OBJECTIVE To detect the underlying mechanism of time window for estrogen(E2)replacement treating cognitive decline.METHODS E2 begun 1 week after the ovariectomy(OVXST)or 3 months after the ovariectomy(OVXLT).Learning ...OBJECTIVE To detect the underlying mechanism of time window for estrogen(E2)replacement treating cognitive decline.METHODS E2 begun 1 week after the ovariectomy(OVXST)or 3 months after the ovariectomy(OVXLT).Learning and memory ability were examined by trace fear memory test and inhibitory avoidance test.LTP and LTD were detected by MED64.High throughput gene expression sequencing and microRNA(miR NA) sequencing were used to detecte the differently expressed genes between OVXSTand OVXLTafter estrogen treatment.RESULTS Subcutaneous injection of E2 improved fear memory formation in both 1 week after ovariectomy(OVXST) mice or 3 months after ovariectomy(OVXLT) mice.However,for fear memory extinction,facilitated by E2 in OVXSTmice,but impaired by E2 in OVXLTmice.Further researches showed in medial prefrontal cortex(mPFC),estrogen facilitates LTD in OVXSTmice but impairs LTD in OVXLTmice.Results of highthroughput sequencings of mR NA and miRNA in mPFC from sham,OVXSTmice,E2 treated OVXST mice,OVXLTmice,and E2 treated OVXLTmice indicated decreased miR-221-5 p expression in OVXLTmice compared with OVXSTmice.In OVXLT mice,miR-221-5 p could be further reduced by E2 treatment.Additionally,miR-221-5 p targeted neuralized E3 ubiquitin protein ligase 1 a/b(Neurl1 a/b) m RNA.Decreased miR-221-5 p will promotes cannabinoid receptor 1(CB1) ubiquitination through up-regulating Neurl1 a/b protein levels in E2 treated OVXLTmice,which disrupted the retrograde endocanabinoids system.Replenishing miR-221-5 p or treating with CB1 agonist rescued the fear extinction impairment in E2 treated OVXLTmice.CONCLUSION These results uncovered a epigenetic change after long term E2 responsible for failure of E2 improving cognitive performance in OVXLTmice,moreover miR-221-5 p and CB1 agonist as potential targets for prolonging the time window for E2 replacement therapy.展开更多
Background Bone morphogenetic protein （BMP） is a member of the superfamily of transforming growth factor-13. Recent studies show that it is an indispensable factor in hematopoiesis. To better characterize the effect...Background Bone morphogenetic protein （BMP） is a member of the superfamily of transforming growth factor-13. Recent studies show that it is an indispensable factor in hematopoiesis. To better characterize the effect of recombinant human BMP （rhBMP）-2 in hematopoiesis, we set out to determine whether rhBMP-2 could promote the proliferation of mesenchymal stem cells （MSCs） and increase the levels of hematopoietic cytokines in MSCs. Methods 2,3-bis （2-methoxy-4-nitro-5-sulfophenyl）-5-（（phenylamino） carbonyl）-2H-tetrazolium hydroxide （XTT）, real-time polymerase chain reaction （PCR） and enzyme-linked immunosorbent assay （ELISA） were used to detect the effects of rhBMP-2 on the proliferation and hematopoietic cytokine levels of MSCs. In addition, MSCs marked with Hoechst33342 were transplanted into BALB/c mice by the intravenous route or intra-bone marrow transplantation, and cluster numbers were counted. Results The XTT test revealed that rhBMP-2 significantly induced proliferation of MSCs in doses ranging from 10 ng/ml to 0.1 mg/ml in a dose-dependent manner. The experiments in vivo showed that there were more clusters of donor cells in bone marrow, spleen, liver and lung of the BMP group than those in the control group after both intra-bone marrow transplantation （P 〈0.001, P 〈0.001, P 〈0.001, and P=0.001, respectively） and intravenous transplantation （P 〈0.001, P 〈0.001, and P 〈0.001 respectively）. The results of real-time PCR and ELISA revealed that rhBMP-2 significantly increased mRNA expressions and protein levels of IL-6, IL-7, IL-11, G-CSF, M-CSF and SCF. Conclusions The treatment with rhBMP-2 promotes the proliferation of MSCs in vivo and in vitro and increases the levels of hematopoietic cytokines in MSCs, which may contribute to the improvement of hematopoietic function.展开更多
