Effects Of Estrogen Replacement On Cerebral Ischemia Injury And Its Mechanism In Ovariectomized Animals

【Background】Stroke has become the leading cause of death and disability in the elderly people.Previously,lots of epidemiological investigation and basic studies have confirmed that estrogen has significant neuroprotective effects against stroke.Compared with men of the same age,the incidence of stroke in postmenopausal women was significantly higher and the prognosis was more worse.However,large-scale clinical trials found that estrogen replacement therapy did not reduce the incidence of stroke in menopausal women,and even exacerbated the nerve injury,and increased the risk of breast cancer and uterine fibroids.Therefore,the researchers returned to basic experiments to deeply explore how to effectively exhibit the estrogen’s neuroprotective effects against stoke and the underlying molecular mechanisms,and put forward a series of hypotheses,including dose-dependent hypothesis,critical period hypothesis and receptor selectivity hypothesis.It has been found that estrogen receptors are highly expressed in astrocyte,which is an important target for estrogen’ s neuroprotective effects.Ndrg2 is a newly identified gene by the biochemical department of our school in 2001.Previous studies have found that NDRG2 is specifically expressed in astrocytes in the brain and is involved in the development of neurodegenerative diseases,but its role in cerebral ischemia is still poorly understood.And a previous study has found that ndrg2 is the target gene for estrogen and estrogen receptor beta.However,the role of ndrg2 in the prevention and treatment of stroke by estrogen replacement therapy has not been explored.【Purpose】1.To explore the dose selection effect of estrogen replacement therapy in prevention and treatment of stroke in OVX animals and the mechanisms;2.To explore whether estrogen replacement therapy exist therapeutic time window in prevention and treatment of stroke in OVX animals and the mechanisms;3.To explore whether activation of estrogen receptor β(ERβ)could exhibit neuroprotective effects against stoke in OVX animals and the mechanisms;4.To explore the role of ndrg2 in the prevention and treatment of stroke in OVX animals by estrogen replacement therapy.【Method】1.In vivo: SD female rats,C57 female mice and ndrg2 gene knockout female mice were received ovariectomy(OVX)surgeries to construct female menopause model.Then the OVX animals were administered with different doses of estradiol and ERβ agonist DPN.Use real-time PCR to detect the expression of ndrg2 mRNA.Use Western blot to detect the expression of NDRG2 and GFAP.Ndrg2 Loxp mice were received lateral ventricle injection of adeno-associated virus to selectively down-regulate ndrg2 expression in adult female rats.Then the normal and OVX female animals received replacement treatment were subjected to MCAO and global cerebral ischemia(GCI)models.Then Garcia and Longa scores was used to detect neurological function,RT-PCR was used to detect the expression of ndrg2 mRNA,Western blot was used to detect the expression of NDRG2 and apoptotic protein.TTC,TUNEL,Nissl and Neun staining were used to detect the nerve injury.2.In vitro: PC12 cell line,primary cultured astrocytes,neurons and their co-cultureswere received different doses of estradiol,ERα agonist PPT and ERβ agonist DPN treatment.Phase contrast microscope was used to observe cell morphological changes,MTT assay was used to detect cell viability,LDH test was used to detect cell damage,flow cytometry was used to detect cell cycle,immunofluorescence was used to detect cell proliferation,real time PCR was used to detect the expression of ndrg2 mRNA,Western blot was used to detect the expression of GFAP and NDRG2.The nerve cells were received oxygen and glucose deprivation(OGD)injury.Then the neuronal apoptosis was detected by flow cytometry,the expression of ndrg2 mRNA was detected by RT-PCR,the expression of NDRG2 and apoptotic protein was detected by Western blot and immunofluorescence.【Result】1.