【作者】 熊焰；

【导师】 蔡光先；

【作者基本信息】 湖南中医药大学 ， 中医内科学， 2014， 博士

【摘要】 研究一阴黄证大鼠IETM模型的建立目的：联合D-半乳糖胺(D-gal)腹腔注射及大黄煎剂灌胃诱导建立肝衰竭IETM阴黄证实验动物模型。方法：SD大鼠68只,随机分为Ⅰ(正常组)、Ⅱ(阳黄证对照组)和Ⅲ(阴黄证组),Ⅰ组6只,Ⅱ、Ⅲ组每组各31只。实验动物分组后,单笼饲养,自由食水,同等条件喂养3天,各组动物称重、测肛温,从第4天起施加处理因素。Ⅱ组大鼠,将其置于湿热造模环境下(造模箱温度30-32℃,R T大于95%)；Ⅲ组大鼠,将其置于寒湿造模环境下(造模箱温度1-6℃,RT大于95%)。Ⅲ组予400%大黄煎剂按照50ml/kg灌胃,2次/天；Ⅱ组予1：1的5%葡糖糖和猪油脂混合液20ml/kg灌胃,2次/天；Ⅰ组予生理盐水灌胃50ml/kg,2次/天。阳黄、阴黄证造模21天。21天后以D-gal1.8g/kg腹腔注射Ⅱ、Ⅲ组建立肝衰竭IETM模型,正常组腹腔注射等量生理盐水,24h、48h分别观察各组大鼠死亡率、肝功能(ALT、AST、TBIL)、肝组织病理改变、门静脉内毒素水平、直肠内粪便肠道菌群水平。结果：1.一般情况：Ⅰ组大鼠无死亡。Ⅱ组、Ⅲ组大鼠死亡率分别为20%、30%。2.肝功能结果：以Ⅰ组作参照,Ⅱ组、Ⅲ组大鼠ALT、AST、TBIL均明显升高,差异有统计学意义(p<0.01),Ⅲ组较Ⅱ组ALT、AST、 TBIL增加更为明显,差异有统计学意义(p<0.05)。3.肝组织病理结果：以Ⅰ组作参照,Ⅱ组、Ⅲ组大鼠肝组织损伤明显加重,各组间比较病理积分,差异有统计学意义(p<0.05)。Ⅱ组、Ⅲ组大鼠肝组织损伤程度达到肝衰竭肝组织损伤诊断水平,肝组织病理积分以Ⅲ组最高,两时间点病理积分组间比较差异无统计学意义p>0.05)。4.血浆内毒素：以Ⅰ组作参照,Ⅱ组、Ⅲ组大鼠血浆内毒素明显升高,差异有统计学意义(p<0.01)；Ⅲ组、Ⅲ组大鼠组间门静脉内毒素比较差异有统计学意义(p<0.05)。5.粪便肠道菌群水平：以Ⅰ组作参照,Ⅱ组、Ⅲ组大鼠肠球菌、肠杆菌均明显增多,差异有统计学意义(p<0.01)；双歧杆菌、乳酸杆菌均减少,差异有统计学意义(p<0.05)；Ⅱ组、Ⅲ组大鼠组间比较,肠球菌、肠杆菌、双歧杆菌差异有统计学意义(p<0.05),乳酸杆菌差异无统计学意义(p>0.05)。结论：联合寒湿造模环境、大黄煎剂灌胃及腹腔注射D-gal方法可诱导建立阴黄证IETM模型。研究二温阳解毒化瘀法对阴黄证肝衰竭IETM大鼠内毒素、细胞因子的影响目的：1.研究温阳解毒化瘀法对阴黄证肝衰竭IETM大鼠IL-6、C-met蛋白免疫表达等表达影响。2.研究温阳解毒化瘀法干预后阴黄证肝衰竭IETM大鼠肠道菌群、血浆内毒素的变化。方法：SD大鼠86只,随机分为阴黄证肝衰竭IETM模型组(Ⅰ组)、温阳解毒化瘀方低剂量组(Ⅱ组)、温阳解毒化瘀方中剂量组(Ⅲ组)、温阳解毒化瘀方高剂量组(Ⅳ组),每组各20只,正常组(Ⅴ组)大鼠6只。Ⅱ、Ⅲ、Ⅳ组大鼠于造模前6天予温阳解毒化瘀方不同剂量灌胃,Ⅰ组大鼠以生理盐水灌胃,2次/天,直至处死。造模21天后I、II、III、IV组予D-gal1.8g/kg腹腔注射、大黄煎剂灌胃建立阴黄证IETM模型；Ⅴ组大鼠正常饮食。观察各组大鼠在造模后24h、48h死亡率,各组大鼠同时对肝功能(ALT、AST、TBIL)、肝脏组织病理、血浆内毒素、肠道菌群、肝组织IL-6、C-met蛋白免疫表达等进行检测。结果：1.死亡率：Ⅰ组大鼠死亡率为32%；Ⅱ、Ⅲ、Ⅳ组大鼠死亡率随温阳解毒化瘀方剂量增加逐渐下降,各组死亡率为23%、20%、15%。2.肝功能：与Ⅰ组比较,Ⅱ、Ⅲ、Ⅳ组大鼠ALT、AST、TBIL均降低,其中Ⅳ组与Ⅰ组组间比较,差异有统计学意义(p<0.01)。3.肝组织病理结果：与Ⅰ组比较,Ⅱ、Ⅲ、Ⅳ组肝组织病理结果显示,损伤均减轻,其中Ⅳ组与Ⅰ组肝组织病理评分,差异有统计学意义(p<0.05)。4.血浆内毒素：与Ⅰ组比较,Ⅱ、Ⅲ、Ⅳ组内毒素水平均减轻,其中Ⅳ组与Ⅰ组组间差异有统计学意义(p<0.05)。5.肠道菌群水平：与Ⅰ组比较,Ⅱ组、Ⅲ、Ⅳ组大鼠肠球菌、肠杆菌均增多,差异有统计学意义(p<0.01)；双歧杆菌、乳酸杆菌均减少,差异有统计学意义(p<0.05)；Ⅱ组、Ⅲ组分别与Ⅳ组大鼠组间比较,肠球菌、肠杆菌、双歧杆菌差异有统计学意义(p<0.05),乳酸杆菌差异无统计学意义(p>0.05)。Ⅱ组、Ⅲ组组间比较差异无统计学意义(p>0.05)。6.肝组织IL-6水平变化：与Ⅰ组比较,Ⅱ组、Ⅲ组、Ⅳ组大鼠IL-6水平明显下降,差异有统计学意义(p<0.05)。7.C-met蛋白免疫组化：与Ⅰ组比较,Ⅱ组、Ⅲ组、Ⅳ组.C-met阳性面积均明显增高,差异有统计学意义(P<0.01)； Ⅳ组与Ⅱ组、Ⅲ组两两比较,Ⅳ组.C-met阳性面积较Ⅱ组、Ⅲ组增高,差异有统计学意义(P<0.05)。结论：1.温阳解毒化瘀方能降低肝衰竭IETM阴黄证大鼠死亡率,改善肝功能、减轻肝组织病理情况。2.温阳解毒化瘀方作用于肝衰竭IETM阴黄证的机制可能是通过(1)降低血浆内毒素的吸收；(2)诱导免疫信号激活；促进肝细胞增生；(3)调节肠道菌群来实现。研究三温阳解毒化瘀法对乙型肝炎相关性肝衰竭阴黄证及阴阳黄证患者的影响目的：通过对入选60例乙型肝炎相关性肝衰竭患者中西结合治疗的分析,评估温阳解毒化瘀法对乙型肝炎相关性肝衰竭非阳黄证患者疗效。方法：入选病例按照中医辨证为非阳黄证组(阴黄证和阴阳黄证),按照随机数字表分为治疗组、对照组,两组患者均予常规保肝、护肝、护胃、改善肝脏微循环、抗病毒、对症支持治疗等,试验组在此基础上予温阳解毒化瘀颗粒剂,对照组在此基础上给予中药模拟剂治疗。用药8周后,对两组患者进行疗效评价。结果：两组治疗后TBIL、PTA、肠道内毒素等组间比较,差异有统计学意义(P<0.05),两组组内治疗前后比较,差异有统计学意义(P<0.05)。结论：温阳解毒化瘀法治疗乙型肝炎相关性肝衰竭患者,可以改善症状,提高疗效。更多还原

