肝胆湿热动物模型的建立和龙胆泻肝丸治疗肝胆湿热的机理研究
发布时间：2018-07-13 18:01
【摘要】： 肝胆湿热动物模型的建立和龙胆泻肝丸治疗 肝胆湿热的机理研究 肝胆湿热为中医常见证候之一，为湿热蕴结肝胆，使肝胆疏泄失常所致，所涉及现代医学的病种大多数是肝胆系疾病，临床上主要表现身目黄疸，胁肋痛。从现代医学的观点来看，身目黄疸是由于胆汁淤积所致。肝内胆汁淤积(intra-haptic cholistasis，IC)是许多肝病共有的基础病变。虽然传统中医学没有肝内胆汁淤积与之相应的明确病名，但根据其临床症状和体征，应当归属于中医学“黄疸”、“积聚”、“臌胀”等病范畴。湿、热、瘀为其基本病因病机，为肝胆疏泄失常所致。 合适的动物模型是进行疾病发病机理、药物筛选、药效评价及其作用机理研究的重要工具。然而，由于目前缺乏理想的肝胆湿热动物模型，使得有关肝胆湿热的病因病机研究以及治疗肝胆湿热药物的筛选、效果评价以及作用机理研究具有很大的困难。中医临床肝胆湿热常有身目黄疸的客观表现，从现代医学的观点来看，身目黄疸是由于胆汁淤积所致。因此肝胆湿热与现代医学中的胆汁淤积有相似之处，故以诱导胆汁淤积的发生作为建立肝胆湿热模型具有可能性。肝细胞和胆小管细胞膜上具有胆汁成分转运分子，它们与胆汁的形成和分泌有关。各种淤胆性肝损害时，肝细胞膜窦面或胆管面转运体蛋白分子的表达或功能将会受到不同的影响，多药耐药蛋白(MDR)和多药耐药相关蛋白(MRP)是两类与胆汁转运功能密切相关的蛋白。近年来的研究表明，MDR或MRP基因缺陷可能与某些淤胆形成有关。 α-萘异硫氰酸酯(ANIT)在动物体内可转化为有肝毒性的代谢产物产生肝毒性，其诱发动物肝损伤的生物化学和病理形态学改变与人类肝内胆汁淤积病变相似。 龙胆泻肝丸(LD)由龙胆、柴胡、黄芩、栀子、泽泻、木通、车前子、当归、地黄、炙甘草组成。具有清肝胆，利湿热的功用，是临床治疗肝胆湿热证的代表方之一。长期以来，，其在临床上治疗肝胆湿热的效果得到公认，但是，对其作用机理未见深入的研究，因此，其治疗肝胆湿热的作用机理尚不清楚。 本研究将根据中医临床肝胆湿热的主要客观表现，参考现代医学的胆汁淤积的形成机理，建立模拟肝胆湿热的动物模型，并对该模型的发病机理进行探讨。在该模型的基础上研究龙胆泻肝丸的治疗肝胆湿热的作用及其机理。 目的：首先采用ANIT建立模拟中医临床的肝胆湿热模型，探讨该模型在胆汁转运代谢方面的分子机制。以该模型为基础，观察龙胆泻肝丸治疗肝胆湿热的作用机理，探讨作用靶点，从而为龙胆泻肝丸治疗肝胆湿热提供科学依据。 方法： 实验一、大鼠肝胆湿热模型的建立 将10只大鼠分为对照组和模型组，每组5只。模型组按100mg／kg的剂量一次性灌胃给予α-萘异硫氰酸酯(ANIT)花生油，对照组灌胃同体积花生油。造模后72h，大鼠一一行胆管插管术。收集4 h内的胆汁，按照收集的时间段计算胆汁分泌量。造模后76h检测血清总胆红素(TBIL)、直接胆红素(DBIL)、丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、碱性磷酸酶(ALP)、总胆固醇(CHO)水平以及肝组织病理变化。此外，将另外5只大鼠分别于灌胃ANIT后12h、24h、48h、72h、96h分别处死一只。另取1只作为正常对照。分别于不同时间点，取出肝脏，提取肝组织总RNA，用RT-PCR方法检测MDR1b及MRP2 mRNA的表达。 实验二、观察龙胆泻肝丸(LD)对肝胆湿热模型大鼠和小鼠的清利肝胆作用 将72只大鼠分为6组，分别为对照组，模型组，胆乐胶囊(DL)组和LD 10、5.0、2.5g生药／kg剂量组。LD各剂量组和DL组每日给药1次，连续给药8天。对照组灌胃同体积花生油。于给药期间的第5天，除了对照组外，其余各组在给药后间隔1 h再灌胃给予ANIT 100 mg／kg(仅一次)。于第8天末次给药后30 min(末次给药前禁食24 h，不禁水)，行胆管插管，收集4 h内的胆汁，按照收集的时间段计算胆汁分泌量。并检测血清AST、ALT、ALP、TBIL、DBIL、CHO、丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽(GSH)水平以及肝组织病理变化。另外72只小鼠按同样方法复制模型，最后检测血清TBIL、DBIL水平。 实验三、观察龙胆泻肝丸对肝胆湿热大鼠MDR1a、MDR1b、MDR2、MRP1、MRP2表达的影响 将20只大鼠分为4组，分别为对照组、模型组、LD 5.0、2.5 g生药／kg剂量组。给药组均于动物处死前8天开始给药，每日灌胃给药1次，连续8天。对照组和模型组灌胃同体积的蒸馏水。其中第5天给药1h后，除对照组给予花生油灌胃外，其他各组大鼠按100mg／kg一次性灌胃ANIT花生油。第8天取出肝脏。提取总RNA，RT-PCR检测MDR1a、MDR1b、MDR2、MRP1、MRP2 mRNA的表达。 实验四、观察龙胆泻肝丸对肝胆湿热大鼠中性粒细胞CD18表达的影响 将20只大鼠分为4组，分别为对照组、模型组、LD 5.0、2.5 g生药／kg。