红芪多糖干预内毒素诱导的葡萄膜炎模型中糖原合成酶3-β的表达及其作用机制
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摘要
目的通过观察红芪多糖(radix hedysari polysaccharide,HPS)和氯化锂(Li Cl)对霍乱弧菌内毒素(lipopolysaccharide,LPS)诱导的葡萄膜炎的抗炎作用,探讨糖原合成酶3-β(glycogen synthase kinase 3-β,GSK3-β)在葡萄膜炎中的作用机制。方法200只Wistar大鼠随机分为4组(n=50):空白对照组(negative control,NC)组、LPS诱导的葡萄膜炎组(LPS组)、HPS治疗组(LPS+HPS组)和Li Cl治疗组(LPS+Li Cl组)。LPS+HPS组腹腔注射400 mg·kg~(-1)HPS,LPS+Li Cl组腹腔注射0. 5 mol·L~(-1)的Li Cl 100μL,对照组和LPS组注射等量PBS。2 h后,LPS组、LPS+HPS组和LPS+Li Cl组每只大鼠足底注射0. 1 mL LPS注射液,NC组注射等体积的PBS。应用临床评分、裂隙灯照相、HE染色等检查评价炎性反应程度; Western blot和RT-PCR检测虹膜睫状体GSK3-β和核因子-κB(neuclear factor-κB,NF-κB) p65表达水平;酶联免疫吸附试验(enzyme-linked immunosorbent as-say,ELISA)检测大鼠前房房水中肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、白细胞介素6(interleukin-6,IL-6)、白细胞介素10(interleukin-10,IL-10)、白细胞介素1β(interleukin-1β,IL-1β)等细胞因子的水平。结果 LPS注射后6 h、12 h、24 h、48 hLPS组的炎症评分分别为(2. 3±0. 2)分、(3. 6±0. 7)分、(3. 9±0. 3)分、(3. 2±0. 4)分,显著高于其他三组(均为P <0. 05),而LPS+HPS组、HPS+Li Cl组和NC组之间差异均无统计学意义(均为P> 0. 05),HPS或Li Cl预处理对LPS诱导的大鼠葡萄膜炎产生了抗炎效果。经过HPS或Li Cl处理,LPS诱导的葡萄膜炎大鼠虹膜睫状体磷酸化GSK3-β水平上调,NF-κB p65表达明显被抑制,前房房水中抗炎因子IL-10水平上调,而TNF-α、IL-6、IL-1β等炎症细胞因子受到抑制。结论 HPS或Li Cl预处理可以抑制LPS诱导的大鼠葡萄膜炎炎性反应,这一抗炎作用与GSK3-β的抑制性磷酸化密切相关。
Objective To evaluate the effect of hedysari polysaccharide (HPS)and lithium chloride (LiCl, a selective glycogen synthase kinase 3-β inhibitor) in endotoxin-induced uveitis (EIU) and to explore this anti-inflammatory mechanism. Methods A total number of 200 Wistar rats were randomly divided into four groups: normal control (NC) group,lipopolysaccharide (LPS)-induced uveitis group (LPS group),HPS-treated group(LPS + HPS group) and LiCl-treated LPS (LPS + LiCl) group. Two hours before LPS injection, LPS + HPS group received intraperitoneal injection of 400 mg· kg~(-1) HPS and LPS + LiCl group received intraperitoneal injection of 100 μL LiCl of 0. 5 mol · L~(-1). LPS group and NC group both was intraperitoneally injected with 100μL saline solution only. After 2 h,the three groups received intraperitoneally injection of 200 μg of LPS in 1 00 μL of sterile saline simultaneously. NC group was intraperitoneally injected with 100 μL saline solution only. Clinical scores slit-lamp photography,hematoxylin and eosin staining were used to determine the degree of inflammatory reaction. The protein levels of GSK3-β and nuclear factor-kappa B (NF-κB) in iris-cilliary body were examined by Western blot. The mRNA expression of GSK3-β and NF-κB was examined by real-time PCR (RT-PCR). Tumor necrosis factor-α (TNF-α), interleukin-10(IL-10), interleukin-6 (IL-6) and interleukin-1 β (IL-1β) in aqueous humor were detected by enzyme-linked immunosorbent assay(ELISA). Results The inflammatory scores of LPS group at 6 h, 1 2 h,24 h and 48 h after LPS injection were 2. 3 ±0.2,3.6 ±0.7,3.9 ±0. 3 and 3.2 ± 0. 4,respectively,which were significantly higher than those in the other three groups (all P < 0. 05). However, there was no significant difference in LPS +LiCl, LPS+HPS and NC groups (all P > 0. 05). Pretreatment with HPS or LiCl both produced an anti-inflammatory effect during endotoxin-induced uveitis. With HPS or LiCl treatment,the level of P-GSK3-β in irisciliary body was upregulated and the expression of NF-κB p65 was significantly suppressed. Also,HPS or LiCl treatment suppressed the production of pro-inflammatory cytokine TNF-α, IL-1β and IL-6, while enhanced the production of anti-inflammatory cytokine IL-10 in the aqueous humor endotoxin-induced uveitis. Conclusion HPS or LiCl pretreatment can suppress intraocular inflammatory responses in rats with endotoxin-induced uveitis. Mechanistically, this anti-inflammatory effect may be related to the inhibitory phosphorylation of GSK3-β.
