自拟和痹方对人类风湿关节炎成纤维样滑膜细胞炎症因子分泌的影响及其机制探讨
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摘要
目的观察和痹方剂对人类风湿关节炎(RA)成纤维样滑膜细胞(FLS)炎症因子分泌的影响,并探讨其机制。方法取FLS,并随机分为6组。激动剂组每孔加含10%PNU-282987药物血清的DMEM培养液,MTX组加含10%甲氨蝶呤(MTX)药物血清的DMEM培养液,低剂量组加含10%低剂量和痹方剂药物血清的DMEM培养液,中剂量组加含10%中剂量和痹方剂药物血清的DMEM培养液,高剂量组加含10%高剂量和痹方剂药物血清的DMEM培养液,模型组加10%DMEM血清的培养液。24 h后取细胞上清液及滑膜细胞。采用ELISA法检测细胞上清液IL-17、高迁移率组蛋白1(HMGB1),Western blotting法检测细胞α7烟碱型乙酰胆碱受体(α7nAChR)、核转录因子-κB亚基p65(NF-κB p65),RT-PCR法检测细胞α7nAchR mRNA。结果与模型组比较,激动剂组、MTX组、中剂量组IL-17、HMGB1表达降低(P均<0.05);与激动剂组比较,MTX组、低剂量组、中剂量组、高剂量组IL-17表达及高剂量组HMGB1表达升高(P均<0.05);与MTX组比较,中剂量组IL-17表达降低(P均<0.05)。与模型组比较,α7nAChR蛋白、mRNA表达升高,p65蛋白表达降低(P均<0.05);与激动剂组比较,低剂量组p65蛋白及中剂量组α7nAChR蛋白、p65蛋白表达升高,低剂量组、高剂量组α7nAChR蛋白及低剂量组、中剂量组、高剂量组α7nAChR mRNA表达降低(P均<0.05);与MTX组比较,低剂量组p65蛋白及中剂量组α7nAChR蛋白、NF-kBp65蛋白、α7nAChR mRNA表达升高(P均<0.05);与低剂量组比较,中剂量组α7nAChR蛋白、mRNA表达升高,中剂量组p65蛋白及高剂量组α7nAChR蛋白、p65蛋白表达降低(P均<0.05);与中剂量组比较,高剂量组α7nAChR、NF-kBp65蛋白表达降低(P均<0.05)。结论中等浓度和痹方剂能抑制RA FLS分泌炎症因子,进而起到治疗作用,机制可能为上调α7nAChR mRNA、蛋白表达,激活CAP,进而调节NF-kB信号通路。
Objective To observe the effect of Hebi recipe on the secretion of inflammatory factors in human rheumatoid arthritis(RA) fibroblast-like synoviocytes(FLSs) and to explore its mechanism.Methods FLSs were taken and randomly divided into 6 groups.In the agonist group,DMEM medium containing 10% PNU-282987 drug serum was added to each well;in the MTX group,DMEM medium containing 10% methotrexate(MTX) drug serum was added;in the lowdose group,DMEM medium containing 10% low-dose Hebi recipe drug serum was added;in the medium-dose group,DMEM medium containing 10% medium-dose Hebi recipe drug serum was added;in the high-dose group,DMEM medium containing 10% high-dose Hebi recipe drug serum was added;and in the model group,10% DMEM serum was added.After 24 h,the cell supernatant and synovial cells were taken.The interleukin(IL)-17 and high mobility group box 1(HMGB1) in the supernatant were detected by enzyme linked immunosorbent assay(ELISA),α7 nicotinic acetylcholine receptor(α7 nAChR) and nuclear transcription factor-κB subunit p65(NF-κB p65) were detected by Western blotting,and the α7 nAchR mRNA was detected by reverse transcription-polymerase chain reaction(RT-PCR).Results Compared with the model group,the expression of IL-17 and HMGB1 in the agonist group,the MTX group and the medium-dose group decreased(both P<0.05).Compared with the agonist group,the expression of IL-17 in the MTX group,the lowdose group,the medium-dose group,the high-dose group,and the expression of HMGB1 in the high-dose group increased(both P<0.05).Compared with the MTX group,the IL-17 expression decreased in the medium-dose group(P<0.05).Compared with the model group,the protein and mRNA expression of α7 nAChR in both the agonist group and the mediumdose group increased,and the expression of p65 protein decreased(all P<0.05).Compared with the agonist group,the expression of p65 protein in the low-dose group and the expression of α7 nAChR protein and p65 protein in the medium-dose group increased,the expression of α7 nAChR protein in the low-dose group and high-dose group,and the expression ofα7 nAChR mRNA in the low-dose group,medium-dose group,and high-dose group decreased(all P<0.05).Compared with the MTX group,the expression of p65 protein in the low-dose group,and α7 nAChR protein,NF-kBp65 protein,andα7 nAChR mRNA in the medium-dose group increased(all P<0.05).Compared with the low-dose group,the protein and mRNA expression of α7 nAChR in the medium-dose group increased,and the expression of p65 protein in the medium-dose group and α7 nAChR protein and p65 protein in the high-dose group decreased(all P<0.05).Compared with the mediumdose group,the protein expression of α7 nAChR and NF-kBp65 in the high-dose group decreased(both P<0.05).Conclusion Moderate concentration of Hebi recipe can inhibit the secretion of inflammatory factors from FLSs of RA,which may play a therapeutic role by up-regulating α7 nAChR mRNA and protein expression,and then regulating NF-kB signal pathway.
