【作者】 胡孔友；

【导师】 余林中；

【作者基本信息】 南方医科大学 ， 中药学， 2009， 硕士

【摘要】 目的:急性肺损伤（Acute lung iniury,ALI）/急性呼吸窘迫综合征（Acuterespiratory distress syndrome,ARDS）是一种常见危重病,病死率极高,严重威胁重症患者的生命并影响其生存质量。ARDS早期的特征性表现为肺毛细血管内皮细胞与肺泡上皮细胞屏障的通透性增高,肺泡与肺间质内积聚大量的水肿液,其中富含蛋白及中性粒细胞为主的多种炎症细胞。目前对于ALI尚缺乏理想的防治手段,成为危重病医学的难点之一。中西医结合是我国防治ALI的特色和优势,一些学者从临床入手,将中医学理论和方法用于全身炎症反应综合征（System inflammatory response syndrome,SIRS）、ALI、ARDS治疗取得较好成绩,相关基础研究也出现良好前景。随着中医温热病病因病机理论与现代病理生理学相关理论的交流和渗透,人们在对ALI发生发展规律的认识与防治研究方面产生了有益的借鉴,如对中医温热病解毒法实质及解毒方药作用机理的深入研究为我们防治ALI提出新的策略与方法。临床SIRS、多器官衰竭功能综合征（Multiple organ dysfunction syndrome,MODS）研究发现,肺脏是最易受损伤的首位靶器官。ALI和ARDS出现最早,发生率也最高。肺功能障碍表现突出,究其原因除开病因对肺组织直接损害因素,可能是由于肺组织构成中的细胞种类丰富,肺循环接纳全部体循环血液,且有全身毛细血管网和最多的内皮结构,血液中的各种有害因子和介质能够与之充分作用,因此易造成ARDS。各种原因引起ALI的发病机制基本相似,主要是以肺泡一毛细血管膜急性损伤为基础的炎症反应。SIRS是ALI的基础,ALI又促进SIRS,而炎症细胞的聚集与活化、炎症介质的合成与过度释放引起的炎症反应失控、免疫功能紊乱是SIRS、ALI发生发展的主要因素。炎症本质上是机体的防御反应,但炎症失控又可损伤自身细胞,炎症是否失控取决于炎症和抗炎反应之间能否达到相对平衡。ALI作为机体过度炎症反应的后果,调控炎症反应则是防止ALI发生发展、降低死亡率的关键所在。目前已有一些药物在临床试用,如糖皮质激素、环氧化酶抑制剂、血栓烷合成酶抑制剂、磷酸二酯酶抑制剂、抗内毒素及细胞因子单克隆抗体或拮抗剂等。但是这些药物在大样本临床试验中并未获得肯定疗效,仍然缺乏一种特异而有效的治疗手段。急性肺损伤的发病,目前多认为巨噬细胞被激活后募集多型核白细胞（PMN）至肺引起炎症反应占主导地位。革兰（氏）阴性杆菌（Gram negative bacilli,GNB）感染后,LPS引发机体SIRS和代偿性抗炎反应综合征（CARS）,过度的SIRS则发展为多脏器功能障碍综合征（MODS）,在肺的表现为ALI。ALI时几乎肺内所有种类的细胞都涉及了这个炎症反应,并释放大量的炎性介质,而肺血管内皮细胞、肺上皮细胞既是效应细胞又是致病因子作用的靶细胞。参与的细胞因子和炎症介质有促炎细胞因子肿瘤坏死因子（TNF-a）、白细胞介素-1（IL-1）、IL-6、血小板活化因子（PAF）和磷脂酶A2（PLA2）等。抗炎细胞因子有IL-4、IL-10、IL-13、IL-1ra等,两者之间的平衡维持机体环境的稳定,若两者失衡,即发生失控性SIRS或过度代偿性炎症反应综合征,均可导致组织或器官功能损害。中药具有多靶点作用,对内毒素性肺损伤的治疗有其独特优势。在中医温热病理论中,“毒”是决定疾病发生、发展与转归的重要因素和病理核心。其既可由外直接侵入,亦可由“六淫”所化。因“毒”所致病证常有发病急、病情重、传变快等特点。中医解毒是中医治疗温热病特别是热毒证的最为常用治疗方法,在长期医疗实践中得到了丰富和发展,产生了清热解毒、凉血解毒、清营化毒、通腑泄毒、益气解毒等具体治法,其应用范围也日益扩大,我们认为对包括ALI在内的诸多热毒证候,早用解毒方法,常能防传杜变、化险为夷,即所谓“解毒不厌早”。中药解毒作用及作用机理现代研究,主要体现在抗病原微生物、抗毒素作用,免疫调节作用,解热、抗炎作用以及改善微循环、抗自由基损伤等方面。凉膈散（载宋《和剂局方》）为温病治疗常用名方,具有清上泻下、解毒排毒之功效。本课题组在以往的研究中,从调控细胞因子产生过程中效应细胞信号转导角度探讨解毒作用机制,发现凉膈散能抑制内毒素诱导的小鼠巨噬细胞CDl4、p38促分裂原活化蛋白激酶（p38MAPK）、核转录因子-κB（NF-κB）表达的增强,同时还能抑制小鼠体内炎症因子TNF-a及IL-6的表达,减轻肝、肺组织损伤。基于以上的认识,我们提出:以温热病理论为指导,运用中医解毒法干预ALI病理过程可能是有效的治疗方法,凉膈散可能是抗内毒素性ALI的有效方剂;凉膈散可能是通过多种有效成分、多信号转导通路、多个作用靶点调控炎症反应,以维护机体炎症、抗炎反应的动态平衡的,而这种综合性治疗作用对于ALI是有益的防治策略。方法:1.受试药物是凉膈散。2.分组及模型制作156只雌性Wistar大鼠随机分为对照组（ig生理盐水,iv生理盐水）;LPS组（ig生理盐水,iv LPS 5mg/kg）;LPS+凉膈散（ig 7.5g生药/kg,iv LPS 5mg/kg）;LPS+凉膈散（ig15g生药/kg,iv LPS 5mg/kg）;LPS+凉膈散（ig,30g生药/kg,iv LPS 5mg/kg）;LPS+地塞米松（ig DEX 0.135mg/kg,iv LPS 5mg/kg）。LPS造模动物,又分为注射LPS后1、2、4、8、16h不同时相处死组,每组6只。灌胃给药,每天一次,共5d,末次给药后次日各组大鼠尾静脉注射内毒素脂多糖LPS 5mg/kg,建立大鼠急性肺损伤模型,然后在相应的时间点处死大鼠,取血和肺组织。3.检测指标3.1动脉血PaO₂3.2肺水含量及肺系数测定3.3肺通透性指数测定3.4肺损伤组织病理形态学3.5 TNF-a、IL-1β、IL-10含量检测3.6 Th1/Th2细胞因子的检测4.统计学分析采用SPSS13.0统计软件。计量资料均用均数±标准差（（?）±s）表示,动脉血PaO₂、湿干重比值及肺系数、肺通透性指数、细胞因子含量检测结果比较运用单因素方差分析（One-way ANOVA）和均数问多重比较,p＜0.05为差异有统计学意义。结果:1.动脉血PaO₂（mmHg）的变化所有实验大鼠的吸氧浓度均为21%,故以动脉血氧分压PaO₂代替氧合指数进行比较,LPS可以诱导大鼠PaO₂显著降低,其中4、8、16h点PaO₂在LPS组较对照组显著降低（p＜0.01）,凉膈散组、地塞米松组较LPS组显著增加（p＜0.05）。2.肺水含量（W/D）的变化LPS可以诱导大鼠肺组织湿质量/干质量值W/D升高,其中4、8、16h点W/D在LPS组较对照组显著增加（P＜0.01）,凉膈散组、地塞米松组较LPS组显著降低（p＜0.05,p＜0.01）。3.肺系数（LI）变化LPS可以诱导大鼠肺重/体重比值升高,其中4、8、16h点LI在LPS组较对照组显著增加（P＜0.01）,凉膈散组、地塞米松组较LPS组显著降低（P＜0.05,p＜0.01）。