| Objective:Hypoxic ischemic encephalopathy(HIE)is a kind of brain lesion caused by various kinds of cerebral ischemia and hypoxia,and the most common is neonatal hypoxic ischemic encephalopathy.But it can also be found in any other age groups.Perinatal asphyxia leads to partial or complete anoxia of brain tissue in fetus or newborn.This phenomenon leads to fetal or neonatal brain tissue damage resulting in hypoxic ischemic encephalopathy.This is a common complication of perinatal asphyxia.Clinically,it often shows the sequelae of nervous system,such as children’s intelligence and behavior disorder,and severe cases can even lead to neonatal death.So far,the pathogenesis of neonatal HIE has not been fully elucidated.The present studys suggest that the occurrence of hypoxic ischemic brain damage(HIBD)is a multifactorial process involved in the synthesis of the pathological process.Studies have shown that thedefined pathophysiology includes energy depletion,oxygen free radicals,Ca2+ excess influx,excitatory aminoacids,and so on.These factors interact with each other,which can induce the apoptosis of brain cells,leading to the destruction of brain tissue and even death.The complexity of its pathophysiological mechanism determines that the treatment of HIE is a very complex process,and there is still lack of specific therapy.How to carry out regular and effective treatment and intervention as soon as possible has become a hot topic.In recent years,many natural products have become a research hotspot because of their neuroprotective effects.Through the research of stachydrine inhibition of histone deacetylase activity(HDAC)of stachydrine in improving cerebral hypoxia ischemia injury in neonatal rats(HIBD)the role and the possible mechanism,so as to provide a theoretical basis and data reference for the clinical diagnosis and treatment of HIE.In this study,through the effect of stachydrine inhibition of histone deacetylase activity(HDAC),we investigate the role and the possible mechanism of stachydrine in improving cerebral hypoxia ischemia injury(HIBD)in neonatal rats,so as to provide a theoretical basis and data reference for the clinical diagnosis and treatment of HIE.Methods:1.Creating animal modelThe 7 days old(P7)Sprague-Dawley(SD)rats in a total of 112,weight(12-16)g,male and female,according to Rice’s method of producing hypoxic ischemic brain damage(HIBD)model,were randomly divided into 4 groups:1)Normal control group(Sham group,n=12):In sham operation group,normal rats,only the left common carotid artery was separated,the ligature was not connected,and the mixed gas was not carried out;2)Negative control group(Con group,n=20):Rats with hypoxic ischemic brain damage were treated only with saline;3)Low dose stachydrine treated group(L group,n=40):Rats with hypoxic ischemic brain damage were given intraperitoneal injection of stachydrine,5mg/kg/d;4)High dose stachydrine treated group(H group,n=40):Rats with hypoxic ischemic brain damage were given intraperitoneal injection of stachydrine,10mg/kg/d.All rats were given a 5 days treatment.2.Isolating neonatal rat brain tissue specimensAfter 12 hours of treatment(P12),the weight and temperature of each neonatal rat were evaluated.After using phenobarbital to anesthetize all of the neonatal rats,the brain tissue was taken after intracardiac perfusion.The isolated brain separated in to two groups and one of them was stored into frozen liquid nitrogen at-70 ℃ for the estimation of protein.Other side of the isolated hemicerebrum was applied to TUNEL staining.3.Evaluation of relevant indicatorsRelated indexes of brain tissue in all neonatal rats,such as infract volume,apoptosis,HDAC activity and biochemical analysis,were detected or evaluated.Specifically as follows:1)Estimation of infract volume:Infracted volume was estimated by using 2,3,5-Triphenyl Tetrazolium Chloride(TTC)in saline solution for the period of 15 min.Analysis of Images of brain sections was achieved by using computerized image analyzer.Infracted area of slices of the brain of same hemisphere added together to get total infract volume.2)Evaluation of apoptosis:The TUNEL staining method was used,and the apoptosis positive cells showed brown yellow.The paraffin sections of brain tissue were observed in high power field.Three high power fields were randomly selected,and the percentages of TUNEL positive cortical cells in total cortical cells were calculated.This method was used to evaluate apoptosis(expressed as%);3)Estimation of HDAC activity:Colorimetric detection was used for the estimation of HDAC activity.The absorbance at 405 nm was observed and the HDAC activity was expressed in terms of the%of control group;4)Estimation of acetylated H3 and H4 expression:Effect of stachydrine on expression of acetylated H3 and H4 was estimated by using westernblot analysis,it was expressed in terms of the%of control group;5)Estimation of biochemical analysis:The supernatant of tissue homogenate was used for the estimation of cytokinins by specific enzyme-linked immunosorbent assay(ELISA)kits and automatic biochemical analyzer.These cytokines include superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px).4.Statistical analysisAll statistical data were analyzed using SPSS 18.0 software.The measurement data was expressed as mean ± SD,and the count data was expressed in numerical and