肝醣合成酶激酶-3 (Glycogen synthase kinase-3，GSK-3)為肝醣代謝過程的重要酵素，可影響細胞分裂、凋亡及胚胎發育期。最近的研究發現在脂多醣(LPS)引發的敗血症模式中，GSK-3b抑制劑的治療不但可抑制促發炎細胞激素的分泌及增加抗發炎細胞激素的濃度，同時也具有改善受感染動物死亡率的效果。不過在較複雜且更貼近臨床的活菌感染模式中，尚不甚了解是否GSK-3b抑制劑的治療也具有同樣的功效。因此在本研究中，我們要探討在A群鏈球菌感染模式中，GSK-3b的抑制是否具保護的效果。我們首先證實在A群鏈球菌感染下的小鼠巨噬細胞株RAW 264.7，其GSK-3b及下游的NF-kB皆產生活化，而GSK-3b活化的機制是透過抑制Akt相關的信息傳導路徑。而透過GSK-3b抑制下，可抑制感染的小鼠巨噬細胞內NF-kB的活化及可誘導型一氧化氮合成脢(iNOS)的合成表現。在A群鏈球菌感染下，小鼠巨噬細胞可以表現高量的細胞激素的產生，在GSK-3b抑制後，腫瘤壞死因子-alpha (TNF-alpha，六小時)、介白素-6 (IL-6，六及十二小時)及趨化激素RANTES的表現量皆有下降的情形。相反的，GSK-3b抑制可提高感染後抗發炎激素介白素-10 (IL-10)的上升。此外也發現一氧化氮(NO)合成下降以及改善細胞的存活率。在動物的感染模式中，我們更證明了透過鋰鹽抑制GSK-3b的活性下，可使血清中TNF-alpha有意義的下降，及使局部組織中TNF-alpha及血清中IL-6呈現下降趨勢。在觀察小鼠存活率方面，單純給予鋰鹽只能提供些微保護效果。因此整體來說，GSK-3b抑制能夠有效的減低A群鏈球菌所引發的發炎反應，但單純提供GSK-3b抑制劑只能部分改善A群鏈球菌動物模式下的存活率。

Glycogen synthase kinase-3 (GSK-3), a key enzyme in glycogen metabolism, is involved in many intracellular functions, such as cell division, apoptosis, or even cell fates during embryonic development. In recent studies of LPS-induced sepsis model, GSK-3b inhibitors can not only suppress the production of pro-inflammatory cytokine but also increase anti-inflammatory cytokine. Besides, they also provide survival advantage in animal sepsis model induced by LPS. But few studies explore the possible role of GSK-3b inhibitors in sepsis induced by bacteria. In the present study, we investigated the effect of GSK-3b inhibition in group A streptococcus (GAS) infection. We first identified that GAS infection could induce the activation of GSK-3band subsequent NF-kB in mouse macrophage cell line RAW 264.7. This was mediated through inhibition of Akt. GSK-3b inhibition blocked NF-kB activity and abolished the expression of iNOS in GAS-infected RAW 264.7 cells. Stimulation with GAS induced pronounced inflammatory cytokine and chemokine production. The levels of TNF-alpha (6 hr), IL-6 (6 and 12 hr), and RANTES were suppressed after GSK-3b inhibition, whereas the production of IL-10, an anti-inflammatory cytokine, was increased. Besides, NO production could also be regulated by GSK-3b and cell viability was improved under GSK-3b inhibition. We further proved that GSK-3 inhibitor, lithium, decreased the level of TNF-alpha in serum and tended to lower TNF-alpha in local area and IL-6 in serum in GAS-infected mice. Mouse survival rate was mildly improved after lithium treatment. These findings demonstrate that GSK-3b inhibition can ameliorate multiple inflammatory responses in GAS-induced sepsis but provide partial benefit in improving mortality.

1.Cunningham MW. Pathogenesis of group A streptococcal infections. Clin Microbiol Rev 2000;13:470-511.
2.Carapetis JR, Steer AC, Mulholland EK, Weber M. The global burden of group A streptococcal diseases. Lancet Infect Dis 2005;5:685-94.