4.肺通透性指数（LPI）的变化LPS可以诱导大鼠肺BLAF蛋白/血浆蛋白比值升高,其中4、8、16h点LPI在LPS组较对照组显著增加（P＜0.01）,凉膈散高、中剂量组、地塞米松组较LPS组显著降低（P＜0.05）。5.肺组织病理改变光镜观察:空白对照组肺泡结构完整,肺泡腔清晰,壁光滑,肺泡腔内无渗出液。各LPS组尾静脉注射LPS 5mg/kg后4h,LPS组大体观察肺,可见肺体积增大,表面有斑点状出血灶。至8h及16h,肺水肿程度明显,肺表面出血点增多,光镜观察见大量炎性细胞浸润并扩散到肺泡腔内,肺泡隔增厚,肺间质及肺泡水肿、问质及肺泡内出血。肺泡膨胀不全或肺泡融合。凉膈散各组镜下显示病变程度较LPS组轻,渗出比模型组明显减少,DEX组损伤伤程度介于LPS组与对照组之间。6.大鼠TNF-a、IL-1β、IL-10含量检测LPS组大鼠TNF-a含量在LPS致伤后1h开始升高,至2h达到最高峰,随后迅速下降;IL-1β含量在LPS致伤后2h显著升高,至8h达到峰值,随后迅速下降;IL-10含量在LPS致伤后2h开始升高。凉膈散各剂量组及地塞米松组与LPS组比较,TNF-a、IL-1β、大鼠肺湿/干重比值显著下降,IL-10明显上升（p＜0.05,P＜0.01）。7.大鼠细胞内Th1/Th2细胞因子含量检测LPS致伤后,LPS组大鼠外周血与BALF中Th1的含量在4、8、16h时间点与对照组比较明显下降,外周血与BALF中Th2的含量在4、8、16h时间点与对照组比较明显升高（P＜0.05,p＜0.01）,提示大鼠尾静脉注射LPS后,Th1向Th2漂移。凉膈散中、高剂量组与地塞米松组的Th1/Th2比例趋于接近正常（p＜0.05,P＜0.01）。结论:1.凉膈散能显著降低内毒素引起的肺组织湿/干重比值,减少肺水含量,降低肺通透性指数;PaO₂较模型组明显提高;病理结果也显示,其能减少炎症细胞的肺内积聚,减少肺泡萎陷及肺实变的发生。2.凉膈散可显著降低内毒素引起的炎症介质TN-a、IL-1β的释放,以及升高抗炎介质IL-10的表达。3.凉膈散能够使Th1/Th2比例趋于接近正常。提示凉膈散对ALI的保护效应可能与促进抗炎介质的表达来抑制炎症介质的释放有关,从而来调节促炎和抗炎反应的动态平衡。因此凉膈散能够有效抑制内毒素导致的炎症反应,对急性肺损伤具有保护作用。更多还原

【Abstract】 Objective Acute lung injury（ALI） and acute respiratory distress syndrome（ARDS） are common disease with high mortality and greatly threaten patients’ health.The earlier symptoms of ARDS reflect,the permeability between pulmonary capillary endothelial cell and alveolar epithelium barrier is increased,and both lung alveolar and interstitium are filled with massive edema fluid,which is rich in protein and a variety of inflammatory cells.At present,as the effective and safe means of prevention and treatment are not available,ALI becomes one of the difficulties in Critical Care Medicine.There are advantages of the integrated traditional Chinese and western medicine to prevent and treat ALI.According to some scholars,Chinese medicine theory and method have shown a potential prospect in treating SIRS,ALI and ARDS.With the development of traditional Chinese medicine theory of seasonal febeile disease pathogenesis and modern pathophysiology,people become aware of the progression of ALI and its prevention and treatment,for example,it provides new strategy and methods to treat and prevent ALI.Clinical investigations have suggested that lung was the most sensitive target organ.ALI and ARDS appear early with the highest incidence.Except for factors directly damage to lung,the reasons for pulmonary dysfunction include a variety of cells in lung tissue,all the blood of systemic circulation,capillary network,a number of endothelial structures and various harmful factors and mediators in blood.Similar to the pathogenesy of ALI caused by various reasons,It is a inflammatory reponse based on acute injury of alveolar-capillary membrane.The basis of ALI is SIRS,and ALI also promotes SIRS.The main cause of SIRS and ALI is the accumulation and activation of inflammatory cells,the excessive synthesis and release of inflammatory mediators,immune dysfunction,and out-of-control inflammation.In essence,inflammation is the defensive reaction,which could damage