Objective To evaluate the effect of hedysari polysaccharide (HPS)and lithium chloride (LiCl, a selective glycogen synthase kinase 3-β inhibitor) in endotoxin-induced uveitis (EIU) and to explore this anti-inflammatory mechanism. Methods A total number of 200 Wistar rats were randomly divided into four groups: normal control (NC) group,lipopolysaccharide (LPS)-induced uveitis group (LPS group),HPS-treated group(LPS + HPS group) and LiCl-treated LPS (LPS + LiCl) group. Two hours before LPS injection, LPS + HPS group received intraperitoneal injection of 400 mg· kg~(-1) HPS and LPS + LiCl group received intraperitoneal injection of 100 μL LiCl of 0. 5 mol · L~(-1). LPS group and NC group both was intraperitoneally injected with 100μL saline solution only. After 2 h,the three groups received intraperitoneally injection of 200 μg of LPS in 1 00 μL of sterile saline simultaneously. NC group was intraperitoneally injected with 100 μL saline solution only. Clinical scores slit-lamp photography,hematoxylin and eosin staining were used to determine the degree of inflammatory reaction. The protein levels of GSK3-β and nuclear factor-kappa B (NF-κB) in iris-cilliary body were examined by Western blot. The mRNA expression of GSK3-β and NF-κB was examined by real-time PCR (RT-PCR). Tumor necrosis factor-α (TNF-α), interleukin-10(IL-10), interleukin-6 (IL-6) and interleukin-1 β (IL-1β) in aqueous humor were detected by enzyme-linked immunosorbent assay(ELISA). Results The inflammatory scores of LPS group at 6 h, 1 2 h,24 h and 48 h after LPS injection were 2. 3 ±0.2,3.6 ±0.7,3.9 ±0. 3 and 3.2 ± 0. 4,respectively,which were significantly higher than those in the other three groups (all P < 0. 05). However, there was no significant difference in LPS +LiCl, LPS+HPS and NC groups (all P > 0. 05). Pretreatment with HPS or LiCl both produced an anti-inflammatory effect during endotoxin-induced uveitis. With HPS or LiCl treatment,the level of P-GSK3-β in irisciliary body was upregulated and the expression of NF-κB p65 was significantly suppressed. Also,HPS or LiCl treatment suppressed the production of pro-inflammatory cytokine TNF-α, IL-1β and IL-6, while enhanced the production of anti-inflammatory cytokine IL-10 in the aqueous humor endotoxin-induced uveitis. Conclusion HPS or LiCl pretreatment can suppress intraocular inflammatory responses in rats with endotoxin-induced uveitis. Mechanistically, this anti-inflammatory effect may be related to the inhibitory phosphorylation of GSK3-β.
引文
[1]RATHINAM S R,NAMPERUMALSAMY P.Global variation and pattern changes in epidemiology of uveitis[J].Indian J Ophthalmol,2007,55(3):173-183.
[2]LU H.Studies on tissue section of uveitis induced by endotoxin extracted from cholera vibrio in Wistar rats[J].Rec Adv Ophthalmol,2002,22(6):384-386.卢弘.霍乱弧菌内毒素诱导大鼠葡萄膜炎的组织切片研究[J].眼科新进展,2002,22(6):384-386.
[3]MEDZHITOV R,PRESTON-HURLBURT P,JANEWAY CA.A human homologue of the Drosophila Toll protein signals activation of adaptive immunity[J].Nature,1997,388(6640):394-397.
[4]MEDZHITOV R,PRESTON-HURLBURT P,KOPP E.My D88 is an adaptor protein in the hToll/IL-1 receptor family signaling pathways[J].Mol Cell,1982,2(2):253-258.
