毒素清对肺炎克雷伯杆菌肺炎所致MODS老龄大鼠JAK/STAT信号转导的干预作用

【作者】 王峰；

【导师】 李建生；

【作者基本信息】 北京中医药大学 ， 中医内科学， 2008， 博士

【摘要】 老年多器官功能障碍综合征（multiple organ dysfunction syndrome,MODS）是临床常见的老年病危重症,是导致老年人死亡的重要原因。炎症介质过度表达是多器官功能衰竭主要发病机制之一,TNF-a、IL-1、IL-6是触发和诱导过度炎症反应的关键促炎因子,是介导SIRS的重要介质,其合成释放与JAK/STAT信号通路密切相关。JAK/STAT信号转导通路在MODS的发病过程中起着极其重要的作用,阻抑JAK/STAT信号通路的活化有助于减轻组织损伤。中医把多器官功能衰竭称为“脏衰证”,认为本病的主要病机是热毒侵袭,气阴不足,瘀血阻滞,脉络不通,脏腑失养,阴竭阳亡,最终导致脏腑衰竭。因此,针对病机拟定清热解毒,活血化瘀,益气养阴为多器官功能障碍的主要治法。据此而研制的毒素清颗粒,具有清热解毒,活血化瘀,益气养阴的功效。动物实验和临床观察表明,毒素清治疗老年肺炎效果肯定,对其它脏器的损伤具有保护作用,可显著提高患者的生活质量和降低死亡率。本研究通过建立多器官功能障碍大鼠模型,研究多器官功能障碍综合征的病理生理特点,观察JAK/STAT信号通路在MODS中的意义,评价毒素清对多器官功能障碍保护作用,探索毒素清对多器官损伤保护作用的机制。1目的基于“肺启动”学说,建立多器官功能障碍大鼠模型;揭示老年多器官功能障碍的病理生理特点,JAK/STAT信号通路变化的意义;评价毒素清对老年多器官功能障碍的保护作用;探索毒素清对MODS对保护作用机制。2方法以SD大鼠为研究对象,老龄大鼠随机分为8组、青年大鼠随机分为2组,以气管插管法制作克雷伯杆菌肺炎老龄大鼠多器官功能障碍模型,造模后48h处死动物。取肺组织,行肺泡灌洗,鉴定细菌种属,并测定肺、心、肝、肾、小肠等组织的含水量。抽取大鼠外周血,观察24h、48h白细胞和中性粒细胞比率、血气指标的变化,采用病理形态学的方法研究大鼠病理变化和微观结构的改变,检测相关的生化指标以及毒素清颗粒对这些变化的影响。采用免疫组织化学的方法观察老龄和青年大鼠肺炎克雷伯杆菌多器官功能障碍肺、心肌、肾脏、小肠IL-1、IL-6、TNF-a、STAT1、STAT3、STAT5、JAK2、MPO蛋白表达的变化,以及毒素清颗粒对这些表达的影响。采用RT-PCR技术研究老龄和青年大鼠肺炎克雷伯杆菌多器官功能障碍肺、心肌、小肠SOCS3mRNA、JAK2mRNA的表达,以及毒素清颗粒对这些表达的影响。3结果3.1成功制备大鼠多器官功能障碍模型青年模型组、老龄模型组大鼠分别与青年对照组、老龄对照组大鼠比较,死亡率上升,组织水肿明显,白细胞总数明显升高,中性粒细胞比率上升;肺组织PaCO₂明显升高,PaO₂明显下降,心肌LDH、CK含量显著升高（P<0.01）。血清ALT、AST明显上升（P<0.05）。小肠绒毛缺失,粘膜下层大量炎性细胞浸润,毛细血管扩张充血。两脏器障碍大鼠占40%、三脏器障碍大鼠占60%。青年模型组、老龄模型组肺、心、肝、小肠等组织病理损伤分别较青年对照组、老龄对照组严重。3.2多器官功能障碍老龄大鼠肺、心、肝、肾、小肠等组织生理病理的特点及JAK/STAT信号通路变化的意义3.2.1老龄模型组死亡率明显高于青年模型组。老龄模型组肝、心、肺等组织含水量、心肌LDH、CK、CK-MB含量以及肝组织ALT、AST含量较青年模型组明显增加。老龄对照组肺、肝、小肠等组织老化现象明显,且老龄模型组肺组织、小肠病理损伤重于青年模型组。青年模型组、老龄模型组肾脏的病理变化以肾小管管腔变窄,上皮细胞水肿为主,BUN、Cr变化不明显,病理损伤均较轻。3.2.2老龄模型组肺组织的（MPO、IL-1、STAT1、STAT5）、心肌（TNF-a、STAT1、STAT5、JAK2）、肾组织（STAT1、STAT3）、小肠（IL-1、STAT1、STAT3）等蛋白表达均较青年模型组明显增强（P<0.05或P<0.01）。3.3毒素清对老龄大鼠多器官功能障碍JAK/STAT信号转导变化的影响及其保护作用机制3.3.1毒素清组能够明显降低PaCO₂,升高PaO₂,减弱MPO、IL-1、IL-6、TNF-a、JAK2、STAT1、STAT3、STAT5、JAK2mNRA的表达,增强SOCS3表达,减轻病理损伤（P<0.01）。毒素清组较洛美沙星组显著减弱MPO、IL-1、TNF-a、STAT1、STAT3的表达（P<0.01）,大鼠死亡只数下降,病理损伤减轻。而AG490组、雷帕霉素组IL-1的表达均弱于毒素清组,但病理损伤与毒素清组无明显差异。AG490合毒素清组、雷帕霉素合毒素清组MPO、IL-1、IL-6、TNF-a表达均弱于毒素清组、AG490组、雷帕霉素组。雷帕霉素合毒素清组还能显著减弱STAT1、STAT3、STAT5的表达,较毒素清组、AG490组、雷帕霉素组有显著意义（P<0.01）。3.3.2毒素清组能够明显降低LDH、CK、CK-MB含量（P<0.01）,减弱IL-1、IL-6、TNF-a、AK2mNRA、STAT1、STAT3、STAT5表达,减轻病理损伤。毒素清组较洛美沙星组显著减弱STAT1、STAT3、STAT5的表达（P<0.01）,大鼠死亡只数低下降,病理损伤减轻。AG490组、雷帕霉素组IL-1、TNF-a、STAT1、STAT3、STAT5的表达方面均弱于毒素清组。AG490合毒素清组、雷帕霉素合毒素清组IL-1、STAT1、STAT3、STAT5的表达方均弱于毒素清组、AG490组、雷帕霉素组。雷帕霉素合毒素清组还能显著减弱IL-6的表达,与毒素清组、AG490组、雷帕霉素组比较有显著意义（P<0.01）。3.3.3老龄大鼠模型组肝组织含水量增加,血清AST、ALT含量升高,病理损伤严重（P<0.01）。毒素清组能够明显降低AST、ALT的含量,且毒素清组、AG490合毒素清组、雷帕霉素合毒素清组的病理损伤较模型组明显减轻（P<0.01）。3.3.4毒素清组能够显著减弱IL-1、IL-6、TNF-a、STAT1、STAT3、STAT5、JAK2的表达（P<0.01,或P<0.05）。毒素清较洛美沙星组能够显著降低IIL-6、TNF-a、JAK2、STAT1、STAT3、STAT5的表达（P<0.01）,病理损伤减轻,大鼠死亡只数下降。AG490合毒素清组IL-1、IL-6、TNF-a、JAK2、STAT1、STAT3、STAT5的表达弱于毒素清组（P<0.01）。雷帕霉素合毒素清组TNF-a、STAT1、STAT3、STAT5的表达弱于毒素清组（P<0.01）,STAT1的表达弱于雷帕霉素组。