三黄茵赤汤抗氧化应激抑制凋亡蛋白防治急性肝衰竭的作用
发布时间：2018-07-17 08:25
【摘要】：目的 急性肝衰竭(acute hepatic failure, AHF)是由多种因素引起的肝细胞严重损害,导致肝脏的合成、代谢、转运和排泄等功能发生严重障碍,继而出现以严重消化系统症状、极度乏力、深度黄疸、凝血功能障碍、肝性脑病和腹水等为主要表现的临床综合征,最终可出现多器官功能衰竭和死亡。AHF来势凶猛,发展迅速,预后很差,死亡率高达70%-80%,是严重危害人类健康的一种疾病。在西医基础综合治疗基本相同的情况下,加用中药保护患者残存的肝细胞和促进其肝脏的再生,提高患者的存活率,就显得尤为重要。由于AHF的症候变化多端,病机错综复杂,给临床的辨证论治带来了诸多困难。但我们认为,急性肝衰竭属于肝脏疾病的极期阶段,其主要的病因病机为“毒瘀胶结,正气衰竭”,中医治疗应以祛邪为主,兼以扶正。AHF属于中医“急黄”“鼓胀”“血证”“肝厥”等范畴,目前对AHF的治疗尚缺乏疗效显著的药物,我们根据南方医院广东省中西医结合肝病重点专科长期的临床经验总结,自拟三黄茵赤汤进行实验研究。本实验通过建立D-氨基半乳糖(D-GalN)联合脂多糖(LPS)诱导的大鼠急性肝衰竭模型,观察三黄茵赤汤对AHF大鼠肝脏内的超氧化物歧化酶(Superoxide Dismutase, SOD)、丙二醛(Malondialdehyde, MDA)和Caspase3的影响,从而探讨其是否通过抗氧化应激抑制凋亡蛋白而达到防治急性肝衰竭的作用,为中医药治疗AHF提供一定的科学理论依据。 方法 1.实验药物 三黄茵赤汤药物组成：大黄、姜黄、黄芪、茵陈蒿、赤芍。 2.实验动物与分组 48只SPF级雄性SD大鼠,体重200～220g,随机分为正常对照组、模型组、三黄茵赤汤高、中、低剂量组、双环醇组,每组8只。 3.造模 大鼠在SPF级条件下适应性饲养1周,1周后预先连续灌胃5天,之后开始造模,造模前禁食12h,不禁水,正常对照组腹腔注射等渗生理盐水,模型组、三黄茵赤汤高、中、低剂量组、双环醇组腹腔注射10μg/kgLPS,半小时后,腹腔注射700mg/kg的D-GalN。造模后治疗组继续灌胃治疗,提供正常饮食。48h后戊巴比妥钠400mg/kg麻醉大鼠,腹主动脉采血,离心,分离血清置-80℃保存,取肝组织,部分浸泡于4%多聚甲醛,部分置于-80℃保存。 4.观察及检测指标 采用全自动生化检测仪检测各组大鼠血清中丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、总胆红素(TBIL)的含量；采用全自动凝血检测仪检测各组大鼠凝血酶原时间(PT)、国际标准比率(INR)、血浆纤维蛋白原(FIB)的水平；利用Nikon Eoipse Ti-s倒置显微镜观察大鼠肝脏病理形态学变化；采用总超氧化物歧化酶(Superoxide Dismutase, SOD)检测试剂盒和丙二醛(Malondialdehyde,MDA)检测试剂盒检测肝脏组织匀浆液中SOD、MDA的含量；免疫组化法检测肝脏中caspase3原位表达情况；采用蛋白质免疫印迹(Western blot)检测肝脏中caspase3的相对表达水平。 5.统计学方法 数据用SPSS13.0软件进行统计学分析,计量资料用均数±标准差(x±s)表示,多样本均数的比较采用One-Way ANOVA方差分析,组间多重比较若符合方差齐性检验时用Tukey法,若不符合则用Dunnett' T3法,计数资料及率的比较用卡方检验。以P0.05均为有统计学意义。 结果 1.肝功能检测结果 与模型组相比,三黄茵赤汤高、中、低剂量组和双环醇组均能降低大鼠血清中ALT、AST以及TBIL的含量,有显著性差异(P0.01),三黄茵赤汤高剂量组与双环醇组相当(P0.05)；中、低剂量组不如双环醇组,亦有统计学差异(P0.05)。 2.凝血功能检测结果 与模型组相比,三黄茵赤汤高、中、低剂量组、双环醇组均能缩短大鼠血清中PT、INR时间和增加FIB的含量,有显著性差异(P0.01),三黄茵赤汤高剂量组与双环醇组相当(P0.05)；中、低剂量组不如双环醇组,亦有统计学差异(P0.05)。 3.肝脏病理形态观察 各组大鼠苏木精—伊红染色法(hematoxylin-eosinstaining, HE染色)的结果显示,正常对照组肝小叶结构清晰,肝细胞排列规则有序；模型组肝细胞成大块、亚大块坏死,肝索紊乱,小叶结构模糊不清,肝窦明显扩张充血并出血,小叶内及汇管区炎性细胞浸润,残存肝细胞水肿变性；给药各组均有改善作用,肿胀减轻,坏死减少,高剂量组与双环醇组相当,中、低剂量组不如双环醇组。 4.肝脏组织匀浆液中SOD、MDA的检测结果 结果显示三黄茵赤汤高、中、低剂量组、双环醇组均能提高大鼠肝脏组织匀浆液中SOD的活性(P0.01)减少MDA的含量(P0.01),三黄茵赤汤高剂量组与双环醇组相当(P0.05)；中、低剂量组不如双环醇组,亦有统计学差异(P0.05)。 5. caspase3原位表达的结果 免疫组化结果显示,caspase3主要在肝细胞胞浆中表达为主,三黄茵赤汤不同剂量组均能减低caspase3的表达水平(P0.05),三黄茵赤汤高剂量组与双环醇组相当(P0.05)；中、低剂量组不如双环醇组,亦有统计学差异(P0.05)。 6.三黄茵赤汤不同剂量对caspase3蛋白表达的影响 Western blot分析显示,三黄茵赤汤不同剂量组均能减低caspase3的表达水平(P0.01),三黄茵赤汤高剂量组与双环醇组相当(P0.05)；中、低剂量组不如双环醇组,亦有统计学差异(P0.05)。 结论 1.三黄茵赤汤具有改善肝脏功能、凝血功能的作用。 2.三黄茵赤汤能提高肝脏中SOD的活性,降低MDA的含量,减少caspase3的表达,抑制肝细胞的凋亡。 3.三黄茵赤汤具有防治急性肝衰竭的作用,其可能的机制是通过抗氧化应激从而抑制caspase3的表达,减少肝细胞的凋亡。
