熟地黄上调木糖基转移酶Ⅰ信号途径促进阴虚大鼠脑内γ-氨基丁酸合成的机制研究  

Study on the Mechanism of Rehmanniae Radix Praeparata in Promoting the Synthesis ofγ-Aminobutyric Acid in the Brain of Yin Deficiency Rats by Up-regulating the XylosyltransferaseⅠSignaling Pathway

作　　者：黎健殷 郭晓纯 张宇婧 王剑[1] LI Jianyin;GUO Xiaochun;ZHANG Yujing;WANG Jian(Guangzhou University of Chinese Medicine,Guangzhou 510006 Guangdong,China)

机构地区：[1]广州中医药大学,广东广州510006

出　　处：《中药新药与临床药理》2024年第8期1170-1180,共11页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：广东省科技厅项目(20194141405058)。

摘　　要：目的基于木糖基转移酶Ⅰ(xylt-1)信号途径探讨熟地黄及其活性成分地黄苷D、梓醇对脑内γ-氨基丁酸(GABA)的调控机制。方法(1)将SD大鼠随机分为正常组、模型组、治疗组(熟地黄组,30 g·kg^(-1)),每组8只,雌雄各半。采用辛温燥湿利湿中药复方(30 g·kg^(-1))灌胃诱导建立阴虚大鼠模型,早晚各1次,连续10 d。造模结束后,治疗组灌胃熟地黄水煎液,早晚各1次,连续10 d。测定记录大鼠体质量并通过旷场行为学实验检测其活跃度、穿格次数及总路程;采用ELISA法检测大鼠血清促性腺激素释放激素(GnRH)、促甲状腺素释放激素(TRH)、促肾上腺皮质激素释放激素(CRH)、黄体生成素(LH)、卵泡刺激素(FSH)水平;qPCR法及全自动毛细管蛋白印迹法检测大鼠脑组织中xylt-1、多配体蛋白聚糖1(SDC-1)、早期生长反应因子1(EGR1)、谷氨酸脱羧酶1(GAD67)mRNA及蛋白表达水平;HPLC法检测脑组织中GABA含量。(2)采用siRNA沉默大鼠高分化肾上腺嗜铬细胞瘤细胞(PC12)的xylt-1基因,细胞分为正常组、阴性对照组、沉默组、沉默+地黄苷D(10μmol·L-1)组、沉默+梓醇(10μmol·L-1)组。采用qPCR法检测细胞中xylt-1、SDC-1、EGR1、GAD67 mRNA表达水平;HPLC法检测细胞内、外液GABA含量;免疫荧光法检测细胞中SDC-1表达水平。(3)采用慢病毒转染PC12细胞过表达xylt-1基因,细胞分为正常组、阴性对照组(空载组)、过表达组。然后采用qPCR法检测细胞中xylt-1、SDC-1、EGR1、GAD67 mRNA表达水平;HPLC法检测细胞内、外液GABA含量。结果(1)与正常组比较,模型组大鼠的体质量显著降低(P<0.01),活跃度显著升高(P<0.01),穿格次数及总路程显著增加(P<0.01);血清LH、FSH、GnRH、CRH、TRH水平均显著升高(P<0.01);海马及大脑皮层组织中的GABA含量明显降低(P<0.05,P<0.01);脑组织中的xylt-1、SDC-1、EGR1、GAD67 mRNA(海马、大脑皮层组织)及蛋白(下丘脑、大脑皮层组织)表达均显著下调Objective To investigate the regulatory mechanism of Rehmanniae Radix Praeparata and its active components rehmannioside D and catalpol onγ-aminobutyric acid(GABA)in the brain based on the xylosy-transferase I(xylt-1)signaling pathway.Methods(1)SD rats were randomly divided into normal group,model group and treatment group(Rehmanniae Radix Praeparata group,30 g·kg^(-1)),with eight rats in each group,half male and half female.The rat model of yin deficiency was established by intragastric administration of traditional Chinese medicine compound(30 g·kg^(-1))with acrid-warm nature and dampness-drying and dampness-draining,once in the morning and once in the evening,for 10 consecutive days.After modeling,the treatment group was given Rehmanniae Radix Praeparata decoction by gavage,once in the morning and once in the evening,for 10 consecutive days.The body mass of rats was measured and recorded,and the activity,crossing times and total distance were detected by open field behavior experiment.The levels of serum gonadotropin-releasing hormone(GnRH),thyrotropin-releasing hormone(TRH),corticotropin-releasing hormone(CRH),luteinizing hormone(LH)and follicle-stimulating hormone(FSH)in rats were detected by ELISA.The mRNA and protein expression levels of xylt-1,multiligand proteoglycan 1(SDC-1),early growth response factor 1(EGR1)and glutamate decarboxylase 1(GAD67)in rat brain tissue were detected by qPCR and automatic capillary Western Blot.The content of GABA in brain tissue was detected by HPLC.(2)The xylt-1 gene of rat well-differentiated adrenal pheochromocytoma cells(PC12)was silenced by siRNA.The cells were divided into normal group,negative control group,silencing group,silencing+rehmannioside D(10μmol·L-1)group,silencing+catalpol(10μmol·L-1)group.The expression levels of xylt-1,SDC-1,EGR1 and GAD67 mRNA in cells were detected by qPCR.The content of GABA in intracellular and extracellular fluid was detected by HPLC.The expression level of SDC-1 in cells was detected by immunofluorescence.(3)PC12 cells were

关 键 词：熟地黄 阴虚证 γ-氨基丁酸 xylt-1/SDC-1/EGR1/GAD67通路 大鼠 

分 类 号：R285.5[医药卫生—中药学]