给药组均于动物处死前8天开始给药，每日灌胃给药1次，连续8天。对照组和模型组灌胃同体积的蒸馏水。其中第5天给药1h后，除对照组花生油灌胃外，其他各组大鼠按100mg／kg一次性灌胃ANIT花生油。第8天眼眶静脉取100μl抗凝血，加入PE标记的CD18抗体，流式细胞仪测中性粒细胞CD18表达，另取血20μl血细胞计数仪测白细胞及粒细胞总数。 结果： 实验一：大鼠肝胆湿热模型的建立 结果显示，模型组给予ANIT 72h后，大鼠胆汁分泌量在胆管插管后4h内一直显著低于对照组(P0.05或P0.01)，而血清TBIL、DBIL、CHO、ALT、AST、ALP水平明显高于对照组。组织形态学观察可见模型组出现肝细胞坏死和胆管损伤。对照组大鼠MDR1b mRNA无明显表达，MRP2 mRNA只有较弱表达。模型组大鼠在造模后48h～96h，MDR1b mRNA表达明显增强，但在造模后24h内表达不明显。造模后12h、24h，MRP2 mRNA表达较弱，而在造模后48h～96h均表达明显。 实验二：龙胆泻肝丸(LD)对肝胆湿热模型大鼠和小鼠的清利肝胆作用 1．胆汁分泌 结果显示，模型组的胆汁分泌量较对照组显著降低(P0.05)。LD各给药组胆汁分泌量不同程度地高于模型组。LD 5.0g生药／kg组、LD2.5g生药／kg组在1h和1.5h时间点的胆汁排泌量较模型组显著增高(P0.05)，其它时间点也有增高趋势，但无统计学意义。 2．血清TBIL、DBIL、CHO水平 模型组大鼠血清TBIL、DBIL、CHO水平均上升，与对照组比较有明显差异(P0.01)。而LD 5.0g生药／kg组、LD2.5g生药／kg组血清TBIL、DBIL、CHO与模型组比较有降低趋势，但无统计学意义。 3．血清ALT、AST、ALP 模型组大鼠血清ALT、AST、ALP升高，与对照组比较有明显差异(P0.01)。LD各剂量组的血清ALT、AST、ALP水平均与模型组比较无差异。 4．血清MDA、SOD、GSH水平 模型组大鼠血清MDA水平升高(与对照组比较P0.01)，血清SOD及GSH水平与对照组比较无明显变化。LD各剂量组血清SOD、GSH水平与模型组比较无明显差异。LD对血清MDA有降低趋势(与对照组比较P0.05)。 5．肝脏组织形态学 各给药组肝细胞坏死不同程度的轻于模型组，其中LD 2.5g生药／kg组分别与模型组相比差异显著(P0.01)。 实验三：龙胆泻肝丸对肝胆湿热大鼠MDR1a、MDR1b、MDR2、MRP1、MRP2表达的影响 1．肝组织MDR1a、MDR1b、MDR 2 mRNA表达 模型组大鼠一次性灌胃ANIT 72h后，肝组织MDR1a、MDR1b mRNA表达显著增加(P0.05，P0.01)。MDR2 mRNA也增加，但未达统计学意义。LD 5.0g生药／kg和LD 2.5g生药／kg对肝胆湿热模型大鼠的肝组织MDR1a、MDR1b、MDR2 mRNA表达无明显影响。 2．肝组织MRP1 mRNA和MRP2 mRNA表达 模型组大鼠一次性灌胃ANIT 72h后，MRP2 mRNA表达下降，但与对照组相比无统计学意义。MRP1 mRNA的表达未见明显变化(P0.05)。LD 5.0g生药／kg和LD 2.5g生药／kg对肝胆湿热模型大鼠的肝组织MRP1、MRP2 mRNA表达无明显影响。 实验四：龙胆泻肝丸对肝胆湿热大鼠中性粒细胞CD18表达的影响 结果显示，LD各给药组白细胞总数以及粒细胞总数有下降趋势，CD18荧光强度明显低于模型组。 结论：通过以上实验，我们认为，通过灌胃ANIT，所建立的模型具有肝内、体内胆汁淤积，肝酶水平增高，肝细胞损伤等病理生理特征，与中医临床上以身目黄疸为主要表现的肝胆湿热证类似，可以作为肝胆湿热模型。该模型的肝脏组织中MDR及MRP mRNA表达发生变化，而MDR及MRP是与胆汁转运代谢有关的分子。说明MDR及MRP参与该肝胆湿热模型的形成。龙胆泻肝丸能增加胆汁流量，加速胆汁排出，但是对胆汁转运代谢分子MDR1a、MDR1b、MDR2、MRP1、MRP2等均无显著影响。结果提示，龙胆泻肝丸治疗肝胆湿热的作用机制可能与增加毛细胆管胆汁流量、减轻脂质过氧化反应、稳定细胞膜、阻抑CD11／CD18介导的中性粒细胞-内皮细胞粘附有关。
[Abstract]:Establishment of animal model of liver and gallbladder damp heat and treatment of long Dan Xie Gan Wan
Study on the mechanism of liver and gallbladder dampness and heat
