OBJECTIVE:To investigate the effect of icariin on proliferation of bone marrow mesenchymal stem cells(BMSCs)in Sprague-Dawley(SD)rats.METHODS:BMSCs were obtained from SD rat bone marrow with differential time adherent...OBJECTIVE:To investigate the effect of icariin on proliferation of bone marrow mesenchymal stem cells(BMSCs)in Sprague-Dawley(SD)rats.METHODS:BMSCs were obtained from SD rat bone marrow with differential time adherent method.Its characteristic was identified through differentiation cell surface antigens and the multi-lineage(osteo/adipo/chondo)differentiation potential.3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)method and 5-Bromo-2-Deoxyuridine(Brd U)incorporation were applied to detect the effect of icariin on BMSCs proliferation.Flow cytometry was used to detect proliferation in-dex of BMSCs.The m RNA level and the distribution ofβ-catenin were evaluated by Real-time Polymerase Chain Reaction(PCR)and Immunofluorescent staining respectively.Western blot was used to detect protein expression levels ofβ-catenin,glycogen synthase kinase-3 beta(GSK-3β),phospho-glycogen synthase kinase-3 beta(p GSK-3β)and cyclin D1.RESULTS:Icariin promoted BMSCs proliferation at the concentration of 0.05-2.0 mg/L.The percentage of Brd U positive cells of BMSCs was increased from40.98%to 70.42%,and the proliferation index value was increased from 8.9%to 17.5%with the treatment of 0.05 mg/L icariin,which significance values were both less than 0.05.Compared with the control group,total and nuclearβ-catenin proteins,as well asβ-catenin m RNA expression,were all increased with icariin treatment.Meanwhile,the phosphorylation level of GSK-3βand cyclin D1 protein expressions were also increased in BMSCs with icariin treatment.CONCLUSION:The findings of the present study demonstrated that low dosage of icariin could promote BMSCs proliferation.The activation of Wnt/β-catenin pathways was involved in this process.展开更多
OBJECTIVE:To explore whether the regulation of matrix metalloproteinase 9(MMP-9)/tissue inhibitors of MMPs(TIMPs)gene expression through histone acetylation is a possible mechanism by which electroacupuncture(EA)prote...OBJECTIVE:To explore whether the regulation of matrix metalloproteinase 9(MMP-9)/tissue inhibitors of MMPs(TIMPs)gene expression through histone acetylation is a possible mechanism by which electroacupuncture(EA)protects blood-brain barrier(BBB)integrity in a middle cerebral artery occlusion(MCAO)rat model.METHODS:Male Sprague-Dawley rats were divided into four groups:the sham group,the MCAO group,the MCAO+EA(MEA)group,and the MCAO+EA+HAT inhibitor(HATi)group.The MCAO model was generated by blocking the middle cerebral artery.EA was applied to Baihui(GV20).Samples were collected 1 or 3 d after reperfusion.Neurological function scores and Evans blue extravasation were employed to evaluate the poststroke injury.The effect of EA on MMP-9/TIMPs gene expression was assessed by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)and chromatin immunoprecipitation(ChIP).RESULTS:Our results showed that EA treatment prominently improved neurological function and ameliorated BBB disruption.The RT-qPCR assay showed that EA reduced the expression of MMP-9 and promoted TIMP-2 mRNA expression,but HATi reversed these effects of EA.In addition,ChIP results revealed that EA decreased the enrichment of H3K9ace/H3K27ace at MMP-9 promoters and notably stimulated the recruitment of H3K9ace/H3K27ace at TIMP-2 promoter.CONCLUSION:EA treatment at Baihui(GV20)regulates the transcription of MMP-9 and TIMP-2 through histone acetylation modification in the acute stage of stroke,which preserves the structural integrity of the BBB in MCAO rats.These findings suggested that the histone acetylation-mediated transcriptional activity of target genes may be a crucial mechanism of EA treatment in stroke.展开更多
基金the the National Natural Science Foundation of China(Study on the Bushen Chinese Medicine on the BMSCs Differentiation,No.81173619,81403479)Natural Science Foundation of the Jiangsu Province of China(Study of Acupuncture on Ischemic Stroke,No.BK20130956)Specialized Research Fund for the Doctoral Program of Higher Education of China(Epigenetic Mechanism of Acupuncture on Brain Injury,No.20133237120002)
