基于生大黄、酒大黄对ICH大鼠的脑保护作用探讨“酒制升提”理论
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摘要
目的:研究生大黄、酒大黄提取液对脑出血(ICH)模型大鼠脑保护作用的差异,探讨大黄"酒制升提"理论的内涵。方法:Wistar雄性大鼠建立ICH模型,用生大黄、酒大黄提取液进行干预,并随机分成6组(空白组、假手术组、模型组、生大黄组、酒大黄组、安宫牛黄丸组);脱氧核糖核苷酸末端转移酶介导的缺口末端标记(TUNEL)检测海马组织神经细胞凋亡的情况,免疫组化法检测出血侧脑组织紧密连接附着蛋白-1(ZO-1)和血管内皮钙黏蛋白-2(VE-cadherin-2)蛋白表达情况,蛋白免疫印迹试验(Western blot)检测出血侧脑组织还原型辅酶Ⅱ(NADPH)氧化酶2(NOX2)和肿瘤坏死因子-α刺激基因6(TSG-6)蛋白的表达,全自动酶标仪检测ICH大鼠血清中还原型谷胱甘肽(GSH)含量;研究生大黄、酒大黄对ICH大鼠脑保护作用的差异。结果:与假手术组比较,模型组大鼠海马组织神经细胞凋亡指数显著增加(P <0. 01),ZO-1表达量显著下降(P <0. 01),NOX2蛋白表达量显著升高(P <0. 01),TSG-6蛋白表达量显著下降(P <0. 01),GSH的含量显著下降(P <0. 01);同模型组比较,酒大黄、生大黄均能显著降低ICH大鼠海马组织的神经细胞凋亡指数(P <0. 01),均显著升高ZO-1蛋白表达(P <0. 01),升高VE-cadherin-2蛋白表达(P <0. 05),显著降低NOX2蛋白的表达(P <0. 01,P <0. 05),显著升高TSG-6蛋白的表达(P <0. 05),显著升高GSH含量(P <0. 01)。结论:生大黄、酒大黄对ICH大鼠模型均具有脑保护作用,但酒大黄的脑保护作用略优于生大黄,为探讨大黄"酒制升提"理论提供了实验依据。
Objective: To explore the difference of effect of raw and wine-processed products of Rhei Radix et Rhizoma extract on brain protection of rats with intracerebral hemorrhage( ICH) and the connotation of the theory of"wine processing could promote efficacy"of Rhei Radix et Rhizoma. Method: Wistar male rats were used to establish the ICH model,extracts of raw and wine-processed products of Rhei Radix et Rhizoma were used for intervention,and these rats were randomly divided into 6 groups( blank group,sham-operated group,model group,raw Rhei Radix et Rhizoma group,wine-processed Rhei Radix et Rhizoma group and Angong Niuhuangwan group). Then the apoptosis of hippocampal neurons was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling( TUNEL) assay. The expression of zonula occludens-1( ZO-1) and vascular endothelial cadherin-2( VE-cadherin-2) in the hemorrhagic brain tissue was detected by immunohistochemical assay. The expression of reduced form of nicotinamide-adenine dinucleotide phosphate( NADPH) oxidase 2( NOX2) and tumor necrosis factor-α stimulated gene-6( TSG-6) in the hemorrhagic brain tissue was detected by Western blot.The content of glutathione( GSH) in the serum of ICH rats was detected by automatic enzyme-linked immunosorbent assay systems. Through these above methods,we investigated the differences between raw products and wine-processed products on brain protection of ICH rats. Result: Compared with the sham-operated group,the apoptosis index of the hippocampal neurons in the model group were increased significantly( P < 0. 01),the expression of ZO-1 was decreased significantly( P < 0. 01), the expression of NOX2 protein was increased significantly( P < 0. 01),the expression of TSG-6 protein was decreased significantly( P < 0. 01),the content of GSH was also decreased significantly( P < 0. 01). Compared with the model group,raw and wine-processed products of Rhei Radix et Rhizoma could reduce the apoptosis index of hippocampal neurons( P < 0. 01),increase the expression of ZO-1( P < 0. 01) and VE-cadherin-2 protein( P < 0. 05),decrease the expression of NOX2( P < 0. 05,P < 0. 01),increase the expression of TSG-6 protein( P < 0. 05) and increase the content of GSH( P < 0. 01) in ICH rats. Conclusion: The raw and wine-processed products of Rhei Radix et Rhizoma all have brain protective effect on ICH rat model,but the protective effect of wine-processed products is slightly better than that of raw products. The result of this study provides experimental basis for exploring the theory of "wine processing could promote efficacy"of this herb.