The liver and gallbladder damp heat is one of the common syndromes in traditional Chinese medicine. It is caused by the damp heat and the liver and gallbladder caused by the liver and gallbladder. Most of the diseases involved in modern medicine are hepatobiliary diseases. The main manifestations of the disease are jaundice and rib pain in the clinic. From the view of modern medicine, the jaundice is caused by cholestasis. Intrahepatic cholestasis (intra-haptic Chol). Istasis, IC) is a common basic pathological change of many liver diseases. Although traditional Chinese medicine does not have a definite name for intrahepatic cholestasis, it should belong to the category of "jaundice", "accumulation", "distension", etc. according to its clinical symptoms and signs. Wet, heat, and blood stasis are the basic pathogenesis of the TCM, which is caused by the disorder of the liver and gallbladder.
The appropriate animal model is an important tool for disease pathogenesis, drug screening, drug efficacy evaluation and its mechanism of action. However, due to the lack of ideal liver and bile hot and hot animal models, the etiology and pathogenesis of liver and gallbladder damp heat, the screening of liver and gallbladder damp heat drugs, the effect evaluation and the mechanism of action are studied. There is a great difficulty. The objective manifestation of jaundice in the TCM clinical liver and gallbladder is often characterized by jaundice. From the point of view of modern medicine, the jaundice is caused by the cholestasis. Therefore, the liver and gallbladder damp heat is similar to the cholestasis in modern medicine. Therefore, it is possible to induce the occurrence of cholestasis as a model of establishing the liver and gallbladder damp heat model. The cell and bile duct cell membranes have bile component transporters, which are related to the formation and secretion of bile. In all kinds of cholestatic liver damage, the expression or function of the protein molecules of the hepatic cell membrane sinus or the bile duct surface transporter will be affected differently. The multidrug resistance protein (MDR) and the multidrug resistance associated protein (MRP) are the two and the bile. Recent studies have shown that MDR or MRP gene defects may be associated with some cholestasis.
