摘要
本文分别观察了人参二醇皂苷对内毒素休克和对失血-内毒素二次打击大鼠肝脏损伤的保护作用,并探讨其机制。结果显示:人参二醇皂苷(PDS)与地塞米松类似,可降低血液黏度,改善微循环,减少氧自由基产生,抑制AST、ALT、ALP、CK、LDH 等血清酶的释放,具有良好的抗休克的作用。进一步的研究发现,PDS 能够抑制内毒素休克和二次打击大鼠肝脏组织CD14 的过度表达、提高I-κB mRNA 的表达水平,减少NF-κB 入核,使含κB 序列的基因转录减弱,进而抑制TNF、IL6 和IL18 的表达,即减少了促炎因子的大量释放,又避免了抗炎因子过量产生所导致的免疫抑制,是实现机体自稳调节的分子机制; PDS 还有调节LPS 导致的AQP8 、9 、1 表达失调的作用。上述发现证明了人参二醇皂苷对肝脏细胞的保护作用,国内外尚未见类似报道,为人参二醇皂苷的开发和临床应用奠定了基础。
The reason that traumatic or infectious shocks as well as others progressed into refractory shocks were related with severe obstruction of microcirculation and severe ischemia-anoxia, caused by disseminated or diffused intravascular coagulation (DIC) besides, with destruction of intestinal mucosal barrier, immune dysfunction and subsequent shifting of bacteria and endotoxin into the blood. After LPS entered blood, they bond to circulating LPS-binding protein (LBP) of which belonging to acute phase protein, and formed LPS-LBP complex which subsequently combined with cell membrane receptor of monocytes and macrophages, CD14 that, in turn, triggered the intracellular signaling transduction pathway mediated by LPS receptor and produced the consequence of intracellular responses including the phosphorylation and degradation of the inhibitory protein IκBαwhich originally presented in the cytoplasm combined with the inactive NF-κB. NF-κB was then activated and released from IκBα, and shifted to the nucleus and bond to κB motif of the target genes, in turn inducing gene expression and a number of inflammatory cytokines produced. When inflammation response failed to be self-controlled because of the overproduction of inflammatory mediators, self-destroyed systemic inflammatory response syndrome (SIRS) induced, which would be the
leading cause of multiple system organ failure (MSOF).
This paper aims at observation of the protective effects of PDS on the liver of two kinds of shocks by building up the pathological models of LPS induced endotoxic shock and hemorrhage–lipopolysaccharides induced two-hit Wistar rats, and exploring the signal transduction system mediated by LPS receptor.
1. The protective effects and mechanism of PDS on liver injury of endotoxic shock in rats
Adult wistar rats were divided randomly into 4 groups: the control group (CTR group) ; the LPS shock model group (LPS group, 5mg/kg) ; Dexamethasone pretherapy +LPS group (DEX group, 2mg/Kg);Panaxadiol saponins pretherapy +LPS group (PDS group, 25mg/Kg). LPS was administered by sublingual vein injection to build up the model of endotoxic shock. When blood pressure reduced to 2/3 of the baseline it was regarded as the start of shock.. DEX and PDS were injected intraperitoneally 10 min before LPS administered. Animals were executed 240min after LPS administered.The results are as follows:
1.1 PDS had the same protective effect as DEX to endotoxic shock rats:①Steadily and permanently improved mean arterial blood pressure( MABP). ②The blood viscosity in DEX group and PDS groups were all significantly lower under various shear rates than that in LPS group. It suggested that PDS had the effect of decreasing blood viscosity, significant improving of microcirculation. ③The levels of serum AST,ALT etc were the highest in LPS group among four groups, and the levels of serum enzyme in PDS and DEX groups were significantly lower compared with that in LPS group; these showed the protective effects of PDS on hepatic cell membrane. ④The technology of reverse transcriptase-polymerase chain reaction (RT-PCR) was used to assay the expression of hepatic AQP1 mRNA : The expression of hepatic AQP1 mRNA in LPS group was significantly lower than that in
Control, DEX and PDS groups; the expression of AQP1mRNA in PDS group was close to that in Control group. The above results hinted that PDS had protective effect on endothelium of capillary vessels in liver. ⑤The protective effect to the histology of liver: The injury of liver tissue was found with light microscope after LPS administered 4h. In LPS group there were expansion and congestion with the central vein of hepatic lobule, and nuclear diffluence, disappearance, and heavy staining in cytoplasm of hepatocytes around the central vein of hepatic lobule; Kupffer cells hyperplasia actively also could be seen in sinusoids. The above signs in PDS and DEX groups were significantly slighter than LPS group. PDS had the same protective effect as DEX to morphology of hepatic cells in endotoxic shock rats.
1.2 The effect of PDS on signal transduction pathway mediated by LPS receptor of liver in endotoxic shock rats
The results of study on several important factors involved in signaling include: ①the expression of CD14 mRNA and protein in LPS group was the highest among four groups as assayed by RT-PCR and western blotting, respectively, while that in DEX and PDS groups it was significantly lower than that in LPS group. The result of Immunohistochemistry showed that Hepatic cell stained with CD14 Ab and Kupffer cells membrane had positive pigmentation in sinusoids; the number of dyed Kupffer cells in LPS group was more than that both in DEX and PDS groups. The above results proved that LPS strongly induced the expression of CD14, but PDS inhibited the expression of CD14. ②The hepatic expression of IκBαmRNA in LPS group was significantly lower than that in PDS and DEX groups , PDS up regulated IκBαmRNA expression in liver. ③PDS inhibited protein expression of NF-κB p65 and NF-κB p50 in cell nucleus: The Western Blotting result showed that, the expression of NF-κB p65 and NF-κB p50 in LPS group were significantly strengthened, the expression in PDS groups having the same tendency as in DEX group was weaker compared with that in LPS group.
