红芪多糖干预内毒素诱导的大鼠葡萄膜炎的机制
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摘要
目的探讨红芪多糖(hedysarum polybotrys saccharide,HPS)在内毒素诱导的大鼠葡萄膜炎发病中的作用及对Toll样受体4(Toll-like receptor 4,TLR-4)信号转导通路中关键分子肿瘤坏死因子受体相关因子6(tumor necrosis factor receptor-associated factor 6,TRAF6)及TIR功能区接头蛋白诱导的干扰素β(TIR-domain-containing adapter-inducing interferon-β,TRIF)表达的影响。方法选用SPF级健康成年Wistar大鼠40只,随机分为内毒素诱导的葡萄膜炎(endotoxin induced uveitis,EIU)模型组、HPS组、HPS+内毒素组、正常对照组(normal control,NC)组,每组10只。EIU模型组大鼠采用皮下注射1 mg·kg~(-1)内毒素的方法建立大鼠急性前葡萄膜炎动物模型。HPS+内毒素组在内毒素注射前1 h给予腹腔注射HPS 400 mg·kg~(-1)。HPS组大鼠只注射HPS 400 mg·kg~(-1)。每2 h用裂隙灯观察大鼠眼前节炎症反应,注射后的0 h、6 h、12 h、18 h、24 h对各组眼部临床炎症反应进行评分。注射后24 h组织病理学检查炎症反应水平。RT-PCR检测核因子-κB、TRAF6及TRIF mRNA的表达。结果 NC组大鼠24 h内眼前节未见炎症反应。注射后24 h,EIU模型组可见虹膜血管扩张明显,瞳孔区大量白色渗出,HPS组仅可见虹膜血管扩张,未见其他眼部炎症反应,HPS+内毒素组大鼠可见虹膜血管扩张和瞳孔缘少量渗出。比较不同组间的大鼠眼部临床炎症反应评分可见,NC组与EIU模型组、HPS+内毒素组、HPS组比较,差异均有统计学意义(均为P<0.001);EIU模型组与HPS+内毒素组、HPS组比较,差异均有统计学意义(均为P<0.05);HPS+内毒素组与HPS组比较,差异有统计学意义(P<0.001)。RT-PCR结果显示,HPS可降低核因子-κB mRNA和TRAF6 mRNA的表达(P<0.001),HPS对TRIF的表达无明显影响(P=0.236)。结论 HPS可能通过抑制TRAF6核酸的表达缓解EIU的炎症反应,HPS对TRIF的表达无明显影响。
Objective To investigate the effect of hedysarum polybotrys saccharide(HPS) on endotoxin-induced uveitis(EIU) in rats and to explore the effect on the expression of tumor necrosis factor receptor-associated factor 6(TRAF6)and TIR-domain-containing adapter-inducing interferon-β(TRIF)in the Toll-like receptor 4(TLR-4) signal transduction pathway in EIU.Methods Experiment select the SPF healthy adult Wistar rats,40 rats were randomly divided into four groups.EIU group,hedysarum polybotrys saccharide treatment(HPS+LPS) group,hedysarum polybotrys saccharide control(HPS) group,normal control(NC) group.Ten rats in each group.EIU was established by subcutaneous injection of 1 mg·kg~(-1) lipopolysaccharide(LPS).HPS(400 mg·kg~(-1)) was given through intraperitoneal injection 1 h before the LPS induction in HPS treatment group.Rats in the HPS control group were only injected with 400 mg·kg~(-1) of HPS.The inflammation of rats was observed every 2 h with slit lamp,and each group was graded at 0 h,6 h,12 h,18 h and 24 h after injection.Histopathological examination of inflammatory was performed 24 h after injection.Real-time quantitative PCR were used to determine the expression of nuclear factor-kappa B(NF-κB),TRAF6 and TRIF.Results No inflammation was observed in NC group within 24 h.24 h after injection,iris blood vessel dilation and a large amount of white exudation was observed in the pupil area in the EIU group.Only iris blood vessel dilation was observed in the HPS group.In the HPS+LPS group,iris blood vessel dilation and a small amount of exudation in the pupil margin were observed.Compared clinical scores of rats in different groups,and the differences were statistically significant among all groups(all P<0.001).Compared with HPS+LPS group and HPS group,the difference of EIU group was statistically significant(both P<0.05).The difference of inflammation between the LPS+HPS group and the HPS group was statistically significant(P<0.001).Real-time quantitative PCR results showed that HPS could reduce the expression of NF-κB and TRAF6 mRNA(both P<0.001),while HPS had no significant effect on the expression of TRIF(P=0.236).Conclusion HPS can restrain the intraocular inflammation in EIU by inhibiting the expression of TRAF6 mRNA.HPS has no significant effect on the expression of TRIF.
