【作者】 黎壮伟；

【导师】 林培政；

【作者基本信息】 广州中医药大学 ， 中医临床基础， 2010， 博士

【摘要】 温病湿热证是临床常见病证,湿热既是中医特有的致病原因,又是具有广泛临床基础的中医病证,涉及多系统、多器官、多组织的病变。岭南气候、地理、饮食习惯及体质因素致使岭南地区湿热病证易发、多发。在岭南地区研究温病湿热证具有重要的意义。目前研究已表明湿热证致病机制与免疫、炎症、水通道蛋白、幽门螺杆菌、胃肠动力功能、内毒素、氧化损伤、微生态、微量元素、血脂代谢及能量代谢等密切相关,并不局限于某一脏腑或某一靶点。本课题通过临床研究与实验研究相结合的思路,从线粒体氧化损伤及能量代谢的角度探讨温病湿热证的发病机制,并采用清热化湿中药进行干预,探讨其部分作用机制。目的：本研究以鼠肝炎病毒(MHV-A59)为生物致病因子,建立“肥甘饮食+外湿热环境+鼠肝炎病毒感染”温病湿热证模型,观察温病湿热证肝线粒体超微结构、超氧化物歧化酶(SOD)、谷胱甘肽过氧化酶(GSH-PX)、丙二醛(MDA)、膜电位、ATP酶活力、ATP含量、四种呼吸链复合物Ⅰ、Ⅱ、Ⅲ、Ⅳ活性,从温病湿热证小鼠肝脏线粒体氧化损伤及能量代谢的角度探讨湿热证的发病机制,并采用清热化湿中药进行干预,反证其致病机制,初步探讨清热化湿法的作用机理。临床探讨外感湿热证患者外周血SOD、GSH-PX和MDA的变化是对实验研究的补充与佐证。方法：1、文献研究,采用评述的方法,整理温病湿热证动物模型的研究现状,湿热证证候本质研究概况,线粒体氧化损伤和能量转换的现代研究,归纳湿热证与线粒体氧化、能量代谢的相关性研究和MHV的研究进展,以期理清本实验的研究思路。2、实验研究,本实验所选用的均为4周龄左右的雄性BALB/c小鼠。①温病湿热证模型的建立：动物随机分为4组：正常对照组(正常饮食、饮水)、单纯湿热组(肥甘饮食+外湿热环境造模)、单纯病毒组(单纯小鼠肝炎病毒MHV-A59造模)和模型组(肥甘饮食+外湿热环境+小鼠肝炎病毒造模),检测小鼠血清TG、肝功能ALT、AST水平及肝组织病理切片,观察动物症状及体征。②在模型成功的基础上探讨温病湿热证的致病机制：动物随机分为4组：正常对照组、单纯湿热组、单纯病毒组和模型组,电镜观察各组线粒体超微结构,检测各组小鼠肝线粒体SOD、GSH-PX、MDA、膜电位、ATP酶活力、ATP含量、四种呼吸链复合物Ⅰ、Ⅱ、Ⅲ、Ⅳ活性的变化。③清热化湿中药的对温病湿热证的干预机制：动物随机分为4组：模型组、蒿芩清胆汤组(模型组用蒿芩清胆汤治疗),甘露消毒丹组(模型组用甘露消毒丹治疗)、西药组(模型组用易善复胶囊治疗)：检测各组小鼠肝线粒体SOD、GSH-PX、MDA、MDA、膜电位、ATP酶活力、ATP含量、四种呼吸链复合物Ⅰ、Ⅱ、Ⅲ、Ⅳ活性的变化。3、临床研究,30例辨证属外感湿热证作为治疗组,病例来源于广州市中医医院门诊,同时期随机检测25例健康体检者作为对照组。比色法测定血清SOD、GSH-PX、MDA水平变化,治疗组运用蒿芩清胆汤治疗3日,复测上述指标,观察蒿芩清胆汤的干预作用。对照组不使用药物治疗。结果：1.温病湿热证动物模型方面,与正常组比较,温病湿热证模型小鼠出现了活动量、饮食量均明显减少、部分动物不进食,粪便及排泄物粘滞或稀溏、明显增多,小鼠体重减轻,肛温升高,出现肢体瘫痪和追尾征,毛发蓬松,无光泽。动物这些症状与体征符合温病湿热证的发病特点。模型组甘油三酯(TG)升高,脂肪代谢紊乱,与正常组和单纯病毒组比较,差异有显著性(P<0.01)。模型组光镜下见小鼠肝脏可见空泡样变、炎性细胞浸润,100%存在小叶内炎症,并可见脂肪样变、点状坏死、灶状坏死、碎屑样坏死。正常组和单纯病毒组小鼠肝脏未见脂肪变性。这些表现基本符合温病湿热证的病理特征。2.线粒体氧化损伤方面,温病湿热证模型组肝脏线粒体SOD活性和GSH-PX活性降低,MDA含量升高,膜电位降低,与正常组、单纯湿热组和单纯病毒组比较,均有显著性差异(P<0.01或P<0.05)。说明温病湿热证动物模型体内脂质过氧化物含量升高,相关抗氧化酶浓度和活性降低,线粒体功能受到损伤。蒿芩清胆汤与甘露消毒丹均能够提高温病湿热证模型组小鼠肝线粒体SOD、GSH-PX活性,提高清除自由基的防御体系功能,降低MDA含量,清除自由基,提高线粒体膜电位,与模型组比较具有显著性差异。(P<0.01)。提示清热化湿中药治疗温病湿热证,通过提高清除自由基的防御体系和减少自由基生成两方面作用,达到提高线粒体抗氧化功能及减少氧化损伤,保护线粒体功能。在与模型组比较,西药易善复在提高SOD、GSH-PX活性及膜电位方面无显著性差异,但在降低MDA含量方面差异有显著性(P<0.05)。本研究结果提示,清热化湿中药干预温病湿热证模型线粒体氧化损伤的机理主要表现在提高SOD、GSH-PX活性及膜电位,提高自由基消除的防御体系,减少自由基生成,降低MDA含量,清除已生成的自由基,提高膜电位。3.线粒体能量代谢方面,与正常组比较,单纯湿热组、模型组ATP酶活力和ATP含量均明显下降。其中,模型组下降最为显著,有非常显著性差异(P<0.01)。ATP酶活性下降提示能量分解和利用减少,ATP含量降低提示能量生成不足。研究结果说明单纯湿热组和模型组能量代谢障碍,ATP生成和分解利用下降。这种现象在模型组表现最为显著。与模型组比较,甘露消毒丹组与蒿芩清胆汤组ATP酶活力和ATP含量均升高最显著(P<0.01),提示清热化湿中药能提高温病湿热证线粒体ATP酶活性,促进能量的合成,促进ATP合成,改善线粒体的能量代谢障碍。模型组呼吸链复合物Ⅰ、Ⅱ、Ⅲ、Ⅳ活性降低,与正常组比较有显著性差异(P<0.01),说明模型组可能存在严重的能量代谢障碍和线粒体功能的障碍。蒿芩清胆汤组和甘露消毒丹组呼吸链复合物Ⅰ、Ⅱ、Ⅲ、Ⅳ活性升高,与模型组比较有显著性差异(P<0.01或P<0.05),提示清热化湿中药治疗温病湿热证,通过提高呼吸链复合物Ⅰ、Ⅱ、Ⅲ、Ⅳ活性,促进ATP的生成和分解利用,能改善线粒体能量代谢。4.临床研究方面,与正常组比较,急性上呼吸道病毒感染湿热证患者外周血清SOD和GSH-PX活性降低(P<0.01),而MDA含量显著升高(P<0.01)；经蒿芩清胆汤治疗后,患者血清SOD和GSH-PX活性显著升高(P<0.01或P<0.05),MDA含量明显降低(P<0.01)。结论：1.以MHV-A59为生物致病因子,结合肥甘饮食及湿热外环境的复合因素构建的动物模型,可以作为温病湿热证动物模型之一,也是对病证结合动物模型的一种新的探索。2.温病湿热证存在线粒体氧化损伤及能量代谢障碍,使用清热化湿中药治疗后能改善氧化损伤,提高膜电位,促进能量的生成和利用,有利于恢复细胞的正常功能。因此,线粒体氧化损伤及能量代谢障碍可能是温病湿热证发病机制之一。3.蒿芩清胆汤和甘露消毒丹均能在一定程度上改善温病湿热证线粒体氧化损伤,保护线粒体的功能,改善能量代谢,反证了温病湿热证的上述致病机制。清热化湿中药治疗温病湿热证具有改善细胞氧化损伤,促进能量代谢功能。4.外感湿热证患者机体存在氧化应激损伤,抗氧化酶系功能降低。蒿芩清胆汤能够改善急性上呼吸道病毒感染湿热证机体氧化损伤。更多还原

