Ginsenoside Rd Improves Brain Injury Through Bdnf By Histone Acetylation Modification In Mice Of Chronic Cerebral Hypoperfusion

Background Sufficient cerebral blood flow is critical to maintain the normal brain functions,while,chronic cerebral hypoperfusion(CCH),characterized as continuing decrease of cerebral blood flow,acts as a common pathological factor in the development of various cerebrovascular diseases and leads to cognitive impairment.However,it is still not entirely clear the pathogenesis of cognitive impairment caused by CCH,moreover,there are no effective drugs and therapy available so far.Therefore,it is still an important issue needed to be solved urgently to look for prevention and treatment strategy for CCH treatment.The pathogenesis of cognitive dysfunction induced by CCH is complex,and the main pathological process includes ischemia-mediated loss of cellular energy,cerebral white matter damage,blood-brain barrier damage,dysfunctions of lipid metabolism and neurovascular unit,as well as cell necrosis/apoptosis.As the smallest functional unit of CNS,neurons in certain number with normal function are the basis of learning and memory.A series of pathophysiological changes including apoptosis and necrosis happened in CCH,lead to the damage of brain function,such as motor dysfunction,cognitive decline and so on.CCH induces neuronal damage especially in the prefrontal cortex(PFC)and hippocampus(Hipp),the most noted brain regions.The two regions are the central brain areas involved in learning and memory,and PFC and Hipp are very sensitive to ischemia and hypoxia,therefore,chronic ischemia leads to the damage of neuronal function and structure of the two regions;the abnormal structures are associated with the ability of learning and memory,thereby causing a decline in the ability of learning and memory,and even dementia.It is generally believed that the damage of neurons in PFC and Hipp is the morphological basis of cognitive impairment in CCH.Therefore,it is important for ischemia therapy to promote neurons survival and supplement of lost neurons.Traditional Chinese medicine has been concentrating on the research and development of drugs and methods for CCH prevention and treatment,and Ginseng(Radix)is one of the commonly used drugs.Ginseng,belongs to Araliaceae family,is commonly used as an alternative or complementary medicine as functional food,and also used in anti-cancer,anti-diabetic and anti-inflammatory treatments;moreover,Ginseng exerts neuroprotection in CNS diseases.Ginsenoside Rd,one of the main active components in Ginseng,has been evidenced exerting protective effects on cerebral ischemia and neurodegenerative diseases.GSRd can prevent memory loss and improve the spatial learning ability of mice,but the underlying mechanisms are still unclear.Studies showed that the neuroprotective effects of GSRd are closely associated with the production of brain-derived neurotrophic factor(BDNF).As an important member of the neurotrophin family,BDNF plays key roles in neural plasticity,neurogenesis and neuronal survival,and it is often released upon hypoxia or ischemia insults to protect the brain from injury.The effects of GSRd including memory improvement and neuroprotection,are mainly due to the upregulation of BDNF,while,the upstream mechanisms of BDNF regulation are still unclear. A variety of mechanisms are involved in the regulation of the expression of target proteins,and the epigenetic modification is considered to be a common upstream regulatory mechanism.Epigenetic modification includes DNA methylation,histone acetylation,chromatin remodeling and non-coding RNA regulation,affecting gene expression,regulating gene transcription,regulating cell cycle and so on.Histone acetylation modification was found to be involved in learning and memory formation.Regulation of histone acetylation is a highly dynamic,mainly through two kinds of enzymes,namely histone deacetylases(HDAC)and histone acetyltransferase(HAT).In general,HDAC inhibited the expression of genes,while HAT promoted genes expression.It is not clear whether epigenetic modification is involved in the regulation of BDNF expression by GSRd in CCH treatment.Objective CCH mice were used to investigate the molecular mechanisms of GSRd by increasing BDNF production and improving the survival of neurons,which is used to treat cognitive dysfunction induced by CCH.Further,in vitro cell culture system of OGD which mimics pathological state of CCH,was used to explore the epigenetic modification of BDNF upregulation and promoted neuronal survival mediated by GSRd,thus improved learning and memory impairment caused by CCH.This study will provide new ideas and theoretical basis for clinical treatment for CCH by GSRd.Methods 1.CCH mice model was established by bilateral carotid artery stenosis(BCAS)using microcoils;Morris water maze was used to measure the ability of mice learning and memory,to evaluate whether CCH led to the cognitive impairment of mice,and whether administration of GSRd for 21 days improved cognitive dysfunction induced by CCH.2.Hematoxylin-Eosin(HE)staining was used to observe the changes of neurons morphology and survival in mice prefrontal cortex(PFC)and Hippocampal CA1 regions with or without GSRd treatment;the expression changes of Cleaved Caspase-3 in apoptosis signaling pathway in CCH mouse hippocampus were detected by Western blot,and the expression levels of Cleaved Caspase-3 were further checked after GSRd administration.3.To explore the potential mechanisms of GSRd in neuroprotection,the changes of BDNF expression in mice from each group were determined by quantitative Real-time PCR(q PCR)and enzyme linked immunosorbent assay(ELISA)methods.4.Oxygen and glucose deprivation(OGD)model in cultured hippocampal neur ons in vitro was established to mimic CCH model,neuron survival and apoptosis were detected by methylthiazolyldiphenyl-tetrazolium bromide(MTT),lactate dehydrogenase(LDH)release,as well as flow cytometry(FCM)methods;the effects of GSRd treatment on cell survival and apoptosis were further checked using the same methods;the expression changes of Caspase-3 in apoptosis signaling pathway were detected