(1)Physiological dose(10 nM or 20 nM)estradiol(E2)promoted PC12 cell proliferation,attenuated OGD-induced PC12 cells damage,pharmacological dose(10 μM or 20 μM)E2 inhibited PC12 cell proliferation,aggravates OGD-induced PC12 cells damage;(2)Physiological dose(6 μg/kg and 20 μg/kg)E2 replacement therapy significantly reduced cerebral ischemic injury in OVX female rats,super physiological dose(50 μg/kg)of E2 replacement therapy did not reduce cerebral ischemic injury in OVX female rats.2.(1)The expression of ERα did not change in both short-term ovariectomy(OVX 1w)and long-term ovarian(OVX 10w)female mice.(2)The expression of ERβ and ERα-Ser118 was significantly decreased in OVX 10 w mice,but not in OVX 1 w mice.(3)Physiological low dose(50 μg/kg)or high dose(100 μg/kg)E2 replacement therapy significantly reduced the cerebral ischemic injury in OVX 1w female mice with,but not in OVX 10 w mice.3.(1)In the female mouse brain,ERβ was mainly co-located with astrocyte marker GFAP and the expression of ERβ was significantly higher than that ERα in primary cultured astrocytes.(2)The expression of GFAP was significantly reduced in OVX mice compared with normal female mice,and 50 μg/kg E2 and 8 mg/kg DPN replacementtherapy significantly increased the expression of GFAP in OVX mice.(3)(2.5 nM,5 nM,10 nM,20 nM)E2 up-regulated the expression of GFAP in primary astrocytes in a dose-dependent manner and peaked at 5 nM and 10 nM;and 10 nM DPN significantly up-regulated GFAP expression in primary astrocytes,but 10 nM PPT did not upregulate the expression of GFAP in primary astrocytes;(4)50 μg/kg E2 and 8 mg/kg DPN replacement therapy significantly reduced the cerebral ischemic injury in short-term OVX mice;(5)Astrocytes pretreatment with 10 nM E2 and 10 nM DPN significantly reduced neuronal apoptosis after OGD.4.(1)E2 significantly up-regulated the expression of ndrg2 mRNA and protein in primary astrocytes with time and dose-dependent effect;(2)10 nM DPN significantly up-regulated the expression of ndrg2 mRNA and protein in primary astrocytes,but 10 nM PPT could not;(3)The expression of ndrg2 was significantly decreased in the brain of OVX mice,and 50 μg/kg E2,100 μg/kg E2 and 8 mg/kg DPN replacement therapy significantly increased the expression of ndrg2 mRNA and protein in OVX female mice,but 2 mg/kg PPT could not.(4)The expression of ndrg2 mRNA and protein in the ischemic penumbra was significantly increased at 6 h after MCAO-reperfusion and reached the peak at 24 h and the nuclear translocation occurred;(5)The expression of ndrg2 mRNA and protein in primary astrocytes was significantly increased at 2 h after OGD-reoxygenation and reached the peak at 6 h,and nuclear translocation occurred;(6)ndrg2 gene knockout(ndrg2-/-)female brain showed a more heavier cerebral ischemic injury compared with wild type(ndrg2+/+)mice;and the expression of NDRG2 in the brain of adult female mice was successfully decreased in ndrg2 Loxp female mice injection with adeno-associated virus,which showed a more heavier cerebral ischemic injury compared with ndrg2 Loxp female mice injection with control adeno-associated virus;(7)50 μg/kg E2 or 8 mg/kg DPN replacement therapy significantly reduced cerebral ischemic injury in ndrg2+/+-OVX mice,but 50 μg/kg E2 or 8 mg/kg DPN replacement therapy could not reduce cerebral ischemic injury in the ndrg2-/--OVX mice.【Conclusion】1.Choose physiological dose of estrogen replacement therapy to prevent and treat stroke in OVX animals;2.The prevention and treatment of stroke by estrogen replacement therapy exists "the best therapeutic time window",which is associated with the significantly decrease of ERβ and/or ERα-Ser118 expression in the brain of long-term ovariectomized animals;3.Selective activation of ERβ by DPN replacement therapy(DRT)significantly reduced cerebral ischemic injury in OVX animals by restoring the activity of astrocytes;and without carcinogenic side effects,selective activation of ERβ is expected to become a new strategy for prevention and treatment of stroke in menopause women;4.Astrocytes ndrg2 is the target gene of estrogen and ERβ;5.Ndrg2 is a key molecule that mediates the neuroprotective effects of estrogen and ERβ against cerebral ischemia.