Objective: To explore the difference of effect of raw and wine-processed products of Rhei Radix et Rhizoma extract on brain protection of rats with intracerebral hemorrhage( ICH) and the connotation of the theory of"wine processing could promote efficacy"of Rhei Radix et Rhizoma. Method: Wistar male rats were used to establish the ICH model,extracts of raw and wine-processed products of Rhei Radix et Rhizoma were used for intervention,and these rats were randomly divided into 6 groups( blank group,sham-operated group,model group,raw Rhei Radix et Rhizoma group,wine-processed Rhei Radix et Rhizoma group and Angong Niuhuangwan group). Then the apoptosis of hippocampal neurons was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling( TUNEL) assay. The expression of zonula occludens-1( ZO-1) and vascular endothelial cadherin-2( VE-cadherin-2) in the hemorrhagic brain tissue was detected by immunohistochemical assay. The expression of reduced form of nicotinamide-adenine dinucleotide phosphate( NADPH) oxidase 2( NOX2) and tumor necrosis factor-α stimulated gene-6( TSG-6) in the hemorrhagic brain tissue was detected by Western blot.The content of glutathione( GSH) in the serum of ICH rats was detected by automatic enzyme-linked immunosorbent assay systems. Through these above methods,we investigated the differences between raw products and wine-processed products on brain protection of ICH rats. Result: Compared with the sham-operated group,the apoptosis index of the hippocampal neurons in the model group were increased significantly( P < 0. 01),the expression of ZO-1 was decreased significantly( P < 0. 01), the expression of NOX2 protein was increased significantly( P < 0. 01),the expression of TSG-6 protein was decreased significantly( P < 0. 01),the content of GSH was also decreased significantly( P < 0. 01). Compared with the model group,raw and wine-processed products of Rhei Radix et Rhizoma could reduce the apoptosis index of hippocampal neurons( P < 0. 01),increase the expression of ZO-1( P < 0. 01) and VE-cadherin-2 protein( P < 0. 05),decrease the expression of NOX2( P < 0. 05,P < 0. 01),increase the expression of TSG-6 protein( P < 0. 05) and increase the content of GSH( P < 0. 01) in ICH rats. Conclusion: The raw and wine-processed products of Rhei Radix et Rhizoma all have brain protective effect on ICH rat model,but the protective effect of wine-processed products is slightly better than that of raw products. The result of this study provides experimental basis for exploring the theory of "wine processing could promote efficacy"of this herb.
引文
[1]黄竹斋.金匮要略方论集注[M].北京:人民卫生出版社,1957:124.
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[5]李丽,肖永庆.大黄饮片炮制前后物质基础变化规律研究[J].中华中医药杂志,2012,27(4):803-813.
[6]赵楠,张晓哲,胡昌江,等.运用代谢组学技术研究发现炮制引起大黄多成分变化[J].中国中药杂志,2014,39(9):1607-1613.
[7]吴育,彭晓清,姜晓燕,等.酒制对大黄中游离蒽醌在大鼠体内组织分布的影响[J].中国中药杂志,2017,42(8):1603-1608.
[8]杨树升.大承气汤及大黄素对大鼠脑出血的治疗作用及对Nrf2/Src/MAPJs通路的影响[D].武汉:湖北中医药大学,2016.
