CCK-8抗内毒素休克时的免疫调节作用及其分子机制初探
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摘要
内毒素休克(endotoxic shock，ES)时由于大量内毒素(endotoxin)诱导产生多种炎症介质的级联反应，使机体处于一种自身难以控制的紊乱状态，可发生多器官功能障碍综合征而最终导致死亡。促炎症细胞因子、氧自由基和一氧化氮(nitric oxide，NO)等多种致炎物质在ES中起主要作用，但确切的发病机制尚未明了，临床救治也较为困难。尽管已经研制出许多抗促炎细胞因子抗体及诱生型NO合酶(inducible NO synthase，iNOS)抑制剂等，但多为单因素抗炎制剂，不能有效地阻断全身性炎症的发展。免疫细胞可分泌各种炎症介质，炎症反应的实质也是一种免疫反应。免疫功能改变在ES发病过程中发挥关键作用。所以寻找具有免疫调节作用的抗炎制剂已成为研究的热点。八肽胆囊收缩素(cholecystokinin-octapeptide，CCK-8)是一种内源性的脑肠肽，近年研究表明CCK-8对免疫系统具有调节作用。本室研究证实，CCK-8对脂多糖(lipopolysaccharide，LPS)诱导的ES有保护作用，但此保护作用是否与其免疫调节作用有关尚无报道。肺脏和脾脏是ES时最易受损的靶器官。肺脏除具有呼吸和代谢功能外，还具有免疫调节作用；脾脏是全身最大的淋巴器官，是ES时最先出现炎症反应的器官之一，具有重要的免疫功能。丝裂原激活蛋白激酶系统(mitogen-activated protein kinase，MAPKs)是多种细胞外刺激激活各种信号途径的交汇点，调节细胞的基本生命活动。在胰腺腺泡细胞CCK可激活MAPK。近几年关于Janus tyrosine kinase／signal transducers and activators of transcription(JAK／STAT)信号通路与MAPK途径的交互应答(cross-talk)正日益受到关注。STAT蛋白可被G蛋白偶联受体激活，而CCK受体作为一种G蛋白偶联受体，是否对JAK／STAT通路具有直接激活作用，尚未见报道。已有报道缺乏STAT3的突变小鼠对ES高度敏感，血清炎性细胞因子如肿瘤坏死因子-α(tumor necrosis factor-α，TNF-α)、白介素-1β(interleukin-1β，IL-1β) 和白介素-6
中文摘要
（nterleukin6，IL＊）浓度增加。新近报道 p3 8 MAPK参与 sTAT丝
氨酸磷酸化从而调节 STAT活性。CCK也可能通过激活 p3 8 MAPK进
而激活STAT，调控基因转录，发挥对ES的免疫调节作用。静息时，
Re皿4 B H聚体与抑制蛋白k Bs结合，在细胞浆中保持无活性。
受到刺激，IKB分子迅速磷酸化并降解，释放NF儿B转位入核，通
过与 K B位点结合调节转录。LPS可激活M4 B，调节许多炎性细胞
因子的转录。丛斌等研究表明CCK七可抑制LPS诱导的大鼠肺组织
W＊B活性。IKB的降解、并从W儿B复合物上解离是W4B激
活的必需过程。CCKS是否通过抑制 LPS诱导的 IK B降解，从而抑制
NF几B激活尚未见报道。本研究在整体、细胞及分子水平，观察了
CCKE对LPS引起的免疫功能改变的调节作用，研究了其受体机制及
MAPK、STAT3和 IK B的信号转导机制，以探讨 CCK七抗 ES时的免
疫调节作用及其分子机制。
！CCK8对内毒素休克大鼠肺脏和脾脏变化的影响
l·ICCK＊减轻ES大鼠肺脏和脾脏形态和功能变化
观察CCK七对LPS引起的大鼠ES的低血压及肺脏和脾脏超微结
构改变的影响。使用生理多道记录仪，观察尾静脉注人 LPS旺 mg／kg tv．）
复制的大鼠 ES模型工PS注入前 10 min尾静脉注入 CCK七K0 11 g／kg
tV．X单独注入CCK七K0 p g／kg tV．）或生理盐水（对照）的四组大鼠
血压的改变，应用光镜观察各组注药后 2 h、6 h、12 h肺脏和脾脏结构
改变。透射电镜观察6h各组肺脏和脾脏超微结构改变。
用试剂盒检测血清、肺脏和脾脏NO／NOS和 MDA／SOD含量及活
性变化；用免疫组化法观察 6 h各组诱导型血红素氧合酶 heme
oxygenase－l，HO－l）在肺脏和脾脏的分布；于LPS注入2 h进行支气
管肺泡灌洗，获支气管肺泡灌洗液（bfoflChO。IVCOlat 13V8gC lllid，
BALF人 从中分离肺泡巨噬细胞，检测吞噬白色念珠菌能力的改变。
实验结果如下：
门）处理前各组血压之间未见显著差异口LPS组 30 min、lh和 2
h分别下降到 7．82士l．07 ha、8．49士0．92 ea和 7．60士0．68 kPa，较对
照组显著降低（P＜0．of人 CCK｛＋LPS组上述各时间点血压较 LPS组
显著升高，分别恢复到 9．33士 1．53 kPa（P＜0＊5）、10．77士 0．82 kPa
2
中文摘要
（P＜0．of）和 10．71士0．45 hP＜（P＜0．of）。CCK－8组与对照组比较各时
间点未见显著差异。
Q）光镜结果显示大鼠注射 LPS后肺泡隔增宽，急性弥漫性炎细
胞浸润，伴肺泡萎陷或消失，轻微的肺不张或肺气肿。CCK七＋LPS组
上述病变有所减轻。LPS组大鼠脾脏可见脾窦扩张充血，红细胞密集，
中性粒细胞弥散浸润。CCK七＋LPS组上述病变有所减轻。CCK七组与
对照组接近。
3）电镜结果显示大鼠注射LPS后 6 h肺毛细血管内 PMN滞留、
嵌塞，内皮细胞肿胀，?