【Abstract】 Background:Damp-heat syndrome of seasonal febrile disease is common clinical disease, involving pathological changes of many systems and organs and tissues. Because of the special climate, geography, food habit and constitution factors in Ling Nan Area, damp-heat disease is common and easy, so it is important significance to study damp-heat syndrome in Ling Nan Area. The current researches demonstrate that pathogenesis of damp-heat syndrome of seasonal febrile disease is not limited to one entrails or target, it is related to immunologic function, inflammation, aquaporins, helicobacter pylori, gastrointestinal motility, endotoxin, oxidative damage, microecology, trace elements, blood lipid metabolism and energy metabolism, etc. On the basis of clinical and experimental research, we explore the pathogenesis of damp-heat syndrome of seasonal febrile disease from oxidative damage and energy metabolism of mitochondria and effects of Chinese herbs.Objective:The research established mouse model of damp-heat syndrome of seasonal febrile disease through richly fatty and sweet diet, humid heat environment and infected by MHV-A59, in which MHV-A59 was used as biological pathogenic factor. The research studied the pathogenesis of damp-heat syndrome of seasonal febrile disease from oxidative damage and energy metabolism of mitochondrion in the model mouse, by observing the ultramicrostructure, superoxide dismutase(SOD), glutathione proxidase (GSH-PX), malondialdehyde(MDA), membrane potential, ATPase activity, ATP concent and activities of respiratory chain complexⅠ、Ⅱ、Ⅲ、Ⅳin the hepatic mitochondrion of mice model of damp-heat syndrome of seasonal febrile disease. The research certificated the pathogenesis of damp-heat syndrome of seasonal febrile disease by treatment with Decoction of Hao qin qing dan and Decoction of Gan lu xiaodu, observed the treatment mechanism of clearing away heat and dampness. It was supplementary and evidence for pathogenesis of damp-heat syndrome of seasonal febrile disease by studing SOD, GSH-PX and MDA of peripheral blood of patients with exogenous damp-heat disease.Methods:1. Literature research:on the basis of the approach of evaluating and reviewing articles, we proposed to arrange the study state of animal model of damp-heat syndrome of seasonal febrile disease, study the survey of essence of damp-heat syndrome, the modern study of oxidative damage and energy metabolism of mitochondrion, sum up the study progression of MHV and the relationship between therapy of clearing away heat and dampness and oxidative damage and energy metabolism of mitochondrion.2. Experimental research:①establishment of damp-heat syndrome of seasonal febrile disease model. four-weeks-old male mice of BALB/c was divided randomly into four groups:control group(normal diet), simple damp-heat syndrome group(richly fatty, sweet diet and humid heat environment), simple virus infection group (normal diet and virus infection) and model group (richly fatty, sweet diet, humid heat environment and infected by MHV-A59), The triglyceride (TG), the indexes of liver function ALT、AST and Hepatic histopathology in mice were detected by biochemistry and ELISA. In the experiment we observed the symptom and sign of mice.②pathogenesis of damp-heat syndrome of seasonal febrile disease:four-weeks-old mice of BALB/c was divided randomly into four groups:control group, simple damp-heat syndrome group, simple virus infection group and model group. The ultramicrostructure of hepatic mitochondria in each group was observed with electronic microscope and the change of SOD, GSH-PX, MDA, membrane potential, ATPase activity, ATP content and activities of respiratory chain complexⅠ、Ⅱ、Ⅲ、Ⅳin the hepatic mitochondrion of mice were detected.