【摘要】： 肝膽濕熱動物模型的建立和龍膽瀉肝丸治療 肝膽濕熱的機理研究 肝膽濕熱為中醫(yī)常見證候之一，為濕熱蘊結(jié)肝膽，使肝膽疏泄失常所致，所涉及現(xiàn)代醫(yī)學的病種大多數(shù)是肝膽系疾病，臨床上主要表現(xiàn)身目黃疸，脅肋痛。從現(xiàn)代醫(yī)學的觀點來看，身目黃疸是由于膽汁淤積所致。肝內(nèi)膽汁淤積(intra-haptic cholistasis，IC)是許多肝病共有的基礎(chǔ)病變。雖然傳統(tǒng)中醫(yī)學沒有肝內(nèi)膽汁淤積與之相應(yīng)的明確病名，但根據(jù)其臨床癥狀和體征，應(yīng)當歸屬于中醫(yī)學“黃疸”、“積聚”、“臌脹”等病范疇。濕、熱、瘀為其基本病因病機，為肝膽疏泄失常所致。 合適的動物模型是進行疾病發(fā)病機理、藥物篩選、藥效評價及其作用機理研究的重要工具。然而，由于目前缺乏理想的肝膽濕熱動物模型，使得有關(guān)肝膽濕熱的病因病機研究以及治療肝膽濕熱藥物的篩選、效果評價以及作用機理研究具有很大的困難。中醫(yī)臨床肝膽濕熱常有身目黃疸的客觀表現(xiàn)，從現(xiàn)代醫(yī)學的觀點來看，身目黃疸是由于膽汁淤積所致。因此肝膽濕熱與現(xiàn)代醫(yī)學中的膽汁淤積有相似之處，故以誘導(dǎo)膽汁淤積的發(fā)生作為建立肝膽濕熱模型具有可能性。肝細胞和膽小管細胞膜上具有膽汁成分轉(zhuǎn)運分子，它們與膽汁的形成和分泌有關(guān)。各種淤膽性肝損害時，肝細胞膜竇面或膽管面轉(zhuǎn)運體蛋白分子的表達或功能將會受到不同的影響，多藥耐藥蛋白(MDR)和多藥耐藥相關(guān)蛋白(MRP)是兩類與膽汁轉(zhuǎn)運功能密切相關(guān)的蛋白。近年來的研究表明，MDR或MRP基因缺陷可能與某些淤膽形成有關(guān)。 α-萘異硫氰酸酯(ANIT)在動物體內(nèi)可轉(zhuǎn)化為有肝毒性的代謝產(chǎn)物產(chǎn)生肝毒性，其誘發(fā)動物肝損傷的生物化學和病理形態(tài)學改變與人類肝內(nèi)膽汁淤積病變相似。 龍膽瀉肝丸(LD)由龍膽、柴胡、黃芩、梔子、澤瀉、木通、車前子、當歸、地黃、炙甘草組成。具有清肝膽，利濕熱的功用，是臨床治療肝膽濕熱證的代表方之一。長期以來，，其在臨床上治療肝膽濕熱的效果得到公認，但是，對其作用機理未見深入的研究，因此，其治療肝膽濕熱的作用機理尚不清楚。 本研究將根據(jù)中醫(yī)臨床肝膽濕熱的主要客觀表現(xiàn)，參考現(xiàn)代醫(yī)學的膽汁淤積的形成機理，建立模擬肝膽濕熱的動物模型，并對該模型的發(fā)病機理進行探討。在該模型的基礎(chǔ)上研究龍膽瀉肝丸的治療肝膽濕熱的作用及其機理。 目的：首先采用ANIT建立模擬中醫(yī)臨床的肝膽濕熱模型，探討該模型在膽汁轉(zhuǎn)運代謝方面的分子機制。以該模型為基礎(chǔ)，觀察龍膽瀉肝丸治療肝膽濕熱的作用機理，探討作用靶點，從而為龍膽瀉肝丸治療肝膽濕熱提供科學依據(jù)。 方法： 實驗一、大鼠肝膽濕熱模型的建立 將10只大鼠分為對照組和模型組，每組5只。模型組按100mg／kg的劑量一次性灌胃給予α-萘異硫氰酸酯(ANIT)花生油，對照組灌胃同體積花生油。造模后72h，大鼠一一行膽管插管術(shù)。收集4 h內(nèi)的膽汁，按照收集的時間段計算膽汁分泌量。造模后76h檢測血清總膽紅素(TBIL)、直接膽紅素(DBIL)、丙氨酸氨基轉(zhuǎn)移酶(ALT)、天冬氨酸氨基轉(zhuǎn)移酶(AST)、堿性磷酸酶(ALP)、總膽固醇(CHO)水平以及肝組織病理變化。