[5]YANG RB,MARK MR,GRAY A.Toll-like receptor-2 mediates lipopolysaccharide-induced cellular signalling[J].Nature,1998,395(6699):284-288.
[6]LU H,CHEN W,ZHAO L.The expression of Toll like receiptor 4 in endotoxin-induced uveitis[J].Ophthalmol CHN,2008,17(1):48-51.卢弘,陈巍,赵丽.Toll样受体4在内毒素诱导的葡萄膜炎中的表达及意义[J].眼科,2008,17(1):48-51.
[7]DUGO L,ABDELRAHMAN M,MURCH O.Glycogen synthase kinase-3beta inhibitors protect against the organ injury and dysfunction caused by hemorrhage and resuscitation[J].Shock,2006,25(5):485-491.
[8]MARTIN M,REHANI K,JOPE R S.Toll-like receptor mediated cytokine production is differentially regulated by glycogen synthase kinase 3[J].Nat Immunol,2005,6(8):777-784.
[9]KLEIN P S,MELTON D A.A molecular mechanism for the effect of lithium on development[J].Proc Natl Acad Sci USA,1996,93(16):8455-8459.
[10]DAVIES S P,REDDY H,CAIVANO M.Specificity and mechanism of action of some commonly used protein kinase inhibitors[J].Biochem J,2000,351(1):95-105.
[11]BALLANGER F,TENAUD I,VOLTEAU C.Anti-inflammatory effects of lithium gluconate on keratinocytes:a possible explanation for efficiency in seborrhoeic dermatitis[J].Arch Dermatol Res,2008,300(5):215-223.
[12]NAHMAN S,BELMAKER R H,AZAB A N.Effects of lithium on lipopolysaccharide-induced inflammation in rat primary glia cells[J].Innate Immun,2012,18(3):447-458.
[13]HOFMANN C,DUNGER N,SCHOLMERICH J.Glycogen synthase kinase 3-beta:a master regulator of toll-like receptor-mediated chronic intestinal inflammation[J].Inflamm Bowel Dis,2010,16(11):1850-1858.
[14]ZHANG P,KATZ J,MICHALEK S M.Glycogen synthase kinase-3beta(GSK3-β)inhibition suppresses the inflammatory response to Francisella infection and protects against tularemia in mice[J].Mol Immunol,2009,46(4):677-687.
[15]ZHANG Q,LEI L S,WU S G.Receptor and signal transduction mechanism involved in biological effects of polysaccharides[J].Chin Trad Herbal Drugs,2005,36(4):614-616.张群,雷林生,吴曙光.多糖类药物作用的受体及信号转导机制的研究进展[J].中草药,2005,36(4):614-616.
[16]QIU Y G,SHI P K,ZHU P,YANG H X,VERMA A,LEI B,LI Q.Angio tensin-converting enzyme 2(ACE2)activator diminazene aceturate ameliorates endotoxin-induced uveitis in mice[J].IOVS,2014,55(6):3816.
[17]ZENG G X,LIU J Y,XIONG J R,LIAO Y H,DAI L H,LI X J,et al.Study on effect of Astragalus polysaccharide for traumatic stress mice cell immunity[J].Chin J Microbiol Immunol,2004,24(12):942-945.
[18]HU F,LI X,ZHAO L,FENG S,WANG C.Antidiabetic properties of purified polysaccharide from Hedysarum polybotrys[J].Can JPhysiol Pharmacol,2010,88(1):64-72.
[19]YU S,ZHANG W,YU J Y,FENG S L,GUO T K,LU H.Inhibiting effect of Radix Hedysari Polysaccharide(HPS)onendotoxin-induceduveitis in rats[J].Internat Immunopharmacol,2014,21:361-368.
[20]LAJAVARDI L,BOCHOT A,CAMELO S.Downregulation of endotoxin-induced uveitis by intravitreal injection of vasoactive intestinal peptide encapsulated in liposomes[J].Invest Ophthalmol Vis Sci,2007,48(7):3230-3238.
[21]BHATTACHERJEE P,WILLIAMS R N,EAKINS K E.An evaluation of ocular inflammation following the injection of bacterial endotoxin ito the rat foot pad[J].Invest Ophthalmol Vis Sci,1983,24(2):196-202.
[22]HU X,PAIK P K,CHEN J.IFN-gamma suppresses IL-10 production and synergizes with TLR2 by regulating GSK3 and CREB/AP-1 proteins[J].Immunity,2006,24(5):563-574.