Objective To observe the effect of Hebi recipe on the secretion of inflammatory factors in human rheumatoid arthritis(RA) fibroblast-like synoviocytes(FLSs) and to explore its mechanism.Methods FLSs were taken and randomly divided into 6 groups.In the agonist group,DMEM medium containing 10% PNU-282987 drug serum was added to each well;in the MTX group,DMEM medium containing 10% methotrexate(MTX) drug serum was added;in the lowdose group,DMEM medium containing 10% low-dose Hebi recipe drug serum was added;in the medium-dose group,DMEM medium containing 10% medium-dose Hebi recipe drug serum was added;in the high-dose group,DMEM medium containing 10% high-dose Hebi recipe drug serum was added;and in the model group,10% DMEM serum was added.After 24 h,the cell supernatant and synovial cells were taken.The interleukin(IL)-17 and high mobility group box 1(HMGB1) in the supernatant were detected by enzyme linked immunosorbent assay(ELISA),α7 nicotinic acetylcholine receptor(α7 nAChR) and nuclear transcription factor-κB subunit p65(NF-κB p65) were detected by Western blotting,and the α7 nAchR mRNA was detected by reverse transcription-polymerase chain reaction(RT-PCR).Results Compared with the model group,the expression of IL-17 and HMGB1 in the agonist group,the MTX group and the medium-dose group decreased(both P<0.05).Compared with the agonist group,the expression of IL-17 in the MTX group,the lowdose group,the medium-dose group,the high-dose group,and the expression of HMGB1 in the high-dose group increased(both P<0.05).Compared with the MTX group,the IL-17 expression decreased in the medium-dose group(P<0.05).Compared with the model group,the protein and mRNA expression of α7 nAChR in both the agonist group and the mediumdose group increased,and the expression of p65 protein decreased(all P<0.05).Compared with the agonist group,the expression of p65 protein in the low-dose group and the expression of α7 nAChR protein and p65 protein in the medium-dose group increased,the expression of α7 nAChR protein in the low-dose group and high-dose group,and the expression ofα7 nAChR mRNA in the low-dose group,medium-dose group,and high-dose group decreased(all P<0.05).Compared with the MTX group,the expression of p65 protein in the low-dose group,and α7 nAChR protein,NF-kBp65 protein,andα7 nAChR mRNA in the medium-dose group increased(all P<0.05).Compared with the low-dose group,the protein and mRNA expression of α7 nAChR in the medium-dose group increased,and the expression of p65 protein in the medium-dose group and α7 nAChR protein and p65 protein in the high-dose group decreased(all P<0.05).Compared with the mediumdose group,the protein expression of α7 nAChR and NF-kBp65 in the high-dose group decreased(both P<0.05).Conclusion Moderate concentration of Hebi recipe can inhibit the secretion of inflammatory factors from FLSs of RA,which may play a therapeutic role by up-regulating α7 nAChR mRNA and protein expression,and then regulating NF-kB signal pathway.
引文
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[9]韩乐,张玲玲,魏伟.胶原三螺旋重复蛋白-1参与疾病作用研究进展[J].中国药理学通报,2017,33(7):904-907.