Endotoxic shock (ES), resulting from cascade reaction of inflammatory mediators induced by excessive endotoxin, leads to uncontrolled disturbance of organisms, multiple organ failure and lethality. Many kinds of inflammatory mediators including pro-inflammatory cytokines, oxygen-derived free radicals and nitric oxide (NO) play an important role in ES, but the exact pathogenesis remains unclear and the treatment is difficult. Many antibodies against pro-inflammatory cytokines and inhibitors of inducible NO synthase (iNOS) have been produced, but most of them are agents targeting one factor and can't efficiently block the development of systemic inflammation. Different kinds of inflammatory mediators are secreted by immunocytes, which suggests that the essence of inflammation is also a kind of immune response. Alterations in immune function are felt to play a pivotal role in the development of ES. So it has become the focus to search for anti-inflammatory agents that have immunomodulatory effect. Cholecystokin
in-octapeptide (CCK-8) is a kind of endogenous brain-gut peptide. Recent studies suggest a regulatory function of CCK-8 in the immune system. Our previous study demonstrated that CCK-8 could protect animals from ES induced by lipopolysaccharide (LPS). However, it is not clear whether this protecting effect of CCK-8 is related to its immunomodulatory properties. Lung and spleen are target organs that are easily damaged during ES. Besides respiratory and metabolic function, lung plays a role in immune system. Spleen, the biggest lymphatic organ in body, has important immune functions. Inflammatory response is first observed in spleen during ES. The mitogen-activated protein kinases (MAPKs) are focal points of discrete
signaling cascades for diverse extracellular stimuli, and function to regulate fundamental cellular processes. CCK activates MAPK in pancreatic acinar cells. Cross-talk has been shown recently between Janus tyrosine kinase/signal transducers and activators of transcription (JAK/STAT) and MAPK pathways. STAT proteins are activated by G-protein coupled receptors, while it has not been reported whether CCK receptors, a kind of G-protein coupled receptors, can activate JAK/STAT pathway. The STAT3 mutant mice were highly susceptible to ES with increased serum concentration of inflammatory cytokines such as tumor necrosis factor-a(TNF-a), interleukin-lp (IL-13) and interleukin-6 (IL-6). It has been demonstrated that p38 MAPK is involved in serine phosphorylation and regulation of STAT activation. CCK may play immunomodulatory role in ES by activating STAT through p38 MAPK and regulating gene transcription. At rest, the heterodimeric Rel/NF- K B complex is located in the cytoplasm bound to an inhibitory factor, I K B. Upon stimulation, I K B is