三黃茵赤湯抗氧化應(yīng)激抑制凋亡蛋白防治急性肝衰竭的作用
發(fā)布時(shí)間：2018-07-17 08:25
【摘要】：目的 急性肝衰竭(acute hepatic failure, AHF)是由多種因素引起的肝細(xì)胞嚴(yán)重?fù)p害,導(dǎo)致肝臟的合成、代謝、轉(zhuǎn)運(yùn)和排泄等功能發(fā)生嚴(yán)重障礙,繼而出現(xiàn)以嚴(yán)重消化系統(tǒng)癥狀、極度乏力、深度黃疸、凝血功能障礙、肝性腦病和腹水等為主要表現(xiàn)的臨床綜合征,最終可出現(xiàn)多器官功能衰竭和死亡。AHF來(lái)勢(shì)兇猛,發(fā)展迅速,預(yù)后很差,死亡率高達(dá)70%-80%,是嚴(yán)重危害人類健康的一種疾病。在西醫(yī)基礎(chǔ)綜合治療基本相同的情況下,加用中藥保護(hù)患者殘存的肝細(xì)胞和促進(jìn)其肝臟的再生,提高患者的存活率,就顯得尤為重要。由于AHF的癥候變化多端,病機(jī)錯(cuò)綜復(fù)雜,給臨床的辨證論治帶來(lái)了諸多困難。但我們認(rèn)為,急性肝衰竭屬于肝臟疾病的極期階段,其主要的病因病機(jī)為“毒瘀膠結(jié),正氣衰竭”,中醫(yī)治療應(yīng)以祛邪為主,兼以扶正。AHF屬于中醫(yī)“急黃”“鼓脹”“血證”“肝厥”等范疇,目前對(duì)AHF的治療尚缺乏療效顯著的藥物,我們根據(jù)南方醫(yī)院廣東省中西醫(yī)結(jié)合肝病重點(diǎn)專科長(zhǎng)期的臨床經(jīng)驗(yàn)總結(jié),自擬三黃茵赤湯進(jìn)行實(shí)驗(yàn)研究。本實(shí)驗(yàn)通過(guò)建立D-氨基半乳糖(D-GalN)聯(lián)合脂多糖(LPS)誘導(dǎo)的大鼠急性肝衰竭模型,觀察三黃茵赤湯對(duì)AHF大鼠肝臟內(nèi)的超氧化物歧化酶(Superoxide Dismutase, SOD)、丙二醛(Malondialdehyde, MDA)和Caspase3的影響,從而探討其是否通過(guò)抗氧化應(yīng)激抑制凋亡蛋白而達(dá)到防治急性肝衰竭的作用,為中醫(yī)藥治療AHF提供一定的科學(xué)理論依據(jù)。 方法 1.實(shí)驗(yàn)藥物 三黃茵赤湯藥物組成：大黃、姜黃、黃芪、茵陳蒿、赤芍。 2.實(shí)驗(yàn)動(dòng)物與分組 48只SPF級(jí)雄性SD大鼠,體重200～220g,隨機(jī)分為正常對(duì)照組、模型組、三黃茵赤湯高、中、低劑量組、雙環(huán)醇組,每組8只。 3.造模 大鼠在SPF級(jí)條件下適應(yīng)性飼養(yǎng)1周,1周后預(yù)先連續(xù)灌胃5天,之后開(kāi)始造模,造模前禁食12h,不禁水,正常對(duì)照組腹腔注射等滲生理鹽水,模型組、三黃茵赤湯高、中、低劑量組、雙環(huán)醇組腹腔注射10μg/kgLPS,半小時(shí)后,腹腔注射700mg/kg的D-GalN。造模后治療組繼續(xù)灌胃治療,提供正常飲食。48h后戊巴比妥鈉400mg/kg麻醉大鼠,腹主動(dòng)脈采血,離心,分離血清置-80℃保存,取肝組織,部分浸泡于4%多聚甲醛,部分置于-80℃保存。 4.觀察及檢測(cè)指標(biāo) 采用全自動(dòng)生化檢測(cè)儀檢測(cè)各組大鼠血清中丙氨酸氨基轉(zhuǎn)移酶(ALT)、天門冬氨酸氨基轉(zhuǎn)移酶(AST)、總膽紅素(TBIL)的含量；采用全自動(dòng)凝血檢測(cè)儀檢測(cè)各組大鼠凝血酶原時(shí)間(PT)、國(guó)際標(biāo)準(zhǔn)比率(INR)、血漿纖維蛋白原(FIB)的水平；利用Nikon Eoipse Ti-s倒置顯微鏡觀察大鼠肝臟病理形態(tài)學(xué)變化；采用總超氧化物歧化酶(Superoxide Dismutase, SOD)檢測(cè)試劑盒和丙二醛(Malondialdehyde,MDA)檢測(cè)試劑盒檢測(cè)肝臟組織勻漿液中SOD、MDA的含量；免疫組化法檢測(cè)肝臟中caspase3原位表達(dá)情況；采用蛋白質(zhì)免疫印跡(Western blot)檢測(cè)肝臟中caspase3的相對(duì)表達(dá)水平。 5.