contributed the improvement of motor ability.Furthermore,6-gingerol played a neuroprotective role by regulating Akt,ERK and NF-κB signaling pathways and H3K27 trimethylation levels.Therefore,6-gingerol can be considered as an effective candidate for neuroprotective treatment of HIE.