AG490组、雷帕霉素组与毒素清组在减弱炎性细胞因子的表达无显著性差异。3.3.5毒素清组显著减弱IL-1、IL-6、TNF-a、STAT1、STAT3、STAT5、JAK2、JAK2mNRA的表达,减轻病理损伤（P<0.01,或P<0.05）。毒素清组较洛美沙星组显著减弱TNF-a、JAK2、STAT1、STAT3、STAT5的表达（P<0.01）,病理损伤减轻。AG490组、雷帕霉素组较毒素清组IL-6、STAT1、STAT5表达均明显减弱,雷帕霉素组还能够显著减弱STAT3的表达,而AG490组明显上调SOCS3mNRA表达,但病理损伤均与毒素清组无明显差异。AG490合毒素清组IL-1、IL-6、JAK2、STAT1、STAT3、STAT5的表达较毒素清组减弱、而SOCS3mNRA的（P<0.01）表达增强;雷帕霉素合毒素清组IL-1、IL-6、TNF-a、STAT1、STAT3、STAT5、JAK2的表达较毒素清组减弱,而SOCS3mNRA的表达增强（P<0.01）,且IL-6、STAT1、STAT3的表达则弱于雷帕霉素组和AG490组。4结论4.1成功复制了肺炎克雷伯杆菌老龄大鼠多器官功能障碍模型。4.2老年具有死亡率高,病理损伤重的特点。4.3 JAK/STAT信号通路是MODS的病理损伤的重要通路之一,该信号通路介导了炎症因子的放大,加重了炎症对机体的损害,导致了多器官功能障碍的发生。4.4毒素清能够使JAK/STAT信号通路活化减弱,降低的IL-1、IL-6、TNF-a表达,从而减轻病理损伤,对多器官损伤起到保护作用。其机制可能是减弱IL-1、IL-6、TNF-a表达,阻抑了JAK/STAT信号转导通路,参与JAK/STAT的负调控机制,并可能通过其他信号通路途径减少了炎性细胞因子的产生,从而对组织损伤起到保护作用。更多还原

【Abstract】 (Multiple organ dysfunction syndrome,MODS) MODS often happens to the aged men in clinical crisis and is an important reason for their death.Mediators of inflammation excessive expression is the main function mechanism of MODS and related to multiple signal passage activation. TNF-a,IL-1 and IL-6 are the key factors to trigger and induce excessive inflammatory response by cascade amplification and the main medium for SIRS , whose composition and release is closely related to JAK/STAT signal pathway. JAK/STAT signal pathway play a very important role in the MODS and blockage it to lessen the organ injury. Chinese medicine call multiple organ failure as Zang-organ failure syndrome.Its pathogenesis is pyrotoxin invasion, haemostasis blockage, qi yin insufficiency, three of which interact to cause negative results. Therefore, clearing away heat and toxic material, promoting blood circulation by removing blood stasis, supplementing qi and nourishing yin are main treatments for multiple organ failure, by which Du Su Qing granule has been developed. Animal experiment and clinical observation suggest the positive effect of Du Su Qing to treat senile pneumonia and Du Su Qing can protect other organs against injury, imrove the patient’s living condition and lower death rate significantly. This experiment duplicates MODS model in rats to explore the pathological and physiological characteristics of rats with MODS and the effects of ageing factor on aged rats in biochemistry and pathology and observe the effects of Du Su Qing on aged rats in biochemistry , pathology and physiology and JAK/STAT passage and their defense mechanism.1 Objective To observe the pathological and physiological characteristics of rats with MODS and the effects of ageing factor on lung, heart, kidney and intestine; To observe the effects of the multiple organ dysfunction in rats on inflammatory cytokine , JAK/STAT signal pathway and explore the relationship between IL-1,IL-6,TNF-a and JAK/STAT signal pathway. To observe of Du Su Qing on multiple organ dysfunction in aged rats on pathology and biochemistry; to probe its defense mechanism to lung, heart, kidney and intestine and possible mechanism ; to observe the effect of Du Su Qing on multiple organ dysfunction in aged rats in JAK/STATsignal passage; to probe its defense protection against multiple organ injury and possible mechamism.2 Method We put the SD aged rats into 8 groups and the SD young rats into 2 groups. Building a multiple dysfunction model by tracheal intubation in klebsiella pneumoniae pneumonia aged rats and kill them 48hours later. We take the lung tissues, do bronch-oalveolar lavage, identify the bacteria type, test the water content in lung, heart, liver, kidney and intestine. Sampleing the rat peripheral blood, observe the ratio between leukocyte and neutrophil, the changes of blood gas index and study the pathological and microstructure changes in rats by the pathomorphological method and measure the related chemical index and the effect of Du Su Qing granule on these changes. We observe the protein expression changes of IL-1, IL-6, TNF-a, STAT1, STAT3,STAT5,JAK2,MPO in lung, heart, kidney, intestine in aged and young rats caused by klebsiella imeumoniae multiple organ dysfunction and the effects of Du Su Qing granule on these expressions.3 Results3.1 Duplicated the model of MODS successfullyThrough the comparison between model group rats and control group rats, leuco- cytes is higher,total white blood cells increase, the ratio of neutrophlic granulocytes rises. PaCO2 in lung is higher , PaO2 is lower. LDH and CK are much higher in heart(P<0.01).Liver function suggests ALT and AST is higher(P<0.05).It is observed that the loss villiof the small intestinal heighten obviously,Inflammatory cell infiltration in subnucosa angiote-lectasis and capillary injection. Bi-visceral dysfunction accounts for 40% in rats, triple-visceral dysfunction is 60%. Pathological injury grade numerical score of lung, heart, liver, intestine in model group is much higher than that in control group. Animal model of the multiple organ dysfunction has been duplicated successfully to the standard of animal model of MODS.3.2 The characteristics of lung, heart, kidney, intestine of MODS aged rats in pathology and physiology and their effects on JAK/STAT signal pathway.The difference of rat lung injury between young control groups and aged control groups is pathologically much higher (P<0.01) and the injury degree of aged model groups is much heavier than that of young model groups (P<0.01). Compared to control groups, MPO, IL-1, IL-6, TNF-a, JAK2, STAT1, STAT3, STAT5 are expressed highly(P<0.01) ,JAK2mNRA expression is up-regulated (P<0.01), MPO, IL-1, STAT1, STAT5 positive cells or the average optical density of protein expression in aged model groups are higher or much higher than that in young model groups (P<0.05 or