1.3. The effect of PDS on down regulate expression of cytokines and production of NOS induced by NF-κB in liver of endotoxic shock rats: ①The expression of hepatic TNFαand IL-6 mRNA: Both expression of TNFαand IL-6 mRNA in LPS group were higher than that in PDS and DEX groups. ②The expression of hepatic IL-18 mRNA and protein: The expression of IL-18 mRNA and protein in LPS group were significantly higher than that in PDS and DEX groups; PDS and DEX had the same effect of down regulated expression of IL-18 mRNA and protein. ③The content of NO2-/NO3-and NOS activity in serum in LPS group was significantly higher than that in PDS and DEX group after endotoxic shock 240 min. These result suggested that PDS and DEX had the same effect to lessen the production of NO·in serum and counteract peroxidization of lipid.
In summary, the protective effects of PDS on endotoxic shock rats: PDS had the same effect as DEX to steadily and permanently maintain mean arterial blood pressure, to lessen serum enzyme release, and strengthen the expression of AQP1, and mitigate the injury of modality and structure in liver. the possible protective mechanisms of PDS were ①decreasing blood viscosity, and improving microcirculation, and elevating blood and oxygen supply under state of endotoxic shock. ②regulating the over expression of signaling transduction pathway mediated by LPS receptor, enhancing self-limitation of production of promoted inflammatory factors and avoiding self-destroyed systemic inflammatory response syndrome (SIRS) induced by overrunning of inflammatory mediators.
2. The protective effect and mechanism of PDS on the liver injury of two-hit rats with hemorrhage -LPS
In order to simulate condition of hemorrhagic shock and subsequently endotoxin (LPS) shift into blood, two-hit models of rats were replicated by resuscitation with 1/2 shed blood plus 2 vol of saline by slow reperfusion after hemorrhagic shock 1h and then LPS (2mg/Kg) injected intraperitoneally.
DEX(2mg/Kg) and PDS(25mg/Kg) were injected intraperitoneally in HLD and HLP groups10 min before LPS administered . Animals were execute at 6h after LPS injected. Adult wistar rats (230g-250g) were randomly divided into 4 groups : the sham operational group(S group); the two-hit groups with hemorrhage-lipopolysaccharides ( hemorrhagic shock/ resuscitation with shed blood and lipopolysaccharides administered, HL group);the Dexamethasone preventive therapy group ( the hemorrhagic shock/ resuscitation / Dexamethasone and lipopolysaccharides administered group, HLD group);the panaxadiol saponins preventive therapy group( the hemorrhagic shock/ resuscitation / panaxadiol saponins and lipopolysaccharides administered group ,HLP group).
2.1The protection of PDS to liver in two-hit rats with hemorrhage-LPS:
2.1.1 PDS had the same effect as DEX on inhibition of release of serum enzymes AST, ALT, ALP, CK and LDH, on protection of hepatic cell membrane and mitigation of cholestasis.
2.1.2 The effect of PDS on expression of AQP8,9,1 in liver: ①The results of Fluorescence-based Real-Time PCR assay showed that , the level of expression of hepatic AQP8 in HL group was the lowest among groups and had a significant difference compared with S, HLD and HLP groups; the level of expression of hepatic AQP 9 mRNA in HL and HLD groups were low, and the level of expression in HLP group was appreciably increasing. ②Application of western blotting assay to test the expression of hepatic AQP8, 9 protein: the expression of AQP8 protein in HL group was significantly lower than that in S group, the expression in HLD and HLP groups were significantly higher than that in HL group, especially in HLP group. The expression of AQP9 protein in HL and HLD groups were significantly lower than that in S group, the expression of AQP9 protein in HLP group was close to that in S group. ③Both PDS and DEX didn’t have protective effect on the expression of hepatic AQP1mRNA. AQP1 expressed mainly on membrane of endothelium of capillary vessels in liver, the membrane of endothelium had
been hurt at stage of hemorrhagic shock, PDS and DEX were gaven after resuscitation, so it couldn’t show the visible protective effect of PDS.
2.1.3 The protective effect of PDS on structure and ultrastructure of liver: with the observation of light microscope , there was obvious injury of hepatocyte with spot necrosis and nuclear diffluence, disappearance, and lymphocyte and monocyte infiltration can be seen in hepatic lobule, and fatty degeneration of hepatocyte also could been seen, the expansion and congestion of the central vein of hepatic lobule, manifolded inflammatory cells in central vein of hepatic lobule all were seen, active hyperplasia of Kupffer cells could also be seen in sinusoids in LPS group. The above signs in HLP groups and HLD group were significantly slighter than that in HL group. With observation of scanning electron microscope, in LPS group there were the degeneration and necrosis of hepatocytes including abnormity nucleus, faintness karyotheca, the swelling of mitochondria and rough surfaced endoplasmic reticulum, and increased lipid drops and lysosomes in cytoplasm; and there were also gotten thin microvilli on the surface of hepatocyte facing the space of sinusoid. There were cavitated necrosis of cells in sinusoid including damaged cytomembrane, cavitated nucleus and cytoplast, and swelled and cavitated mitochondria in Kupffer cells. The above signs in HLP and HLD groups were significantly slighter than that in HL group.