3.Wessels MR, Moses AE, Goldberg JB, DiCesare TJ. Hyaluronic acid capsule is a virulence factor for mucoid group A streptococci. Proc Natl Acad Sci U S A 1991;88:8317-21.
4.Fischetti VA. Streptococcal M protein. Sci Am 1991;264:58-65.
5.Berggard K, Johnsson E, Morfeldt E, Persson J, Stalhammar-Carlemalm M, Lindahl G. Binding of human C4BP to the hypervariable region of M protein: a molecular mechanism of phagocytosis resistance in Streptococcus pyogenes. Mol Microbiol 2001;42:539-51.
6.Jenkins HT, Mark L, Ball G, et al. Human C4b-binding protein, structural basis for interaction with streptococcal M protein, a major bacterial virulence factor. J Biol Chem 2006;281:3690-7.
7.Ji Y, McLandsborough L, Kondagunta A, Cleary PP. C5a peptidase alters clearance and trafficking of group A streptococci by infected mice. Infect Immun 1996;64:503-10.
8.Lottenberg R, Broder CC, Boyle MD. Identification of a specific receptor for plasmin on a group A streptococcus. Infect Immun 1987;55:1914-8.
9.Tsai WH, Chang CW, Chuang WJ, et al. Streptococcal pyrogenic exotoxin B-induced apoptosis in a549 cells is mediated by a receptor- and mitochondrion-dependent pathway. Infect Immun 2004;72:7055-62.
10.Kuo CF, Wu JJ, Tsai PJ, et al. Streptococcal pyrogenic exotoxin B induces apoptosis and reduces phagocytic activity in U937 cells. Infect Immun 1999;67:126-30.
11.Tsai WH, Chang CW, Lin YS, et al. Streptococcal pyrogenic exotoxin B-induced apoptosis in A549 cells is mediated through alpha(v)beta(3) integrin and Fas. Infect Immun 2008;76:1349-57.
12.Stevens DL. Invasive group A streptococcus infections. Clin Infect Dis 1992;14:2-11.
13.Norrby-Teglund A, Chatellier S, Low DE, McGeer A, Green K, Kotb M. Host variation in cytokine responses to superantigens determine the severity of invasive group A streptococcal infection. Eur J Immunol 2000;30:3247-55.
14.Norrby-Teglund A, Pauksens K, Norgren M, Holm SE. Correlation between serum TNF alpha and IL6 levels and severity of group A streptococcal infections. Scand J Infect Dis 1995;27:125-30.
15.Mollick JA, Chintagumpala M, Cook RG, Rich RR. Staphylococcal exotoxin activation of T cells. Role of exotoxin-MHC class II binding affinity and class II isotype. J Immunol 1991;146:463-8.
16.Kapur V, Majesky MW, Li LL, Black RA, Musser JM. Cleavage of interleukin 1 beta (IL-1 beta) precursor to produce active IL-1 beta by a conserved extracellular cysteine protease from Streptococcus pyogenes. Proc Natl Acad Sci U S A 1993;90:7676-80.
17.Muller-Alouf H, Alouf JE, Gerlach D, Ozegowski JH, Fitting C, Cavaillon JM. Comparative study of cytokine release by human peripheral blood mononuclear cells stimulated with Streptococcus pyogenes superantigenic erythrogenic toxins, heat-killed streptococci, and lipopolysaccharide. Infect Immun 1994;62:4915-21.
18.Hackett SP, Stevens DL. Superantigens associated with staphylococcal and streptococcal toxic shock syndrome are potent inducers of tumor necrosis factor-beta synthesis. J Infect Dis 1993;168:232-5.
19.Embi N, Rylatt DB, Cohen P. Glycogen synthase kinase-3 from rabbit skeletal muscle. Separation from cyclic-AMP-dependent protein kinase and phosphorylase kinase. Eur J Biochem 1980;107:519-27.
20.Dugo L, Collin M, Thiemermann C. Glycogen synthase kinase 3beta as a target for the therapy of shock and inflammation. Shock 2007;27:113-23.
21.Woodgett JR. Molecular cloning and expression of glycogen synthase kinase-3/factor A. EMBO J 1990;9:2431-8.