P<0.01).The pathological injury in model groups is obvious, and we can see cardiac muscle fibers disorder and a small or large amount of inflammatory cellular infiltration, LDH,CK,CK-MB is higher in the enzyme of the cardiac (p<0.01). IL-1, IL-6, TNF-a, JAK2, STAT1, STAT3, STAT5 in model groups are highly expressed (p<0.01), JAK2mNRA expression is up-regulated (p<0.01), compared to young model groups, TNF-a,STAT1,STAT5,JAK2 positive cells or the average optical density of protein expression in aged model groups are higher or much higher than that in young model groups (P<0.05 or P<0.01).The difference of rat liver injury between young control groups and aged control groups is pathologically higher (P<0.05) and pathological changes are main liver cell vacuolar-degeneration. ALTand AST are much higher in model groups(p<0.01).Model groups show certain injuries, especially in nephric tubele.There is no obvious difference in the changes of BUN and Cr between model groups and control groups. IL-1,IL-6,TNF-a,JAK2,STAT1,STAT3,STAT5 are highly expressed(P<0.01)in model groups. The protein expression of IL-6,STAT3,STAT5 in aged control group is higher or much higher than that in young control group (P<0.05 or P<0.01),IL-1,STAT1,STAT3 positive cells or the average optical density of protein expression in aged model groups are higher or much higher than that in young model groups (P<0.05 or P<0.01).3.3 The effects of Du Su Qing on lung, heart, liver, intestine of MOD aged rats in pathology and physiology and its effects on JAK/STAT signal pathway.The difference of rat lung injuries in aged control groups is pathologically much higher (P<0.01), total white blood cells increase,lung water content is much higher, PaCO2 is higher and PaO2 is much lower (P<0.01) in serum. IL-1,IL-6,TNF-a,JAK2,STAT1,STAT3,STAT5 are highly expressed(P<0.01)in aged model groups. AK2mNRA expression is up-regulated (p<0.01), all dose groups show PaCO2 is lower, PaO2 higher(p<0.01), injury degrees are improved(p<0.01), The expression of MPO,IL-1,IL-6 and TNF-a is much lower (P<0.01) ,expression of JAK2m(except for Lomefloxacin) STAT1,STAT3,STAT5 is down–regulated. Du Su Qing group, AG490group , AG490unit group ,Rapamycin group can down-regulate JAK2mNRA expression ( P < 0.01 ) , up-regulate SOCS3mNRA expression.The rat death rate in Du Su Qing group is lower than that in Lomefloxacin group, injury degree score is lowered and Du Su Qing group is optimal to Lomefloxacin group in lowering MPO,IL-1,TNF-a,STAT1,STAT3 and STAT5 expressin(P<0.01), but AG 490 union group and rapanycin group are optimal to Du Su Qing group, AG490 group and Rapamycin group in up-regulating MPO,IL-1,IL-6 and TNF-a protein expression.Rapamycin group can obviously lower STAT1,STAT3,STAT5 expression which is more significant(p<0.01) compared to Du Su Qing group, AG490 group and