[23]DOBLE B W,WOODGETT J R.GSK3:tricks of the trade for a multi-tasking kinase[J].J Cell Sci,2003,116(7):1175-1186.
[24]BEUREL E,MICHALEK S M,JOPE R S.Innate and adaptive immune responses regulated by glycogen synthase kinase-3(GSK3)[J].Trends Immunol,2010,31(1):24-31.
[25]CAVAILLON J M,ADIB-CONQUY M.Bench-to-bedside review:endotoxin tolerance as a model of leukocyte reprogramming in sepsis[J].Crit Care,2006,10(5):233.
[26]DE SARNO P,AXTELL R C,RAMAN C.Lithium prevents and ameliorates experimental autoimmune encephalomyelitis[J].J Immunol,2008,181(1):338-345.
[27]OHTANI M,NAGAI S,KONDO S.Mammalian target of rapamycin and glycogen synthase kinase 3 differentially regulate lipopolysaccharide-induced interleukin-12 production in dendritic cells[J].Blood,2008,112(3):635-643.
[28]CHEN K,WU Y,ZHU M.Lithium chloride promotes host resistance against Pseudomonas aeruginosa keratitis[J].Mol Vis,2013,19:1502-1514.
[29]LIU Y,ZHANG Q Y,ZHAO Y Y.Saponins from the roots of Hedysarum polybotrys[J].Biochem Systemat Ecol,2007,35:389-391.
[30]YANG M,CHAN G C,DENG R,NG M H,CHENG S W,LAU CP,ET A L.An herbal decoction of Radix astragali and Radix angelicae sinensis promotes hematopoiesis and thrombopoiesis[J].JEthnopharmacol,2009,124(1):87-97.
[31]LAN Z F,ZHANG Z L,CHENG G Q,WANG F L,XI S F.Effects of radix hedysari polysaccharides on immunological function and transplanted tumors in mice[J].Acta Pharmacol Sin,1987,8(3):275-277.
[2]LU H.Studies on tissue section of uveitis induced by endotoxin extracted from cholera vibrio in Wistar rats[J].Rec Adv Ophthalmol,2002,22(6):384-386.卢弘.霍乱弧菌内毒素诱导大鼠葡萄膜炎的组织切片研究[J].眼科新进展,2002,22(6):384-386.
[3]MEDZHITOV R,PRESTON-HURLBURT P,JANEWAY CA.A human homologue of the Drosophila Toll protein signals activation of adaptive immunity[J].Nature,1997,388(6640):394-397.
[4]MEDZHITOV R,PRESTON-HURLBURT P,KOPP E.My D88 is an adaptor protein in the hToll/IL-1 receptor family signaling pathways[J].Mol Cell,1982,2(2):253-258.
[5]YANG RB,MARK MR,GRAY A.Toll-like receptor-2 mediates lipopolysaccharide-induced cellular signalling[J].Nature,1998,395(6699):284-288.
[6]LU H,CHEN W,ZHAO L.The expression of Toll like receiptor 4 in endotoxin-induced uveitis[J].Ophthalmol CHN,2008,17(1):48-51.卢弘,陈巍,赵丽.Toll样受体4在内毒素诱导的葡萄膜炎中的表达及意义[J].眼科,2008,17(1):48-51.
[7]DUGO L,ABDELRAHMAN M,MURCH O.Glycogen synthase kinase-3beta inhibitors protect against the organ injury and dysfunction caused by hemorrhage and resuscitation[J].Shock,2006,25(5):485-491.
[8]MARTIN M,REHANI K,JOPE R S.Toll-like receptor mediated cytokine production is differentially regulated by glycogen synthase kinase 3[J].Nat Immunol,2005,6(8):777-784.
[9]KLEIN P S,MELTON D A.A molecular mechanism for the effect of lithium on development[J].Proc Natl Acad Sci USA,1996,93(16):8455-8459.
[10]DAVIES S P,REDDY H,CAIVANO M.Specificity and mechanism of action of some commonly used protein kinase inhibitors[J].Biochem J,2000,351(1):95-105.
[11]BALLANGER F,TENAUD I,VOLTEAU C.Anti-inflammatory effects of lithium gluconate on keratinocytes:a possible explanation for efficiency in seborrhoeic dermatitis[J].Arch Dermatol Res,2008,300(5):215-223.
[12]NAHMAN S,BELMAKER R H,AZAB A N.Effects of lithium on lipopolysaccharide-induced inflammation in rat primary glia cells[J].Innate Immun,2012,18(3):447-458.