【Abstract】 Study one Yin yellow card IETM model in ratsObjectives:Joint D-galactosamine (D-gal) intraperitoneal injection and rhubarb decoction lavage induced liver failure IETM Yin yellow card experimental animal model.Methods:68SD rats, were randomly divided into three groups, Ⅰ (normal control group), Ⅱ(yang yellow card group)and Ⅲ(Yin yellow card group). Normal group6, Ⅱ, Ⅲ groups each group only31. After experimental animal group, single cage, the free water, the same article3days, each animal weighing, measuring the anus temperature, from4days of the processing factors. Ⅱ group of rats, made mould to place it in hot and humid environment, Ⅲ group of rats, puts the cold dampness building environment. Group Ⅲ to400%rhubarb decoction according to the50ml/kg to fill the stomach,2times/day, Ⅱ group to1:15%sugar and pig oil mix20ml/kg to fill the stomach,2times/day; I set to saline to fill the stomach,2times/day. Hot and humid, cold dampness syndrome made mould for21days.21days will be D-gal intraperitoneal injection of1.8g/kg group Ⅱ and Ⅲ IETM liver failure model is established. By intraperitoneal injection of normal group amount of saline solution, After24h,48h were observed between groups of rats mortality, Liver function(ALT、AST、TBIL), Pathological changes of the liver tissue, Endotoxin level of portal vein, Rectum waste levels of intestinal flora. Results:1. General situation:group I rats without death. II, III group rats mortality respectively:20%,30%.2. Results of liver function:As a reference in group I, II and III group rats ALT, AST and TBIL were significantly increased, the difference was statistically significant (p<0.01). As a reference in group III, II group of ALT, AST and TBIL increased more obviously, the difference was statistically significant (p<0.05).3. Liver tissue pathology results:as a reference in group I, group III in the rat liver tissue damage is aggravating, obviously pathological integral is compared between each group, the difference was statistically significant (p<0.05). II, III group in the rat liver tissue damage to liver failure diagnosis of liver tissue damage level and liver tissue pathology the highest score in group III, comparing differences between two time points pathological integral group has no statistical significance (p>0.05).4. Portal vein endotoxin:As a reference in group I, II, III group rats with portal vein endotoxin obviously increases, the difference was statistically significant (p<0.01); Ⅱ, III group rats portal vein endotoxin comparative differences between groups was statistically significant (p<0.05).5.Excrement and urine levels of intestinal flora:As a reference in group I, enterococcus, Ⅱ, III group rats enterbacteriaceae were significantly increased, the difference was statistically significant (p<0.01); Bifidobacterium and lactobacillus were reduced, the difference was statistically significant (p<0.05);Ⅱ, III group comparison between groups of