【作者】 赵文霞；

【导师】 薛博瑜；

【作者基本信息】 南京中医药大学 ， 中医内科学， 2012， 博士

【摘要】 非酒精性脂肪性肝病(NAFLD)发病率逐年增加,非酒精性脂肪性肝炎(NASH)是其向肝硬化、肝功能衰竭发展的重要环节,已成为目前肝病领域的新挑战。目前尚无专用于治疗NASH的药物,中医药在治疗NASH方面具有一定的优势,导师在多年临床研究基础上积累了丰富的经验,制定了化痰祛湿活血方有效方剂,经临床应用效果良好。为进一步明确其临床疗效和作用机制,开展本研究。目的：探讨化痰祛湿活血方治疗NASH的临床疗效和作用机制,为临床治疗NASH提供依据。方法：①临床研究：采用多中心、随机、阳性对照临床研究方法,从4个分中心共收集NASH患者202例,分为治疗组和对照组,两组在健康教育、饮食运动基础上,治疗组使用化痰祛湿活血方颗粒剂1剂/d；对照组使用多烯磷脂酰胆碱(易善复)胶囊,456mmg/次,3次/d；疗程3个月,随访3个月。观察治疗、随访后患者血清ALT、TG、 AST、TC、肝/脾CT值、症状体征、体重指数等的变化；评价化痰祛湿活血方治疗NASH患者的临床疗效。②实验研究：根据NASH的发病机理,选择与其相关的血清ALT、AST、TG、TC.肝匀浆游离脂肪酸(FFA),肝指数、肝脏病理组织学,肝组织细胞色素P4502E1(cytochrome P4502E1, CYP2E1)和过氧化物酶体增殖物激活受体α(peroxisome proliferator-activated receptor-a, PPARa)基因表达等作为观察指标。具体的实验过程是：取SD雄性大鼠72只,随机分为空白组、模型组、多烯磷脂酰胆碱胶囊组(对照组)和化痰祛湿活血方高(高剂量组)、中(中剂量组)、低(低剂量组)剂量组。使用高脂饲料联合腹腔注射四环素进行造模,第7周实验结束时处死全部实验动物,腹主动脉采血；之后迅速取出肝脏称肝湿重,留取肝脏标本。观察肝湿重、肝指数、病理组织学变化(光学显微镜、透射电镜),检测血清谷丙转氨酶(ALT)、谷草转氨酶(AST)、甘油三酯(TG)、总胆固醇(TC)、肝匀浆FFA含量、肝组织CYP2E1、PPARα基因表达情况(RT-PCR法检测)等指标。结果：临床及实验研究结果分述如下：1.临床研究：①治疗组与对照组在治疗前后降低血清ALT及TG水平均有显著性差别(P<0.05),说明两组在保肝降酶、降TG方面均具有明显的作用。两组间治疗后比较治疗组在降低ALT、TG方面优于对照组,差异有显著性意义(P<0.05)。两组在改善肝/脾CT值治疗前后比较有显著差异(P<0.05),两组间治疗后比较也有显著差异(P<0.05)。②两组在减轻体重指数(BMI)方面,治疗组、对照组组内治疗前后比较有显著性差异(P<0.05),治疗后两组比较无显著性差异(P>0.05),说明治疗组、对照组均可有效减轻体重指数。治疗组、对照组组内治疗前后比较症状和体征积分有显著性差异(P<0.05)。治疗组治疗后在改善肢体困重、胁胀胁痛、脘腹胀满、食少纳呆、大便溏薄症状方面优于对照组,差异有显著性(P<0.05)。2.实验研究结果显示：①各用药组与模型组比较：各用药组肝指数、血清ALT、AST、 TC、TG和肝匀浆FFA含量均有不同程度下降,以化痰祛湿活血方高、中剂量组疗效最优(P<0.01或P<0.05)。化痰祛湿活血方高、中、低剂量组及对照组肝组织CYP2E1基因表达水平均有显著降低(P<0.01)。化痰祛湿活血方高、中剂量组PPAR α基因表达水平显著升高(P<0.01)。②化高、中剂量组与对照组相比：高剂量组、中剂量组的肝湿重、病理组织学改善明显；血清ALT、AST、TC、TG、肝匀浆FFA含量显著降低(P<0.05)；高剂量组、中剂量组抑制CYP2E1基因表达效果明显优于对照组(P<0.01或P<0.05)；化痰祛湿活血方高、中剂量组的PPAR α基因表达水平较对照组显著升高(P<0.05)化高、中剂量组与低剂量组相比有显著性差异(P<0.01或P<0.05)。化痰祛湿活血方高、中剂量组之间比较无显著性差异(P>0.05)。③病理学观察：光学显微镜显示：化痰祛湿活血方高、中剂量组与模型组脂肪变性比较有非常显著性差异(P<0.01),高剂量组与对照组比较有显著性差异(P<0.05),高剂量组与中剂量组比较有显著性差异(P<0.05),高剂量组与低剂量组比较有非常显著性差异(P<0.01),中剂量组与低剂量组比较有显著性差异(P<0.05)。透射电镜显示：与模型组相比,对照组、高剂量组、中剂量组均可升高肝细胞线粒体体密度(Vv)、比膜面(δm)、粗面内质网比膜面(δm),有显著性差异(P<0.01),均可降低肝细胞内脂滴体密度(Vv)、线粒体比表面(δ),有显著性差异(P<0.01)。高剂量组优于中剂量组和对照组(P<0.01),中剂量组与对照组效果相当。结论：①化痰祛湿活血方治疗非酒精性脂肪性肝炎痰湿瘀阻证患者在降低血清ALT、TG,提高肝／脾CT值,改善症状体征方面优于多烯磷脂酰胆碱胶囊,与多烯磷脂酰胆碱胶囊均有降低患者BMI作用。化痰祛湿活血方适用于非酒精性脂肪性肝炎(痰湿瘀阻证)的患者,尚未发现不良反应,是一种有效安全的方药。②采用高脂饲料联合四环素腹腔注射,造模时间短,成功率高,是一种较为理想的大鼠NASH造模方法。化痰祛湿活血方各剂量组均可有效降低NASH模型大鼠的肝湿重、肝指数、血清ALT、AS、TC、TG及肝匀浆FFA水平,改善病理组织学变化,具有显著的抗脂肪肝作用；化痰祛湿活血方可激活PPAR α mRNA表达,纠正肝脏脂质代谢紊乱；同时可抑制CYP2E1mRNA表达,阻止脂质过氧化反应。化痰祛湿活血方在增强大鼠PPAR α的表达的同时又可抑制CYP2E1表达,故不会加重脂质过氧化作用对肝脏的损伤,而是最终发挥了降低血脂、保肝降酶、阻止或减轻脂肪肝形成的作用,这可能是化痰祛湿活血方防治NASH的重要作用机制之一。化痰祛湿活血方可显著改善肝脏脂肪沉积,其治疗NASH的机制可能是通过增加线粒体嵴和线粒体数量,改善粗面内质网脱颗粒现象,保护肝细胞线粒体和内质网,恢复其形态结构和功能,增强肝细胞的能量代谢,从而达到治疗NASH的作用。更多还原

【Abstract】 With the morbidity of non-alcohol fatty liver disease increasing year by year, NASH is critical in the development of liver fibrosis and liver failure, which has became a new challenge in the field of liver diseases research. At present, there is still no specific medicine for NASH, but the Chinese traditional medicine has its advantage on NASH. With many years of clinic research and experience, a recipe of HTQF (hua tan qu shi huo xue fang) has been formulated by our tutor and that has a good effect. What’s more, after years of clinical application, the effect is inspiring. To further clarify