22.Plyte SE, Hughes K, Nikolakaki E, Pulverer BJ, Woodgett JR. Glycogen synthase kinase-3: functions in oncogenesis and development. Biochim Biophys Acta 1992;1114:147-62.
23.Eldar-Finkelman H, Seger R, Vandenheede JR, Krebs EG. Inactivation of glycogen synthase kinase-3 by epidermal growth factor is mediated by mitogen-activated protein kinase/p90 ribosomal protein S6 kinase signaling pathway in NIH/3T3 cells. J Biol Chem 1995;270:987-90.
24.Stambolic V, Woodgett JR. Mitogen inactivation of glycogen synthase kinase-3 beta in intact cells via serine 9 phosphorylation. Biochem J 1994;303 ( Pt 3):701-4.
25.Sutherland C, Leighton IA, Cohen P. Inactivation of glycogen synthase kinase-3 beta by phosphorylation: new kinase connections in insulin and growth-factor signalling. Biochem J 1993;296 ( Pt 1):15-9.
26.Hoeflich KP, Luo J, Rubie EA, Tsao MS, Jin O, Woodgett JR. Requirement for glycogen synthase kinase-3beta in cell survival and NF-kappaB activation. Nature 2000;406:86-90.
27.Beg AA, Sha WC, Bronson RT, Ghosh S, Baltimore D. Embryonic lethality and liver degeneration in mice lacking the RelA component of NF-kappa B. Nature 1995;376:167-70.
28.Li Q, Van Antwerp D, Mercurio F, Lee KF, Verma IM. Severe liver degeneration in mice lacking the IkappaB kinase 2 gene. Science 1999;284:321-5.
29.Schwabe RF, Brenner DA. Role of glycogen synthase kinase-3 in TNF-alpha-induced NF-kappaB activation and apoptosis in hepatocytes. Am J Physiol Gastrointest Liver Physiol 2002;283:G204-11.
30.Takada Y, Fang X, Jamaluddin MS, Boyd DD, Aggarwal BB. Genetic deletion of glycogen synthase kinase-3beta abrogates activation of IkappaBalpha kinase, JNK, Akt, and p44/p42 MAPK but potentiates apoptosis induced by tumor necrosis factor. J Biol Chem 2004;279:39541-54.
31.Sanchez JF, Sniderhan LF, Williamson AL, Fan S, Chakraborty-Sett S, Maggirwar SB. Glycogen synthase kinase 3beta-mediated apoptosis of primary cortical astrocytes involves inhibition of nuclear factor kappaB signaling. Mol Cell Biol 2003;23:4649-62.
32.Martin M, Rehani K, Jope RS, Michalek SM. Toll-like receptor-mediated cytokine production is differentially regulated by glycogen synthase kinase 3. Nat Immunol 2005;6:777-84.
33.Gratz N, Siller M, Schaljo B, et al. Group A streptococcus activates type I interferon production and MyD88-dependent signaling without involvement of TLR2, TLR4, and TLR9. J Biol Chem 2008;283:19879-87.
34.Loof TG, Goldmann O, Medina E. Immune recognition of Streptococcus pyogenes by dendritic cells. Infect Immun 2008;76:2785-92.
35.Ojaniemi M, Glumoff V, Harju K, Liljeroos M, Vuori K, Hallman M. Phosphatidylinositol 3-kinase is involved in Toll-like receptor 4-mediated cytokine expression in mouse macrophages. Eur J Immunol 2003;33:597-605.
36.Gelman AE, LaRosa DF, Zhang J, et al. The adaptor molecule MyD88 activates PI-3 kinase signaling in CD4+ T cells and enables CpG oligodeoxynucleotide-mediated costimulation. Immunity 2006;25:783-93.
37.Dugo L, Collin M, Allen DA, et al. GSK-3beta inhibitors attenuate the organ injury/dysfunction caused by endotoxemia in the rat. Crit Care Med 2005;33:1903-12.
38.Wang JE, Dahle MK, Yndestad A, et al. Peptidoglycan of Staphylococcus aureus causes inflammation and organ injury in the rat. Crit Care Med 2004;32:546-52.