phosphorylated and degraded, free NF- K B then translocates into the nucleus where it binds to K B site to regulate transcription. NF- K. B can be activated by LPS and regulate transcription of many pro-inflammatory cytokines. Cong et al proved that CCK-8 inhibited LPS-induced NF- K B activity in lung of rats. The degradation of I K B and dissociation from NF-K B complex are essential process for activation of NF- K B. There is no report about whether CCK-8 inhibits NF- K B activity through inhibiting LPS-induced degradation of I K B. The present study was designed to observe the regulatory effect of CCK-8 on changes of immune functions induced by LPS, the receptor mechanism and signal transduction mechanism involving MAPK, STAT3 and I K B at different levels including in vivo and in vitro, in order to study its immunomodulatory effect on reversing ES and its molecular mechanisms. 1 Effect of CCK-8 on changes in lung and spleen of ES rats 1.1 CCK-8 attenuates morphologic and functional changes in lung and spleen of ES rats
To observe the effect of CCK-8 on decrease of mean arterial pressure (MAP) induced by LPS and ultrastructure changes in lung and spleen of ES rats, animals were randomly assigned to four groups injected different agents via tail
中文摘要
（nterleukin6，IL＊）浓度增加。新近报道 p3 8 MAPK参与 sTAT丝
氨酸磷酸化从而调节 STAT活性。CCK也可能通过激活 p3 8 MAPK进
而激活STAT，调控基因转录，发挥对ES的免疫调节作用。静息时，
Re皿4 B H聚体与抑制蛋白k Bs结合，在细胞浆中保持无活性。
受到刺激，IKB分子迅速磷酸化并降解，释放NF儿B转位入核，通
过与 K B位点结合调节转录。LPS可激活M4 B，调节许多炎性细胞
因子的转录。丛斌等研究表明CCK七可抑制LPS诱导的大鼠肺组织
W＊B活性。IKB的降解、并从W儿B复合物上解离是W4B激
活的必需过程。CCKS是否通过抑制 LPS诱导的 IK B降解，从而抑制
NF几B激活尚未见报道。本研究在整体、细胞及分子水平，观察了
CCKE对LPS引起的免疫功能改变的调节作用，研究了其受体机制及
MAPK、STAT3和 IK B的信号转导机制，以探讨 CCK七抗 ES时的免
疫调节作用及其分子机制。
！CCK8对内毒素休克大鼠肺脏和脾脏变化的影响
l·ICCK＊减轻ES大鼠肺脏和脾脏形态和功能变化
观察CCK七对LPS引起的大鼠ES的低血压及肺脏和脾脏超微结
构改变的影响。使用生理多道记录仪，观察尾静脉注人 LPS旺 mg／kg tv．）
复制的大鼠 ES模型工PS注入前 10 min尾静脉注入 CCK七K0 11 g／kg
tV．X单独注入CCK七K0 p g／kg tV．）或生理盐水（对照）的四组大鼠
血压的改变，应用光镜观察各组注药后 2 h、6 h、12 h肺脏和脾脏结构
改变。透射电镜观察6h各组肺脏和脾脏超微结构改变。
用试剂盒检测血清、肺脏和脾脏NO／NOS和 MDA／SOD含量及活
性变化；用免疫组化法观察 6 h各组诱导型血红素氧合酶 heme
oxygenase－l，HO－l）在肺脏和脾脏的分布；于LPS注入2 h进行支气
管肺泡灌洗，获支气管肺泡灌洗液（bfoflChO。IVCOlat 13V8gC lllid，
BALF人 从中分离肺泡巨噬细胞，检测吞噬白色念珠菌能力的改变。
实验结果如下：
门）处理前各组血压之间未见显著差异口LPS组 30 min、lh和 2
h分别下降到 7．82士l．07 ha、8．49士0．92 ea和 7．60士0．68 kPa，较对
照组显著降低（P＜0．of人 CCK｛＋LPS组上述各时间点血压较 LPS组
显著升高，分别恢复到 9．33士 1．53 kPa（P＜0＊5）、10．77士 0．82 kPa
2
中文摘要
（P＜0．of）和 10．71士0．45 hP＜（P＜0．of）。CCK－8组与对照组比较各时
间点未见显著差异。
Q）光镜结果显示大鼠注射 LPS后肺泡隔增宽，急性弥漫性炎细
胞浸润，伴肺泡萎陷或消失，轻微的肺不张或肺气肿。CCK七＋LPS组
上述病变有所减轻。LPS组大鼠脾脏可见脾窦扩张充血，红细胞密集，
中性粒细胞弥散浸润。CCK七＋LPS组上述病变有所减轻。CCK七组与
对照组接近。
3）电镜结果显示大鼠注射LPS后 6 h肺毛细血管内 PMN滞留、
嵌塞，内皮细胞肿胀，?