2.2 The effects of PDS to the signaling pathway mediated by LPS receptors in two-hit rats
Similar to endotoxic shock , ①after LPS injected intraperitoneally 6 h ,the expression of CD14 mRNA and protein in HL group was higher than that in S group, while that in HLP group was a little bit lower than that in HL group. ②The expression of IκB in HL group is significantly lower than that in S group, while that in HLP group was close to S group and higher than that in HL group; therefore speculation was be given that PDS had the protective effects on inhibitory protein IκB from degradation. ③The expression of NF-κB p65 in HL group is significantly strengthened while in HLP and HLD groups were getting weak but higher than that of S group. ④The effect of
引文
[1] Su GL,Klein RD,Aminlari A,et al. Kupffer cell activation by lipopolysaccharide in rats :role for lipopolysacccharide binding protein and toll-like receptor 4. Hepatology,2000,31:932-936.
[2] 张顺财主编. 内毒素基础与临床. 科学出版社2003: 1-436.
[3] 罗海波,鲍行豪.细菌内毒素研究进展(第一版).人民卫生出版社,1983: 1-373
[4] Bayston KF,Cohen J. Bacterial endotoxin and current concepts in the diagnosis and treatment of endotoxamia. J Med Microbiol,1990,31:73-83.
[5] Mitov IG, Terziiski DG. Immunoprophyiaxis and immunotherapy of gram-negative sepsis and shock with antibodies to core glycolipids and lipid A of bacterial lipopolysaccharides. Infection, 1991, 19:383-390.
[6] Jawetz E, Melnick J.l., Adelberg E. A著,邓瑞麟译.医学微生物学(第一版) ,人民卫生出版社,1 983: 270-272.
[7] 邱世翠,曲云,胡涛英等.滨州医学院学报1992, 15(3): 211-212.
[8] Birgitt Stein, Petra Frank, Wilhelm Schmitz1,et al. Endotoxin and Cytokines Induce Direct Cardiodepressive Effects in Mammalian Cardiomyocytes via Induction of Nitric Oxide Synthase. J Mol Cell Cardiol 1996, 28: 1631-1639.
[9] Torre-Amione G, Kapadia S, Lee J,et al. Tumor necrosis factor-alpha and tumor necrosis factor receptors in the failing human heart. Circulation 1996,93:704-11.
[10] Roig E, Orus J, Pare C, et al. Serum interleukin-6 in congestive heart failure secondary to idiopathic dilated cardiomyopathy. Am J Cardiol 1998,82:688–90.
[11] R.S. Hotchkiss, P.E. Swanson, B.D. Freeman,et al. Apoptotic cell death in patients with sepsis, shock and multiple organ dysfunction, Crit. Care Med. 27 (1999) 1230–1251.
[12] A. Ayala, Y.X. Xu, C.A. Ayala,et al. Increased mucosal B-lymphocyte apoptosis during polymicrobial sepsis is a Fas ligand but not an endotoxin mediated process, Blood 91 (1998) 1362–1372.
[13] Alfred Ayala., Joanne L. Lomas, Patricia S. Grutkoski, Chun-Shiang Chung. Pathological aspects of apoptosis in severe sepsis and shock? The International Journal of Biochemistry & Cell Biology 35 (2003) 7–15
[14] Francoise, MA.,Luc, DL.,Adjuvants. Immunology Today. 1993, 14(6): 281
[15] [27]李桂杰,朱瑞良,郭宝聚. 细菌内毒素的研究进展. 山东农业大学学报1998, 30(2) 193-198
[16] 陆松敏,杨鹤鸣,刘建仓,等.内毒素、失血性休克大鼠线粒体质子跨膜转运和H+-ATP 酶的改变.中华创伤杂志,1998,14:28-31.
[17] Kurzawa R, Gombowski W, Wenda-Rócewicka L Evaluation of mouse blastocysts grown in media enriched with insulin-like growth factor I and II, epidermal growth factor and tumor necrosis factor alpha. Folia Histochemica et Cytobiologica,2001, 39 :245-251.
[18] 王密,杨柯,赵丹,等.人参二醇组皂甙对游泳训练大鼠LPO 和SOD 的影响[J].白求恩医科大学学报,2001,27(4):358-360.
[19]刘媛媛.人参二醇组皂甙对内毒素休克鼠组织过氧化脂质的影响[J]. 白求恩医科大学学报,1990,16(4):342-345.
[20] 王本祥.人参的研究.天津科学技术出版社,1985,
[21] 徐琳,林清华,赵雪俭,等.人参二醇皂甙对失血性休克犬单胺类递质和单胺氧化酶的影响.中国病理生理杂志,1990,6 (2):69
[22] 刘媛媛,赵雪俭,李扬,等.人参二醇组皂甙对内毒素性休克犬保护作用机制的实验研究.中国药理学学报,1992,8(1):6
[23] 杨柯,赵丹,赵雪俭,等.人参二醇组皂甙及游泳训练对大鼠股四头肌α-肌动蛋白mRNA表达的影响.中国病理生理杂志,2002,18(8):942-945
[24] 朱建明.人参皂甙的抗衰老作用研究进展.中国医药信息,1998,2:18-20
[25] 袁茂昆,赵洪序,宋翔翎,姜亦忠.人参二醇组皂甙对缺血心肌再灌注损伤的保护作用及其浓度-效应关系. 白求恩医科大学学报,1997,23(5):492-493
[26] 张冬梅等. 人参茎叶总甙对大鼠海马糖皮质激素受体的影响. 白求恩医科大学学报1988, 增刊: 59
[27] 赵文海,乔利民,王卫兵.肾上腺皮质激素所致股骨头无菌性坏死的预防和治疗.长春中医学院学报,1994,10(40):31-32
[28] 王心生,陈凤苞,许振华等.早期激素性股骨头缺血性坏死的实验研究.河南医科大学学报,1994,23(3):243-245
[29] Chelvarajan RL, Collins SM, Van Willigen JM, Bondada S.The unresponsiveness of aged mice to polysaccharide antigens is a result of a defect in macrophage function. J Leukoc Biol. 2005 Apr,77(4):503-12. Epub 2005 Jan 3.