[Abstract]:objective
Acute liver failure (acute hepatic failure, AHF) is a serious damage to liver cells caused by a variety of factors, leading to serious disorders in the liver synthesis, metabolism, transport and excretion, and then the major manifestations of severe digestive system symptoms, extreme fatigue, deep jaundice, blood coagulation dysfunction, hepatic encephalopathy and ascites, etc. In the end, multiple organ failure and death.AHF will eventually appear fierce, rapid development, poor prognosis and a high mortality rate of 70%-80%. It is a serious harm to human health. Under the basic comprehensive treatment of Western medicine, traditional Chinese medicine is used to protect the remaining liver cells and promote the regeneration of the liver and improve the patients. The survival rate is particularly important. Because the symptoms of AHF are varied and the pathogenesis is complicated, it brings many difficulties to the clinical syndrome differentiation and treatment. However, we think that the acute liver failure is the stage of the liver disease, its main pathogenesis is "toxic stasis cementation, positive gas exhaustion", and the traditional Chinese medicine treatment should be mainly dispelling evil. AHF belongs to the category of "acute yellow", "bulge", "blood syndrome", "liver syncope" and so on. At present, the treatment of AHF is still lack of effective drugs. According to the long-term clinical experience of the key specialist in the combination of traditional Chinese and Western medicine in the Southern Hospital of Guangdong, we have made a study of Sanhuang Yin Chi decoction. This experiment was established by the establishment of D- amino galactose (D -GalN) combined with lipopolysaccharide (LPS) induced acute liver failure model in rats, the effects of three Huang Yin Chi Decoction on superoxide dismutase (Superoxide Dismutase, SOD), malondialdehyde (Malondialdehyde, MDA) and Caspase3 in the liver of AHF rats were observed to explore whether it could prevent acute liver failure by inhibiting apoptosis protein by anti oxidative stress. It provides a scientific theoretical basis for the treatment of AHF by Chinese medicine.