Endotoxic shock (ES), resulting from cascade reaction of inflammatory mediators induced by excessive endotoxin, leads to uncontrolled disturbance of organisms, multiple organ failure and lethality. Many kinds of inflammatory mediators including pro-inflammatory cytokines, oxygen-derived free radicals and nitric oxide (NO) play an important role in ES, but the exact pathogenesis remains unclear and the treatment is difficult. Many antibodies against pro-inflammatory cytokines and inhibitors of inducible NO synthase (iNOS) have been produced, but most of them are agents targeting one factor and can't efficiently block the development of systemic inflammation. Different kinds of inflammatory mediators are secreted by immunocytes, which suggests that the essence of inflammation is also a kind of immune response. Alterations in immune function are felt to play a pivotal role in the development of ES. So it has become the focus to search for anti-inflammatory agents that have immunomodulatory effect. Cholecystokin
in-octapeptide (CCK-8) is a kind of endogenous brain-gut peptide. Recent studies suggest a regulatory function of CCK-8 in the immune system. Our previous study demonstrated that CCK-8 could protect animals from ES induced by lipopolysaccharide (LPS). However, it is not clear whether this protecting effect of CCK-8 is related to its immunomodulatory properties. Lung and spleen are target organs that are easily damaged during ES. Besides respiratory and metabolic function, lung plays a role in immune system. Spleen, the biggest lymphatic organ in body, has important immune functions. Inflammatory response is first observed in spleen during ES. The mitogen-activated protein kinases (MAPKs) are focal points of discrete
signaling cascades for diverse extracellular stimuli, and function to regulate fundamental cellular processes. CCK activates MAPK in pancreatic acinar cells. Cross-talk has been shown recently between Janus tyrosine kinase/signal transducers and activators of transcription (JAK/STAT) and MAPK pathways. STAT proteins are activated by G-protein coupled receptors, while it has not been reported whether CCK receptors, a kind of G-protein coupled receptors, can activate JAK/STAT pathway. The STAT3 mutant mice were highly susceptible to ES with increased serum concentration of inflammatory cytokines such as tumor necrosis factor-a(TNF-a), interleukin-lp (IL-13) and interleukin-6 (IL-6). It has been demonstrated that p38 MAPK is involved in serine phosphorylation and regulation of STAT activation. CCK may play immunomodulatory role in ES by activating STAT through p38 MAPK and regulating gene transcription. At rest, the heterodimeric Rel/NF- K B complex is located in the cytoplasm bound to an inhibitory factor, I K B. Upon stimulation, I K B is phosphorylated and degraded, free NF- K B then translocates into the nucleus where it binds to K B site to regulate transcription. NF- K. B can be activated by LPS and regulate transcription of many pro-inflammatory cytokines. Cong et al proved that CCK-8 inhibited LPS-induced NF- K B activity in lung of rats. The degradation of I K B and dissociation from NF-K B complex are essential process for activation of NF- K B. There is no report about whether CCK-8 inhibits NF- K B activity through inhibiting LPS-induced degradation of I K B. The present study was designed to observe the regulatory effect of CCK-8 on changes of immune functions induced by LPS, the receptor mechanism and signal transduction mechanism involving MAPK, STAT3 and I K B at different levels including in vivo and in vitro, in order to study its immunomodulatory effect on reversing ES and its molecular mechanisms. 1 Effect of CCK-8 on changes in lung and spleen of ES rats 1.1 CCK-8 attenuates morphologic and functional changes in lung and spleen of ES rats
To observe the effect of CCK-8 on decrease of mean arterial pressure (MAP) induced by LPS and ultrastructure changes in lung and spleen of ES rats, animals were randomly assigned to four groups injected different agents via tail