此外，將另外5只大鼠分別于灌胃ANIT后12h、24h、48h、72h、96h分別處死一只。另取1只作為正常對照。分別于不同時間點，取出肝臟，提取肝組織總RNA，用RT-PCR方法檢測MDR1b及MRP2 mRNA的表達。 實驗二、觀察龍膽瀉肝丸(LD)對肝膽濕熱模型大鼠和小鼠的清利肝膽作用 將72只大鼠分為6組，分別為對照組，模型組，膽樂膠囊(DL)組和LD 10、5.0、2.5g生藥／kg劑量組。LD各劑量組和DL組每日給藥1次，連續(xù)給藥8天。對照組灌胃同體積花生油。于給藥期間的第5天，除了對照組外，其余各組在給藥后間隔1 h再灌胃給予ANIT 100 mg／kg(僅一次)。于第8天末次給藥后30 min(末次給藥前禁食24 h，不禁水)，行膽管插管，收集4 h內(nèi)的膽汁，按照收集的時間段計算膽汁分泌量。并檢測血清AST、ALT、ALP、TBIL、DBIL、CHO、丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽(GSH)水平以及肝組織病理變化。另外72只小鼠按同樣方法復(fù)制模型，最后檢測血清TBIL、DBIL水平。 實驗三、觀察龍膽瀉肝丸對肝膽濕熱大鼠MDR1a、MDR1b、MDR2、MRP1、MRP2表達的影響 將20只大鼠分為4組，分別為對照組、模型組、LD 5.0、2.5 g生藥／kg劑量組。給藥組均于動物處死前8天開始給藥，每日灌胃給藥1次，連續(xù)8天。對照組和模型組灌胃同體積的蒸餾水。其中第5天給藥1h后，除對照組給予花生油灌胃外，其他各組大鼠按100mg／kg一次性灌胃ANIT花生油。第8天取出肝臟。提取總RNA，RT-PCR檢測MDR1a、MDR1b、MDR2、MRP1、MRP2 mRNA的表達。 實驗四、觀察龍膽瀉肝丸對肝膽濕熱大鼠中性粒細胞CD18表達的影響 將20只大鼠分為4組，分別為對照組、模型組、LD 5.0、2.5 g生藥／kg。給藥組均于動物處死前8天開始給藥，每日灌胃給藥1次，連續(xù)8天。對照組和模型組灌胃同體積的蒸餾水。其中第5天給藥1h后，除對照組花生油灌胃外，其他各組大鼠按100mg／kg一次性灌胃ANIT花生油。第8天眼眶靜脈取100μl抗凝血，加入PE標記的CD18抗體，流式細胞儀測中性粒細胞CD18表達，另取血20μl血細胞計數(shù)儀測白細胞及粒細胞總數(shù)。 結(jié)果： 實驗一：大鼠肝膽濕熱模型的建立 結(jié)果顯示，模型組給予ANIT 72h后，大鼠膽汁分泌量在膽管插管后4h內(nèi)一直顯著低于對照組(P0.05或P0.01)，而血清TBIL、DBIL、CHO、ALT、AST、ALP水平明顯高于對照組。組織形態(tài)學觀察可見模型組出現(xiàn)肝細胞壞死和膽管損傷。對照組大鼠MDR1b mRNA無明顯表達，MRP2 mRNA只有較弱表達。模型組大鼠在造模后48h～96h，MDR1b mRNA表達明顯增強，但在造模后24h內(nèi)表達不明顯。造模后12h、24h，MRP2 mRNA表達較弱，而在造模后48h～96h均表達明顯。 實驗二：龍膽瀉肝丸(LD)對肝膽濕熱模型大鼠和小鼠的清利肝膽作用 1．膽汁分泌 結(jié)果顯示，模型組的膽汁分泌量較對照組顯著降低(P0.05)。LD各給藥組膽汁分泌量不同程度地高于模型組。LD 5.0g生藥／kg組、LD2.5g生藥／kg組在1h和1.5h時間點的膽汁排泌量較模型組顯著增高(P0.05)，其它時間點也有增高趨勢，但無統(tǒng)計學意義。 2．血清TBIL、DBIL、CHO水平 模型組大鼠血清TBIL、DBIL、CHO水平均上升，與對照組比較有明顯差異(P0.01)。