Alpha naphthyl thiocyanate (ANIT) can be transformed into a hepatotoxic metabolite in animals to produce liver toxicity. The biochemical and pathomorphological changes in animal liver injury are similar to those of human intrahepatic cholestasis.
LD is composed of gentian, bupleurum, Radix Scutellariae, Scutellaria, gardenia, gardenia, Alisma orientalis, wood Tong, Radix Angelicae, Radix Angelicae, Radix Rehmanniae and licorice. It is one of the representatives of liver and gallbladder damp heat syndrome, which has long been recognized in clinical treatment of liver and gallbladder damp heat. However, the mechanism of its action has not been studied deeply. Therefore, the mechanism of its treatment of liver and gallbladder damp heat is not clear.
This study will be based on the main objective manifestation of the liver and gallbladder damp heat in traditional Chinese medicine, and refer to the formation mechanism of the cholestasis of modern medicine, establish an animal model to simulate the damp heat of the liver and gallbladder, and discuss the pathogenesis of the model. On the basis of this model, the effect and mechanism of the treatment of the damp heat of the liver and gallbladder by the long gentian Xie Gan pill are studied.
Objective: first, to establish the liver and gallbladder damp heat model with ANIT, and to explore the molecular mechanism of the model in the bile transport and metabolism. Based on this model, we observe the mechanism of the action of long gentian Xie Gan pill to treat the damp heat of the liver and gallbladder, and explore the target target, thus providing a scientific basis for the treatment of the liver and gallbladder damp heat.
Method:
Experiment 1, establishment of rat liver and gallbladder damp heat model
10 rats were divided into the control group and the model group, with 5 rats in each group. The model group was given the alpha naphthyl thiocyanate (ANIT) peanut oil at the dose of 100mg / kg, and the control group was given the same volume of peanut oil. The bile duct intubation in the rats was performed one by one after the model 72h. The bile in 4 h was collected and the amount of bile secretion was calculated according to the time period of collection. After the model, 76 of the bile was calculated. H detected serum total bilirubin (TBIL), direct bilirubin (DBIL), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total cholesterol (CHO) level and pathological changes of liver tissue. In addition, the other 5 rats were separated from the gastric ANIT 12h, 24h, 48H, 72h, and another 1 were taken as normal. The liver was extracted at different time points, and the total RNA of liver tissue was extracted. The expression of MDR1b and MRP2 mRNA was detected by RT-PCR.
Experiment two, to observe the effect of long Dan Xie Gan pill (LD) on liver and gallbladder dampness heat rats and mice.
72 rats were divided into 6 groups: the control group, the model group, the DL group and the LD 10,5.0,2.5g drug / kg dose group, each dose group and the DL group 1 times daily and the continuous administration of the drug for 8 days. The control group was given the same volume of peanut oil. In addition to the control group, the other groups were given ANIT at the interval of 1 h after the administration, except the control group. 100 mg / kg (only once). At the end of the eighth day, 30 min after the last dose (before the last dose of 24 h, can not help water), bile duct intubation, collect bile in 4 h, and calculate the bile secretion according to the time period of collection. And detect serum AST, ALT, ALP, TBIL, DBIL, CHO, malondialdehyde (MDA), superoxide dismutase, glutathione level and liver tissue The other 72 mice were duplicated by the same method, and the serum levels of TBIL and DBIL were detected finally.
Experiment three, to observe the effect of long Dan Xie Gan Pill on expression of MDR1a, MDR1b, MDR2, MRP1 and MRP2 in rats with liver and gallbladder damp heat.
20 rats were divided into 4 groups: the control group, the model group, the LD 5.0,2.5 g drug / kg dose group. The administration group was given the medicine 8 days before the death of the animals, and the medicine was administered daily for 8 days for 8 days. The control group and the model group were given the same volume of distilled water. After the medicine was given to the control group, the other groups were given the peanut oil and the other groups were given to the stomach. ANIT peanut oil was administered once a day by 100mg / kg. The liver was extracted on the eighth day. Total RNA was extracted. RT-PCR was used to detect MDR1a, MDR1b, MDR2, MRP1, MRP2 mRNA expression.
Experiment four, to observe the effect of long Dan Xie Gan Pill on the expression of CD18 in neutrophils of rats with liver and gallbladder dampness heat.