its own cells when out-of-control,whether inflammation keep relative balance depends on response between inflammation and anti-inflammatory.ALI as the consequences of excessive inflammatory response,the key to reduce mortality and prevent the occurrence of ALI was regulation inflammatory response.So far,many medicines are in clinical trials,such as glucocorticoids,cyclooxygenase inhibitors,thromboxane synthetase inhibitors,phosphodiesterase inhibitor,anti-endotoxin and cytokines,such as monoclonal antibodies or antagonists et al.However,these drugs has not had positive effects in clinical trials of large sample,so it still lacks a specific and effective treatment.The pathogenesis of ALI was not yet clear,but the recognition that macrophages were activated to raise many nuclear leukocyte（PMN） caused to the pulmonary inflammatory response plays a dominant role.SIRS and compensatory anti-inflammatory response syndrome（CARS） induced LPS after GNB infection, excessive SIRS is the development of multiple organ dysfunction syndrome（MODS） which can induce ALI.When acute lung injury happened,intrapulmonary almost all types of cells are involved in the inflammatory response and the release of many inflammatory mediators,however,pulmonary vascular endothelial cells and lung epithelial cells is the effector cell as well as target cell which is a pathogenic factor. There are two tyes of cytokines:proinflammatory cytokines include:TNF-α,IL-1, IL-6,PAF,and PLA2,etc.and anti-inflammatory cytokines:IL-4,IL-10,IL-13, IL-1ra and so on,whose balance can maintain the stability of environment.But unbalance in two types of cytokines would lead to further amplification of inflammation cascade and eventually caused tissue or organ lesion. It have many targets,and the traditional Chinese medicine has unique advantages on lung injury induced by LPS.In Warm Disease theory,toxin is an important factor and pathological core in determination of the occurrence, development and turnover of disease.It either direct intrusion from outside,or influence by the "six exopathogens".The main characteristics of the syndrome caused by "toxin" are as follows:acute pathogenic,heavy pathogenetic condition,faster transmission of a disease and so on.Chinese medicine deintoxication is the most frequent treatment for Warm Disease especially syndrome pyretic toxicity.It has been enriched and developed at the long-term medical practice,which results in methods for a heat-clearing and detoxifying,cooling blood detoxification,cooling yingfen detoxification,Tongfu detoxification,increasing gas detoxification,etc,and the scope of its application have been expanded.We believe that detoxification methods used early could alleviate the condition in ALI including many pyretic toxicity syndromes,the so-called "detoxification appropriate early." Research of Chinese medicine detoxification role and mechanism mainly embodied in the original disease-resistant microorganisms,the anti-toxin,the immune regulatory,the relieve