[30] Duval DL, Sieg DJ, Billings RE: Regulation of hepatic nitric oxide synthase by reactive oxygen intermediates and glutathione. Arch Biochem Biophys 1995, 316:699.
[31] Lakics V, Vogel SN. Lipopolysaccharide and ceramide use divergent signaling pathways to induce cell death in murine macrophages. J Immunol. 1998,161:2490-2500
[32] 苏踊跃,许波,罗向东.内毒素和IFNγ对巨噬细胞的体外调变作用. 重庆医学,2002,31(6):488-489.
[33] Okada S, Zhang H, Hatano M, Tokuhisa T: A physiologic role of Bcl-xL induced in activated macrophages.
[34] Thurman R G,Bradford B U,Limuro Y,et al. Role of kupffer cells,endotoxin and free radicals in hepatotoxicity due to prolonged alcohol con-sumption:studies in female and male rats[J]. J Nutr,1 977,1 2 7(5suppl) :903~906.
[35] SakaguchiS,KandaN,HsuCC,et al. Lipid peroxide for-mation and membrane damage in endotoxin-poisoned mice.Microbio lImmunol,1981,25(3):229~244.
[36] 陈宁,王玉芬,潘焰. 氧自由基与多器官衰竭. 中国病理生理杂志1991, 7(5): 477~481.
[37] 高洪, 陈利平, 唐兆新, 陈万芳. 内毒素诱生自由基致膜损伤机理的研究进展.动物医学进展1999,20(3):1-5.
[38] Palmer R M J,Ferriage A G,Mocada S.Nitric oxide release accountsfor the biological activity of endothelium relaxing factor.Nature1987, 327: 524 -530.
[39] Thiemermann C. and Vane J. R. (1990) Inhibition of nitric oxide synthesis reduces the hypotension induced by bacterial lipopolysaccharide in the rat. Eur. J. Pharmacol. 182, 591-595.
[40]Julou-Schaeffer G, Gray G, Fleming, et al.Loss of vascular responsiveness induced by endotoxin involves the L-arginine pathway. Am. J. Physiol. 259, H1038-H1043.
[41] Rees D. D., Cellek S., Pahner R. M. J. and Moncada S. (1990) DEXamethasone prevents the induction of nitric oxide synthase and the associated effects on the vascular tone: an insight into endotoxic shock, l~iochem.Biophys. Res. Commun. 173, 541-547.
[42] Cantoni L, Di Nicolantonio F, Barelli D, et al .Modulation of cerebellar and hepatic nitric oxide synthase by exogenous arginine and endotoxin. Nitric Oxide. 2001 Apr,5(2):198-207.
[43] Thiemermann C. Nitric Oxide and Septic Shock. Gen. Pharmac 1997, 29(2): 159-166.
[44] 谢平初,陆家齐,孙大铭,等.猪内毒素休克外周与内脏微循环灌注的动 态变化.中国危重病急救医学1999, 11(12): 718-720.
[45] Lin HI, Wang D, Leu FJ, etal.Ischemia and reperfusion of liver induces eNOS and iNOS expression: effects of a NO donor and NOS inhibitor .Chin J Physiol. 2004 Sep 30,47(3):121-7.
[46] Stolz DB, Zamora R, Vodovotz Y,et al .Peroxisomal localization of inducible nitric oxide synthase in hepatocytes. Hepatology. 2002 Jul,36(1):81-93.
[47] Olinga P, Merema MT, de Jager MH, et al. Rat liver slices as a tool to study LPS-induced inflammatory response in the liver. J Hepatol. 2001 Aug,35(2):187-94.
[48]Ulevitch RJ, Tobias PS. Recognition of endotoxin by cells leading to transmembrane signaling.Curr Opin Immunol, 1994,6:125-130.
[49]Ulevitch RJ, Tobias PS. Receptor-dependent mechanisms of cell stimulation by bacterial endotoxin. Annu Rev Immunol, 1995,13:437-457.
[50] Wright SD, Ramos RA,Tobias PS, et al. CD14 serves as the celluar receptor for complexes of lipopolysaccharide with lipopolysaccharide binding protein. Science, 1990, 249: 1431-1433.
[51] Mark M. Wurfel, Eric Hailman, and Samuel D. Wright. Soluble CD14 Acts as a Shuttle in the Neutralization of Lipopolysaccharide (LPS) by LPS-binding Protein and Reconstituted High Density Lipoprotein . The Rockefeller University Press,1995,181 :1743-1754.
[52] Schroder NW, Opitz B,Lamping N et al . Involvement of lipopolysaccharide binding protein,CD14,and Toll-like receptors in the initiation of innate immune responses by Treponema Glycolipods[J ] . J Immunol,2000 ,165 (5) :268322693.
[53] 王新颖. 内毒素激活细胞信号传导机制的研究进展. 肠外与肠内营养2001, 8(3): 177-184.
[54] Cowan DB, Poutias DN, Del Nido PJ, et al. CD14-independent activation of cardiomyocyte signal transduction by bacterial endotoxin [J]. Am J Physiol Heart Circ Physiol 2000, 279(2 ): H619-629.
[55] Lien E,Ingalls R.Toll like receptors.Crit Care Med,2002,30:1-11.
[56] 冯俊明,史景泉,刘友生,等.大鼠枯否细胞CD14表达的实验研究.中国病理生理杂志,2004,20(2):162-165.
[57] Landmann R,Knopf H,Link S,et al.Human monocyte CD14 is upregulated by lipopolysaccharide.Infect Immun, 1996,64:1763-1769.