20 rats were divided into 4 groups: the control group, the model group, the LD 5.0,2.5 g drug / kg. administration group were given the medicine 8 days before the animals were executed, and the medicine was administered daily for 8 days for 8 days. The control group and the model group were given the same volume of distilled water. Among the fifth days after the drug was given to the peanut oil, the rats in the other groups were treated with 100mg / kg one. ANIT peanut oil was intragastric intragastric. Eighth day eye orbital vein was treated with 100 L anticoagulant, CD18 antibody labeled with PE, CD18 expression of neutrophils was measured by flow cytometry, and the total number of leukocyte and granulocyte was measured by blood cell counting instrument of 20 micron L.
Result:
Experiment 1: establishment of rat liver and gallbladder damp heat model
The results showed that after the model group was given ANIT 72h, the bile secretion of the rats was significantly lower than the control group (P0.05 or P0.01) after the bile duct intubation, while the serum TBIL, DBIL, CHO, ALT, AST, ALP levels were significantly higher than those of the control group. The histopathological observation showed that the hepatocyte necrosis and bile duct injury were found in the model group. No obvious table of the rats in the control group was found. The expression of MRP2 mRNA was only weak. The expression of 48h to 96h and MDR1b mRNA in the model group were obviously enhanced, but the expression in 24h was not obvious after the model building. The expression of 12h, 24h, MRP2 mRNA was weak after model building.
Experiment two: the effect of long Dan Xie Gan pill (LD) on liver and gallbladder dampness heat rats and mice.
1. bile secretion
The results showed that the bile secretion of the model group was significantly lower than that of the control group (P0.05) the bile secretion of each group in.LD was higher than that of the model group.LD 5.0g raw drug / kg group. The bile excretion of the LD2.5g raw drug / kg group at 1H and 1.5h time points was significantly higher than that in the model group (P0.05), and the other time points also increased, but there was no statistical significance. Righteousness.
2. serum TBIL, DBIL, CHO levels
The serum levels of TBIL, DBIL and CHO in the model group were all increased, compared with the control group (P0.01), while LD 5.0g / kg group, LD2.5g raw drug / kg group serum TBIL, DBIL, CHO and model group had a lower trend, but no statistical significance.
3. serum ALT, AST, ALP
The serum levels of ALT, AST and ALP in the model group were higher than those in the control group (P0.01) the serum ALT, AST and ALP levels of all.LD groups were not different from those in the model group.
4. serum MDA, SOD, GSH levels
The level of serum MDA in the model group was increased (compared with that of the control group P0.01). The serum SOD and GSH levels were not significantly different from the control group. There was no significant difference in the serum SOD between the.LD and the.LD groups. There was no significant difference in the GSH level between the model group and the model group (.LD against the control group) (compared with the control group, P0.05).
5. histomorphology of liver
The necrosis of hepatocytes in each drug group was lighter than that in the model group, and there was significant difference in the LD 2.5G crude drug / kg group compared with the model group (P0.01).
Experiment three: effects of long Dan Xie Gan Wan on expression of MDR1a, MDR1b, MDR2, MRP1 and MRP2 in rats with liver and gallbladder dampness heat
1. expression of MDR1a, MDR1b, and MDR 2 mRNA in liver tissue
The expression of MDR1a and MDR1b mRNA in the liver tissue was significantly increased (P0.05, P0.01).MDR2 mRNA also increased in model group rats after ANIT 72h, but there was no significant effect on the expression of the liver tissues of the rats of the liver and gallbladder damp heat model.
Expression of MRP1 mRNA and MRP2 mRNA in 2. liver tissues
The expression of MRP2 mRNA decreased in the rat model group after ANIT 72h, but there was no significant difference in.MRP1 mRNA expression compared with the control group (P0.05).LD 5.0g new drug / kg and LD 2.5G, and there was no obvious effect on the liver tissues of the rat model of liver and gallbladder damp heat.
Experiment four: the effect of long Dan Xie Gan Pill on the expression of CD18 in neutrophils of rats with liver and gallbladder dampness heat
The results showed that the total number of leukocytes and the total number of granulocytes in each group of LD decreased, and the fluorescence intensity of CD18 was significantly lower than that of the model group.
