黄芩苷对脂多糖刺激下人牙周膜细胞IKKα表达影响的初步研究
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摘要
目的观察黄芩苷对脂多糖(LPS)刺激下人牙周膜细胞NF-κB非经典途径IKKα表达的影响。方法酶消化法体外分离、培养、鉴定人牙周膜细胞(h PDLCs);CCK-8检测黄芩苷对h PDLCs增殖的影响;实验分空白对照组(NC)、LPS组、LPS+黄芩苷1~4组;酶联免疫吸附试验检测炎性因子肿瘤坏死因子(TNF)-α和白细胞介素(IL)-1β的分泌,蛋白质免疫印迹法(Western Blot)、反转录-聚合酶链反应(RT-PCR)检查IKKα蛋白及m RNA的表达。结果 0.001~50 mg/L黄芩苷对h PDLCs增殖起促进作用;ELISA结果显示,LPS组与NC组相比,IL-1β、TNF-α分泌量增加,LPS+黄芩苷1~4组随着黄芩苷浓度的增加炎症因子分泌量减少;Western Blot结果显示IKKα蛋白的表达在LPS组较NC组高,LPS+黄芩苷1~4组较LPS组低,且RT-PCR结果与Western Blot结果相一致。结论黄芩苷介导了LPS刺激下人牙周膜细胞中NF-κB非经典途径IKKα表达下调,对牙周膜细胞的炎症损伤起保护作用。
Objective To investigate the effect of Baicalin on the expression of NF-κB non-classical pathway IKKα in LPS-stimulated human periodontal ligament cells(h PDLCs). Methods The h PDLCs were in vitro isolated,cultured and identified by enzyme digestion method. The effect of Baicalin on the proliferation of h PDLCs was deter-mined by CCK-8 assay. This study included the blank control group(NC), LPS group and LPS+Baicalin1-4 group.The secretions of the inflammatory factors TNF-α and IL-1β were determined by ELISA. Western blot and RT-PCR were used to examine the expression of IKKα protein and m RNA expression, respectively. Results Baicalin could promote the proliferation of h PDLCs with a concentration between 0.001~50 mg/L. The findings of ELISA showed that the secretion of IL-1β and TNF-α was increased in the LPS group compared with those in the NC group. The secre-tions of the inflammatory factors were decreased with the increase of Baicalin concentration in the LPS +Baicalin1-4group. The findings of Western Blot showed that the expression of IKKα protein was higher in the LPS group than that in the NC group and LPS+Baicalin1-4 group, and the findings of RT-PCR were consistent with those of the Western Blot. Conclusion Baicalin mediates the down-regulation of NF-κB non-classical pathway IKKα expression in the LPS-stimulated human periodontal ligament cells, and protects against inflammatory injury of periodontal ligament cells.
Objective To investigate the effect of Baicalin on the expression of NF-κB non-classical pathway IKKα in LPS-stimulated human periodontal ligament cells(h PDLCs). Methods The h PDLCs were in vitro isolated,cultured and identified by enzyme digestion method. The effect of Baicalin on the proliferation of h PDLCs was deter-mined by CCK-8 assay. This study included the blank control group(NC), LPS group and LPS+Baicalin1-4 group.The secretions of the inflammatory factors TNF-α and IL-1β were determined by ELISA. Western blot and RT-PCR were used to examine the expression of IKKα protein and m RNA expression, respectively. Results Baicalin could promote the proliferation of h PDLCs with a concentration between 0.001~50 mg/L. The findings of ELISA showed that the secretion of IL-1β and TNF-α was increased in the LPS group compared with those in the NC group. The secre-tions of the inflammatory factors were decreased with the increase of Baicalin concentration in the LPS +Baicalin1-4group. The findings of Western Blot showed that the expression of IKKα protein was higher in the LPS group than that in the NC group and LPS+Baicalin1-4 group, and the findings of RT-PCR were consistent with those of the Western Blot. Conclusion Baicalin mediates the down-regulation of NF-κB non-classical pathway IKKα expression in the LPS-stimulated human periodontal ligament cells, and protects against inflammatory injury of periodontal ligament cells.
引文
[1]边专.口腔生物学[M].4版.北京:人民卫生出版社,2014:135-136.
[2]王勤涛.牙周病学[M].北京:人民卫生出版社,2011:177-178.
[3]欧阳玉玲,吴亚菲.核转录因子-kappa B与牙周炎[J].国际口腔医学杂志,2007,34(5):345-347.
[4]Wang HZ,Wang HH,Huang SS,et al.Inhibitory effect of baicalin on collagen-induced arthritis in rats through the nuclear factorκB pathway[J].J Pharmacol Exp Ther,2014,350(2):435-443.
[5]Dong P,Zhang Y,Gu J,et al.Wogonin,an active ingredient of Chinese herb medicine Scutellaria baicalensis,inhibits the mobility and invasion of human gallbladder carcinoma GBC-SD cells by inducing the expression of maspin[J].J Ethnopharmaeol,2011,137(3):1373-1380.