rats, enterococcus, e. coli and bifidobacteria difference was statistically significant (p<0.05), the difference of lactobacillus has no statistical significance (p>0.05).Conclussion:Combined cold dampness building environment, rhubarb decoction lavage and intraperitoneal injection of D-gal method to induce Yin yellow card IETM model is set up. Study two This toxin and blood endotoxin of IETM Yin yellow card liver failure rats, the influence of cytokinesObjectives:1.Research explored blood detoxification method of IETM Yin yellow card liver failure rat IL-6, C-met immune protein expression and expression.2. Study this toxin and blood Yin yellow card to liver failure after intervention IETM rat intestinal flora, the change of plasma endotoxin.Methods:86SD rats, Were randomly divided into Yin yellow card to liver failure IETM model group (group I), explored the detoxification of blood low dose group (group II), explored the detoxification of blood in the dose group (group III), explored the detoxifyication of blood high dose group (group IV), each group of20, Normal6(group V) in rats. II, III, IV group6days prior to the building of rats with different blood party explored detoxified dose lavage, I group of rats with normal saline lavage,2times/day, until death. Building21days in group I, II, III, IV to D-gal intraperitoneal injection of1.8g/kg Yin yellow card IETM model is set up; V group rats normal diet. Observed between groups of rats in24h,48h after building mortality, groups of rats and liver function (ALT, AST,TBIL) and liver tissue pathology, plasma endotoxin, intestinal flora, IL-6, C-the liver tissue tested met the immune protein expression and so on.Results: 1.Mortality:I group rats mortality rate of32%; Ⅱ, Ⅲ, Ⅳ group rats mortality declined gradually with the increase of this blood detoxification party dose, groups of mortality was23%,20%and15%, respectively.2. Liver function:compared with group Ⅰ, Ⅱ, Ⅲ, IV group rats ALT, AST, TBIL is reduced, the IV group and the comparison between the way, I was statistically significant difference (p<0.01).3. Liver tissue pathology results:compared with group Ⅰ, Ⅱ, Ⅲ, Ⅳ group, according to the results of liver tissue pathology damage reduce, only IV group and the group I liver histopathologic grading, the difference was statistically significant (p<0.05).4. Plasma endotoxin:compared with group Ⅰ, Ⅱ, Ⅲ, IV group, the level of endotoxin was reduced, only high dose group and the difference between model way was statistically significant (p<0.05).5. Fecal intestinal flora level:compared with group Ⅰ, Ⅱ, Ⅲ, Ⅳ group rat intestinal bacteria, e. coli were increased, the difference was statistically significant(p<0.01); BifidoBacterium and lactobacillus were reduced, the difference was