Conclusion: through the above experiments, we think that the model established by gavage of ANIT has the pathological and physiological characteristics such as liver, cholestasis in the body, the increase of liver enzyme and liver cell injury. It is similar to the liver and gallbladder damp heat syndrome, which is the main manifestation of jaundice in the clinic of traditional Chinese medicine. It can be used as the liver and gallbladder damp heat model. In this model, the liver tissue is MDR And the expression of MRP mRNA changes, and MDR and MRP are related to the bile transport and metabolism. It shows that MDR and MRP are involved in the formation of the liver and gallbladder damp heat model. The long gentian Xie Gan pill can increase the bile flow and accelerate the bile discharge, but it has no significant influence on the bile transport and metabolic molecules MDR1a, MDR1b, MDR2, MRP1, MRP2 and so on. The mechanism of treatment of liver and gallbladder damp heat may be related to increasing the bile flow of capillary bile duct, reducing lipid peroxidation, stabilizing cell membrane and blocking the adhesion of CD11 / CD18 mediated neutrophil endothelial cell adhesion.
【学位授予单位】：中国中医科学院
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R-332;R285.5
本文编号：2120286
[Abstract]:Establishment of animal model of liver and gallbladder damp heat and treatment of long Dan Xie Gan Wan
Study on the mechanism of liver and gallbladder dampness and heat
The liver and gallbladder damp heat is one of the common syndromes in traditional Chinese medicine. It is caused by the damp heat and the liver and gallbladder caused by the liver and gallbladder. Most of the diseases involved in modern medicine are hepatobiliary diseases. The main manifestations of the disease are jaundice and rib pain in the clinic. From the view of modern medicine, the jaundice is caused by cholestasis. Intrahepatic cholestasis (intra-haptic Chol). Istasis, IC) is a common basic pathological change of many liver diseases. Although traditional Chinese medicine does not have a definite name for intrahepatic cholestasis, it should belong to the category of "jaundice", "accumulation", "distension", etc. according to its clinical symptoms and signs. Wet, heat, and blood stasis are the basic pathogenesis of the TCM, which is caused by the disorder of the liver and gallbladder.
The appropriate animal model is an important tool for disease pathogenesis, drug screening, drug efficacy evaluation and its mechanism of action. However, due to the lack of ideal liver and bile hot and hot animal models, the etiology and pathogenesis of liver and gallbladder damp heat, the screening of liver and gallbladder damp heat drugs, the effect evaluation and the mechanism of action are studied. There is a great difficulty. The objective manifestation of jaundice in the TCM clinical liver and gallbladder is often characterized by jaundice. From the point of view of modern medicine, the jaundice is caused by the cholestasis. Therefore, the liver and gallbladder damp heat is similar to the cholestasis in modern medicine. Therefore, it is possible to induce the occurrence of cholestasis as a model of establishing the liver and gallbladder damp heat model. The cell and bile duct cell membranes have bile component transporters, which are related to the formation and secretion of bile. In all kinds of cholestatic liver damage, the expression or function of the protein molecules of the hepatic cell membrane sinus or the bile duct surface transporter will be affected differently. The multidrug resistance protein (MDR) and the multidrug resistance associated protein (MRP) are the two and the bile. Recent studies have shown that MDR or MRP gene defects may be associated with some cholestasis.
Alpha naphthyl thiocyanate (ANIT) can be transformed into a hepatotoxic metabolite in animals to produce liver toxicity. The biochemical and pathomorphological changes in animal liver injury are similar to those of human intrahepatic cholestasis.
LD is composed of gentian, bupleurum, Radix Scutellariae, Scutellaria, gardenia, gardenia, Alisma orientalis, wood Tong, Radix Angelicae, Radix Angelicae, Radix Rehmanniae and licorice. It is one of the representatives of liver and gallbladder damp heat syndrome, which has long been recognized in clinical treatment of liver and gallbladder damp heat. However, the mechanism of its action has not been studied deeply. Therefore, the mechanism of its treatment of liver and gallbladder damp heat is not clear.
This study will be based on the main objective manifestation of the liver and gallbladder damp heat in traditional Chinese medicine, and refer to the formation mechanism of the cholestasis of modern medicine, establish an animal model to simulate the damp heat of the liver and gallbladder, and discuss the pathogenesis of the model. On the basis of this model, the effect and mechanism of the treatment of the damp heat of the liver and gallbladder by the long gentian Xie Gan pill are studied.
