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（10 μmol·L-1）组、沉默+梓醇（10 μmol·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（P＜0.05，P＜0.01）。与模型组比较， 治疗组大鼠经熟地黄干预后的体质量明显升高（P＜0.05），活跃度显著下降（P＜0.01），总路程明显缩短（P＜ 0.05）；血清 LH、FSH、GnRH、CRH、TRH 水平均显著下降（P＜0.01）；海马及大脑皮层组织中的 GABA 含量 明显升高（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）表达均显著上调（P＜0.05，P＜0.01）。（2）与阴性对照组比较，沉默组细胞的 xylt-1、 SDC-1、EGR1、GAD67 mRNA 表达显著下调（P＜0.01）；细胞内、外液中的 GABA 含量明显降低（P＜0.05， P＜0.01）；细胞中 SDC-1 表达显著下调（P＜0.01）。与沉默组比较，沉默+地黄苷 D 组细胞的 xylt-1mRNA 表达 上调，但差异无统计学意义（P＞0.05），沉默+梓醇组细胞的 xylt-1 mRNA 表达明显上调（P＜0.05）；沉默+地黄 苷 D 组、沉默+梓醇组细胞的 SDC-1、EGR1、GAD67 mRNA 表达显著上调（P＜0.05，P＜0.01），细胞内、外 液中的 GABA 含量显著升高（P＜0.01），细胞中 SDC-1 表达显著上调（P＜0.05，P＜0.01）。（3）与正常组及阴性 对照组比较，过表达组细胞的 xylt-1 mRNA 表达显著上调（P＜0.01）。与阴性对照组比较，过表达组细胞的 SDC-1、EGR1、GAD67 mRNA 表达显著上调（P＜0.01），细胞内、外液中的 GABA 含量显著升高（P＜0.01）。 结论 脑内可能存在调节 GABA 合成的 xylt-1/SDC-1/EGR1/GAD67 通路，熟地黄可能通过上调该通路提高脑 内 GABA 水平。

Objective To investigate the regulatory mechanism of Rehmanniae Radix Praeparata and its active components rehmannioside D and catalpol on γ -aminobutyric acid （GABA） in the brain based on the xylosytransferase 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 （10 μmol·L-1） group，silencing + catalpol （10 μmol·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 transfected with lentivirus to overexpress xylt-1 gene. The cells were divided into normal group，negative control group （empty vector group） and overexpression group. The mRNA expression levels of xylt-1，SDC-1，EGR1 and GAD67 in cells were detected by qPCR. Results （1） Compared with the normal group，the body mass of the rats in the model group was significantly decreased （P＜0.01），the activity was significantly increased （P＜0.01），and the number of crossings and the total distance were significantly increased （P＜0.01）. The levels of serum LH，FSH，GnRH，CRH and TRH were significantly increased （P＜0.01）. The content of GABA in hippocampus and cerebral cortex was significantly decreased （P＜ 0.05，P＜0.01）. The expression of xylt-1，SDC-1，EGR1 and GAD67 mRNA （hippocampus，cerebral cortex） and protein （hypothalamus， cerebral cortex） in brain tissue were significantly down-regulated （P＜0.05， P＜ 0.01）. Compared with the model group，the body mass of the rats in the treatment group was significantly increased （P＜0.05），the activity was significantly decreased （P＜0.01），and the total distance was significantly shortened （P＜0.05）. The levels of serum LH，FSH，GnRH，CRH and TRH were significantly decreased （P＜0.01）. The content of GABA in hippocampus and cerebral cortex was significantly increased （P＜0.05，P＜0.01）. The content of GABA in hippocampus and cerebral cortex was significantly increased （P＜0.05，P＜0.01）. The expressions of xylt-1，SDC-1，EGR1 and GAD67 mRNA （hippocampus，cerebral cortex） and protein （hypothalamus，cerebral cortex） in brain tissue were significantly up-regulated （P＜0.05， P＜0.01）. （2） Compared with the negative control group，the mRNA expressions of xylt-1，SDC-1，EGR1 and GAD67 in the silencing group was significantly down-regulated （P＜0.01）. The content of GABA in intracellular and extracellular fluid was significantly decreased （P＜0.05，P＜0.01）. The expression of SDC-1 in cells was significantly down-regulated （P＜0.01）. Compared with the silencing group，the expression of xylt-1 mRNA in the silencing + rehmannioside D group was up-regulated，but the difference was not statistically significant （P＞0.05）. The mRNA expression of xylt-1 in the silencing + catalpol group was significantly up-regulated （P＜0.05）. The mRNA expressions of SDC-1，EGR1 and GAD67 in the silencing + rehmannioside D group and the silencing + catalpol group was significantly up-regulated （P＜0.05， P＜0.01），the content of GABA in the intracellular and extracellular fluid was significantly increased （P＜0.01）， and the expression of SDC-1 in the cells was significantly up-regulated （P＜0.05，P＜0.01）. （3） Compared with the normal group and the negative control group，the mRNA expression of xylt-1 in the overexpression group was significantly up-regulated （P＜0.01）. Compared with the negative control group，the mRNA expressions of SDC-1， EGR1 and GAD67 in the overexpression group was significantly up-regulated （P＜0.01），and the GABA content in the intracellular and extracellular fluid was significantly increased （P＜0.01）. Conclusion There may be a xylt-1/ SDC-1/EGR1/GAD67 pathway that regulates GABA synthesis in the brain. Rehmanniae Radix Praeparata may increase GABA levels in the brain by up-regulating this pathway.

R285.5

广东省科技厅项目（20194141405058）