Method
1. experimental drugs
The composition of Sanhuang Decoction is rhubarb, Jiang Huang, astragalus, Artemisia capillaris, and Radix Paeoniae Rubra.
2. experimental animals and groups
48 SPF male SD rats weighing 200 to 220g were randomly divided into normal control group, model group, Sanhuang Yin Chi Tang high, middle, low dose group, bicyclic alcohol group, 8 rats in each group.
3. mold making
The rats were fed for 1 weeks under the condition of SPF, and after 1 weeks, the rats were given the stomach for 5 days in advance, then the model began to be made, and then the model was made. The fasting before the model was 12h, and the normal control group was injected with isotonic saline. The model group, the middle, low dose group, and the double ring alcohol group were injected with the abdominal injection of 10 mu g/kgLPS in the model group. After half an hour, the D-GalN. model of 700mg/kg was injected intraperitoneally. The post treatment group continued to gavage, providing a normal diet.48h after pentobarbital sodium 400mg/kg anesthetized rats, abdominal aorta blood collection, centrifugation, separation of serum and -80 C preservation, liver tissue, partially soaked in 4% polyformaldehyde, part of the -80 preservation.
4. observation and detection index
The content of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBIL) in the serum of each group was detected by an automatic biochemical analyzer, and the level of prothrombin time (PT), international standard ratio (INR) and plasma fibrinogen (FIB) were detected by automatic coagulant detector, and Nikon E was used. Oipse Ti-s inverted microscope was used to observe the pathological changes of liver in rats; the content of SOD and MDA in liver homogenate was detected by Superoxide Dismutase (SOD) detection kit and Malondialdehyde (MDA) detection kit, and the expression of Caspase3 in liver was detected by immunization. The relative expression level of Caspase3 in liver was detected by Western blot.
5. statistical method
The data were analyzed statistically with SPSS13.0 software, and the measured data were expressed with mean standard deviation (x + s). One-Way ANOVA variance analysis was used for the comparison of all the average numbers. If Tukey was used in the test of homogeneity of variance, Dunnett'T3 method was used, and the comparison of count data and rate was tested with the Tukey method. Statistical significance.
Result
1. results of liver function test
Compared with the model group, the high level of Sanhuang Yin Chi decoction, middle, low dose group and double ring alcohol group could reduce the content of ALT, AST and TBIL in the serum of rats, and there were significant differences (P0.01). The high dose group of Sanhuang Yin Chi decoction was equivalent to that of the double ring alcohol group (P0.05), and the low dose group was not as good as that of the double ring alcohol group (P0.05).
2. detection results of coagulation function
Compared with the model group, the high, middle, low dose group and double ring alcohol group could shorten the PT, INR time and increase the content of FIB in the rat serum, and there were significant differences (P0.01). The high dose group of Sanhuang Yin Chi decoction was equivalent to that of the double ring alcohol group (P0.05), and the low dose group was not like the double ring alcohol group, and there were also statistical differences (P0.05).
3. pathological morphological observation of the liver
The results of hematoxylin eosin staining (hematoxylin-eosinstaining, HE staining) showed that the structure of hepatic lobule in the normal control group was clear and the liver cells were arranged regularly. The liver cells in the model group were large, submajor necrosis, hepatic cord disorder, blurred structure of lobular lobule, hepatic sinusoid dilation and bleeding, intramedullary and confluence inflammation All the groups were improved, the swelling was reduced, and the necrosis was reduced. The high dose group was equivalent to the double ring alcohol group, and the lower dose group was not as good as the double ring alcohol group.
Detection results of SOD and MDA in 4. liver homogenate
The results showed that the high, middle, low dose group and double ring alcohol group could improve the activity of SOD in the homogenate fluid of the rat liver (P0.01) to reduce the content of MDA (P0.01), and the high dose group of Sanhuang Yin Chi decoction was equivalent to that of the double ring alcohol group (P0.05), and the low dose group was not as good as that of the double ring alcohol group, and there were also statistical differences (P0.05).