Objective: first, to establish the liver and gallbladder damp heat model with ANIT, and to explore the molecular mechanism of the model in the bile transport and metabolism. Based on this model, we observe the mechanism of the action of long gentian Xie Gan pill to treat the damp heat of the liver and gallbladder, and explore the target target, thus providing a scientific basis for the treatment of the liver and gallbladder damp heat.
Method:
Experiment 1, establishment of rat liver and gallbladder damp heat model
10 rats were divided into the control group and the model group, with 5 rats in each group. The model group was given the alpha naphthyl thiocyanate (ANIT) peanut oil at the dose of 100mg / kg, and the control group was given the same volume of peanut oil. The bile duct intubation in the rats was performed one by one after the model 72h. The bile in 4 h was collected and the amount of bile secretion was calculated according to the time period of collection. After the model, 76 of the bile was calculated. H detected serum total bilirubin (TBIL), direct bilirubin (DBIL), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total cholesterol (CHO) level and pathological changes of liver tissue. In addition, the other 5 rats were separated from the gastric ANIT 12h, 24h, 48H, 72h, and another 1 were taken as normal. The liver was extracted at different time points, and the total RNA of liver tissue was extracted. The expression of MDR1b and MRP2 mRNA was detected by RT-PCR.
Experiment two, to observe the effect of long Dan Xie Gan pill (LD) on liver and gallbladder dampness heat rats and mice.
72 rats were divided into 6 groups: the control group, the model group, the DL group and the LD 10,5.0,2.5g drug / kg dose group, each dose group and the DL group 1 times daily and the continuous administration of the drug for 8 days. The control group was given the same volume of peanut oil. In addition to the control group, the other groups were given ANIT at the interval of 1 h after the administration, except the control group. 100 mg / kg (only once). At the end of the eighth day, 30 min after the last dose (before the last dose of 24 h, can not help water), bile duct intubation, collect bile in 4 h, and calculate the bile secretion according to the time period of collection. And detect serum AST, ALT, ALP, TBIL, DBIL, CHO, malondialdehyde (MDA), superoxide dismutase, glutathione level and liver tissue The other 72 mice were duplicated by the same method, and the serum levels of TBIL and DBIL were detected finally.
Experiment three, to observe the effect of long Dan Xie Gan Pill on expression of MDR1a, MDR1b, MDR2, MRP1 and MRP2 in rats with liver and gallbladder damp heat.
20 rats were divided into 4 groups: the control group, the model group, the LD 5.0,2.5 g drug / kg dose group. The administration group was given the medicine 8 days before the death of the animals, and the medicine was administered daily for 8 days for 8 days. The control group and the model group were given the same volume of distilled water. After the medicine was given to the control group, the other groups were given the peanut oil and the other groups were given to the stomach. ANIT peanut oil was administered once a day by 100mg / kg. The liver was extracted on the eighth day. Total RNA was extracted. RT-PCR was used to detect MDR1a, MDR1b, MDR2, MRP1, MRP2 mRNA expression.
Experiment four, to observe the effect of long Dan Xie Gan Pill on the expression of CD18 in neutrophils of rats with liver and gallbladder dampness heat.
20 rats were divided into 4 groups: the control group, the model group, the LD 5.0,2.5 g drug / kg. administration group were given the medicine 8 days before the animals were executed, and the medicine was administered daily for 8 days for 8 days. The control group and the model group were given the same volume of distilled water. Among the fifth days after the drug was given to the peanut oil, the rats in the other groups were treated with 100mg / kg one. ANIT peanut oil was intragastric intragastric. Eighth day eye orbital vein was treated with 100 L anticoagulant, CD18 antibody labeled with PE, CD18 expression of neutrophils was measured by flow cytometry, and the total number of leukocyte and granulocyte was measured by blood cell counting instrument of 20 micron L.
Result:
Experiment 1: establishment of rat liver and gallbladder damp heat model
The results showed that after the model group was given ANIT 72h, the bile secretion of the rats was significantly lower than the control group (P0.05 or P0.01) after the bile duct intubation, while the serum TBIL, DBIL, CHO, ALT, AST, ALP levels were significantly higher than those of the control group. The histopathological observation showed that the hepatocyte necrosis and bile duct injury were found in the model group. No obvious table of the rats in the control group was found. The expression of MRP2 mRNA was only weak. The expression of 48h to 96h and MDR1b mRNA in the model group were obviously enhanced, but the expression in 24h was not obvious after the model building. The expression of 12h, 24h, MRP2 mRNA was weak after model building.