文摘OBJECTIVE To detect the underlying mechanism of time window for estrogen(E2)replacement treating cognitive decline.METHODS E2 begun 1 week after the ovariectomy(OVXST)or 3 months after the ovariectomy(OVXLT).Learning and memory ability were examined by trace fear memory test and inhibitory avoidance test.LTP and LTD were detected by MED64.High throughput gene expression sequencing and microRNA(miR NA) sequencing were used to detecte the differently expressed genes between OVXSTand OVXLTafter estrogen treatment.RESULTS Subcutaneous injection of E2 improved fear memory formation in both 1 week after ovariectomy(OVXST) mice or 3 months after ovariectomy(OVXLT) mice.However,for fear memory extinction,facilitated by E2 in OVXSTmice,but impaired by E2 in OVXLTmice.Further researches showed in medial prefrontal cortex(mPFC),estrogen facilitates LTD in OVXSTmice but impairs LTD in OVXLTmice.Results of highthroughput sequencings of mR NA and miRNA in mPFC from sham,OVXSTmice,E2 treated OVXST mice,OVXLTmice,and E2 treated OVXLTmice indicated decreased miR-221-5 p expression in OVXLTmice compared with OVXSTmice.In OVXLT mice,miR-221-5 p could be further reduced by E2 treatment.Additionally,miR-221-5 p targeted neuralized E3 ubiquitin protein ligase 1 a/b(Neurl1 a/b) m RNA.Decreased miR-221-5 p will promotes cannabinoid receptor 1(CB1) ubiquitination through up-regulating Neurl1 a/b protein levels in E2 treated OVXLTmice,which disrupted the retrograde endocanabinoids system.Replenishing miR-221-5 p or treating with CB1 agonist rescued the fear extinction impairment in E2 treated OVXLTmice.CONCLUSION These results uncovered a epigenetic change after long term E2 responsible for failure of E2 improving cognitive performance in OVXLTmice,moreover miR-221-5 p and CB1 agonist as potential targets for prolonging the time window for E2 replacement therapy.
基金This work was supported by a grant from the National Natural Science Foundation of China （No. 30470528）.Acknowledgments： We thank Prof. ZHANG Shao-zhang and Prof. LU Fan （senior laboratorian） in the Fourth Military Medical University, China for their technical instructions and Prof. PU Qin for providing the rhBMP-2.
文摘Background Bone morphogenetic protein （BMP） is a member of the superfamily of transforming growth factor-13. Recent studies show that it is an indispensable factor in hematopoiesis. To better characterize the effect of recombinant human BMP （rhBMP）-2 in hematopoiesis, we set out to determine whether rhBMP-2 could promote the proliferation of mesenchymal stem cells （MSCs） and increase the levels of hematopoietic cytokines in MSCs. Methods 2,3-bis （2-methoxy-4-nitro-5-sulfophenyl）-5-（（phenylamino） carbonyl）-2H-tetrazolium hydroxide （XTT）, real-time polymerase chain reaction （PCR） and enzyme-linked immunosorbent assay （ELISA） were used to detect the effects of rhBMP-2 on the proliferation and hematopoietic cytokine levels of MSCs. In addition, MSCs marked with Hoechst33342 were transplanted into BALB/c mice by the intravenous route or intra-bone marrow transplantation, and cluster numbers were counted. Results The XTT test revealed that rhBMP-2 significantly induced proliferation of MSCs in doses ranging from 10 ng/ml to 0.1 mg/ml in a dose-dependent manner. The experiments in vivo showed that there were more clusters of donor cells in bone marrow, spleen, liver and lung of the BMP group than those in the control group after both intra-bone marrow transplantation （P 〈0.001, P 〈0.001, P 〈0.001, and P=0.001, respectively） and intravenous transplantation （P 〈0.001, P 〈0.001, and P 〈0.001 respectively）. The results of real-time PCR and ELISA revealed that rhBMP-2 significantly increased mRNA expressions and protein levels of IL-6, IL-7, IL-11, G-CSF, M-CSF and SCF. Conclusions The treatment with rhBMP-2 promotes the proliferation of MSCs in vivo and in vitro and increases the levels of hematopoietic cytokines in MSCs, which may contribute to the improvement of hematopoietic function.
