中药兔耳风提取物ainsliadimer A对坏死性小肠结肠炎的作用和机制研究
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摘要
目的:Ainsliadimer A是提取于中药兔耳风的一种内酯类化合物,前期研究已经发现其具有抑制NF-kB信号活性和抑制肿瘤生长的功能。已知的NF-kB信号通路在介导坏死性小肠结肠炎的炎症反应中发挥重要作用,因此,拟研究ainsliadimer A对坏死性小肠结肠炎的作用并对机制进行探讨。方法:通过LPS刺激人肠上皮细胞(FHC)、大鼠小肠隐窝上皮细胞(IEC-6)诱导肠细胞炎症模型,在不同浓度ainsliadimer A处理后,qPCR检测IL-6、TNF-α的mRNA水平,ELISA检测上清中IL-6水平,检测其对炎症因子水平的影响;对FHC和IEC-6细胞处理不同浓度ainsliadimer A后, CCK-8法和划痕实验分别检测其对细胞增殖、迁移的影响,评估其对坏死性小肠结肠炎的肠细胞修复功能;通过免疫印迹检测ainsliadimer A处理后,细胞内NF-kB信号的活性,初步探讨ainsliadimer A的作用机制。结果:Ainsliadimer A不同浓度(5,10μmol·L~(-1))皆可有效降低FHC和IEC-6细胞中IL-6、TNF-α的水平,同时,显著恢复LPS引起的肠细胞增殖、迁移抑制,免疫印迹显示ainsliadimer A显著降低了NF-kB信号的活性。结论:Ainsliadimer A可有效降低LPS引起的炎症因子表达和释放,且恢复了肠细胞的迁移和增殖,表现出明显的肠修复作用;机制研究初步揭示其通过下调NF-kB信号活性来降低炎症因子水平,缓解炎症反应,研究在细胞水平证实了ainsliadimer A对坏死性小肠结肠炎的缓解作用,为坏死性小肠结肠炎的药物研发和治疗提供了新线索。
OBJECTIVE Ainsliadimer A is a structurally unique compound isolated from Ainsliaea macrocephala. Previous studies have demonstrated the ability of ainsliadimer A in inhibiting NF-kB signaling and repressing tumor growth. The NF-kB activity is known as a crucial factor in the pathogenesis of necrotizing enterocolitis(NEC). We therefore intend to investigate the function of ainsliadimer A in NEC and clarify the mechanism. METHODS LPS was used to stimulate human intestinal epithelial cells(FHC) and rat intestinal crypt epithelial cells(IEC-6) to induce intestinal inflammation. After treatment with different concentrations of ainsliadimer A, mRNA levels of IL-6 and TNF-α were determined by qPCR. IL-6 level in the supernatant was measured by ELISA to detect its effect on the level of inflammatory factors. After treatment of FHC and IEC-6 cells with different concentrations of ainsliadimer A, CCK-8 assay and scratch assay were used to detect its effect on cell proliferation and migration, respectively, to assess its enterocyte repair function in necrotizing enterocolitis; the activity of NF-kB signal in cells after treatment with ainsliadimer A was detected by immunoblotting to preliminarily investigate the mechanism of action of ainsliadimer A.RESULTS Different dose of ainsliadimer A(5 μM, 10 μM) effectively reduced the levels of IL-6 and significantly restored LPS-induced inhibition of intestinal cell proliferation and migration. Immunoblotting showed that ainsliadimer A significantly reduced the activity of NF-kB signaling. CONCLUSION Ainsliadimer A can effectively reduce the expression and release of inflammatory factors induced by LPS, and restore the migration and proliferation of intestinal cells which exhibited obvious intestinal repair effect. The mechanism study reveals that it reduces the level of inflammatory factors and alleviates inflammatory response by downregulating NF-kB signaling activity. The study confirms the alleviating effect of ainsliadimer A on necrotizing enterocolitis at the cellular level, providing a new clue for the drug development and treatment of necrotizing enterocolitis.