39.Wray GM, Foster SJ, Hinds CJ, Thiemermann C. A cell wall component from pathogenic and non-pathogenic gram-positive bacteria (peptidoglycan) synergises with endotoxin to cause the release of tumour necrosis factor-alpha, nitric oxide production, shock, and multiple organ injury/dysfunction in the rat. Shock 2001;15:135-42.
40.Kuo CF, Wu JJ, Lin KY, et al. Role of streptococcal pyrogenic exotoxin B in the mouse model of group A streptococcal infection. Infect Immun 1998;66:3931-5.
41.Kuo CF, Luo YH, Lin HY, et al. Histopathologic changes in kidney and liver correlate with streptococcal pyrogenic exotoxin B production in the mouse model of group A streptococcal infection. Microb Pathog 2004;36:273-85
42.Rodionova E, Conzelmann M, Maraskovsky E, et al. GSK-3 mediates differentiation and activation of proinflammatory dendritic cells. Blood 2007;109:1584-92.
43.Kuure S, Popsueva A, Jakobson M, Sainio K, Sariola H. Glycogen synthase kinase-3 inactivation and stabilization of beta-catenin induce nephron differentiation in isolated mouse and rat kidney mesenchymes. J Am Soc Nephrol 2007;18:1130-9.
44.Timmer AM, Timmer JC, Pence MA, et al. Streptolysin O promotes group A Streptococcus immune evasion by accelerated macrophage apoptosis. J Biol Chem 2009;284:862-71.
45.Goldmann O, Rohde M, Chhatwal GS, Medina E. Role of macrophages in host resistance to group A streptococci. Infect Immun 2004;72:2956-63.
46.Ghosh S, May MJ, Kopp EB. NF-kappa B and Rel proteins: evolutionarily conserved mediators of immune responses. Annu Rev Immunol 1998;16:225-60.
47.Bonizzi G, Karin M. The two NF-kappaB activation pathways and their role in innate and adaptive immunity. Trends Immunol 2004;25:280-8.
48.Tsai PJ, Chen YH, Hsueh CH, et al. Streptococcus pyogenes induces epithelial inflammatory responses through NF-kappaB/MAPK signaling pathways. Microbes Infect 2006;8:1440-9.
49.Cross DA, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 1995;378:785-9.
50.Fernandes D, Assreuy J. Nitric oxide and vascular reactivity in sepsis. Shock 2008;30 Suppl 1:10-3.
51.Christ EA, Meals E, English BK. Streptococcal pyrogenic exotoxins A (SpeA) and C (SpeC) stimulate the production of inducible nitric oxide synthase (iNOS) protein in RAW 264.7 macrophages. Shock 1997;8:450-3.
52.Goldmann O, Chhatwal GS, Medina E. Immune mechanisms underlying host susceptibility to infection with group A streptococci. J Infect Dis 2003;187:854-61.
53.Cohen P, Frame S. The renaissance of GSK3. Nat Rev Mol Cell Biol 2001;2:769-76.
54.Dugo L, Collin M, Cuzzocrea S, Thiemermann C. 15d-prostaglandin J2 reduces multiple organ failure caused by wall-fragment of Gram-positive and Gram-negative bacteria. Eur J Pharmacol 2004;498:295-301.
55.Dugo L, Abdelrahman M, Murch O, Mazzon E, Cuzzocrea S, Thiemermann C. Glycogen synthase kinase-3beta inhibitors protect against the organ injury and dysfunction caused by hemorrhage and resuscitation. Shock 2006;25:485-91.
56.Zhang P, Katz J, Michalek SM. Glycogen synthase kinase-3beta (GSK3beta) inhibition suppresses the inflammatory response to Francisella infection and protects against tularemia in mice. Mol Immunol 2009;46:677-87.
57.Li Q, Verma IM. NF-kappaB regulation in the immune system. Nat Rev Immunol 2002;2:725-34.
58.Baeuerle PA, Baichwal VR. NF-kappa B as a frequent target for immunosuppressive and anti-inflammatory molecules. Adv Immunol 1997;65:111-37.
59.Tak PP, Firestein GS. NF-kappaB: a key role in inflammatory diseases. J Clin Invest 2001;107:7-11.
