银杏二萜内酯K对D-半乳糖诱导的小鼠原代星形胶质细胞抗衰老作用  

Ginkgolide K inhibits D-galactose-induced senescence in murine primary astrocytes

作　　者：操娇娇 杜雨芯 张倩霞 吴伟 杨颖博 肖保国[4] 肖伟[1,3] 王振中[1,3] CAO Jiao-jiao;DU Yu-xin;ZHANG Qian-xia;WU Wei;YANG Ying-bo;XIAO Bao-guo;XIAO Wei;WANG Zhen-zhong(Nanjing University of Chinese Medicine,Nanjing 210000,China;Institute of Chinese Materia Medica,Shanghai University of Chinese Medicine,Shanghai 200120,China;National Key Laboratory on Technologies for Chinese Medicine Pharmaceutical Process Control and Intelligent Manufacture,Jiangsu Kanion Pharmaceutical Co.,Ltd.,Lianyungang 222001,China;Institute of Neurology,Huashan Hospital,Fudan University,Shanghai 200040,China)

机构地区：[1]南京中医药大学,江苏南京210000 [2]上海中医药大学中药研究所,上海200120 [3]中药制药过程与智能制造技术全国重点实验室,江苏康缘药业股份有限公司,江苏连云港222001 [4]上海复旦大学神经病学研究所,上海200040

出　　处：《中草药》2023年第11期3578-3585,共8页Chinese Traditional and Herbal Drugs

基　　金：2021年国家中医药管理局岐黄学者项目。

摘　　要：目的研究银杏二萜内酯K(ginkgolide K,GK)对D-半乳糖诱导的小鼠原代星形胶质细胞抗衰老作用及可能机制。方法以小鼠原代星形胶质细胞为研究对象,建立D-半乳糖诱导的衰老模型,设置对照组、模型组和GK组,GK(50μg/mL)预处理3 h后,以150 mmol/L D-半乳糖造模6 d,采用β-半乳糖苷酶染色法检测衰老星形胶质细胞;Western blotting法测定衰老标志蛋白(p21、p53和Lamin B1)、沉默信息调节因子1(silent information regulator 1,SIRT1)、脑源性神经营养因子(brain-derived neurotrophic factor),BDNF、自噬标志蛋白微管相关蛋白1轻链3-Ⅱ/I(microtubule associated protein 1light chain 3-Ⅱ/I,LC3-Ⅱ/I)和自噬选择性底物p62表达;ELISA检测衰老相关分泌表型[白细胞介素-6(interleukin-6,IL-6)、IL-1β、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)];免疫荧光检测增殖标志分子Ki67表达。结果与对照组比较,D-半乳糖显著诱导原代星形胶质细胞β-半乳糖苷酶阳性表达(P<0.001),促进衰老标志蛋白p21、p53的上调和Lamin B1的下调(P<0.05、0.01),减少增殖标志分子Ki67的表达(P<0.01),表明成功构建了公认的细胞衰老模型。与模型组比较,GK显著减少β-半乳糖苷酶阳性表达(P<0.001),下调p21、p53和上调Lamin B1蛋白的表达(P<0.05、0.01),增加Ki67的表达(P<0.01),表明GK具有抗衰老作用。此外,GK显著促进BDNF蛋白表达(P<0.05),减少TNF-α的分泌(P<0.05),上调SIRT1和自噬标志蛋白LC3-Ⅱ/I表达(P<0.05、0.01),减少p62蛋白表达(P<0.01)。结论GK可有效抑制原代星形胶质细胞的衰老,其作用机制可能与上调SIRT1、BDNF表达和促进细胞自噬有关,从而多维度协同达到抗细胞衰老的效应。Objective To study the anti-aging effect and possible mechanism of ginkgolide K(GK)on murine primary astrocytes induced by D-galactose.Methods The aging model induced by D-galactose was established in murine primary astrocytes,and the aging astrocytes were set up in control group,model group and GK group.After pretreatment with GK(50μg/mL)for 3 h,the aging astrocytes were modeled with 150 mmol/L D-galactose for 6 d,andβ-galactosidase staining was used to detect the aging astrocytes.Western blotting was used to detect the aging marker proteins(p21,p53 and Lamin B1),silent information regulator 1(SIRT1),brain-derived neurotrophic factor(BDNF),autophagy marker protein microtubule associated protein 1 light chain 3-II/I(LC3-II/I)and autophagy selective substrate p62 expressions.ELISA was used to detect aging-related secretory phenotype[interleukin-6(IL-6),IL-1β,tumor necrosis factor-α(TNF-α)];The expression of Ki67 was detected by immunofluorescence.Results Compared with control group,D-galactose significantly induced the positive expression ofβ-galactosidase in primary astrocytes(P<0.001),promoted the up-regulation of aging marker proteins p21 and p53 and the down-regulation of Lamin B1(P<0.05,0.01),and reduced the expression of proliferation marker molecule Ki67(P<0.01),which indicated that a recognized cell aging model was successfully established.Compared with model group,GK significantly reduced the positive expression ofβ-galactosidase(P<0.001),down-regulated the expressions of p21,p53 and up-regulated Lamin B1 protein expression(P<0.05,0.01),and increased the expression of Ki67(P<0.01),indicating that GK had anti-aging effect.In addition,GK significantly promoted the expression of BDNF protein(P<0.05),decreased the secretion of TNF-α(P<0.05),increased the expressions of SIRT1 and autophagy marker protein LC3-Ⅱ/I/I(P<0.05,0.01),and decreased the expression of p62 protein(P<0.01).Conclusion GK can effectively inhibit the aging of primary astrocytes,and its mechanism may be related to up-regulating the

关 键 词：银杏二萜内酯K 原代星形胶质细胞 D-半乳糖 衰老 自噬 沉默信息调节因子1 脑源性神经营养因子 

分 类 号：R285.5[医药卫生—中药学]