而LD 5.0g生藥／kg組、LD2.5g生藥／kg組血清TBIL、DBIL、CHO與模型組比較有降低趨勢，但無統(tǒng)計學意義。 3．血清ALT、AST、ALP 模型組大鼠血清ALT、AST、ALP升高，與對照組比較有明顯差異(P0.01)。LD各劑量組的血清ALT、AST、ALP水平均與模型組比較無差異。 4．血清MDA、SOD、GSH水平 模型組大鼠血清MDA水平升高(與對照組比較P0.01)，血清SOD及GSH水平與對照組比較無明顯變化。LD各劑量組血清SOD、GSH水平與模型組比較無明顯差異。LD對血清MDA有降低趨勢(與對照組比較P0.05)。 5．肝臟組織形態(tài)學 各給藥組肝細胞壞死不同程度的輕于模型組，其中LD 2.5g生藥／kg組分別與模型組相比差異顯著(P0.01)。 實驗三：龍膽瀉肝丸對肝膽濕熱大鼠MDR1a、MDR1b、MDR2、MRP1、MRP2表達的影響 1．肝組織MDR1a、MDR1b、MDR 2 mRNA表達 模型組大鼠一次性灌胃ANIT 72h后，肝組織MDR1a、MDR1b mRNA表達顯著增加(P0.05，P0.01)。MDR2 mRNA也增加，但未達統(tǒng)計學意義。LD 5.0g生藥／kg和LD 2.5g生藥／kg對肝膽濕熱模型大鼠的肝組織MDR1a、MDR1b、MDR2 mRNA表達無明顯影響。 2．肝組織MRP1 mRNA和MRP2 mRNA表達 模型組大鼠一次性灌胃ANIT 72h后，MRP2 mRNA表達下降，但與對照組相比無統(tǒng)計學意義。MRP1 mRNA的表達未見明顯變化(P0.05)。LD 5.0g生藥／kg和LD 2.5g生藥／kg對肝膽濕熱模型大鼠的肝組織MRP1、MRP2 mRNA表達無明顯影響。 實驗四：龍膽瀉肝丸對肝膽濕熱大鼠中性粒細胞CD18表達的影響 結(jié)果顯示，LD各給藥組白細胞總數(shù)以及粒細胞總數(shù)有下降趨勢，CD18熒光強度明顯低于模型組。 結(jié)論：通過以上實驗，我們認為，通過灌胃ANIT，所建立的模型具有肝內(nèi)、體內(nèi)膽汁淤積，肝酶水平增高，肝細胞損傷等病理生理特征，與中醫(yī)臨床上以身目黃疸為主要表現(xiàn)的肝膽濕熱證類似，可以作為肝膽濕熱模型。該模型的肝臟組織中MDR及MRP mRNA表達發(fā)生變化，而MDR及MRP是與膽汁轉(zhuǎn)運代謝有關(guān)的分子。說明MDR及MRP參與該肝膽濕熱模型的形成。龍膽瀉肝丸能增加膽汁流量，加速膽汁排出，但是對膽汁轉(zhuǎn)運代謝分子MDR1a、MDR1b、MDR2、MRP1、MRP2等均無顯著影響。結(jié)果提示，龍膽瀉肝丸治療肝膽濕熱的作用機制可能與增加毛細膽管膽汁流量、減輕脂質(zhì)過氧化反應(yīng)、穩(wěn)定細胞膜、阻抑CD11／CD18介導(dǎo)的中性粒細胞-內(nèi)皮細胞粘附有關(guān)。
[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.
【學位授予單位】：中國中醫(yī)科學院
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R-332;R285.5

【引證文獻】

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1 錢靜娟;劉霞英;華忠;周秀琳;徐滿琴;張海燕;;銀翹漱口液在慢性乙型肝炎肝膽濕熱證患者口腔管理中的應(yīng)用[J];現(xiàn)代中西醫(yī)結(jié)合雜志;2013年05期

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