③The pathogenesis of treating damp-heat syndrome of seasonal febrile disease with the decoction of clearing away heat and dampness:four-weeks-old mice of BALB/c were divided into four groups randomly, model group, Hao qin qing dan decoction group (mice of model group were treated by Decoction of Hao qin qing dan), Gan lu xiao du decoction group(mice of model group were treated by Decoction of Gan lu xiao du), western medicine group (mice of model group were treated by Polyene Phosphatidylcholine Capsules).The change of SOD, GSH-PX,MDA, membrane potential, ATPase activity, ATP content and activities of respiratory chain complexⅠ、Ⅱ、Ⅲ、Ⅳin the hepatic mitochondrion of mice were detected3. Clinical research:The 30 patients of exogenous damp-heat syndrome as the test group were from out-patient clinic of guangzhou hospital of Chinese medicine. There were 25 healthy people undergoing medical examination as control group. The level of SOD, GSH-PX and MDA of patients and healthy people were detected by colorimetry. The patients were treated by Decoction of Hao qin qing dan for three days. Then we detected the level of SOD, GSH-PX and MDA again. Healthy people without treatment.Results:1.In the research of establishment of damp-heat syndrome of seasonal febrile disease model:Compared to control group, the mice in model group showed some features such as drinking less water, eat less food, viscous and more plentiful excretion, weight loss, rectal temperature going up, bulky hair, etc. These signs and symptoms were closely related to the features of damp-heat syndrome of seasonal febrile disease. The level of TG obviously risen up compared to control group and simple virus infection group (p＜0.01). The liver of the mice under microscope in model group showed vacuolating chang, inflammatory cell infiltration,100% leaflet inflammation, hepatic steatosis, necrosi. Hepatic steatosis was not existing in control group and simple virus infection group. These pathological changes were closely related to basically pathological features of damp-heat syndrome of seasonal febrile disease.2. In the research of oxidative damage of mitochondria:The level of SOD, GSH-PX and membrane potential decline and MDA risen up in model group obviously compared to control group, simple damp-heat syndrome group and simple virus infection group (P＜0.01 or P＜0.05). These show that lipid peroxidation content risen up and relative antioxidase activity decreased, mitochondrion function was injured in damp-heat syndrome of seasonal febrile disease model. Decoction of Hao qin qing dan and Decoction of Gan lu xiao du could improve the activities of SOD, GSH-PX and membrane potential in the hepatic mitochondria of damp-heat syndrome of seasonal febrile disease, improve the function of defense system scavenging free radicals, at the same time,they reduced MDA content and scavenged the free radicals. The difference was significant compared to model group (p＜0.01) These findings indicate that Chinese herbs of clearing away heat and dampness can reinforce mitochondria function of antioxidation and reduce the injure of oxidation to protect mitochondrion though improving the function of defense system scavenging free radicals and reducing the production of free radicals. There is no significant difference in improving the activities of SOD, GSH-PX and membrane potential in the hepatic mitochondrion between model group and western medicine group, but western medicine group obviously ruduce MDA content compared to model group(P＜0.05). The research result indicate that the mechanism of Chinese herbs clearing away heat and dampness was to improve the activities of SOD, GSH-PX and mitochondrial membrane potential, improve the function of defense system, reduce