[6]Zhou YJ,Wang H,Sui HH,et al.Inhibitory effect of baicalin on allergic response in ovalbumin-induced allergic rhinitis guinea pigs and lipopolysaccharide-stimulated human mast cell[J].Inflamm Res,2016,65(8):603-612.
[7]Pei Z,Wang B,Zhang F,et al.Response of human periodontal ligament cells to baicalin[J].J Periodontol,2014,85(9):1283-1290.
[8]葛志华,张莹,孙立新.黄芩苷对内毒素作用的人牙周膜细胞增殖和分泌肿瘤坏死因子-α的影响[J].实用口腔医学杂志,2010,26(4):534-537.
[9]李玲.黄芩苷对牙周炎大鼠牙移动保持阶段VEGF、BMP-2及IL-1β表达的影响[J].中国生化药物杂志,2015,40(3):32-35.
[10]王国芳,吴织芬,牛兵.淫羊藿苷,黄芩苷和大黄素联合作用对牙周炎大鼠牙周组织中RANKL和OPG表达的影响[J].时珍国医国药,2011,22(10):2556-2558.
[11]Feng J,Guo C,Zhu Y,et al.Baicalin down regulates the expression of TLR4 and NFk B-p65 in colon tissue in mice with colitis induced by dextran sulfate sodium[J].Int J Clin Med,2014,7(11):4063-4072.
[12]Yang W,Li H,Cong X,et al.Baicalin attenuates lipopolysaccharide induced inflammation and apoptosis of cow mammary epithelial cells by regulating NF-κB and HSP72[J].Int Immunopharmacol,2016,40:139-145.
[13]Wang GF,Wu ZF,Wan L,et al.Influence of baicalin on the expression of receptor activator of nuclear factor-κB ligand in cultured human periodontal ligament cells[J].Pharmacology,2006,77(2):71-77.
[14]胡永亮,宋伦.IKK激酶的NF-κB非相关性功能研究进展[J].生物技术通讯,2015,27(2):268-271.
[15]谢林艳,闫兆,刘洪泱,等.支气管哮喘免疫耐受的新视角:核因子κB非经典途径[J].中华微生物学和免疫学杂志,2014,34(1):69-74.
[16]Luo JL,Tan W,Ricono JM,et al.Nuclear cytokine-activated IKKalpha controls prostate cancer metastasis by repressing Maspin[J].Nature,2007,446(7136):690-694.
[17]徐德立.IKK-α的研究进展[J].细胞生物学杂志,2006,28(3):392-394.
[18]Kim YS,Shin SI,Kang KL,et al.Nicotine and lipopolysaccharide stimulate the production of MMPs and prostaglandin E2by hypoxia-inducible factor-1αup-regulation in human periodontal ligament cells[J].J Periodontal Res,2012,47(6):719-728.
[19]余科,吴湘楠,刘文佳,等.LPS刺激小鼠骨细胞RANKL/OPG和IL-6表达的实验研究[J].口腔医学研究,2016,32(3):220-223.
[20]徐婧秋,吴丽萍.脂多糖和白细胞介素-1β对人牙周膜细胞中诱导型一氧化氮合酶表达的影响[J].华西口腔医学杂志,2011,29(4):415-419.
[21]Luo WC,Wang Y,Jin L.Baicalin downregulates Porphyromonas gingivalis lipopolysaccharide-upregulated IL-6 and IL-8 expression in human oral keratinocytes by negative regulation of TLR signaling[J].PLo S One,2012,7(12):e51008.
[22]张江琳,王浩宇,李丹,等.复方黄芩片对人牙周膜细胞及牙周炎牙槽骨OPG/RANKL表达的影响[J].口腔医学研究,2015,31(11):1064-1068.
[23]Yu FY,Huang SG,Zhang HY,et al.Effects of baicalin in CD4+CD29+T cell subsets of ulcerative colitis patients[J].World J Gastroenterol,2014,20(41):15299-15309.
[24]陈小萍,李明慧,张永振,等.LPS耐受单核细胞中p50抑制IKKα结合于IL-1β启动子区[J].中华微生物学和免疫学杂志,2010,30(4):291-296.
[25]Hoshino K,Sugiyama T,Matsumoto M,et al.IκB kinase-αis critical for interferon-αproduction induced by Toll-like receptors 7and 9[J].Nature,2006,440(7086):949-953.
[26]Lawrence T,Bebien M,Liu GY,et al.IKKalpha limits macrophage NF-kappa B activation and contributes to the resolution of inflammation[J].Nature,2005,434(7037):1138-1143.
[2]王勤涛.牙周病学[M].北京:人民卫生出版社,2011:177-178.
[3]欧阳玉玲,吴亚菲.核转录因子-kappa B与牙周炎[J].国际口腔医学杂志,2007,34(5):345-347.
[4]Wang HZ,Wang HH,Huang SS,et al.Inhibitory effect of baicalin on collagen-induced arthritis in rats through the nuclear factorκB pathway[J].J Pharmacol Exp Ther,2014,350(2):435-443.