文摘OBJECTIVE:To investigate the effect of icariin on proliferation of bone marrow mesenchymal stem cells(BMSCs)in Sprague-Dawley(SD)rats.METHODS:BMSCs were obtained from SD rat bone marrow with differential time adherent method.Its characteristic was identified through differentiation cell surface antigens and the multi-lineage(osteo/adipo/chondo)differentiation potential.3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)method and 5-Bromo-2-Deoxyuridine(Brd U)incorporation were applied to detect the effect of icariin on BMSCs proliferation.Flow cytometry was used to detect proliferation in-dex of BMSCs.The m RNA level and the distribution ofβ-catenin were evaluated by Real-time Polymerase Chain Reaction(PCR)and Immunofluorescent staining respectively.Western blot was used to detect protein expression levels ofβ-catenin,glycogen synthase kinase-3 beta(GSK-3β),phospho-glycogen synthase kinase-3 beta(p GSK-3β)and cyclin D1.RESULTS:Icariin promoted BMSCs proliferation at the concentration of 0.05-2.0 mg/L.The percentage of Brd U positive cells of BMSCs was increased from40.98%to 70.42%,and the proliferation index value was increased from 8.9%to 17.5%with the treatment of 0.05 mg/L icariin,which significance values were both less than 0.05.Compared with the control group,total and nuclearβ-catenin proteins,as well asβ-catenin m RNA expression,were all increased with icariin treatment.Meanwhile,the phosphorylation level of GSK-3βand cyclin D1 protein expressions were also increased in BMSCs with icariin treatment.CONCLUSION:The findings of the present study demonstrated that low dosage of icariin could promote BMSCs proliferation.The activation of Wnt/β-catenin pathways was involved in this process.
基金the National Natural Science Foundation of China:the Role of Intestinal Flora-Treg/γδT Cell-IL-17 Signaling in the Neuroprotective Effect of Electroacupuncture on Ischemic Brain Injury(No.81774403)the Natural Science Foundation of the Jiangsu Province of China:Study on the Mechanism of Acupuncture Antistroke Immune Inflammatory Response Based on Intestinal Treg/γδT Cell-IL-17 Signaling Pathway(No.BK20171492)+2 种基金the Postgraduate Scientific Research Innovation Practice Project of the Jiangsu Province of China:a Study based on the Butyric Acid-HDAC-Foxp3 Pathway to Explore the Regulatory Effect of Acupuncture on Intestinal Treg in Rats with Stroke(No.KYCX21_1715)a Study on the Anti-brain Injury of Electroacupuncture Based on Intestinal Microbiota-Treg/γδT Cell-IL-17 Pathway(No.KYCX21_1716)the Key University Science Research Project of Jiangsu Province:the Role of Preactivation of the Treg Immune Response in the Mechanism of Anti-stroke Neuroinflammatory Response in Acupuncture Pretreatment(No.22KJA360003)。
文摘OBJECTIVE:To explore whether the regulation of matrix metalloproteinase 9(MMP-9)/tissue inhibitors of MMPs(TIMPs)gene expression through histone acetylation is a possible mechanism by which electroacupuncture(EA)protects blood-brain barrier(BBB)integrity in a middle cerebral artery occlusion(MCAO)rat model.METHODS:Male Sprague-Dawley rats were divided into four groups:the sham group,the MCAO group,the MCAO+EA(MEA)group,and the MCAO+EA+HAT inhibitor(HATi)group.The MCAO model was generated by blocking the middle cerebral artery.EA was applied to Baihui(GV20).Samples were collected 1 or 3 d after reperfusion.Neurological function scores and Evans blue extravasation were employed to evaluate the poststroke injury.The effect of EA on MMP-9/TIMPs gene expression was assessed by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)and chromatin immunoprecipitation(ChIP).RESULTS:Our results showed that EA treatment prominently improved neurological function and ameliorated BBB disruption.The RT-qPCR assay showed that EA reduced the expression of MMP-9 and promoted TIMP-2 mRNA expression,but HATi reversed these effects of EA.In addition,ChIP results revealed that EA decreased the enrichment of H3K9ace/H3K27ace at MMP-9 promoters and notably stimulated the recruitment of H3K9ace/H3K27ace at TIMP-2 promoter.CONCLUSION:EA treatment at Baihui(GV20)regulates the transcription of MMP-9 and TIMP-2 through histone acetylation modification in the acute stage of stroke,which preserves the structural integrity of the BBB in MCAO rats.These findings suggested that the histone acetylation-mediated transcriptional activity of target genes may be a crucial mechanism of EA treatment in stroke.