The results of 5. Caspase3 in situ expression
The results of immunohistochemical staining showed that Caspase3 was mainly expressed in the cytoplasm of liver cells, and the expression level of Caspase3 was reduced in the different dosage groups of Sanhuang Yin Chi Decoction (P0.05). The high dose group of Sanhuang Yin Chi decoction was equivalent to that of the double ring alcohol group (P0.05), and the low dose group was not as good as the double ring alcohol group, and there were also statistical differences (P0.05).
6. the effect of different doses of Sanhuang Yin Decoction on the expression of Caspase3 protein
Western blot analysis showed that the different dosage groups of Sanhuang Yin Chi decoction could reduce the expression level of Caspase3 (P0.01), and the high dose group of Sanhuang Yin Chi decoction was equivalent to that of the double ring alcohol group (P0.05), and the low dose group was not as good as that of the double ring alcohol group, and there were also statistical differences (P0.05).
conclusion
1. Sanhuang Yin decoction has the function of improving liver function and coagulation function.
2. Sanhuang Yin decoction can increase the activity of SOD in liver, reduce the content of MDA, reduce the expression of Caspase3, and inhibit the apoptosis of hepatocytes.
3. the effect of Sanhuang Yin Chi Decoction on prevention and treatment of acute liver failure, its possible mechanism is to inhibit the expression of Caspase3 through antioxidant stress and reduce the apoptosis of liver cells.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R259
本文编号：2129793
[Abstract]:objective
Acute liver failure (acute hepatic failure, AHF) is a serious damage to liver cells caused by a variety of factors, leading to serious disorders in the liver synthesis, metabolism, transport and excretion, and then the major manifestations of severe digestive system symptoms, extreme fatigue, deep jaundice, blood coagulation dysfunction, hepatic encephalopathy and ascites, etc. In the end, multiple organ failure and death.AHF will eventually appear fierce, rapid development, poor prognosis and a high mortality rate of 70%-80%. It is a serious harm to human health. Under the basic comprehensive treatment of Western medicine, traditional Chinese medicine is used to protect the remaining liver cells and promote the regeneration of the liver and improve the patients. The survival rate is particularly important. Because the symptoms of AHF are varied and the pathogenesis is complicated, it brings many difficulties to the clinical syndrome differentiation and treatment. However, we think that the acute liver failure is the stage of the liver disease, its main pathogenesis is "toxic stasis cementation, positive gas exhaustion", and the traditional Chinese medicine treatment should be mainly dispelling evil. AHF belongs to the category of "acute yellow", "bulge", "blood syndrome", "liver syncope" and so on. At present, the treatment of AHF is still lack of effective drugs. According to the long-term clinical experience of the key specialist in the combination of traditional Chinese and Western medicine in the Southern Hospital of Guangdong, we have made a study of Sanhuang Yin Chi decoction. This experiment was established by the establishment of D- amino galactose (D -GalN) combined with lipopolysaccharide (LPS) induced acute liver failure model in rats, the effects of three Huang Yin Chi Decoction on superoxide dismutase (Superoxide Dismutase, SOD), malondialdehyde (Malondialdehyde, MDA) and Caspase3 in the liver of AHF rats were observed to explore whether it could prevent acute liver failure by inhibiting apoptosis protein by anti oxidative stress. It provides a scientific theoretical basis for the treatment of AHF by Chinese medicine.
Method
1. experimental drugs
The composition of Sanhuang Decoction is rhubarb, Jiang Huang, astragalus, Artemisia capillaris, and Radix Paeoniae Rubra.
2. experimental animals and groups
48 SPF male SD rats weighing 200 to 220g were randomly divided into normal control group, model group, Sanhuang Yin Chi Tang high, middle, low dose group, bicyclic alcohol group, 8 rats in each group.
3. mold making
The rats were fed for 1 weeks under the condition of SPF, and after 1 weeks, the rats were given the stomach for 5 days in advance, then the model began to be made, and then the model was made. The fasting before the model was 12h, and the normal control group was injected with isotonic saline. The model group, the middle, low dose group, and the double ring alcohol group were injected with the abdominal injection of 10 mu g/kgLPS in the model group. After half an hour, the D-GalN. model of 700mg/kg was injected intraperitoneally. The post treatment group continued to gavage, providing a normal diet.48h after pentobarbital sodium 400mg/kg anesthetized rats, abdominal aorta blood collection, centrifugation, separation of serum and -80 C preservation, liver tissue, partially soaked in 4% polyformaldehyde, part of the -80 preservation.