fever,the anti-inflammatory effects,improving the microcirculation,and anti-free radical damage and so on.Lianggesan（contained Song "He Ji Ju Fang"） is the common famous complex prescription of warm disease,with the effect of kiyokami、purgation and detoxication and elimination of toxicant.In previous studies,we studied the mechanism of detoxification from the signal transduction of effector cells in the production of cytokines regulation,in which we found Lianggesan can inhibit the enhancement of expression of endotoxin-induced murine macrophage CD14, p38MAPK,nuclear transcription factor—κB（NF-κB）,while inhibition of the expression of inflammatory cytokines TNF-αand IL-6 in mice,reducing the liver, lung tissue damage.Based on the above understanding,we propose:guided by the theory of epidemic febrile disease,the use of traditional Chinese medicine detoxification method may be an effective method to intervent ALI pathological process.Lianggesan maintains the dynamic equilibrium of systemic inflammatory and anti-inflammatory response through many effective components,multi-signal transduction pathway and inflammatory response regulation by a number of effect target,while this comprehensive therapeutic effects are beneficial to treat ALI.Methods:1.group and model makingWistar rats were randomly divided into six groups,the control group,the acute lung injury group（Group LPS）,LPS+Lianggesan three different treatment groups（group7.5,15,30g/crude drug/kg）,LPS+DEX treatment group（group 0.135mg/kg）.Each LPS group was subdivided into five small groups according to the time of endotoxin administrated at 1,2,4,8h and 16h,6 in every group.The course of intragastric administration in all groups lasted 5 days.After the last dose of the drugs, rat ALI model was established by injection of lipopolysaccharide 5mg/kg into tail.All rats were killed corresponding time,the tissue and blood were observed.3.Detection index3.1 Arterial blood PaO₂3.2 Wet-to-dry weight ratio and lung index determination3.3 Lung permeability index determination3.4 Situation of histopathology of lung3.5 Content of TNF-α,IL-1β,IL-10 determination3.6 Th1/Th2 cytokine determination4.Statistics analysisData are expressed as means±SE（（?）±s）.The consequence of Arterial blood PaO₂,Wet-to-dry weight ratio,lung index,Lung permeability index and Content of cytokine was analyzed by One-way ANOVA and multiple comparison,and P value less than 0.05 was considered to be statistically significant. Results1.The change of arterial blood PaO₂（mmHg）Concentration of oxygen inhalation is 21%in all rats.Comparison by PaO₂ instead of oxygenation index.In LPS group the levels of PaO₂ at 4、8、16h was significantly decreased in comparison with the control group（P＜0.01）.and PaO₂ was increased in DEX and three doses of Lianggesan groups compared with the LPS group（P＜0.05）2.The change of wet-to-dry weight ratioIn LPS group the ratio of W/D at 4、8、16h was significantly increased in comparison with the control group（P＜0.01）.and W/D was