Objective To investigate the effect of hedysarum polybotrys saccharide(HPS) on endotoxin-induced uveitis(EIU) in rats and to explore the effect on the expression of tumor necrosis factor receptor-associated factor 6(TRAF6)and TIR-domain-containing adapter-inducing interferon-β(TRIF)in the Toll-like receptor 4(TLR-4) signal transduction pathway in EIU.Methods Experiment select the SPF healthy adult Wistar rats,40 rats were randomly divided into four groups.EIU group,hedysarum polybotrys saccharide treatment(HPS+LPS) group,hedysarum polybotrys saccharide control(HPS) group,normal control(NC) group.Ten rats in each group.EIU was established by subcutaneous injection of 1 mg·kg~(-1) lipopolysaccharide(LPS).HPS(400 mg·kg~(-1)) was given through intraperitoneal injection 1 h before the LPS induction in HPS treatment group.Rats in the HPS control group were only injected with 400 mg·kg~(-1) of HPS.The inflammation of rats was observed every 2 h with slit lamp,and each group was graded at 0 h,6 h,12 h,18 h and 24 h after injection.Histopathological examination of inflammatory was performed 24 h after injection.Real-time quantitative PCR were used to determine the expression of nuclear factor-kappa B(NF-κB),TRAF6 and TRIF.Results No inflammation was observed in NC group within 24 h.24 h after injection,iris blood vessel dilation and a large amount of white exudation was observed in the pupil area in the EIU group.Only iris blood vessel dilation was observed in the HPS group.In the HPS+LPS group,iris blood vessel dilation and a small amount of exudation in the pupil margin were observed.Compared clinical scores of rats in different groups,and the differences were statistically significant among all groups(all P<0.001).Compared with HPS+LPS group and HPS group,the difference of EIU group was statistically significant(both P<0.05).The difference of inflammation between the LPS+HPS group and the HPS group was statistically significant(P<0.001).Real-time quantitative PCR results showed that HPS could reduce the expression of NF-κB and TRAF6 mRNA(both P<0.001),while HPS had no significant effect on the expression of TRIF(P=0.236).Conclusion HPS can restrain the intraocular inflammation in EIU by inhibiting the expression of TRAF6 mRNA.HPS has no significant effect on the expression of TRIF.
引文
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[9] CUI X M,WANG Z P,ZHANG Z H.Astragalus polysaccharide enhanced the killing effect of LAK cells on bladder tumor cells[J].Pharmacol Clin Trad Chin Med,1998,14(2):19-20.崔笑梅,王志平,张志华.红芪多糖增强LAK细胞对膀胱肿瘤瘤细胞的杀伤作用[J].中药药理与临床,1998,14(2):19-20.
[10] CHEN H Q,ZHANG X R,KIM O.Modification and degradation of traf6 in the activation of NF-κB pathway[J].Acta Univ Meds Nanjing,2004,24(1):59-62.陈华群,张锡然,KIM O.TRAF6在NF-κB信号通路中的修饰与降解[J].南京医科大学学报,2004,24(1):59-62.
[11] NAITO A,YOSHIDA H,NISHIOKA E,SATO H M,AZUMA S,YAMAMOTO T,et al.TRAF6-deficient mice display hypohidrotic ectodermal dysplasia[J].Proc Natl Acad Sci USA,2002,99(13):8766-8771.