the clinical efficacy and therapeutic mechanism, this study is implemented.Objective:to implore the clinical efficacy and therapeutic mechanism of HTQF. to provide the theoretic evidence for the clinic application of HTQF.Methods:the study was divided into two parts.1. Clinical research:A multi-center randomized controlled clinical study was done on202NASH patients from four sub-centers which were divided into the treatment group and the control group. Base on the health education, diet and exercise, the treatment group was administrated HTQF one dose per day and the control group polyene phosphatidylcholine three times per day. The course of the study was three months and the follow-up was three months too. After that the ALT, AST, TC, FFA of the liver homogenate, the ratio of liver/spleen CT values, the signs and symptoms, Body Mass index were observed to evaluate the clinical efficacy of HTQF on NASH.2.experimental study:the ALT, AST, TG, TC, FFA of the liver homogenate, the liver histopathology, the liver cytochrome P4502E1, the PPARa-mRNA were detected as the observation targets according to the NASH pathogenesis.72male rats were divided into6groups randomly, the normal group, the model group, the polyene phosphatidylcholine group, the HTQF high, medium and low dose group. NASH rat model was duplicated with high fat diet and tetracycline intraperitoneal injection. The animals were sacrificed after7weeks. After the blood collected from the abdominal aorta, the liver wet weights were measured and the liver specimens kept. The liver index and the histopathological changes (light microscopy and electron microscopy) were observed. The ALT, AST, TC, FFA of the liver homogenate, the liver cytochrome P4502E1, the PPARa-mRNA and so on were investigated too. Result:the results of the clinical research and experimental study are displaced as follow.1. Clinical research①The levels of serum ALT and TG, in treatment and control group, declined significantly after therapy (P<0.05), which indicated that the two groups were all effective in protecting liver function, reducing enzymes and decreasing TG, while the former groups were superior to the latter group (P<0.05). The ratio of liver/spleen CT values, in treatment and control group, were reduced significantly after therapy (P<0.05) and the former groups were superior to the latter group too (P<0.05)②The BMI, in treatment and control group, declined significantly after therapy (P<0.05), but groups comparison showed no difference in statistics (P>0.05),which indicated that the two groups were all effective in decreasing BMI. Integration of signs and symptoms in treatment and control group declined significantly after therapy (P<0.05) and the former groups were more effective in ameliorating symptoms, such as heavy sensation in the limbs and body, hypochondriac distention and pain, abdominal fullness and distention, eating less, anorexia and loose stool (P<0.05)2. Experimental study①Treatment and model group comparison The content of liver index, serum ALT, AST, TC, TG and liver homogenate FFA, in treatment and control group, declined significantly at different level, after therapy (P<0.05), with the high-dose and medium-dose HTQF