OBJECTIVE Ainsliadimer A is a structurally unique compound isolated from Ainsliaea macrocephala. Previous studies have demonstrated the ability of ainsliadimer A in inhibiting NF-kB signaling and repressing tumor growth. The NF-kB activity is known as a crucial factor in the pathogenesis of necrotizing enterocolitis(NEC). We therefore intend to investigate the function of ainsliadimer A in NEC and clarify the mechanism. METHODS LPS was used to stimulate human intestinal epithelial cells(FHC) and rat intestinal crypt epithelial cells(IEC-6) to induce intestinal inflammation. After treatment with different concentrations of ainsliadimer A, mRNA levels of IL-6 and TNF-α were determined by qPCR. IL-6 level in the supernatant was measured by ELISA to detect its effect on the level of inflammatory factors. After treatment of FHC and IEC-6 cells with different concentrations of ainsliadimer A, CCK-8 assay and scratch assay were used to detect its effect on cell proliferation and migration, respectively, to assess its enterocyte repair function in necrotizing enterocolitis; the activity of NF-kB signal in cells after treatment with ainsliadimer A was detected by immunoblotting to preliminarily investigate the mechanism of action of ainsliadimer A.RESULTS Different dose of ainsliadimer A(5 μM, 10 μM) effectively reduced the levels of IL-6 and significantly restored LPS-induced inhibition of intestinal cell proliferation and migration. Immunoblotting showed that ainsliadimer A significantly reduced the activity of NF-kB signaling. CONCLUSION Ainsliadimer A can effectively reduce the expression and release of inflammatory factors induced by LPS, and restore the migration and proliferation of intestinal cells which exhibited obvious intestinal repair effect. The mechanism study reveals that it reduces the level of inflammatory factors and alleviates inflammatory response by downregulating NF-kB signaling activity. The study confirms the alleviating effect of ainsliadimer A on necrotizing enterocolitis at the cellular level, providing a new clue for the drug development and treatment of necrotizing enterocolitis.
引文
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[7] Wang J,Liu YT,Xiao L,et al.Anti-inflammatory effects of apigenin in lipopolysaccharide-induced inflammatory in acute lung injury by suppressing COX-2 and NF-kB pathway [J].Inflammation,2014,37(6):2085-2090.
[8] Caplan MS,Miller Catchpole R,Kaup S,et al.Bifidobacterial supplementation reduces the incidence of necrotizing enterocolitis in a neonatal rat model [J].Gastroenterol,1999,117(3):577-583.
[9] Jilling T,Simon D,Lu J,et al.The roles of bacteria and TLR4 in rat and murine models of necrotizing enterocolitis [J].J Immunol,2006,177(5):3273-3282.
[10] Akira S,Takeda K,Kaisho T.Toll-like receptors:critical proteins linking innate and acquired immunity [J].Nat Immunol,2001,2(8):675-680.
[11] Neal MD,Sodhi CP,Dyer M,et al.A critical role for TLR4 induction of autophagy in the regulation of enterocyte migration and the pathogenesis of necrotizing enterocolitis [J].J Immunol,2013,190(7):3541-3551.
[2] Neu J,Walker WA.Necrotizing enterocolitis [J].New Engl J Med,2011,364(3):255-64.
[3] Wu ZJ,Xu XK,Shen YH,et al.Ainsliadimer A,a new sesquiterpene lactone dimer with an unusual carbon skeleton from Ainsliaea macrocephala [J].Organic Letters,2008,10(12):2397-2400.
[4] Dong T,Li C,Wang X,et al.Ainsliadimer A selectively inhibits IKKα/β by covalently binding a conserved cysteine [J].Nat Commun,2015,6:6522.
[5] Dignass AU.Mechanisms and modulation of intestinal epithelial repair [J].Inflamm Bowel Dis,2001,7(1):68-77.
[6] Zhao W,Sun Z,Wang S,et al.Wnt1 participates in inflammation induced by lipopolysaccharide through upregulating scavenger receptor A and NF-kB [J].Inflammation,2015,38(4):1700-1706.
[7] Wang J,Liu YT,Xiao L,et al.Anti-inflammatory effects of apigenin in lipopolysaccharide-induced inflammatory in acute lung injury by suppressing COX-2 and NF-kB pathway [J].Inflammation,2014,37(6):2085-2090.
[8] Caplan MS,Miller Catchpole R,Kaup S,et al.Bifidobacterial supplementation reduces the incidence of necrotizing enterocolitis in a neonatal rat model [J].Gastroenterol,1999,117(3):577-583.
[9] Jilling T,Simon D,Lu J,et al.The roles of bacteria and TLR4 in rat and murine models of necrotizing enterocolitis [J].J Immunol,2006,177(5):3273-3282.
[10] Akira S,Takeda K,Kaisho T.Toll-like receptors:critical proteins linking innate and acquired immunity [J].Nat Immunol,2001,2(8):675-680.
[11] Neal MD,Sodhi CP,Dyer M,et al.A critical role for TLR4 induction of autophagy in the regulation of enterocyte migration and the pathogenesis of necrotizing enterocolitis [J].J Immunol,2013,190(7):3541-3551.