the production of free radicals, remove the producted free radicals in treatment of damp-heat syndrome of seasonal febrile disease.3. In the research of energy metabolism:The level of ATPase activity and ATP content obviously decline in model group and simple damp-heat syndrome group compared to control group, specially in model group (P＜0.01).The decreasement of ATPase activity indicates that energy decomposition and utilization decrease. The decreasement of ATP content indicates that energy product Insufficiently. The research result indicate that energy metabolize disorderly and the production and utilization of ATP decrease in damp-heat syndrome of seasonal febrile disease. Chinese herbs clearing away heat and dampness could improve the ATPase activity and ATP content obviously compared to model group (P＜0.01), improve energy metabolic disorders and promote the production of energy.Activities of respiratory chain complexⅠ、Ⅱ、Ⅲ、Ⅳin the hepatic mitochondrion of mice from model of damp-heat syndrome of seasonal febrile disease decline obviously compared to control group (p＜0.01), which illustrate that serious energy metabolic disorders and dysfunction of mitochondrion may esist in damp-heat syndrome of seasonal febrile disease. Activities of complexⅠ、Ⅱ、Ⅲ、Ⅳin Hao qin qing dan decoction group and Gan lu xiao du decoction group obviously risen up compared to model group (p＜0.05 or p＜0.01). The research result indicate that Chinese herbs clearing away heat and dampness could improve the activities of complexⅠ、Ⅱ、Ⅲ、Ⅳ, in order to promote the production and utilization of energy, improve the energy metabolism of mitochondrion.4. clinical research:Compared to healthy people, the level of Peripheral blood SOD, GSH-PX of patients obviously decreased (p＜0.01) and MDA rised up (p＜0.01), there were significant differences. The level of Peripheral blood SOD, GSH-PX of patients who treated by decoction of Hao qin qing dan obviously rised (p＜0.01 or p＜0.05), and MDA obviously decreased (p＜0.01).Conclusion:1.A new mouse model of damp-heat syndrome of seasonal febrile disease was duplicated by richly fatty and sweet diet, humid heat environment and MHV-A59. MHV-A59 was a biological pathogenic factor in the model which possessed characteristics of the illness and syndrome.2.Oxidative damage and energy metabolic disorders exist in damp-heat syndrome of seasonal febrile disease. Chinese herbs clearing away heat and dampness could reduce oxidative damage and improve production and utilization of energy, which was in favor of recovery of normal function of cell. So, oxidative damage and energy metabolic disorders in mitochondrion may be one of the pathogenesis of damp-heat syndrome of seasonal febrile disease.3. Both decoction of Hao qin qing dan and decoction of Gan lu xiao du could reduce oxidative damage of mitochondrion to a certain extent in the damp-heat syndrome of seasonal febrile disease, protect the function of mitochondrion, improve the energy metabolism. These proof the pathogenesis of damp-heat syndrome of seasonal febrile disease discussed above. Chinese herbs of clearing away heat and dampness could reduce oxidative damage of mitochondrion and promote the production and utilization of energy.4.Oxidative damage and antioxidant enzyme system function deficiency exists in the body with damp-heat syndrome of seasonal febrile disease. Decoction of Hao qin qing dan could reduce the oxidative damage in treating viral upper respiratory infection of damp-heat syndrome更多还原