統(tǒng)計(jì)學(xué)方法 數(shù)據(jù)用SPSS13.0軟件進(jìn)行統(tǒng)計(jì)學(xué)分析,計(jì)量資料用均數(shù)±標(biāo)準(zhǔn)差(x±s)表示,多樣本均數(shù)的比較采用One-Way ANOVA方差分析,組間多重比較若符合方差齊性檢驗(yàn)時(shí)用Tukey法,若不符合則用Dunnett' T3法,計(jì)數(shù)資料及率的比較用卡方檢驗(yàn)。以P0.05均為有統(tǒng)計(jì)學(xué)意義。 結(jié)果 1.肝功能檢測(cè)結(jié)果 與模型組相比,三黃茵赤湯高、中、低劑量組和雙環(huán)醇組均能降低大鼠血清中ALT、AST以及TBIL的含量,有顯著性差異(P0.01),三黃茵赤湯高劑量組與雙環(huán)醇組相當(dāng)(P0.05)；中、低劑量組不如雙環(huán)醇組,亦有統(tǒng)計(jì)學(xué)差異(P0.05)。 2.凝血功能檢測(cè)結(jié)果 與模型組相比,三黃茵赤湯高、中、低劑量組、雙環(huán)醇組均能縮短大鼠血清中PT、INR時(shí)間和增加FIB的含量,有顯著性差異(P0.01),三黃茵赤湯高劑量組與雙環(huán)醇組相當(dāng)(P0.05)；中、低劑量組不如雙環(huán)醇組,亦有統(tǒng)計(jì)學(xué)差異(P0.05)。 3.肝臟病理形態(tài)觀察 各組大鼠蘇木精—伊紅染色法(hematoxylin-eosinstaining, HE染色)的結(jié)果顯示,正常對(duì)照組肝小葉結(jié)構(gòu)清晰,肝細(xì)胞排列規(guī)則有序；模型組肝細(xì)胞成大塊、亞大塊壞死,肝索紊亂,小葉結(jié)構(gòu)模糊不清,肝竇明顯擴(kuò)張充血并出血,小葉內(nèi)及匯管區(qū)炎性細(xì)胞浸潤(rùn),殘存肝細(xì)胞水腫變性；給藥各組均有改善作用,腫脹減輕,壞死減少,高劑量組與雙環(huán)醇組相當(dāng),中、低劑量組不如雙環(huán)醇組。 4.肝臟組織勻漿液中SOD、MDA的檢測(cè)結(jié)果 結(jié)果顯示三黃茵赤湯高、中、低劑量組、雙環(huán)醇組均能提高大鼠肝臟組織勻漿液中SOD的活性(P0.01)減少M(fèi)DA的含量(P0.01),三黃茵赤湯高劑量組與雙環(huán)醇組相當(dāng)(P0.05)；中、低劑量組不如雙環(huán)醇組,亦有統(tǒng)計(jì)學(xué)差異(P0.05)。 5. caspase3原位表達(dá)的結(jié)果 免疫組化結(jié)果顯示,caspase3主要在肝細(xì)胞胞漿中表達(dá)為主,三黃茵赤湯不同劑量組均能減低caspase3的表達(dá)水平(P0.05),三黃茵赤湯高劑量組與雙環(huán)醇組相當(dāng)(P0.05)；中、低劑量組不如雙環(huán)醇組,亦有統(tǒng)計(jì)學(xué)差異(P0.05)。 6.三黃茵赤湯不同劑量對(duì)caspase3蛋白表達(dá)的影響 Western blot分析顯示,三黃茵赤湯不同劑量組均能減低caspase3的表達(dá)水平(P0.01),三黃茵赤湯高劑量組與雙環(huán)醇組相當(dāng)(P0.05)；中、低劑量組不如雙環(huán)醇組,亦有統(tǒng)計(jì)學(xué)差異(P0.05)。 結(jié)論 1.三黃茵赤湯具有改善肝臟功能、凝血功能的作用。 2.三黃茵赤湯能提高肝臟中SOD的活性,降低MDA的含量,減少caspase3的表達(dá),抑制肝細(xì)胞的凋亡。 3.三黃茵赤湯具有防治急性肝衰竭的作用,其可能的機(jī)制是通過(guò)抗氧化應(yīng)激從而抑制caspase3的表達(dá),減少肝細(xì)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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R259
本文編號(hào)：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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R259
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