Mongersen在牙周炎症微环境中抗炎作用的体外研究
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摘要
目的:探讨Smad7反义寡核苷酸mongersen的体外抗炎作用。方法:使用LipofectamineTM2000载mongersen转染小鼠巨噬细胞RAW264.7,之后将RAW264.7与牙周炎的主要致病菌牙龈卟啉单胞菌的脂多糖(Porphyromonas gingivlis lipopolysaccharide,Pg-LPS)共培养,以模拟牙周炎症微环境,共培养12h后,采用流式细胞术、Western blot、qRT-PCR分别检测转染效率、Smad7蛋白的表达情况及巨噬细胞相关细胞因子的表达。结果:流式细胞术检测转染效率可达76.57%;Western blot结果显示mongersen可明显抑制Smad7蛋白的表达;qRT-PCR结果显示加入mongersen后肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-6(interleukin-6,IL-6)、白细胞介素-1β(interleukin-1β,IL-1β)等促炎因子基因表达明显降低。结论:mongersen可通过抑制Smad7蛋白的表达发挥抗炎作用。
Objective:To investigate the anti-inflammatory effect of mongersen,a Smad7 antisense oligonucleotide,in vitro.Methods:LipofectamineTM 2000 contained mongersen was used to tansfect mouse macrophage(RAW264.7).Then RAW264.7 cells were co-cultured with Pg-LPS which was the main pathogen of periodontitis to simulate the periodontal inflammatory environment.After 12 hours,flow cytometry,western blot,and qRT-PCR were used to detect the transfection efficiency and expression of Smad7 protein and macrophages-related cytokines.Results:The transfection efficiency was 76.57%by flow cytometry.Mongeren could inhibit the protein expression of Smad7 by western blot.The expression of TNF-a,IL-6,IL-1β,and other pro-inflammatory cytokines were significantly reduced after adding mongersen.Conclusion:Mongersen can inhibit inflammatory by inhibiting the protein expression of Smad7.
Objective:To investigate the anti-inflammatory effect of mongersen,a Smad7 antisense oligonucleotide,in vitro.Methods:LipofectamineTM 2000 contained mongersen was used to tansfect mouse macrophage(RAW264.7).Then RAW264.7 cells were co-cultured with Pg-LPS which was the main pathogen of periodontitis to simulate the periodontal inflammatory environment.After 12 hours,flow cytometry,western blot,and qRT-PCR were used to detect the transfection efficiency and expression of Smad7 protein and macrophages-related cytokines.Results:The transfection efficiency was 76.57%by flow cytometry.Mongeren could inhibit the protein expression of Smad7 by western blot.The expression of TNF-a,IL-6,IL-1β,and other pro-inflammatory cytokines were significantly reduced after adding mongersen.Conclusion:Mongersen can inhibit inflammatory by inhibiting the protein expression of Smad7.
引文
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[4] Danese S,Fiorino G,Peyrin-Biroulet L.Targeting SMAD7in Crohn's disease by Mongersen:therapeutic revolution under way[J].Gastroenterology,2015,149(4)∶1121-1123
[5] Chen W,Ten Dijke P.Immunoregulation by members of the TGF-βsuperfamily[J].Nat Rev Immunol,2016,16(12)∶723-740
[6] MohanKumar K,Namachivayam K,Chapalamadugu KC,et al.Smad7interrupts TGF-βsignaling in intestinal macrophages and promotes inflammatory activation of these cells during necrotizing enterocolitis[J].Pediatr Res,2016,79(6)∶951-961
[7] Lim R,Barker G,Lappas M.SMAD7regulates proinflammatory and prolabor mediators in amnion and myometrium[J].Biol Reprod,2017,97(2)∶288-301
[8] Hajishengallis G.Periodontitis:from microbial immune subversion to systemic inflammation[J].Nat Rev Immunol,2015,15(1)∶30-44
[9] Lam RS,O'Brien-Simpson NM,Lenzo JC,et al.Macrophage depletion abates porphyromonas gingivalis-induced alveolar bone resorption in mice[J].J Immunol,2014,193(5)∶2349-2362
[10] Maekawa T,Krauss JL,Abe T,et al.Porphyromonas gingivalis manipulates complement and TLR signaling to uncouple bacterial clearance from inflammation and promote dysbiosis[J].Cell Host Microbe,2014,15(6)∶768-778