statistically significant (p<0.05); Ⅱ, Ⅲ and IV respectiveely compared between group rats, enterococcus, e. coli and bifidobacteria difference was statistically significant (p<0.05), the difference of lactobacillus has no statistical significance (p>0.05). Group Ⅱ, Ⅲ, comparing differences between way has no statistical significance (p>0.05).6. IL-6level changes:the liver tissue compared with group Ⅰ, Ⅱ, Ⅲ, Ⅳ group rats IL-6levels increased significantly, the difference was statistically significant (p<0.05).7. C-met protein immunohistochemical:compared with group Ⅰ, Ⅱ, Ⅲ, IV group. C-met positive area were significantly increased, the difference was statistically significant (P<0.01); IV group compared with two Ⅱ, Ⅲ, IV group. C-met positive area is higher than Ⅱ, Ⅲ, the difference was statistically significant (P<0.05).Conclussion:1. Using this blood detoxification reduce liver failure IETM Yin yellow card mortality in rats, improve liver function, reduce liver tissue pathological situation.2. This blood detoxification side effects on liver failure mechanism of IETM Yin yellow card may be by (1) reduce the absorption of the plasma endotoxin;(2) inducing immune signal activation; To promote the increment of liver cell;(3) regulating intestinal flora; To implement. Study three Wenyang Jiedu Huayu method for patients with hepatitis B liver failure correlation Yin yellow card and Yin Yang yellow cardObjectives:Through selected60cases of patients with hepatitis B liver failure correlation the analysis of the combined therapy of Chinese and western, evaluate this toxin and blood in patients with hepatitis B liver failure correlation Yin yellow card and Yin Yang yellow card curative effect.Methods:Selected cases according to syndrome differentiation of TCM into the Yin yellow card and Yin Yang yellow card groups. And according to random number table is divided into the experimental group and control group, two groups of patients were in normal liver, protection of liver, stomach, improve the hepatic microcirculation, antiviral, symptomatic treatment, etc.on the basis of the experimental group to this blood detoxification granules, the control group on the basis of the simulation agent treatment with traditional Chinese medicine. After eight weeks, to evaluate curative effect of two groups of patients.Results:The two groups after treatment of liver TBIL, PTA, gut endotoxin comparison between groups, such as difference was statistically significant (P>0.05), inside two way comparison before and after the treatment, the difference was statistically significant (P<0.05).Conclussion:Combining this detoxification treated patients with hepatitis B liver failure correlation method, can improve symptoms, improve curative effect.更多还原