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目的:观察黄连解毒汤水提物及3个有效部位(总生物碱、总黄酮、总环烯醚萜)对中动脉栓塞(MCAO)大鼠丘脑的保护作用。方法:线栓法建立MCAO大鼠模型,雄性SD大鼠随机分成假手术组、模型组、黄连解毒汤水提物(800 mg·kg-1)、总生物碱组(44 mg·kg-1)、总黄酮组(50 mg·kg-1)、总环烯醚萜组(80 mg·kg-1),连续ig给药7 d,每天给药1次。HE染色观察丘脑病理学改变;高效液相-色谱荧光法检测丘脑谷氨酸(Glu)和γ-氨基丁酸(GABA)含量;免疫荧光单标染色法检测丘脑谷氨酸脱羧酶(GAD65)的表达;免疫荧光双标染色法检测星形胶质细胞纤维酸性蛋白(glial fibrillary acidic protein,GFAP)、缝隙连接蛋白43(Connexin43,Cx43)表达。结果:中动脉栓塞7 d大鼠丘脑部分神经元变性,和假手术组相比,丘脑Glu,GABA含量降低(P<0.05),GAD65表达减弱(P<0.05),GFAP,Cx43表达增强(P<0.01)。黄连解毒汤水提取物及3个部位均可不同程度减轻丘脑神经元变性程度。总黄酮可明显上调丘脑GAD65表达,降低GFAP,Cx43蛋白表达,差异较模型组明显(P<0.01),促进丘脑Glu,GABA含量恢复至正常水平。黄连解毒汤水提取物、总生物碱组、总环烯醚萜组大鼠丘脑Cx43蛋白表达较模型组明显上调(P<0.01),水提物组大鼠GAD65表达也较模型组明显增高(P<0.05)。黄连解毒汤水提取物、总生物碱组、总环烯醚萜组大鼠丘脑GFAP蛋白表达较模型组没有显著性差异,GABA含量明显低于假手术组(P<0.05)。结论:中动脉栓塞后,丘脑可出现神经细胞变性,递质氨基酸含量减少,星形胶质细胞异常活化,GFAP,Cx43蛋白表达异常。黄连解毒汤总黄酮可减轻脑缺血后丘脑神经细胞损伤,其作用与抑制星形胶质细胞异常活化,调节GFAP,GAD65蛋白表达关系密切。
Objective: To observe the protective effects of water extract of Huanglian Jiedu Decoction and three effective sites (total alkaloids, total flavonoids, total iridoid) on the thalamus of rats with middle cerebral artery occlusion (MCAO). Methods: MCAO rat model was established by thread occlusion. Male Sprague-Dawley rats were randomly divided into sham operation group, model group, water extract of Huanglian Jiedu Decoction (800 mg · kg -1), total alkaloid group (44 mg · kg -1) , Total flavonoids group (50 mg · kg -1) and total iridoid group (80 mg · kg -1) were administrated once daily for 7 days. The pathological changes of thalamus were observed by HE staining. The levels of glutamate (Glu) and γ-aminobutyric acid (GABA) in the thalamus were detected by high performance liquid chromatography-fluorescence spectroscopy. The content of glutamate decarboxylase (GAD65) The expression of glial fibrillary acidic protein (GFAP) and connexin43 (Cx43) were detected by immunofluorescence double staining. Results: The content of Glu and GABA in the hypothalamus decreased (P <0.05), the expression of GAD65 decreased (P <0.05), the expression of GFAP and Cx43 increased (P <0.01). Coptis decoction soup extract and three parts can reduce the degree of thalamic neuron degeneration to varying degrees. Total flavonoids significantly increased the expression of GAD65 in the thalamus and decreased the expression of GFAP and Cx43, which was significantly higher than that in the model group (P <0.01), and promoted the levels of Glu and GABA in the thalamus to return to normal levels. The expression of Cx43 protein in the hypothalamus of rats treated with Huanglian Jiedu decoction, total alkaloids group and total iridoid group was significantly higher than that of model group (P <0.01), and the expression of GAD65 in water extract group was significantly higher than that of model group (P <0.05). The expression of GFAP protein in the hypothalamus of Coptis chinensis decoction soup, total alkaloid group and total iridoid group was not significantly different from that of the model group, and the content of GABA was significantly lower than that of the sham operation group (P <0.05). CONCLUSION: After thalamic embolization, the thalamus may degenerate, the content of amino acids in the transmitter decreases, the astrocytes activate abnormally, and the expressions of GFAP and Cx43 are abnormal. The total flavonoids of Coptis chinensis decoction can relieve the damage of thalamic neurons after cerebral ischemia, and its effect is related to inhibiting the activation of astrocytes and regulating the expression of GFAP and GAD65.