Experiment two: the effect of long Dan Xie Gan pill (LD) on liver and gallbladder dampness heat rats and mice.
1. bile secretion
The results showed that the bile secretion of the model group was significantly lower than that of the control group (P0.05) the bile secretion of each group in.LD was higher than that of the model group.LD 5.0g raw drug / kg group. The bile excretion of the LD2.5g raw drug / kg group at 1H and 1.5h time points was significantly higher than that in the model group (P0.05), and the other time points also increased, but there was no statistical significance. Righteousness.
2. serum TBIL, DBIL, CHO levels
The serum levels of TBIL, DBIL and CHO in the model group were all increased, compared with the control group (P0.01), while LD 5.0g / kg group, LD2.5g raw drug / kg group serum TBIL, DBIL, CHO and model group had a lower trend, but no statistical significance.
3. serum ALT, AST, ALP
The serum levels of ALT, AST and ALP in the model group were higher than those in the control group (P0.01) the serum ALT, AST and ALP levels of all.LD groups were not different from those in the model group.
4. serum MDA, SOD, GSH levels
The level of serum MDA in the model group was increased (compared with that of the control group P0.01). The serum SOD and GSH levels were not significantly different from the control group. There was no significant difference in the serum SOD between the.LD and the.LD groups. There was no significant difference in the GSH level between the model group and the model group (.LD against the control group) (compared with the control group, P0.05).
5. histomorphology of liver
The necrosis of hepatocytes in each drug group was lighter than that in the model group, and there was significant difference in the LD 2.5G crude drug / kg group compared with the model group (P0.01).
Experiment three: effects of long Dan Xie Gan Wan on expression of MDR1a, MDR1b, MDR2, MRP1 and MRP2 in rats with liver and gallbladder dampness heat
1. expression of MDR1a, MDR1b, and MDR 2 mRNA in liver tissue
The expression of MDR1a and MDR1b mRNA in the liver tissue was significantly increased (P0.05, P0.01).MDR2 mRNA also increased in model group rats after ANIT 72h, but there was no significant effect on the expression of the liver tissues of the rats of the liver and gallbladder damp heat model.
Expression of MRP1 mRNA and MRP2 mRNA in 2. liver tissues
The expression of MRP2 mRNA decreased in the rat model group after ANIT 72h, but there was no significant difference in.MRP1 mRNA expression compared with the control group (P0.05).LD 5.0g new drug / kg and LD 2.5G, and there was no obvious effect on the liver tissues of the rat model of liver and gallbladder damp heat.
Experiment four: the effect of long Dan Xie Gan Pill on the expression of CD18 in neutrophils of rats with liver and gallbladder dampness heat
The results showed that the total number of leukocytes and the total number of granulocytes in each group of LD decreased, and the fluorescence intensity of CD18 was significantly lower than that of the model group.
Conclusion: through the above experiments, we think that the model established by gavage of ANIT has the pathological and physiological characteristics such as liver, cholestasis in the body, the increase of liver enzyme and liver cell injury. It is similar to the liver and gallbladder damp heat syndrome, which is the main manifestation of jaundice in the clinic of traditional Chinese medicine. It can be used as the liver and gallbladder damp heat model. In this model, the liver tissue is MDR And the expression of MRP mRNA changes, and MDR and MRP are related to the bile transport and metabolism. It shows that MDR and MRP are involved in the formation of the liver and gallbladder damp heat model. The long gentian Xie Gan pill can increase the bile flow and accelerate the bile discharge, but it has no significant influence on the bile transport and metabolic molecules MDR1a, MDR1b, MDR2, MRP1, MRP2 and so on. The mechanism of treatment of liver and gallbladder damp heat may be related to increasing the bile flow of capillary bile duct, reducing lipid peroxidation, stabilizing cell membrane and blocking the adhesion of CD11 / CD18 mediated neutrophil endothelial cell adhesion.
【学位授予单位】：中国中医科学院
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R-332;R285.5
【引证文献】
相关期刊论文 前1条
1 钱静娟;刘霞英;华忠;周秀琳;徐满琴;张海燕;;银翘漱口液在慢性乙型肝炎肝胆湿热证患者口腔管理中的应用[J];现代中西医结合杂志;2013年05期
本文编号：2120286
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