Rapamycin group. AG490 group and Rapamycin group are optimal to Du Su Qing group in lowering IL-1 expression.In aged model group rat myocardial injury has significant difference (p<0.01), water content is higher, LDH,CK and CK-MB in serum rise(P<0.01), rats IL-1, IL-6, TNF-a, JAK2, STAT1,STAT3 and STAT5 in aged model groups are high expressed (P<0.01), JAK2mNRA is up-regulated(P<0.01), each dose group can obviously lower LDH and CK-MB, improve injury degree(P<0.01), and reduce the expression of IL-1,IL-6(except for Lomefloxacin ) and TNF-a, down–regulate the protein expression of STAT1,STAT3,STAT5(except for Lomefloxacin). All dose group can down-regulate JAK2mNRA gen (P<0.01),AG490uniou group can up-regulate SOCS3mNRA gene expression. The rat death rate in Du Su Qing group is lower than that in Lomefloxacin group, injury degree score is lowered, but which have no significance in statistics; Compared to Lomefloxacin group, Du Su Qing grop can significantly lower the expression of STAT1,STAT3 and STAT5(P<0.01). AG490 union group and Rapamycin group is optimal to Du Su Qinggrop,AG490grop and Rapamycin grop in down-regulating the protein of IL-1,STAT1,STAT3 and STAT5. Besides, Rapamycin union group can significantly lower IL-6 expression, which is more significant(p<0.01) compared to Du Su Qing group,AG490 group and Rapamycin group. AG490 group and Rapamycin group are optimal to Du sSu Qing group in reducing the expression of IL-1,TNF-a,STAT1,STAT3 and STAT5. The difference of rat liver injuries in aged control groups is pathologically much higher (P<0.01), water content increases, ASTand ALT in serum rises,. All dose grops can obviously lower LDH,CK and CK-MB. Compared to model group,AG490 pathological injury score is significant (P<0.01); the pathological injury scores of remaining groups tend to decrease, but compared to model group there is no significance. All dose groups have no obvious differences in lowering LDH,CK and CK-MB.Rat kidney injuries in aged model groups pathologically worsen, death rate rises, water content increases, BUN and Cr (P<0.01) in serum decrease , but which is insignificant compared to control group. IL-1, IL-6, TNF-a, JAK2, STAT1, STAT3, STAT5 are highly expressed(P<0.01)in aged model groups. AK2mNRA is up-regulated (p<0.01);all dose groups can lower BUN and Cr, improve pathological injury degree which is not significant compared to model group. Compared to model grops, Lomefloxacin groups show no difference in decreasing the expression of IL-1,IL-6,TNF-a and JAK2, but the remaining group can significantly reduce the expression of IL-1,IL-6,TNF-a, down-regulate the protein expression of STAT1,STAT3,STAT5 and JAK2.The rat death rate in Du Su Qing group is lower than that in Lomefloxacin group, pathological injury scores decrease, which are not significant statistically; compared to