[58] Zieglet-Heitbrock HWL,Ulevitch RJ. CD14,cell surface receptor and differentiation marker. Immunol Today 1993,14:121.
[59] Bazil V,Strominger JL,Shedding as a mechanism of downmodulation of CD14 on stimulated human monocyte.J Immunol 1991, 147(5):1567.
[60] Fearns C,Kravchenko VV,Ulevich RJ,et al,Murine CD14 gene expression in vivo:extramyeloid synthesis and regulation by lipopolysaccharide. J Exp Med 1995, 181: 857.
[61] Cosention G,Soprana E,Thienes CP,IL-13 down-regulates CD14 expression and TNFαsecretion on normal human monocytes.J Immun,1995,155:3145.
[62] Lauener RP,Goyert SU,Geha RS,et al.Interleukin down-regulate the expression of CD14 in normal human monocytes.Eur J Immuol, 1990, 20: 2375.
[63] Hojman H,Lounsbury D,Harres H,Immunodepressive effects of LPS on monocyte CD14 in vivo.J Surg Res,1997,69:7
[64] Gwozdzinski K, Pieniazek A,Kaca W. Lipopolysaccharide from Proteus mirabilis O29 induces changes in red blood cell membrane lipids and proteins[J ] . Int J Biochem Cell Biol,2003 ,35 (3) :333 -3381 .
[65] Wang S C,Klein R D,Wahl WL,et al . Tissue coexpression of LBP and CD14 mRNA in a mouse model of sepsis[J ] . J Surg Res,1998 ,76 (1) :67-73.
[66]万志红,陆松敏,陈惠孙.内毒素对巨噬细胞膜CD14 表达的影响及与膜脂微环境关系.第三军医大学学报,2003,25(17):1536-1539.
[67]Annelies Verbon, Pascale E. P. Dekkers, Tessa ten Hove,et al IC14, an Anti-CD14 Antibody, Inhibits Endotoxin-Mediated Symptoms and Inflammatory Responses in Humans. The Journal of Immunology, 2001, 166: 3599-3605.
[68] Furusako S, Takahashi T, Mori S, et al. Protection of mice from LPS induced shock by CD14 antisense oligonucleotide [J]. Acta Med Okayama 2001, 55(2): 105.
[69] Axtelle T, Pribble J. IC14, a CD14 specific monoclonal antibody, isapotential treatment for patients with severe sepsis [J]. J Endotoxin Res 2001, 7(4): 310
[70] Iwami KI,Matsuguchi T ,Masuda A et al . Cutting edge : naturally occurring soluble form of mouse Toll-like receptor 4 inhibits lipopolysaccharide signaling [ J ] . J Immunol,2000,165 ( 12) :66822 6686.
[71] Remick D. Applied molecular biology of sepsis. J Crit Care. 1995, 10(4):198-212.
[72] Heumann D,Roger T. Initial responses to endotoxins and Gram -negative bacteria [J ] . Clin Chim Acta,2002 ,323 (1 -2) :59 -72.
[73] Arbour NC,Lorenz E,Schutte BC,et al. TLR4 mutations are associated with endotoxin hyporesponsiveness in humans. Nat Genet , 2000 , 25 : 187 -191 .
[74] Schr?der OWJ, Opitz B, Schumann RR,et al. Involvement of lipopolysaccharide binding protein,CD14,and toll-like receptors in the initiation of innate immune responses by Treponema Glycolipids[J]. Immunol,2000,165:2683-2693.
[75]Orange JS, Geha RS .Finding NEMO: genetic disorders of NF-κB activation[J]. J Clin Invest, 2003 ,112 (7): 983–985.
[76] Martin TR.Recognition of bacterial endotoxin in the lungs. Am J Respir Cell Mol Biol,2000,23:128-132.
[77]李永旺,麻莉,毛宝龄,等.内毒素诱导的TLR4-MD2信号传导通路.中国药理学通报,2002,18:121-124.
[78] Derek S,Jong S,Edward A. Sepsis:current concepts in intracellular signaling. Int j Biochem,2002,1302:1-7.
[79] Diks SH, van Deventer SJ and Peppelenbosch MP. Lipopolysaccharide recognition, internalisation, signalling and other cellular effects. J Endotoxin Res ,2001,7: 335-348.
[80] Hugh M. Paterson, Thomas J. Murphy, Elizabeth J. Purcell, et al. Lederer Injury Primes the Innate Immune System for Enhanced Toll-Like Receptor Reactivity. The Journal of Immunology, 2003, 171: 1473-1483.
[81] Diks SH, van Deventer SJ and Peppelenbosch MP Lipopolysaccharide recognition, internalisation, signalling and other cellular effects. J Endotoxin Res.2001,7: 335-348.
[82] Hoffmann, A, Leung, TH, & Baltimore, D. Genetic analysis of NF-kappaB/Rel transcription factors defines functional specificities . EMBO J 2003, 22:5530-5539.
[83]WANG Gang,ZHAO Min and WANG En-hua.Effects of glycine and methylprednisolone on hemorrhagic shock in rats.Chin Med J 2004, 117(9): 1334-1341 .
[84] Byrne AT, Southgate J, Brison DR, Leese HJEffects of insulin-like growth factorsI and II on tumour-necrosis-factor-alpha-induced apoptosis in early murine embryos.Reproduction Fertility and Development ,2002 , 14: 79-83.
[85] Wirtz PH, von Kanel R, Kunz-Ebrecht S, et al.Reduced glucocorticoid sensitivity of monocyte interleukin-6 release in male employees with high plasma levels of tumor necrosis factor-alpha. Life Sci. 2004 May 21,75(1):1-10.