引文
1 Shi Y, Li HQ, Pan J et al. Evidence of increased endogenous carbon monoxide production in newborn rat endotoximia. Chin Med Sci J, 1997, 12:212～215
2 凌亦凌，黄善生，王乐丰，等．八肽胆囊收缩素抗内毒素休克的实验研究．生理学报，1996，48(4)：390～394
3 凌亦凌，黄善生，王乐丰，等．胆囊收缩素抗内毒素休克与肺代谢。中国病理生理杂志，1995，11(7)：815～815
4 凌亦凌，黄善生，张君岚，等．胆囊收缩素对内毒素性休克大鼠SOD、MDA及吞噬细胞发光的影响。中国病理生理杂志，1997，13(5)：483～486
5 孟爱宏，凌亦凌，王殿华，等．八肽胆囊收缩素减轻肿瘤坏死因子诱导的离体兔肺动脉反应性变化及内皮细胞损伤。生理学报，2000，52(6)：502～506
6 Wu GH, Zhang YW, Wu ZH. Modulation of postoperative immune and inflammatory response by immune-enhancing enteral diet in gastrointestinal cancer patients. World J Gastroenterol, 2001, 7(3) : 357-362
7 Mailman D, Guntuku S, Bhuiyan MB, et al. Organ sites of lipopolysaccharide-induced nitric oxide production in the anesthetized rat. Nitric Oxide, 2001, 5(3) : 243-51
8 Nadler EP, Dickinson EC, Beer-Stolz D, et al. Scavenging nitric oxide reduces hepatocellular injury after endotoxin challenge. Am J Physiol, 2001,281:G173-181
9 Kang JL, Lee K and Castranova V. Nitric oxide up-regulates DNA-binding activity of nuclear factor-K B in macrophages stimulated with silica and inflammatory stimulants. Mol Cell Bioche, 2000,215:1-9
10 Lee RP, Wang D, Kao SJ et al. The lung is the major site that produces nitric oxide to induce acute pulmonary oedema in endotoxin shock. Clin Exp Pharmacol Physiol, 2001, 28:315-320
11 Otterbein LE, Choi AMK. Heme oxygenase: colors of defense against cellular stress. Am J Physiol, 2000, 279: L1029-1037
12 Ou HS, Yang J, Dong LW et al. Role of endogenous carbon monoxide in the pathogenesis of hypotension during septic shock. Acta Physiologica Sinica, 1999, 51(1) : 1-6
13 Schipper HM, Chertkow H, Mehindate K et al. Evaluation of heme oxygenase-1 as a systemic biological marker of sporadic AD. Neurology, 2000,54:1297-1304
14 Suttner DM and Dennery PA. Reversal of HO-1 related cytoprotection with increased expression is due to reactive iron. FASEB J, 1999, 13: 1800-1809
15 Carrasco M, Monica DR, Angel H et al. Inhibition of human neutrophil functions by sulfated and nonsulfated cholecystokinin octapeptides. Peptides, 1997, 18:415-422
2 凌亦凌，黄善生，王乐丰，等．八肽胆囊收缩素抗内毒素休克的实验研究．生理学报，1996，48(4)：390～394
3 凌亦凌，黄善生，王乐丰，等．胆囊收缩素抗内毒素休克与肺代谢。中国病理生理杂志，1995，11(7)：815～815
4 凌亦凌，黄善生，张君岚，等．胆囊收缩素对内毒素性休克大鼠SOD、MDA及吞噬细胞发光的影响。中国病理生理杂志，1997，13(5)：483～486
5 孟爱宏，凌亦凌，王殿华，等．八肽胆囊收缩素减轻肿瘤坏死因子诱导的离体兔肺动脉反应性变化及内皮细胞损伤。生理学报，2000，52(6)：502～506
6 Wu GH, Zhang YW, Wu ZH. Modulation of postoperative immune and inflammatory response by immune-enhancing enteral diet in gastrointestinal cancer patients. World J Gastroenterol, 2001, 7(3) : 357-362
7 Mailman D, Guntuku S, Bhuiyan MB, et al. Organ sites of lipopolysaccharide-induced nitric oxide production in the anesthetized rat. Nitric Oxide, 2001, 5(3) : 243-51
8 Nadler EP, Dickinson EC, Beer-Stolz D, et al. Scavenging nitric oxide reduces hepatocellular injury after endotoxin challenge. Am J Physiol, 2001,281:G173-181
9 Kang JL, Lee K and Castranova V. Nitric oxide up-regulates DNA-binding activity of nuclear factor-K B in macrophages stimulated with silica and inflammatory stimulants. Mol Cell Bioche, 2000,215:1-9
10 Lee RP, Wang D, Kao SJ et al. The lung is the major site that produces nitric oxide to induce acute pulmonary oedema in endotoxin shock. Clin Exp Pharmacol Physiol, 2001, 28:315-320
11 Otterbein LE, Choi AMK. Heme oxygenase: colors of defense against cellular stress. Am J Physiol, 2000, 279: L1029-1037
12 Ou HS, Yang J, Dong LW et al. Role of endogenous carbon monoxide in the pathogenesis of hypotension during septic shock. Acta Physiologica Sinica, 1999, 51(1) : 1-6
13 Schipper HM, Chertkow H, Mehindate K et al. Evaluation of heme oxygenase-1 as a systemic biological marker of sporadic AD. Neurology, 2000,54:1297-1304
14 Suttner DM and Dennery PA. Reversal of HO-1 related cytoprotection with increased expression is due to reactive iron. FASEB J, 1999, 13: 1800-1809
15 Carrasco M, Monica DR, Angel H et al. Inhibition of human neutrophil functions by sulfated and nonsulfated cholecystokinin octapeptides. Peptides, 1997, 18:415-422