the most effective (P<0.01, P<0.05). The expression of CYP2E1-mRNA in hepatic tissue were all depressed in high-dose, medium-dose and low-dose HTQF and Polyene group (P<0.01), while the expression of PPARa-mRNA in hepatic tissue were elevated in high-dose and medium-dose HTQF group (P<0.01)②High-dose, medium-dose HTQF and Polyene group comparison When compared with Polyene group, several indexs changed significantly, which included ameliorated liver wet weight and histopathology, decreased level of serum ALT, AST, TC, TG and liver homogenate FFA, depressed CYP2E1-mRNA and elevated PPARa-mRNA(P<0.05), with no significantly different among the two groups of HTQF (P>0.05)③pathology The degree of steatosis, under light microscope, had significant difference between high-dose or medium-dose HTQF group and model group (P<0.01). There were significant differences when comparing either positive control (P<0.05), medium-dose HTQF group (P<0.05) or low-dose HTQF group (P<0.01) with high-dosed HTQSHXF group. There was significant difference between middle-dose and low-dose HTQF group too (P<0.05). Under electron microscope, there were significant difference of the elevated liver cells mitochondria bulk density (Vv), membrane density (δm) and rough endoplasmic reticulum membrane density (δm) when positive control group, high-dosed or middle-dosed HTQSHXF group compared with model group (P<0.05).There were significant difference of the decreased lipid drops bulk density (Vv) and mitochondrion specific surface (δ) too. While high-dose HTQF was superior to the medium-dose HTQF group and Polyene control group (P<0.01) with no significant difference between the latter two groups.Conclusion:As regarding to reducing the ALT, TG of the serum, raising the ratio of liver/spleen CT values, improving the clinical symptoms, the HTQF group was superior to the polyene group on NASH of phlegm and stasis. HTQF and polyene both can relieve the BMI. By far there are no side effect of HTQF on NASH of phlegm and stasis and HTQF is a safe and effective recipe. Using high fat diet and tetracycline intraperitoneal injection to duplicate NASH rat model is an ideal method, which is time saving and has high success rate. By reducing the wet liver weight, decreasing the index of liver, lowering the ALT, AST, TC, TG of serum and the FFA of liver homogenate, and improving the liver histopathology, HTQF has the good effect of anti-fatty liver. HTQF can induce the expression of PPARa-mRNA and correct the lipid metabolism disorders. At the same time HTQF can suppress the expression of CYP2E2-mRNA to prevent the lipid peroxidation. By protecting liver damage through preventing lipid peroxidation, HTQF can lower blood lipids, protect liver function and reduce the lever of liver transminases, so as to resist the formation of fatty liver, which may be one of its mechanism on NASH. HTQF can reduce the liver fat deposition. By increasing the mitochondria cristae and its number, improving the degranulation of rough endoplasmic reticulum, protecting the mitochondria and endoplasmic reticulum, restoring its morphology and function, enhancing the energy metabolism of the liver cells, HTQF owns the curative effect on NASH.更多还原