BVT-14225通过抑制11β-羟基类固醇脱氢酶1还原活性促进神经干细胞增殖和迁移  

Effect of BVT-14225 on proliferation and migration of neural stem cells by inhibiting reduction activity of 11β-hydroxysteroid dehydrogenase type 1

作　　者：马君梅 南海函[2] 胡志妍 陈依尔 崔燕华 陈星驰 李莉 苏颖 梅虹霞 缪项慧 张旭彤[1] 林函[1,3] MA Jun-mei;NAN Hai-han;HU Zhi-yan;CHEN Yi-er;CUI Yan-hua;CHEN Xing-chi;LI Li;SU Ying;MEI Hong-xia;MIAO Xiang-hui;ZHANG Xu-tong;LIN Han(Department of Anesthesiology and Perioperative Medicine,the Second Affiliated Hospital and Yuying Children′s Hospital of Wenzhou Medical University,Wenzhou 325027,China;School of Laboratory Medicine and Life Science,Wenzhou Medical University,Wenzhou 325035,China;Zhejiang Key Laboratory of Anesthesiology,Wenzhou 325035,China;Wenzhou People′s Hospital,Wenzhou 325000,China)

机构地区：[1]温州医科大学附属第二医院,育英儿童医院麻醉与围术期医学科,浙江温州325027 [2]温州医科大学检验医学院(生命科学学院),浙江温州325035 [3]浙江省麻醉学重点实验室,浙江温州325035 [4]温州市人民医院,浙江温州325000

出　　处：《中国药理学与毒理学杂志》2020年第4期278-288,共11页Chinese Journal of Pharmacology and Toxicology

基　　金：国家科技重大专项(2020ZX09201002);国家自然科学基金(30800323);浙江省自然科学基金(LY14H090015);浙江省自然科学基金(LQ19H090014);温州市科技局项目(Y20180067);温州市科技局项目(Y20170149)。

摘　　要：目的探讨BVT-14225(BVT)通过抑制11β-羟基类固醇脱氢酶1(11β-HSD1)的还原活性对神经干细胞(NSC)增殖、分化和迁移的影响。方法取孕15 d SD胎大鼠的大脑皮质,分离培养原代NSC,采用免疫荧光染色法对NSC标志物巢蛋白和性别决定基因相关转录因子2(Sox2)进行染色,鉴定原代细胞;逆转录聚合酶链反应及核酸凝胶电泳法检测11β-HSD1 mRNA在NSC上的表达。细胞随机分为5组:细胞对照组(培养48 h)、溶剂对照组(0.1%DMSO孵育48 h)、DHC模型组(DHC 10.0μmol·L^-1处理48 h)、BVT对照组(BVT 10.0μmol·L^-1处理48 h)和BVT干预组(BVT 10μmol·L^-1处理1 h后再给予DHC10.0μmol·L^-1,继续处理48 h)。5-乙炔基-2’脱氧尿嘧啶核苷(EdU)细胞增殖试剂盒检测NSC的增殖能力;乳酸脱氢酶(LDH)释放实验检测细胞毒性;超高效液相色谱-质谱(UPLC-MS)检测11β-HSD1酶的还原活性;Western印迹法检测11β-HSD1蛋白表达水平;胶质纤维酸性蛋白(GFAP)和神经元核抗原(NeuN)抗体免疫荧光染色检测NSC的分化能力;划痕实验检测NSC的迁移能力。结果巢蛋白和Sox2免疫染色鉴定原代培养细胞为NSC,核酸凝胶电泳条带图证明11β-HSD1 mRNA在NSC定性表达。LDH释放实验结果显示,DHC 10.0μmol·L^-1和BVT 10.0μmol·L^-1对NSC均无毒性作用。UPLC-MS实验结果显示,BVT 10.0μmol·L^-1能显著降低11β-HSD1的还原活性(P<0.01)。Western印迹结果显示,DHC 10.0μmol·L^-1和BVT 10.0μmol·L^-1均不改变11β-HSD1蛋白表达水平。EdU实验结果显示,DHC 10.0μmol·L^-1可抑制NSC增殖(P<0.01),但BVT 10.0μmol·L^-1预处理可显著缓解DHC 10.0μmol·L^-1对NSC增殖的抑制(P<0.05)。GFAP和NeuN的免疫荧光染色实验结果表明,DHC 10.0μmol·L^-1和BVT 10.0μmol·L^-1均不影响NSC分化。划痕实验结果显示,DHC 10.0μmol·L^-1不影响NSC的迁移,但BVT 10.0μmol·L^-1预处理可显著促进NSC迁移(P<0.05)。结论糖皮质激素浓度显著升高时,BVT可通过抑制NSC 11β-HSD1的还原�OBJECTIVE To investigate the effect of BVT-14225(BVT)on proliferation,differentiation and migration of neural stem cells(NSCs)by inhibiting reduction activity of 11β-hydroxysteroid dehydrogenase type 1(11β-HSD1).METHODS Primary NSCs were isolated and cultured from the fetal rat cerebrum cortex of SD rats at 14-16 d of gestation(E14-E16).Primary cells were identified by immunofluorescence staining of NSC marker—nestin and SRY-box transcription factor-2(Sox2).Reverse transcription PCR and nucleic acid gel electrophoresis were used to detect the expression of 11β-HSD1 mRNA in NSCs.The cells were randomly divided into five groups:cell control group(NSCs were cultured for 48 h),DMSO group(NSCs were treated with 0.1%DMSO for 48 h),model group(NSCs were treated with DHC 10.0μmol·L^-1 for 48 h),BVT control group(NSCs were treated with BVT10.0μmol·L^-1 for 48 h)and intervention group(NSCs were treated with DHC 10μmol·L^-1 for 48 h after pretreatment of NSCs with BVT 10.0μmol·L^-1 for 1 h).5-Ethynyl-2′deoxyuracilnucleoside(EdU)cell proliferation kit was used to detect NSC proliferation.Lactate dehydrogenase(LDH)release assay was used to detect cytotoxicity.Ultra performance liquid chromatography-mass spectrometry assay(UPLC-MS)was used to detect the reduction activity of 11β-HSD1,and Western blotting was used to detect the expression of 11β-HSD1 protein.Immunofluorescence staining of glial fibrillary acidic protein(GFAP)and neuronal nuclei(NeuN)was used to detect the induced NSC differentiation in vitro.The scratch assay was used to detect NSC migration.RESULTS The primary cultured cells were identified as NSCs by the immunofluorescence staining of nestin and Sox2.And nucleic acid gel electrophoresis assay indicated that 11β-HSD1 mRNA was qualitatively expressed in NSCs.LDH assay indicated that neither DHC 10.0μmol·L^-1 nor BVT 10.0μmol·L^-1 had significant toxic effect on NSCs.UPLC-MS assay indicated that BVT 10.0μmol·L^-1 could significantly mitigate the reduction activity of 11β-HSD1(P<0.01).Wes

关 键 词：BVT-14225 神经干细胞 11Β-羟基类固醇脱氢酶1 糖皮质激素 

分 类 号：R963[医药卫生—微生物与生化药学]