[13]HOFMANN C,DUNGER N,SCHOLMERICH J.Glycogen synthase kinase 3-beta:a master regulator of toll-like receptor-mediated chronic intestinal inflammation[J].Inflamm Bowel Dis,2010,16(11):1850-1858.
[14]ZHANG P,KATZ J,MICHALEK S M.Glycogen synthase kinase-3beta(GSK3-β)inhibition suppresses the inflammatory response to Francisella infection and protects against tularemia in mice[J].Mol Immunol,2009,46(4):677-687.
[15]ZHANG Q,LEI L S,WU S G.Receptor and signal transduction mechanism involved in biological effects of polysaccharides[J].Chin Trad Herbal Drugs,2005,36(4):614-616.张群,雷林生,吴曙光.多糖类药物作用的受体及信号转导机制的研究进展[J].中草药,2005,36(4):614-616.
[16]QIU Y G,SHI P K,ZHU P,YANG H X,VERMA A,LEI B,LI Q.Angio tensin-converting enzyme 2(ACE2)activator diminazene aceturate ameliorates endotoxin-induced uveitis in mice[J].IOVS,2014,55(6):3816.
[17]ZENG G X,LIU J Y,XIONG J R,LIAO Y H,DAI L H,LI X J,et al.Study on effect of Astragalus polysaccharide for traumatic stress mice cell immunity[J].Chin J Microbiol Immunol,2004,24(12):942-945.
[18]HU F,LI X,ZHAO L,FENG S,WANG C.Antidiabetic properties of purified polysaccharide from Hedysarum polybotrys[J].Can JPhysiol Pharmacol,2010,88(1):64-72.
[19]YU S,ZHANG W,YU J Y,FENG S L,GUO T K,LU H.Inhibiting effect of Radix Hedysari Polysaccharide(HPS)onendotoxin-induceduveitis in rats[J].Internat Immunopharmacol,2014,21:361-368.
[20]LAJAVARDI L,BOCHOT A,CAMELO S.Downregulation of endotoxin-induced uveitis by intravitreal injection of vasoactive intestinal peptide encapsulated in liposomes[J].Invest Ophthalmol Vis Sci,2007,48(7):3230-3238.
[21]BHATTACHERJEE P,WILLIAMS R N,EAKINS K E.An evaluation of ocular inflammation following the injection of bacterial endotoxin ito the rat foot pad[J].Invest Ophthalmol Vis Sci,1983,24(2):196-202.
[22]HU X,PAIK P K,CHEN J.IFN-gamma suppresses IL-10 production and synergizes with TLR2 by regulating GSK3 and CREB/AP-1 proteins[J].Immunity,2006,24(5):563-574.
[23]DOBLE B W,WOODGETT J R.GSK3:tricks of the trade for a multi-tasking kinase[J].J Cell Sci,2003,116(7):1175-1186.
[24]BEUREL E,MICHALEK S M,JOPE R S.Innate and adaptive immune responses regulated by glycogen synthase kinase-3(GSK3)[J].Trends Immunol,2010,31(1):24-31.
[25]CAVAILLON J M,ADIB-CONQUY M.Bench-to-bedside review:endotoxin tolerance as a model of leukocyte reprogramming in sepsis[J].Crit Care,2006,10(5):233.
[26]DE SARNO P,AXTELL R C,RAMAN C.Lithium prevents and ameliorates experimental autoimmune encephalomyelitis[J].J Immunol,2008,181(1):338-345.
[27]OHTANI M,NAGAI S,KONDO S.Mammalian target of rapamycin and glycogen synthase kinase 3 differentially regulate lipopolysaccharide-induced interleukin-12 production in dendritic cells[J].Blood,2008,112(3):635-643.
[28]CHEN K,WU Y,ZHU M.Lithium chloride promotes host resistance against Pseudomonas aeruginosa keratitis[J].Mol Vis,2013,19:1502-1514.
[29]LIU Y,ZHANG Q Y,ZHAO Y Y.Saponins from the roots of Hedysarum polybotrys[J].Biochem Systemat Ecol,2007,35:389-391.
[30]YANG M,CHAN G C,DENG R,NG M H,CHENG S W,LAU CP,ET A L.An herbal decoction of Radix astragali and Radix angelicae sinensis promotes hematopoiesis and thrombopoiesis[J].JEthnopharmacol,2009,124(1):87-97.
[31]LAN Z F,ZHANG Z L,CHENG G Q,WANG F L,XI S F.Effects of radix hedysari polysaccharides on immunological function and transplanted tumors in mice[J].Acta Pharmacol Sin,1987,8(3):275-277.
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