by Western blot to evaluate the neuroprotective effects of GSRd.5.The expression changes of BDNF in cultured neurons upon OGD injury with or without GSRd treatment were detected by q PCR and ELISA methods.6.To explore the upstream mechanisms of epgenetic modification in BDNF expression,the expression levels of p300/CBP,acetylation of histone H3(Ac-H3)and HDAC2 in hippocampus from each group with or without GSRd treatment were checked using Western blot.7.OGD model of cultured neurons in vitro was further used to confirm the effects of GSRd on the expression of Ac-H3 and HDAC2,and to analyze the upstream regulation of BDNF expression.8.Using chromatin immunoprecipitation(Ch IP),we observed whether GSRd-mediated increase of BDNF expression was through promoting the binding of Ac-H3 with promoter IV of BDNF,or reducing that of HDAC2;further,we observed whether cultured neurons pretreated with MS-275,an HDAC inhibitor,can enhance the above effects,and thus affected the expression of BDNF.Results 1.The data from Morris water maze test showed that CCH by BCAS led to mice learning and memory dysfunction compared with sham group,characterized as extend latency to find platform,shortened swimming distance in target quadrant,decreased numbers of crossing platform(P < 0.01);while,GSRd administration for 21 days significantly improved the performance of learning and memory dysfunction in CCH mice compared with CCH models(P < 0.05 or P < 0.01).2.HE staining results indicated that neuron configuration in PFC and CA1 of CCH mice was not clear,nuclear pyknosis showed up in cells as well as a large number of apoptotic cells in these regions(P < 0.01);the neuron configuration was recovered similar to normal after GSRd administration,and the percent of apoptotic cells decreased compared with CCH models;the expression of Cleaved Caspase-3 increased in CCH hippocampus,while GSRd administration decreased Cleaved Caspase-3 expression in CCH mice(P < 0.05 or P < 0.01).3.The expression levels of both m RNA and protein of BDNF decreased in CCH hippocampus compared with sham group by q PCR and ELISA(P < 0.01);however,all these changes were reversed after GSRd treatment in CCH mice compared with CCH models treated with vehicle.4.OGD injury led to neuron apoptosis and decreased neuron survival by MTT,LDH and FCM methods(P < 0.01),GSRd promoted cell survival in a concentration-dependent manner which suffered from OGD insult(P < 0.05 or P < 0.01);at the same time,GSRd treatment reduced the increased expression of Cleaved Caspase-3 under OGD,these results indicated that GSRd exerted neuroprotection in vitro also.5.Using OGD model in vitro,we also observed and confirmed that GSRd increased BDNF expression in cutured neurons upon OGD insults(P < 0.05 or P < 0.01),and this was consistent with what observed in vivo,this result suggested that GSRd may exert neuroprotection by increasing the expression of BDNF.6.The upstream mechanisms of epigenetic modification in BDNF expression were determined by Western blot analysis,and the result showed that p300/CBP and Ac-H3 levels in CCH hippocampus decreased significantly compared with sham group(P < 0.01),while the expression levels of HDAC2 increased after CCH injury(P < 0.01);GSRd treatment reversed all the changes above,which were the upstream proteins regulating BDNF expression.7.The epigenetic modification of BDNF expression were further determined by OGD model in vitro(P < 0.01),and the result showed that the expression levels of p300/CBP and Ac-H3 decreased,while HDAC2 expression increased significantly under OGD injury(P < 0.01);GSRd treatment reversed all the changes above.8.Ch IP results showed that the binding of Ac-H3 with promoter IV of BDNF decreased while that of HDAC increased upon OGD insult,thus resulted in the decreased expression of BDNF;GSRd-mediated increase of BDNF expression was through promoting the binding of Ac-H3 with promoter IV of BDNF,or reducing that of HDAC2;further,we observed that the above effects were enhanced in cultured neurons pretreated with MS-275,an HDAC inhibitor.Therefore,the data indicated that the expression of BDNF was regulated by epigenetic modification upon OGD injury.Conclusion 1.GSRd administration improved mice learning and memory deficits induced by CCH,and reduced neuron apoptosis in PFC and Hipp of CCH mice,and the effects of GSRd were associated with increased BDNF expression in CCH hippocampus;the expression of BDNF was regulated by P300/CBP,Ac-H3 and HDAC2 of epigenetic modification.Therefore,GSRd regulated the expression of BDNF through epigenetic mechanisms,and protected neurons from CCH damage,thus improved the ability of spatial learning and memory.2.Exposure to OGD/R injury led to loss of neuronal activity,and GSRd had neuroprotective effects on cultured neurons.GSRd decreased the OGD/R-induced loss of cell viability and LDH release by upregulating the expression of BDNF,which was consistent with the morphological analysis and Caspase-3 expression related to apoptosis.OGD/R injury increased HDAC2 expression and reduced Ac-H3 expression,which were accompanied by decreased expression of BDNF;GSRd reversed these changes above,suggesting that GSRd promoted neuronal survival through the reconstruction of Ac-H3 and HDAC2 balance which were involved in BDNF regulation.3.BDNF promoter IV was regulated by epigenetic mechanisms,and MS-275,an HDAC inhibitor,had a synergistic effect with GSRd in CCH treatment.All data collected indicated that,GSRd provided neuroprotection by regulating BDNF expression through epigenetic mechanisms in CCH mice.We found that GSRd could improve cognitive dysfunction in CCH mice,and BDNF mediated the neuroprotective effects of GSRd both in vivo(CCH)and in vitro(OGD/R).The molecular mechanism of GSRd neuroprotection was the upregulation of BDNF by epigenetic modification.However,we cannot exclude that GSRd may exert neuroprotective effects through other ways.Epigenetic regulation may be a potential target for therapeutic intervention in CCH related diseases.Epigenetic regulation may be a representative of neuroprotective pathway and a potential target for therapeutic intervention in CCH related diseases.