[5]Dong P,Zhang Y,Gu J,et al.Wogonin,an active ingredient of Chinese herb medicine Scutellaria baicalensis,inhibits the mobility and invasion of human gallbladder carcinoma GBC-SD cells by inducing the expression of maspin[J].J Ethnopharmaeol,2011,137(3):1373-1380.
[6]Zhou YJ,Wang H,Sui HH,et al.Inhibitory effect of baicalin on allergic response in ovalbumin-induced allergic rhinitis guinea pigs and lipopolysaccharide-stimulated human mast cell[J].Inflamm Res,2016,65(8):603-612.
[7]Pei Z,Wang B,Zhang F,et al.Response of human periodontal ligament cells to baicalin[J].J Periodontol,2014,85(9):1283-1290.
[8]葛志华,张莹,孙立新.黄芩苷对内毒素作用的人牙周膜细胞增殖和分泌肿瘤坏死因子-α的影响[J].实用口腔医学杂志,2010,26(4):534-537.
[9]李玲.黄芩苷对牙周炎大鼠牙移动保持阶段VEGF、BMP-2及IL-1β表达的影响[J].中国生化药物杂志,2015,40(3):32-35.
[10]王国芳,吴织芬,牛兵.淫羊藿苷,黄芩苷和大黄素联合作用对牙周炎大鼠牙周组织中RANKL和OPG表达的影响[J].时珍国医国药,2011,22(10):2556-2558.
[11]Feng J,Guo C,Zhu Y,et al.Baicalin down regulates the expression of TLR4 and NFk B-p65 in colon tissue in mice with colitis induced by dextran sulfate sodium[J].Int J Clin Med,2014,7(11):4063-4072.
[12]Yang W,Li H,Cong X,et al.Baicalin attenuates lipopolysaccharide induced inflammation and apoptosis of cow mammary epithelial cells by regulating NF-κB and HSP72[J].Int Immunopharmacol,2016,40:139-145.
[13]Wang GF,Wu ZF,Wan L,et al.Influence of baicalin on the expression of receptor activator of nuclear factor-κB ligand in cultured human periodontal ligament cells[J].Pharmacology,2006,77(2):71-77.
[14]胡永亮,宋伦.IKK激酶的NF-κB非相关性功能研究进展[J].生物技术通讯,2015,27(2):268-271.
[15]谢林艳,闫兆,刘洪泱,等.支气管哮喘免疫耐受的新视角:核因子κB非经典途径[J].中华微生物学和免疫学杂志,2014,34(1):69-74.
[16]Luo JL,Tan W,Ricono JM,et al.Nuclear cytokine-activated IKKalpha controls prostate cancer metastasis by repressing Maspin[J].Nature,2007,446(7136):690-694.
[17]徐德立.IKK-α的研究进展[J].细胞生物学杂志,2006,28(3):392-394.
[18]Kim YS,Shin SI,Kang KL,et al.Nicotine and lipopolysaccharide stimulate the production of MMPs and prostaglandin E2by hypoxia-inducible factor-1αup-regulation in human periodontal ligament cells[J].J Periodontal Res,2012,47(6):719-728.
[19]余科,吴湘楠,刘文佳,等.LPS刺激小鼠骨细胞RANKL/OPG和IL-6表达的实验研究[J].口腔医学研究,2016,32(3):220-223.
[20]徐婧秋,吴丽萍.脂多糖和白细胞介素-1β对人牙周膜细胞中诱导型一氧化氮合酶表达的影响[J].华西口腔医学杂志,2011,29(4):415-419.
[21]Luo WC,Wang Y,Jin L.Baicalin downregulates Porphyromonas gingivalis lipopolysaccharide-upregulated IL-6 and IL-8 expression in human oral keratinocytes by negative regulation of TLR signaling[J].PLo S One,2012,7(12):e51008.
[22]张江琳,王浩宇,李丹,等.复方黄芩片对人牙周膜细胞及牙周炎牙槽骨OPG/RANKL表达的影响[J].口腔医学研究,2015,31(11):1064-1068.
[23]Yu FY,Huang SG,Zhang HY,et al.Effects of baicalin in CD4+CD29+T cell subsets of ulcerative colitis patients[J].World J Gastroenterol,2014,20(41):15299-15309.
[24]陈小萍,李明慧,张永振,等.LPS耐受单核细胞中p50抑制IKKα结合于IL-1β启动子区[J].中华微生物学和免疫学杂志,2010,30(4):291-296.
[25]Hoshino K,Sugiyama T,Matsumoto M,et al.IκB kinase-αis critical for interferon-αproduction induced by Toll-like receptors 7and 9[J].Nature,2006,440(7086):949-953.
[26]Lawrence T,Bebien M,Liu GY,et al.IKKalpha limits macrophage NF-kappa B activation and contributes to the resolution of inflammation[J].Nature,2005,434(7037):1138-1143.