[12] WANG C,DENG L,HONG M,AKKARAJU G R,INOUE J,CHEN Z J.TAK1 is a ubiquitin-dependent kinase of MKK and IKK[J].Nature,2001,412(6844):346-351.
[13] LI S,LU H,HU X F,CHEN W,XU Y Z,WANG J.Expression of TLR4-MyD88 and NF-κB in the iris during endotoxin-induced uveitis[J].Mediators Inflamm,2010,2010:748218.
[14] KAWAI T,AKIRA S.The role of pattern-recognition receptors in innate immunity:update on Toll-like receptors[J].Nat Immunol,2010,11(5):373-384.
[2] WANG J,LU H,HU X,XU Z,LI S.Nuclear factor translocation and acute anterior uveitis[J].Mol Vis,2011,17:170-176.
[3] LU Y C,YEH W C,OHASHI P S.LPS/TLR4 signal transduction pathway[J].Cytokine,2008,42(2):145-151.
[4] LIU R Y,ZHAO H M,ZHOU F,HUANG X Y,LV A P,LUO X J.Astragalus polysaccharide regulates the expression of small intestinal mucosal lymphocyte cytokines in mice[J].Chin J Bas CHN Med,2008,14(9):692-693.刘端勇,赵海梅,周枫,黄小英,吕爱平,罗晓建.黄芪多糖调节小鼠小肠黏膜淋巴细胞因子的表达[J].中国中医基础医学杂志,2008,14(9):692-693.
[5] 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.曾广仙,刘俊英,熊金蓉,廖奕华,代丽红,李杏娟,等.黄芪多糖调节创伤应激小鼠免疫功能的研究[J].中华微生物学和免疫学杂志,2004,24(12):942-945.
[6] HU F,LI X,ZHAO L,FENG S,WANG C.Antidiabetic properties of purified polysaccharide from Hedysarum polybotrys[J].Can J Physiol Pharmacol,2010,88(1):64-72.
[7] QIU Y,SHIL P K,ZHU P,YANG H.Angiotensin-converting enzyme 2 (ace2) activator diminazene aceturate ameliorates endotoxin-induced uveitis in mice[J].Invest Ophthalmol Vis Sci,2014,55(6):3809-3818.
[8] YU S,ZHANG W,YU J,FENG S,GUO T,HONG L.Inhibiting effect of Radix Hedysari Polysaccharide (HPS) onendotoxin-induceduveitis in rats[J].Int Immunopharmacol,2014,21(2):361-368.
[9] CUI X M,WANG Z P,ZHANG Z H.Astragalus polysaccharide enhanced the killing effect of LAK cells on bladder tumor cells[J].Pharmacol Clin Trad Chin Med,1998,14(2):19-20.崔笑梅,王志平,张志华.红芪多糖增强LAK细胞对膀胱肿瘤瘤细胞的杀伤作用[J].中药药理与临床,1998,14(2):19-20.
[10] CHEN H Q,ZHANG X R,KIM O.Modification and degradation of traf6 in the activation of NF-κB pathway[J].Acta Univ Meds Nanjing,2004,24(1):59-62.陈华群,张锡然,KIM O.TRAF6在NF-κB信号通路中的修饰与降解[J].南京医科大学学报,2004,24(1):59-62.
[11] NAITO A,YOSHIDA H,NISHIOKA E,SATO H M,AZUMA S,YAMAMOTO T,et al.TRAF6-deficient mice display hypohidrotic ectodermal dysplasia[J].Proc Natl Acad Sci USA,2002,99(13):8766-8771.
[12] WANG C,DENG L,HONG M,AKKARAJU G R,INOUE J,CHEN Z J.TAK1 is a ubiquitin-dependent kinase of MKK and IKK[J].Nature,2001,412(6844):346-351.
[13] LI S,LU H,HU X F,CHEN W,XU Y Z,WANG J.Expression of TLR4-MyD88 and NF-κB in the iris during endotoxin-induced uveitis[J].Mediators Inflamm,2010,2010:748218.
[14] KAWAI T,AKIRA S.The role of pattern-recognition receptors in innate immunity:update on Toll-like receptors[J].Nat Immunol,2010,11(5):373-384.