decreased in DEX and three doses of Lianggesan groups compared with the LPS group（P＜0.05,P＜0.01）3.The change of lung index.In LPS group the levels of lung index at 4、8、16h was significantly increased in comparison with the control group（P＜0.01）.and LI was decreased in DEX and three doses of Lianggesan groups compared with the LPS group（P＜0.05,P＜0.01）4.The change of lung permeability indexIn LPS group the levels of lung permeability index at 4、8、16h was significantly increased in comparison with the control group（P＜0.01）.and LPI was decreased in DEX and high dose and mid-dose Lianggesan groups compared with the LPS group （P＜0.05）.5.Situation of histopathology of lungLight microscopic observation:In blank control group,pulmonary alveoli presented continuous structure and intact wall,without obvious exudation.After 4h whole lung observation in LPS groups:lung volume increased,punctate hemonhage on the surface.The manifestation of pulmonary edema and bleeding point on the surface increasing at 8,16h.interalveolar septum thickening,hemorrhage,edema and infiltration of lots of inflammatory cells were observed.The lesion degree and effusion were weaker in DEX and three doses of Lianggesan groups than LPS group.6.Content of TNF-α、IL-1β、IL-10 determinationIn LPS group the levels of TNF-αbegan to increase at 1h,which reached the peaks at 2 h,then declined significantly;IL-1βexpression tended to elevate at 2 h, peaked at 8 h,then declined dramatically;IL-10 expression significantly enhanced at 2 h.But the level of TNF-α,IL-1βwere significantly decreased and level of IL-10 was increased in DEX and three doses of Lianggesan groups compared with the LPS group（P＜0.05,P＜0.01）.7.Th1/Th2 cytokine determinationDuring or after 4h observation in LPS groups,we determined Th1 in peripheral blood and in BALF was significantly decreased and Th2 was significantly increased in comparison with the control group（P＜0.05,P＜0.01）,indicating that LPS makes CD4+T cells drift from Th1 to Th2,and the ratio of Th1/Th2 cytokine is close to a normal level in DEX and high dose and mid-dose Lianggesan groups（P＜0.05,P＜0.01）.Conclusion1.Lianggesan could significantly decreased wet-to-dry weight ratio,lung permeability and increased PaO₂ induced by LPS.Results from light microscopy showed that it could reduce inflammatory cell accumulation、alveoli of lung deteleotasis and consolidation of lung.2.Lianggesan could significantly decreased the expression of mediators of inflammation TNF-α,IL-1βand increased the expression of IL-10 induced by LPS. 3.Lianggesan could significantly enhanced the content of Th1 and decreased the expression of Th2 induced by LPS,and the ratio of Th1/Th2 cytokine is close to a normal level.The results suggest that Lianggesan plays a protective role in the endotoxin-induced ALI.The mechanisms of actions may be attributed to its anti-inflammatory activity that inhibits the release of pro-inflammatory cytokines, rectification imbalance between inflammatory and anti-inflammatory.更多还原