[86] Lee A. Denson,Ram K. Menon, Angel Shaufl,et al.TNFαdownregulates murine hepatic growth hormone receptor expression by inhibiting Sp1 and Sp3 binding. J Clin Invest. 2001 June 1, 107(11): 1451–1458.
[87] Cusumano, V., G. Mancuso, F. Genovese, et al. Neonatal hypersusceptibility to endotoxin correlates with increased tumor necrosis factor production in mice. J. Infect. Dis. 1997. 176:168–176.
[88] Baud V, Karin M. Signal transduction by tumor necrosis factor and its relatives. Trends in Cell Biol 2000, 11:372-377. 22 .
[89] Ning BT, Tang YM, Shen HQ, et al.Preparation and characterization of a directly labeled mouse anti-human CD14 monoclonal antibody ZCH-2F9-FITC. Zhejiang Da Xue Xue Bao Yi Xue Ban. 2005 Mar,34(2):167-71.
[90] 朱参胜,欧阳为明,刘雪松,等.抗人TNF 单克隆抗体对TNF 诱导的NFκB 核转位的抑制作用.细胞与分子免疫学杂志,2004,20(5):585-587.
[91] Zhang Y, Wang C, Zhang Y, Sun M.C6 glioma cells retrovirally engineered to express IL-18 and Fas exert FasL-dependent cytotoxicity against glioma formation. Biochem Biophys Res Commun. 2004 Dec 24,325(4):1240-5.
[92] Finotto S, Siebler J, Hausding M, et al. Severe hepatic injury in interleukin 18 (IL-18) transgenic mice: a key role for IL-18 in regulating hepatocyte apoptosis in vivo. Gut. 2004 Mar,53(3):392-400.
[93] Gracie, J. A., Robertson, S. E. and McInnes, I. B., Interleukin-18. J. Leukoc. Biol. 2003. 73: 213–224.
[94] Leung, B. P., Culshaw, S., Gracie, J. A., et al. A role for IL-18 in neutrophil activation. J. Immunol. 2001. 167: 2879–2886.
[95] Okamura, H., Nagata, K., Komatsu, T., et al.A novel costimulatory factor forgamma interferon induction found in the livers of mice causes endotoxic shock. Infect. Immun. 1995.63: 3966–3972.
[96] Tsutsui H,Kayagaki N,Kuida K, et al. Caspase 1 independent,Fas/ Fas ligand-mediated IL-18 secretion from macrophages cause acute liver injury in mice[J ] . Immun ,1999 ,11 :359 –366.
[97] Matsui, K., Yoshimoto, T., Tsutsui, H., et al. Propionibacterium acnes treatment diminishes CD4+ NK1.1+ T cells but induces type I T cells in the liver by induction of IL-12 and IL-18 production from Kupffer cells.J. Immunol. 1997. 159: 97–106.
[98] Tsutsui H, Matsui K, Okamura H, Nakanishi K.Pathophysiological roles of interleukin-18 in inflammatory liver diseases. Immunol Rev. 2000
Apr,174:192-209.
[99] Vankayalapati R, Wizel B, Weis SE.Production of interleukin-18 in human tuberculosis[J].J Infect Dis,2000,182(1):234-239.
[100] 毛宝龄. 深入探讨全身炎症反应的失控与调控. 中华内科杂志,1997,36(1):34.
[101]Kellum JA, Song M, Venkataraman R.Hemoadsorption removes tumor necrosis factor, interleukin-6, and interleukin-10, reduces nuclear factor-kappaB DNA binding, and improves short-term survival in lethal endotoxemia. Crit Care Med. 2004 Mar,32(3):801-805.
[102]张青,徐剑铖,毛宝龄,等.内毒素致伤大鼠肺组织TNFα、IL-6的mRNA表达及NFIL-6 活化研究.中国危重病急救医学,2001, 13(9):523-526.
[103]Blackwell TS,Christman.Sepsis and cytokines:current status.British Journal of Anaesthesia,1996,77110-77117.
[104] Masyuk AI, Marinelli RA, LaRusso NF. Water transport by epithelia of the digestive tract. Gastroenterology 2002,122:545-562.
[105]Kozono D, Yasui M, King LS, Agre P. Aquaporin water channels: atomic structure molecular dynamics meet clinical medicine. J Clin Invest 2002,109:1395-1399.
[106]Jiao G, Li E, Yu R.Decreased expression of AQP1 and AQP5 in acute injured lungs in rats. Chin Med J (Engl)2002 Jul,115(7):963-7.
[107]Su X, Song Y, Jiang J, Bai C.The role of aquaporin-1 (AQP1) expression in a murine model of lipopolysaccharide-induced acute lung injury. Respir Physiol Neurobiol. 2004 Aug 20,142(1):1-11.
[108]Lai KN, Leung JC, Metz CN,et al.Role for macrophage migration inhibitory factor in acute respiratory distress syndrome. J Pathol. 2003 Apr,199(4):496-508.
[109]Ohinata A, Nagai K, Nomura J, et al.Lipopolysaccharide changes the subcellular distribution of aquaporin 5 and increases plasma membrane water permeability in mouse lung epithelial cells. Biochem Biophys Res Commun. 2005 Jan 21,326(3):521-6.)
[110]Grinevich V, Knepper MA, Verbalis J,et al.Acute endotoxemia in rats induces down-regulation of V2 vasopressin receptors and aquaporin-2 content in the kidney medulla. Kidney Int. 2004 Jan,65(1):54-62.)
[111]Tsukaguchi H, Shayakul C, Berger UV, et al. Molecular characterization of a broad selectivity neutral solute channel. J Biol Chem 1998, 273:24737-24743.
[112]Elkjaer M-L, Vajda Z, Nejsum LN, et al. Immunolocalization of AQP9 in liver, epididymis, testis, spleen, and brain. Biochem Biophys Res Commun 2000,276:1118-1128.
