| Estrogen and its receptor GPR30 have been revealed to exert neuroprotective effects against cerebral stroke and neurodegenerative diseases.However,their roles and mechanisms following ischemic stroke after long-term E2 eprivation(LTED)were still not completely elucidated.Some scholars suggested that its possible mechanisms included the decline of ER? leading to hippocampal-specific insensitivity to E2,neurons began to switch their preferred energy source from glucose to ketones leading to an overall decreased amount of available ATP and the activity of choline acetyltransferase was lost during LTED leading to a lack of acetylcholine(ACh).Our study mainly confirmed the role and mechanism of GPR30 in the loss of E2 neuroprotection during the critical period hypothesis.The critical period hypothesis was that estrogen should be administered in premenopausal or postmenopausal women immediately.If estrogen was administered in postmenopause women after a long time,the neuroprotective effect would be obviously attenuated or even disappeared.Four-vessel occlusion and ovariectomized models in rat were established.The present study confirmed that E2 and G1 exerted marked neuroprotective effects against cerebral ischemi in short-term E2 deprivation(STED)rats following GCI,but exerted no neuroprotection in LTED rats.In our study,the different expression of GPR30 was found in hippocampal CA1 region between STED and LTED rats.The possible mechanisms of downregulation of GPR30 were revealed in vitro experiments.And in rats that received E2 treatment for the whole ovariectomy period(10 week),E2 prevented a decrease in the GPR30 protein and exerted robust neuroprotection in the CA1 region of LTED rats.Finally,the mechanisms of downregulation of GPR30 were also verified in aged female rats.Our study results further elucidated the mechanisms of loss of E2 neuroprotection during the critical period hypothesis and provided a novel therapeutic target against ischemic stroke for perimenopausal patients in clinical settings.The experiment was divided into three parts.Part 1: Neuroprotective effects of E2 and G1 in STED and LTED rats.Objectives: To successfully construct rat models of ovariectomized and global cerebral ischemia(GCI)and to observe whether E2 and G1 exert neuroprotective effects in STED and LTED rats.Methods:(1)Adult female Sprague-Dawley rats were randomly divided into four groups: sham-operated group 1,1-week ovariectomized group,sham-operated group 2 and 10-week ovariectomized group.Bilateral ovariectomy was performed in ovariectomized group,and then serum estradiol concentration was assayed in sham-operated group 1 and 1-week ovariectomized group after one week and sham-operated group 2 and 10-week ovariectomized group after ten weeks.The same surgical procedures were performed for rats in sham-operated group,however,the bilateral ovaries were not resected.(2)Adult female Sprague-Dawley rats were randomly divided into three groups: sham-operated group,10 min ischemia group and 20 min ischemia group.In the ischemia group,the vertebral arteries were electrocauterized with a monopolar coagulator and the common carotid arteries(CCA)were clipped for 10 min or 20 min after 24 h and rats were subsequently reperfused.The suiviving neurons were detected in the hippocamp CA1 region among the three group rats.The same surgical procedures were performed for rats in sham-operated groups,however,the four vessels were not occluded.(3)The rats of STED and LTED were randomly divided into four groups respectively: sham-operated group,placebo group,E2 group and G1 group.The suiviving neurons were detected in the hippocamp CA1 region among the four group rats after Nissl staining for brain slices following GCI.Results:(1)The serum E2 concentration on the 1st,3rd,5th and 7th days decreased significantly in the 1-week ovariectomized group compared with sham-operated group 1 and the serum E2 concentration on 7th day was less than 5 pg/mL in the 1-week ovariectomized group.The serum E2 concentration at 1,3,5,and 10 weeks decreased significantly in the 10-week ovariectomized group compared with sham-operated group 2 and the serum E2 concentration at 7 week was less than 1 pg/mL in the 10-week ovariectomized group.(2)Most of the neurons in the hippocampal CA1 region died,the pyramidal cells injuried and reduced severely and karyopyknosis occurred in the 10 min ischemia group compared with sham-operated group.The injury of neurons in the 20 min ischemia group was more severe than in the 10 min ischemia group.The normal structure and nissl bodies disappeared and karyolysis occurred in most cells,only leaving a small number of intact neurons.(3)E2 or Gl exerted a marked neuroprotective effect on the hippocampal CA1 region and effectively reduced the damage of hippocampus neurons,but revealed no obvious neuroprotection in LTED rats.Conclusions: The ovariectomized model can significantly reduce the level of estradiol and the GCI model can lead to significant damage of neurons in the hippocampus.Furthermore,we found that E2 or G1 effectively reduced the damage of hippocampus neurons and exerted robust neuroprotection in STED rats,but exerted no obvious neuroprotection in LTED rats.Part 2: The study on mechanisms of GPR30 in the loss of E2-mediated neuroprotection in the critical period hypothesis.Objectives: To explore the role of GPR30 on the loss of E2-mediated neuroprotection following LTED rats and the mechanisms of downregulation of GPR30.Methods:(1)Adult female Sprague-Dawley rats were randomly divided into sham-operated group(Sham),short-term E2 deprivation group(STED)and long-term E2 eprivation group(LTED).GPR30 immunoreactivity in the hippocampus were detected by immunohistochemistry among Sham,STED and LTED rats and the expression of GPR30 in the hippocampal CA1 region was detected by western blot among Sham,STED and LTED rats.