4. observation and detection index
The content of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBIL) in the serum of each group was detected by an automatic biochemical analyzer, and the level of prothrombin time (PT), international standard ratio (INR) and plasma fibrinogen (FIB) were detected by automatic coagulant detector, and Nikon E was used. Oipse Ti-s inverted microscope was used to observe the pathological changes of liver in rats; the content of SOD and MDA in liver homogenate was detected by Superoxide Dismutase (SOD) detection kit and Malondialdehyde (MDA) detection kit, and the expression of Caspase3 in liver was detected by immunization. The relative expression level of Caspase3 in liver was detected by Western blot.
5. statistical method
The data were analyzed statistically with SPSS13.0 software, and the measured data were expressed with mean standard deviation (x + s). One-Way ANOVA variance analysis was used for the comparison of all the average numbers. If Tukey was used in the test of homogeneity of variance, Dunnett'T3 method was used, and the comparison of count data and rate was tested with the Tukey method. Statistical significance.
Result
1. results of liver function test
Compared with the model group, the high level of Sanhuang Yin Chi decoction, middle, low dose group and double ring alcohol group could reduce the content of ALT, AST and TBIL in the serum of rats, and there were significant differences (P0.01). The high dose group of Sanhuang Yin Chi decoction was equivalent to that of the double ring alcohol group (P0.05), and the low dose group was not as good as that of the double ring alcohol group (P0.05).
2. detection results of coagulation function
Compared with the model group, the high, middle, low dose group and double ring alcohol group could shorten the PT, INR time and increase the content of FIB in the rat serum, and there were significant differences (P0.01). The high dose group of Sanhuang Yin Chi decoction was equivalent to that of the double ring alcohol group (P0.05), and the low dose group was not like the double ring alcohol group, and there were also statistical differences (P0.05).
3. pathological morphological observation of the liver
The results of hematoxylin eosin staining (hematoxylin-eosinstaining, HE staining) showed that the structure of hepatic lobule in the normal control group was clear and the liver cells were arranged regularly. The liver cells in the model group were large, submajor necrosis, hepatic cord disorder, blurred structure of lobular lobule, hepatic sinusoid dilation and bleeding, intramedullary and confluence inflammation All the groups were improved, the swelling was reduced, and the necrosis was reduced. The high dose group was equivalent to the double ring alcohol group, and the lower dose group was not as good as the double ring alcohol group.
Detection results of SOD and MDA in 4. liver homogenate
The results showed that the high, middle, low dose group and double ring alcohol group could improve the activity of SOD in the homogenate fluid of the rat liver (P0.01) to reduce the content of MDA (P0.01), and the high dose group of Sanhuang Yin Chi decoction was equivalent to that of the double ring alcohol group (P0.05), and the low dose group was not as good as that of the double ring alcohol group, and there were also statistical differences (P0.05).
The results of 5. Caspase3 in situ expression
The results of immunohistochemical staining showed that Caspase3 was mainly expressed in the cytoplasm of liver cells, and the expression level of Caspase3 was reduced in the different dosage groups of Sanhuang Yin Chi Decoction (P0.05). The high dose group of Sanhuang Yin Chi decoction was equivalent to that of the double ring alcohol group (P0.05), and the low dose group was not as good as the double ring alcohol group, and there were also statistical differences (P0.05).
6. the effect of different doses of Sanhuang Yin Decoction on the expression of Caspase3 protein
Western blot analysis showed that the different dosage groups of Sanhuang Yin Chi decoction could reduce the expression level of Caspase3 (P0.01), and the high dose group of Sanhuang Yin Chi decoction was equivalent to that of the double ring alcohol group (P0.05), and the low dose group was not as good as that of the double ring alcohol group, and there were also statistical differences (P0.05).
conclusion
1. Sanhuang Yin decoction has the function of improving liver function and coagulation function.
2. Sanhuang Yin decoction can increase the activity of SOD in liver, reduce the content of MDA, reduce the expression of Caspase3, and inhibit the apoptosis of hepatocytes.
3. the effect of Sanhuang Yin Chi Decoction on prevention and treatment of acute liver failure, its possible mechanism is to inhibit the expression of Caspase3 through antioxidant stress and reduce the apoptosis of liver cells.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R259
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