[113]Garcia F, Kierbel A, Larocca MC,et al. The water channel aquaporin-8 is mainly intracellular in rat hepatocytes and its plasma membrane insertion is stimulated by cyclic AMP. J Biol Chem 2001,276:12147-12152.
[114]Calamita G, Mazzone A, Bizzoca A,et al. Expression and immunolocalization of the aquaporin-8 water channel in rat gastrointestinal tract. Eur J Cell Biol 2001,80:711-719.
[115]Nicchia GP, Frigeri A, Nico B,et al. Tissue distribution and membrane localization of aquaporin-9 water channel: evidence for sex-linked differences in liver. J Histochem Cytochem 2001,49:1547-1556.
[116] Elkjaer ML, Nejsum LN, Gresz V,et al. Immunolocalization of aquaporin-8 in rat kidney, gastrointestinal tract, testis, and airways. Am J Physiol Renal Physiol 2001,281: F1047-F1057.
[117]Tani T, Koyama Y, Nihei K, et al. Immunolocalization of aquaporin-8 in rat digestive organs and testis. Arch Histol Cytol 2001,64:159-168.
[118]Huebert RC, Splinter PL, Garcy′a F,et al. Expression and localization of aquaporin water channels in rat hepatocytes. Evidence for a role in canalicular bile secretion. J Biol Chem 2002,277:22710-22717.
[119]Ferri D, Mazzone A, Liquori GE,et al. Ontogeny, distribution, and possible functional implications of an unusual aquaporin, AQP8, in mouse liver. Hepatology: 2003 Oct,38(4):947-57.
[120]Masyuk AI, Marinelli RA, LaRusso NF. Water transport by epithelia of the digestive tract. Gastroenterology 2002,122:545-562.
[121]Masyuk AI, Marinelli RA, LaRusso NF. Water transport by epithelia of the digestive tract. Gastroenterology 2002,122:545-562.
[122]Huebert RC, Splinter PL, Garcy′a F, et al. Expression and localization of aquaporin water channels in rat hepatocytes. Evidence for a role in canalicular bile secretion. J Biol Chem 2002,277:22710-22717.
[123]Fabiana García, Arlinet Kierbel, M. Cecilia Larocca, et al.The Water Channel Aquaporin-8 Is Mainly Intracellular in Rat Hepatocytes, and Its Plasma Membrane Insertion Is Stimulated by Cyclic AMP. J. Biol. Chem., Vol. 276, Issue 15, 12147-12152, April 13, 2001.
[124] Sergio A. Gradilone, Fabiana García, Robert C. Huebert,et al. Glucagon induces the plasma membrane insertion of functional aquaporin-8 water channels in isolated rat hepatocytes.Hepatology 2003 ,37(6):1435 -1441.
[125] Carreras FI, Gradilone SA, Mazzone A,et al. Rat hepatocyte aquaporin-8 water channels are down-regulated in extrahepatic cholestasis. Hepatology: 2003 May,37(5):1026-33.
[126]Ferri D, Mazzone A, Liquori GE, et al.. Ontogeny, distribution, and possible functional implications of an unusual aquaporin, AQP8, in mouse liver. Hepatology: 2003 Oct,38(4):947-57.
[127]Calamita G, Ferri D, Gena P,et al. The inner mitochondrial membrane has aquaporin-8 water channels and is highly permeable to water. J Biol Chem .2005 Mar 4.
[128]Yang B, Song Y, Zhao D, Verkman AS. PHENOTYPE ANALYSIS OF AQUAPORIN-8 NULL MICE. Am J Physiol Cell Physiol 2005 Jan 12.
[129]Nihei K, Koyama Y, Tani T, et al.Immunolocalization of aquaporin-9 in rat hepatocytes and Leydig cells. Arch Histol Cytol. 2001 Feb, 64 (1): 81 -8.
[130]Nicchia GP, Frigeri A, Nico B, et al.Tissue distribution and membrane localization of aquaporin-9 water channel: evidence for sex-linked differences in liver. J Histochem Cytochem. 2001 Dec,49(12):1547-56.
[131]JenniferM. Carbrey, Daniel A. Gorelick-Feldman, David Kozono, et al.Aquaglyceroporin AQP9: Solute permeation and metabolic control of expression in liver.Physiology, 2003 March 4, 100(5): 2945–2950.
[132]Kuriyama H, Shimomura I, Kishida K, et al.Coordinated regulation of fat-specific and liver-specific glycerol channels, aquaporin adipose and aquaporin 9. Diabetes. 2002 Oct,51(10):2915-21.
[133]Nielsen, S., Smith, B. L., Christensen, E. I. et al. (1993). Distribution of the aquaporin CHIP in secretory and resorptive epithelia and capillary endothelia. Proceedings of the National Academy of Sciences of the USA 90, 7275-7279).
[134]Roberts, S. K., Yano, M., Ueno, Y., et al. (1994). Cholangiocytes express the aquaporin CHIP and transport water via a channel-mediated mechanism. Proceedings of the National Academy of Sciences of the USA 91, 13009-13013).
[135] Marinelli RA, Pham L, Agre P, LaRusso NF.Secretin promotes osmotic water transport in rat cholangiocytes by increasing aquaporin-1 water channels in plasma membrane. Evidence for a secretin-induced vesicular translocation of aquaporin-1. J Biol Chem. 1997,272(20):12984-8.
[136] Akiba T, Ota T, Fushimi K, et al.Water channel AQP1, 3, and 4 in the human peritoneum and peritoneal dialysate. Adv Perit Dial. 1997,13:3-6.