60.Obligado SH, Ibraghimov-Beskrovnaya O, Zuk A, Meijer L, Nelson PJ. CDK/GSK-3 inhibitors as therapeutic agents for parenchymal renal diseases. Kidney Int 2008;73:684-90.
61.Doble BW, Woodgett JR. GSK-3: tricks of the trade for a multi-tasking kinase. J Cell Sci 2003;116:1175-86.
62.Cross DA, Culbert AA, Chalmers KA, Facci L, Skaper SD, Reith AD. Selective small-molecule inhibitors of glycogen synthase kinase-3 activity protect primary neurones from death. J Neurochem 2001;77:94-102.
63.Meijer L, Skaltsounis AL, Magiatis P, et al. GSK-3-selective inhibitors derived from Tyrian purple indirubins. Chem Biol 2003;10:1255-66.
64.Stambolic V, Ruel L, Woodgett JR. Lithium inhibits glycogen synthase kinase-3 activity and mimics wingless signalling in intact cells. Curr Biol 1996;6:1664-8.
65.Russell JA. Management of sepsis. N Engl J Med 2006;355:1699-713.
66.Abraham E, Laterre PF, Garbino J, et al. Lenercept (p55 tumor necrosis factor receptor fusion protein) in severe sepsis and early septic shock: a randomized, double-blind, placebo-controlled, multicenter phase III trial with 1,342 patients. Crit Care Med 2001;29:503-10.
67.Abraham E, Wunderink R, Silverman H, et al. Efficacy and safety of monoclonal antibody to human tumor necrosis factor alpha in patients with sepsis syndrome. A randomized, controlled, double-blind, multicenter clinical trial. TNF-alpha MAb Sepsis Study Group. JAMA 1995;273:934-41.
68.Fisher CJ, Jr., Dhainaut JF, Opal SM, et al. Recombinant human interleukin 1 receptor antagonist in the treatment of patients with sepsis syndrome. Results from a randomized, double-blind, placebo-controlled trial. Phase III rhIL-1ra Sepsis Syndrome Study Group. JAMA 1994;271:1836-43.
69.Huang WC, Lin YS, Wang CY, et al. Glycogen synthase kinase-3 negatively regulates anti-inflammatory interleukin-10 for lipopolysaccharide-induced iNOS/NO biosynthesis and RANTES production in microglial cells. Immunology 2008.
70.Beurel E, Jope RS. Glycogen synthase kinase-3 promotes the synergistic action of interferon-gamma on lipopolysaccharide-induced IL-6 production in RAW264.7 cells. Cell Signal 2009;21:978-85.
71.Lin CF, Tsai CC, Huang WC, et al. IFN-gamma synergizes with LPS to induce nitric oxide biosynthesis through glycogen synthase kinase-3-inhibited IL-10. J Cell Biochem 2008;105:746-55.
72.Chan MM, Cheung BK, Li JC, Chan LL, Lau AS. A role for glycogen synthase kinase-3 in antagonizing mycobacterial immune evasion by negatively regulating IL-10 induction. J Leukoc Biol 2009.
73.Watcharasit P, Bijur GN, Zmijewski JW, et al. Direct, activating interaction between glycogen synthase kinase-3beta and p53 after DNA damage. Proc Natl Acad Sci U S A 2002;99:7951-5.
74.Loberg RD, Vesely E, Brosius FC, 3rd. Enhanced glycogen synthase kinase-3beta activity mediates hypoxia-induced apoptosis of vascular smooth muscle cells and is prevented by glucose transport and metabolism. J Biol Chem 2002;277:41667-73.
75.Bijur GN, De Sarno P, Jope RS. Glycogen synthase kinase-3beta facilitates staurosporine- and heat shock-induced apoptosis. Protection by lithium. J Biol Chem 2000;275:7583-90.
76.Forde JE, Dale TC. Glycogen synthase kinase 3: a key regulator of cellular fate. Cell Mol Life Sci 2007;64:1930-44.
77.Stevens DL, Bryant AE, Hackett SP, et al. Group A streptococcal bacteremia: the role of tumor necrosis factor in shock and organ failure. J Infect Dis 1996;173:619-26.