percentage.Statistical analysis of data was done by t-test,one-way ANOVA,or LSD post.hoc analysis.p<0.05 was considered significant statistically.Results:1.Effect of stachydrine on Infract volumeThe infract volume(224 mm2)of brain tissue in Con group was significantly higher than that in Sham group(20 mm2),and the difference was statistically significant(p<0.01).It was observed that hypoxia condition results in increased infract volume.The infract volume in the group of treatment with stachydrine(L group:89 mm2;H group:53 mm2)was significantly lower than that in Con group(p<0.01).However the infract volume at subcortex region remain unchanged in all the animals of cerebral ischemia induced injury in neonatal rat model(p>0.05).2.Effect of stachydrine on apoptosis in cerebral ischemic nenonatal ratsThe apoptosis rate of the brain tissues in Con group was 23.8%,and that was 3.5%in Sham group.The difference was statistically significant(p<0.01).These results indicated that the apoptosis of brain cells was significantly increased in hypoxic neonatal rats Whereas,staining were found to be faint or less visible in the group treated with stachydrine(L group:11.4%;H group:5.6%)compared to Con group,and the difference was statistically significant(p<0.01).3.Effect of Stachydrine on HDAC activityThe HD AC activity was about 238%in Con group.And it was decreased to 179%in L group after the treatment with stachydrine,while decreased in H group to more than 136%.It was observed that stachydrine decreases the activity of HDAC enzyme in dose dependent manner in the brain tissues of ischemia induced injury in nenonatal rats.The difference was statistically significant(p<0.01).Moreover the effect of stachydrine on the expression of acetylated H3 and H4 protein in the brain tissues of ischemic neonatal rats was determines in this study.There were significant(p<0.01)increases in the expression of acetylated H3 and 4 proteins with the stachydrine treatment in cerebral tissues of ischemic neonatal rats compared to negative control group.4.Estimation of Cytokines in the brain tissuesThe level of TNF-a(169 pg/100 mg)in Con group was significantly higher than that in Sham group(48 pg/100 mg),and the difference was statistically significant(p<0.01).The level of TNF-a in the group of treatment with stachydrine(L group:98 pg/100 mg;H group:71 pg/100 mg)was significantly lower than that in Con group(p<0.01).Moreover the level of IL-10 found to be increased significantly(p<0.05,p<0.01)in stachydrine treated group of negative control group.Effect of satchydrine on the TNF-a and IL-10 were found to be in a dose dependent manner(p<0.01,(p<0.01).5.Estimation of oxidative stress in the brain tissuesMarkers of oxidative stress parameters such as SOD and GSH in the brain tissues of hypoxia induced cerebral injured neonatal rats were found to be decreased compared to control group of rats.In Sham group,the level of SOD was 2.67±0.23 U/100 mg,and the level of GSH-PX was 192.7±8.3 U/100 mg.In Con group,they were 0.79±0.12 U/100 mg and 96.8±5.9 U/100 mg,the difference was statistically significant(p<0.01).It was observed that treatment with the stachydrine significantly increases(p<0.01)in the SOD and GSH-PX level in the brain tissues.The level of SOD was 1.73±0.19 U/100 mg in L group,and it was 2.18±0.20 U/100 mg in H group.The level of GSH-PX was 130.6±7.2 U/100 mg in L group,and it was 169.2±8.1 U/100 mg in H group.Conclusions:1.Hypoxia can lead to cerebral infarction in neonatal rats,and the volume ofcerebral infarction was significantly reduced after the treatment with stachydrine.This shows that the neuroprotective effect of stachydrine.2.By reducing the apoptosis rate of the brain cells in the neonatal rats,stachydrine can improve the brain damage induced by hypoxia ischemia.3.Stachydrine can decrease the activity of HDAC enzyme in brain tissue of HIBD rats,and it is suggested that this may be the mechanism of its action.Furthermore,it was found that the inhibitory effect of Stachydrine on HDAC activity was dose dependent.Moreover the effect of stachydrine on the expression of acetylated H3 and H4 protein in the brain tissues of ischemic neonatal rats was determines in this study.4.Stachydrine ameliorates the hopoxia induced cerebral injury by significant reduction in the level of TNF-a,and can significantly increase the level of IL-10 in the brain tissues which confirms the brain injury induced by cerebral ischemia.To some extent,it can reduce the damage of cytokines to cells and the inhibition of mononuclear phagocyte function.Similarly,we found that the effect of satchydrine on the TNFa and IL-10 were to be in a dose dependent manner.This finding suggests that stachydrine can protect brain tissue from further damage.5.Stachydrine can significantly increases the SOD and GSH-PX level in the brain tissues,and can protect brain cells from endogenous superoxide anion.It may be one of the mechanisms of improving brain injury.In this study,we confirmed the neuroprotective effect of stachydrine in HIBD rats,which can significantly improve the degree of brain injury.This study provided a good theoretical basis for the treatment of HIE.We further found that the mechanism of neuroprotection may be to inhibit the activity of HDAC and decrease the oxidative stress and inflammatory reaction. |
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