[9]高志清.大黄保留灌肠治疗急性期脑出血的临床观察[J].内蒙古中医药,2017,36(1):57-58.
[10]袁梦果,李健香,顾恒,等.大黄治疗脑出血的脑保护作用及其机制研究进展[J].中华中医药学刊,2017,35(7):1766-1768.
[11]李跃,孙景波,黄燕.益脑康胶囊对脑梗死大鼠溶栓后血脑屏障的保护作用及对ZO-1蛋白表达的影响[J].中国实验方剂学杂志,2013,19(11):268-271.
[12]樊凯芳,李晓亮,梁晓东,等.三化汤对大鼠脑缺血再灌注后血脑屏障损伤的保护作用[J].中国实验方剂学杂志,2012,18(7):181-184.
[13]许兵,张俞,杜久林.血脑屏障的研究进展[J].生理学报,2016,68(3):306-322.
[14]李东辉.TSG-6对脑出血大鼠血脑屏障的保护作用[J].神经损伤与功能重建,2016,11(6):476-479.
[15]冯晓帆,柳春,赵丹玉,等.苦荞麦总黄酮对软脂酸诱导的脐静脉内皮细胞NOX4蛋白表达的影响[J].中国实验方剂学杂志,2016,22(9):106-110.
[16]戚之琳,刘银华,齐世美,等.红景天苷通过抑制NOX2-ROS-MAPKs信号途径保护H2O2诱导的PC12细胞凋亡[J].南方医科大学学报,2017,37(2):178-183.
[2]王好古.汤液本草[M].北京:人民卫生出版社,1956:33.
[3]曾大安.酒制升提质疑[J].中药通报,1985,10(3):19-20.
[4]张学顺.也谈“酒制升提”[J].中药通报,1987,12(3):154-155.
[5]李丽,肖永庆.大黄饮片炮制前后物质基础变化规律研究[J].中华中医药杂志,2012,27(4):803-813.
[6]赵楠,张晓哲,胡昌江,等.运用代谢组学技术研究发现炮制引起大黄多成分变化[J].中国中药杂志,2014,39(9):1607-1613.
[7]吴育,彭晓清,姜晓燕,等.酒制对大黄中游离蒽醌在大鼠体内组织分布的影响[J].中国中药杂志,2017,42(8):1603-1608.
[8]杨树升.大承气汤及大黄素对大鼠脑出血的治疗作用及对Nrf2/Src/MAPJs通路的影响[D].武汉:湖北中医药大学,2016.
[9]高志清.大黄保留灌肠治疗急性期脑出血的临床观察[J].内蒙古中医药,2017,36(1):57-58.
[10]袁梦果,李健香,顾恒,等.大黄治疗脑出血的脑保护作用及其机制研究进展[J].中华中医药学刊,2017,35(7):1766-1768.
[11]李跃,孙景波,黄燕.益脑康胶囊对脑梗死大鼠溶栓后血脑屏障的保护作用及对ZO-1蛋白表达的影响[J].中国实验方剂学杂志,2013,19(11):268-271.
[12]樊凯芳,李晓亮,梁晓东,等.三化汤对大鼠脑缺血再灌注后血脑屏障损伤的保护作用[J].中国实验方剂学杂志,2012,18(7):181-184.
[13]许兵,张俞,杜久林.血脑屏障的研究进展[J].生理学报,2016,68(3):306-322.
[14]李东辉.TSG-6对脑出血大鼠血脑屏障的保护作用[J].神经损伤与功能重建,2016,11(6):476-479.
[15]冯晓帆,柳春,赵丹玉,等.苦荞麦总黄酮对软脂酸诱导的脐静脉内皮细胞NOX4蛋白表达的影响[J].中国实验方剂学杂志,2016,22(9):106-110.
[16]戚之琳,刘银华,齐世美,等.红景天苷通过抑制NOX2-ROS-MAPKs信号途径保护H2O2诱导的PC12细胞凋亡[J].南方医科大学学报,2017,37(2):178-183.