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[12]丁从珠,汪悦,姚瑶,等.青藤碱治疗类风湿性关节炎抑制骨破坏的研究进展[J].南京中医药大学学报,2011(1):98-100.
[13]张艳艳,邢洁,庞爱梅,等.和痹方灌胃对类风湿关节炎大鼠的治疗作用及机制探讨[J].山东医药,2017,57(2):37-39.
[14]王建,庞爱梅,姜萍.和痹方联合甲氨蝶呤对CIA大鼠Th炎症平衡及血清OPG、RANKL表达的影响[J].时珍国医国药,2017,28(6):1320-1322.
[15]Metawi SA.Serum and synovial fluid levels of interleukin-17 in correlation with disease activity in patients with RA[J].Clin Rheumatol,2011,30(9):1201-1207.
[16]Ryu S,Lee JH,Kim SI.IL-17 increased the production of vascular endothelial growth factor in rheumatoid arthritis synoviocytes[J].Clin Rheumatol,2006,25(1):16-20.
[17]郭惠芳,刘淑霞,马丽艳,等.类风湿关节炎患者血清中高迁移率族蛋白1的作用及其与MMP2、TIMP2的关系[J].中国现代医学杂志,2010,20(2):192-195.
[2]张艳艳,刘伟,张超,等.基于胆碱能抗炎通路的类风湿关节炎治疗机制与中医药作用机制研究进展[J].山东中医杂志,2018(12):1038-1040.
[3]姜萍,张琳英,戴玲玲,等.和痹方治疗类风湿关节炎疗效观察[J].中国中西医结合杂志,2016(1):24-28.
[4]张艳艳,王建,姜萍,等.和痹方联合MTX治疗肝脾失调型早期类风湿关节炎疗效及对患者血清MMP-3及RANK/RANKL/OPG表达的影响[J].中国中西医结合杂志,2016(10):1197-1201.
[5]Waring JF,Abel S,Li J,et al.Analysis of gene expression profiles in rat hippocampus following treatment with nicotine and anα7 nAChR selective agonist[J].Neurosci Res,2008,60(3):266-274.
[6]黄继汉,黄晓晖,陈志扬,等.药理试验中动物间和动物与人体间的等效剂量换算[J].中国临床药理学与治疗学,2004,9(9):1069-1072.
[7]丁娟,王志军,董晓薇,等.RA滑膜成纤维细胞的原代培养及鉴定[J].现代生物医学进展,2012,12(36):7008-7011.
[8]刘宣,王炎,李丹光,等.丹参酮ⅡA对COX-2激活Wnt/β-catenin信号通路介导的人肠癌细胞VEGF表达的调控作用[J].中华中医药杂志,2013(1):108-112.
[9]韩乐,张玲玲,魏伟.胶原三螺旋重复蛋白-1参与疾病作用研究进展[J].中国药理学通报,2017,33(7):904-907.
[10]Han L,Zhang LL,Wei W.Progress in role of collagen triple helix repeat containing-1 involved in diseases[J].Chin Pharmacol Bull,2017,33(7):904-907.
[11]张利.白芍的药理作用及现代研究进展[J].中医临床研究,2014(29):25-26.
[12]丁从珠,汪悦,姚瑶,等.青藤碱治疗类风湿性关节炎抑制骨破坏的研究进展[J].南京中医药大学学报,2011(1):98-100.
[13]张艳艳,邢洁,庞爱梅,等.和痹方灌胃对类风湿关节炎大鼠的治疗作用及机制探讨[J].山东医药,2017,57(2):37-39.
[14]王建,庞爱梅,姜萍.和痹方联合甲氨蝶呤对CIA大鼠Th炎症平衡及血清OPG、RANKL表达的影响[J].时珍国医国药,2017,28(6):1320-1322.
[15]Metawi SA.Serum and synovial fluid levels of interleukin-17 in correlation with disease activity in patients with RA[J].Clin Rheumatol,2011,30(9):1201-1207.
[16]Ryu S,Lee JH,Kim SI.IL-17 increased the production of vascular endothelial growth factor in rheumatoid arthritis synoviocytes[J].Clin Rheumatol,2006,25(1):16-20.
[17]郭惠芳,刘淑霞,马丽艳,等.类风湿关节炎患者血清中高迁移率族蛋白1的作用及其与MMP2、TIMP2的关系[J].中国现代医学杂志,2010,20(2):192-195.