Lomefloxacin group, Du Su Qing grop can significantly lower the expression of IL-1, IL-6, TNF-a, JAK2, STAT1, STAT3 and STAT5(P<0.01).AG490group , Rapamycin group and Du Su Qing group show no significant difference in lowering the protein expression of inflammatory cytokines.Rat intestine injuries in aged model groups worsen pathologically, death rate rises, water content increases.IL-1,IL-6,TNF-a,JAK2,STAT1,STAT3 and STAT5 in rats are highly expressed(P<0.01)in aged model groups. AK2mNRA and SOCS3mNRA are up-regulated (p<0.01); all dose groups can improve the intestine pathological injury degree, which has significant difference from model group(P<0.05或P<0.01). Model groups and Lomefloxacin groups show no difference in the decrease the expression of IL-6 and JAK2, but the rest groups can see the significant decrease of the expression of IL-1,IL-6 and TNF-a , the down-regulation of the protein expression of STAT1,STAT3,STAT5 and JAK2 and the down-regulation of JAK2mNRA(P<0.01 orP<0.05).The rat death rate in Du Su Qing group is lower than that in Lomefloxacin group and pathological injury scores decrease, which are not significant statistically; compared to Lomefloxacin group, Du Su Qing grop can significantly lower the expression of IL-1, IL-6, JAK2, STAT1, STAT3 and STAT5(P<0.01).AG490 union group is optimal to Rapamycin group and Du Su Qing group in down-regulating the protein expression of IL-1, IL-6, JAK2, STAT1, STAT3,STAT5 and SOCS3mNRA expression (P<0.01) and optimal to Rapamycin group in down-regulating IL-1 protein expression and SOCS3mNRA gene expression. Rapamycin group is optimal to Du Su Qing group and Lomefloxacin group in lowering the expression of IL-1, IL-6, TNF-a, STAT1, STAT3,STAT5,JAK2 and SOCS3mNRA (P<0.01) and optimal to Rapamycin group and AG490 group in down-regulating IL-6,STAT1and STAT3 protein expression. AG490 group and Rapamycin group are optimal to Du Su Qing group and Lomefloxacin group in lowering IL-6,STAT1,STAT5 protein expression. In addition, compared to Du Su Qing group, Rapamycin group can significantly lower STAT3 expression and AG490 group can significantly lower SOCS3mNRA expression.4 ConclusionMultiple organ dysfunction model in rats with klebsiella imeumoniae was successfully duplicated. Compared to young rat model, rat death rate is higher and pathological injury degree is more serious. Inflammatory cytokines played an important role in MODS pathological process. JAK/STAT signal passage mediated the amplification of imflammatory, and increased inflammatory injury to organs, caused MODS. Du Su Qing lowered the related cytokine expression, weakened JAK/STAT signal passage activation. Its defense mechanism to MODS probably blocked JAK/STAT signal passage, and had synergistic function with blocker to up-regulate the cytokine expression and protect the organs from injuries.更多还原