[137] Marinelli RA, Tietz PS, Pham LD, et al.Secretin induces the apical insertion of aquaporin-1 water channels in rat cholangiocytes. Am J
Physiol. 1999 J,276(1):280-6.
[138] Talbot NC, Garrett WM, Caperna TJ.Analysis of the expression of aquaporin-1 and aquaporin-9 in pig liver tissue: comparison with rat liver tissue. Cells Tissues Organs. 2003,174(3):117-28.
[139] 全竹富..抗内毒素核心糖脂抗体:防治革兰氏阴性菌感染的研究现状[J] . 国外医学·外科学分册,1995 ,22(5) :284.
[140]Elsbach P , Weiss J , Doerfler M, et al . The bactericidal/ permeability increasing protein of neutrophils is a potent antibacterial and anti-endotoxin agent in vitro and in vivo[J] . Prog Clin Biol Res ,1994 ,388 : 41.
[141] Natanson C , Hoffmea WD , Suffredini AF , et al . Selected treatment strategies for septic shock based on proposed mechanisms of pathogenesis[J] . Ann Intern Med , 1994 , 120(9):771.
[142]赵永亮. 特异抗体治疗内毒素血症. 国外医学外科学分册2002, 29(3): 132-135.
[143]Moncada S. and Higgs A. The L-arginine-nitric oxide pathway. N. Eng. J. Med. 1993, 329: 2202-2212.
[144] 冉瑞琼,吴裕灯,付华,等. 糖皮质激素对人胃癌细胞肿瘤坏死因子受体的调节[J ] . 中国免疫学杂志,1997 ,13(2) :81.
[145]王钧,孙滨,刘林英,等. 地塞米松对内毒素肺损伤绵羊血浆、肺淋巴液神经肽和皮质醇含量的影响[J] . 中国病理生理杂志,1999 ,15 (11) :1006.
[146]Haddad EB,Selmon M,Koto H,et al. Ozone induction of cytokine induced neutrophil chemoattractant and nuclear factor-kappa B in rat lung: inhibition by corticosteroids. Febs Lett,1996,379:265-268.
[147] 常青. 山莨菪碱对内毒素血症大鼠肠系膜血管内皮细胞与白细胞粘附力的影响[J ] . 中国病理生理杂志,1995 ,11(5) :471.
[148]刘庆增,王金兰.人参抗内毒素作用研究.天津医药,1998,10(4):11-15.
[149]刘媛媛,许昶,吕美德,等. 人参二醇皂甙对内毒素性休克保护作用机制的实验研究.中国药理学通报,1992,8(1):60-62.
[150] 林桦,李扬,赵雪俭,等.人参二醇皂甙对休克细胞的保护作用及其机理的实验研究.白求恩医科大学学报,1992,18(2):86-88.
[151]赵克森.休克微循环与细胞流变学变化.病理生理学报,1980,1(2):47.
[152]Bone RC, Balk RA, Cerra FB,et al. Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. Chest, 1992, 101:1028.
[153] Hinshaw L. B. and Beller B. K. Septic shock 1: physiological aspects. In Shock: The Reversible Stage of Dying (Edited by Hardaway R. M.) 1988, pp. 178–185. PSG Publishing, Littleton, MA.
[154] Traber D. L., Schlag G., Redl H., Strohmair W. and Traber L. D. Pulmonary microvascular changes during hyperdynamic sepsis in an ovine model. Circ. Shock 1987, 22: 185–193.
[155] Lefer A. M. Significance of lipid mediators in shock states. Circ.Shock 1989, 27: 3–12.
[156] Chang S. W., Feddersen C.O., Henson P. M. and Voelkel N. F. Platelet-activating factor mediates hemodynamic changes and lung injury in endotoxin-treated rats. J. Clin. Invest. 1987, 79(5): 1498-1509.
[157] Dobrowsky R. T., Voyksner R. D. and Olson N. C. Effect of SRI 63-675 on hemodynamics and blood PAF levels during porcine endotoxemia. Am. J. Physiol. 1991: 260: H1455–1465.
[158] Cannon J. G., Tompkins R. G., Gelfand J. A., et al. Circulating Interleukin-1 and tumor necrosis factor in septic shock and experimental endotoxin fever. J. Infect. Dis. 1990, 161: 79–84.
[159] Dinarello C. A. Biology of interleukin 1. FASEB J. 1988, 2: 108–115.
[160] Flohe′L. and Giertz H. Endotoxins, arachidonic acid and superoxid formation. In Perspectives on Bacterial Pathogenesis and Host Defense (Edited by Urbascheck B.) pp. 123–131. University of Chicago Press, Chicago. 1991
[161] Hack C. E., De Groot E. R., Felt-Borsma J.et al. A. Increased plasma levels of interleukin-6 in sepsis. Blood 1989, 74: 1704–1710.
[162]Howard M., Muchamel T., Andrade S. and Menon S. Interleukin 10 protects mice from lethal endotoxemia. J. Exp. Med. 1993, 177: 1205–1208.
[163]Klosterhalfen B, Ho¨rstmann-Jungemann K, Vogel P, et al. Hemodynamic variables and plasma levels of PGI2, TxA2 and IL-6 in aporcine model of recurrent endotoxemia. Circ. Shock 1991, 35: 237–244.
[164] Fritz H., Jochum M., Daswald K. H, et al. Granulocyte proteinases as mediators of unspecific proteolysis in inflammation: a review. In Proteinases in Inflammation and Tumor Invasion (Edited by Tschesche H.) Walter de Gruyter, Berlin. 1986 pp. 1–23.
[165] West R. L., Elovitz M. J. and Hardaway R. M. Blood coagulation factors activity in experimental endotoxemia. Ann. Surg. 1966, 163, 567–572.