(2)The mRNA levels of GPR30 in the hippocampal CA1 region were detected by qPCR among Sham,STED and LTED rats and the ubiquitination of GPR30 in the hippocampal CA1 region was detected by co-immunoprecipitation among Sham,STED and LTED rats.(3)Adult female Sprague-Dawley rats that received E2 or placebo treatment for the whole ovariectomy period(10 week)and received E2 or placebo treatment at 10 weeks post-ovariectomy were randomly divided into three groups respectively: control group,pla group and E2 group.The hippocampal CA1 tissue was isolated from the decapitated brain,and the protein and RNA of GPR30 were extracted and detected by western blot and qPCR respectively.(4)Adult female Sprague-Dawley rats that received E2 or placebo treatment for the whole ovariectomy period(10 week)and received E2 or placebo treatment at 10 weeks post-ovariectomy were randomly divided into five groups respectively: sham-operated group,pla group,E2 group,E2+G15 group and G15 group.The suiviving neurons were detected in the hippocamp CA1 region among the five group rats after Nissl staining for brain slices following GCI.(5)Adult female Sprague-Dawley rats that received E2 or placebo treatment for the whole ovariectomy period(10 week)and received E2 or placebo treatment at 10 weeks post-ovariectomy were randomly divided into three groups respectively: sham-operated group,pla group and E2 group.The apoptotic neurons were detected in the hippocampal CA1 region among the three group rats after tunel staining for brain slices following GCI.Results:(1)Immunofluorescence staining showed that GPR30 decreased slightly in hippocampal CA1 region of STED group but decreased significantly in hippocampal CA1 region of LTED group compared with Sham group.Furthermore,western blotting analysis revealed that GPR30 expression levels in the CA1 region of the hippocampus were significantly reduced in LTED rats compared with STED rats.(2)Co-immunoprecipitation showed that there was no significant change of GPR30 ubiquitination among the Sham,STED and LTED groups.The qPCR results demonstrated that GPR30 mRNA expression levels within STED rats were >2 times higher compared with those in LTED rats.(3)E2 treatment initiated at 10 weeks post-ovariectomy revealed no effects in preventing reductions in GPR30 protein expression levels within the CA1 region of the hippocampus and did not exhibit robust neuroprotection within LTED rats following GCI.In rats that received E2 treatment for the whole ovariectomy period(10 week),E2 prevented a decrease in the GPR30 protein and mRNA expression levels and exhibited marked neuroprotective effects in LTED rats,but the administration of G15 at the end of E2 treatment attenuated the neuroprotective effects of E2.(4)While the number of apoptotic cells was were only slightly decreased by E2 treatment that was initiated at 10 weeks post-ovariectomy compared with the Pla(GCI)group in LTED rats,the reduction observed following treatment with E2 immediately post-ovariectomy and maintained for 10 weeks was greater in LTED rats.Conclusions: These results indicated GPR30 decreased in the CA1 region of the hippocampus in LTED rats compared with STED rats.The decrease of GPR30 was primarily attributable to the decreased mRNA levels.E2 treatment initiated immediately post-ovariectomy and maintained for 10 weeks can prevent the decrease of GPR30 and mRNA levels and can exhibit marked neuroprotective effects in LTED rats.Part 3: The expression levels of GPR30 in the CA1 region of the hippocampus and the mechanisms on whether E2 and Gl exert neuroprotective effects on naturally aging female rats.Objectives: To detect whether the expression of GPR30 decreased in hippocampal CA1 region in naturally aging female rats,the mechanisms of downregulation of GPR30,and whether E2 and G1 exerted neuroprotective effects on naturally aging female rats.Methods:(1)The rats were divided into the young rats and the aging rats.The expression of ER?,ER? and GPR30 in hippocampal CA1 region were detected by western blot between 3-and 24-month-old rats.(2)qPCR and co-immunoprecipitation were used to detect mRNA levels and the ubiquitination of GPR30 in the hippocampal CA1 region between 3-and 24-month-old rats respectively.(3)The naturally aging female rats were randomly divided into four groups: sham-operated group,pla group,E2 group and G1 group.The suiviving and apoptotic neurons were detected respectively in the hippocampal CA1 region among the four group rats after Nissl and tunel staining for brain slices following GCI.Results:(1)Western blot analysis of GPR30 revealed that the protein expression levels of GPR30,ER? and ER? were decreased significantly within 24-month-old rats compared with 3-month-old rats.(2)The qPCR results revealed that the mRNA expression levels of GPR30 within the CA1 region of the hippocampus of 24-month-old rats were decreased to ~50% of the levels in 3-month-old female rats;however,the ubiquitination of GPR30 revealed no significant differences between 3-and 24-month-old rats.(3)Nissl and tunel staining results of the CA1 region of the hippocampus and quantification of neuronal survival and apoptosis demonstrated that G1 or E2 did not exhibit neuroprotection within the 24-month-old female rats.Conclusions: It was further verified that the expression of GPR30 was decreased in 24-month-old female rats,and the decrease of GPR30 was mainly caused by the decreased mRNA levels.E2 or G1 did not exert marked neuroprotection in the hippocampal CA1 region of 24-month-old female rats. |
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