钩藤中钩藤碱对肿瘤坏死因子-α诱导人星型胶质细胞炎症模型作用机制研究
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摘要
目的研究钩藤中钩藤碱对肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)诱导人星型胶质细胞(Human Astrocytes,HA)的炎症模型的作用机制.方法:体外培养HA,用TNF-α诱导HA造模,将细胞分为正常组、模型组、钩藤碱组、依达拉奉组.采用噻唑蓝(MTT)法检测钩藤碱对HA增殖能力的影响,用逆转录PCR法(RT-PCR)检测一氧化氮合酶(nitric oxide synthase,NOS)mRNA、白细胞介素-23(interleukin-23,IL-23)mRNA、白细胞介素-6(interleukin-6,IL-6)mRNA的表达,用分光光度法检测一氧化氮(nitric oxide,NO)、超氧化物歧化酶(superoxide dismutase,SOD)、丙二醛(malondialdehyde,MDA)的表达,用蛋白免疫印迹法(Western blot)检测p-mTOR,mTOR,p-Stat3和Stat3的蛋白表达.结果与正常组比较,32ng·mL-1的TNF-α对星形胶质细胞有明显抑制作用(P<0.001);400μmol·L~(-1)的钩藤碱对星形胶质细胞活力有明显抑制作用(P<0.001).与模型组相比,200μmol·L~(-1)的钩藤碱对模型细胞有明显的增殖作用(P<0.01);钩藤碱组和依达拉奉组NOS mRNA,NO和SOD都显著上升(P<0.001),IL-23mRNA,IL-6mRNA和MDA都显著下降(P<0.001);钩藤碱组p-mTOR/mTOR和p-Stat3/Stat3都显著下降(P<0.01).结论钩藤有效成分钩藤碱具有抑制TNF-α诱导HA炎症的损伤作用,可能是通过下调mTOR/STAT3信号通路增加氧化相关因子NOS,NO,SOD的表达,减少氧化相关因子MDA以及炎症相关因子IL-23和IL-6的表达.
Objective This study aims to study the mechanism of rhynchophylline in the inflammation model of human astrocyte(HA)induced by tumor necrosis factor-α(TNF-α).Method HA was cultured in vitro,was induced by TNF-α,and the cells were divided into normal group,model group,rhynchophylline group and edaravone group.The effect of rhynchophylline on the proliferative capacity of HA was detected by MTT assay.Nitric oxide synthase(NOS)mRNA and interleukin-23(IL-23)mRNA,interleukin-6(IL-6)mRNA expression were detected by reverse transcription-PCR(RT-PCR).Determination of nitric oxide(NO),superoxide dismutase(SOD)and malondialdehyde(MDA)by spectrophotometry.The protein expression of p-mTOR,mTOR,p-Stat3 and Stat3 were detected by Western blot.Result Compared with the normal group,32 ng·mL-1 TNF-αsignificantly inhibited astrocytes viability(P<0.001)and 400μmol·L~(-1) rhynchophylline significantly inhibited astrocyte viability(P<0.001).Compared with the model group,200μmol·L~(-1) of rhynchophylline had significant proliferation effects on model cells(P<0.01);NOS mRNA,NO and SOD in the rhyndamine group and captopril group were significantly increased(P<0.001);IL-23 mRNA,IL-6 mRNA and MDA in the rhyndamine group and edaravone group were significantly decreased(P<0.001);p-mTOR/mTOR and p-Stat3/Stat3 were significantly decreased in the rhyndamine group(P<0.01)Conclusion:The active constituent of Uncaria sinensis,rhynchophylline,has the inhibitory effect of inhibiting inflammation of TNF-α-induced HA,possibly by down-regulating mTOR/STAT3 signaling pathway to increse the expression of NOS,NO and SOD in oxidative related factors,reduce the expression of MDA in oxidative related factors and IL-23 and IL-6 in inflammation-related factors.
Objective This study aims to study the mechanism of rhynchophylline in the inflammation model of human astrocyte(HA)induced by tumor necrosis factor-α(TNF-α).Method HA was cultured in vitro,was induced by TNF-α,and the cells were divided into normal group,model group,rhynchophylline group and edaravone group.The effect of rhynchophylline on the proliferative capacity of HA was detected by MTT assay.Nitric oxide synthase(NOS)mRNA and interleukin-23(IL-23)mRNA,interleukin-6(IL-6)mRNA expression were detected by reverse transcription-PCR(RT-PCR).Determination of nitric oxide(NO),superoxide dismutase(SOD)and malondialdehyde(MDA)by spectrophotometry.The protein expression of p-mTOR,mTOR,p-Stat3 and Stat3 were detected by Western blot.Result Compared with the normal group,32 ng·mL-1 TNF-αsignificantly inhibited astrocytes viability(P<0.001)and 400μmol·L~(-1) rhynchophylline significantly inhibited astrocyte viability(P<0.001).Compared with the model group,200μmol·L~(-1) of rhynchophylline had significant proliferation effects on model cells(P<0.01);NOS mRNA,NO and SOD in the rhyndamine group and captopril group were significantly increased(P<0.001);IL-23 mRNA,IL-6 mRNA and MDA in the rhyndamine group and edaravone group were significantly decreased(P<0.001);p-mTOR/mTOR and p-Stat3/Stat3 were significantly decreased in the rhyndamine group(P<0.01)Conclusion:The active constituent of Uncaria sinensis,rhynchophylline,has the inhibitory effect of inhibiting inflammation of TNF-α-induced HA,possibly by down-regulating mTOR/STAT3 signaling pathway to increse the expression of NOS,NO and SOD in oxidative related factors,reduce the expression of MDA in oxidative related factors and IL-23 and IL-6 in inflammation-related factors.
引文
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[24]吕婧.mTOR信号通路在慢性痛形成和维持中的作用及机制[D].广州:广州医科大学,2015.
[2]Qin L,Wu X,Block M L,et al.Systemic LPS causes chronic neuroinflammation and progressive neurodege neration[J].Glia,2007,55(5):453-462.
[3]Terrando N,Monaco C,Ma D,et al.Tumor necrosis factor-alpha triggers a cytokine cascade yielding postoperative cognitive decline[J].Proc Natl Acad Sci USA,2010,107(47):20518-20522.
[4]付惠群,王天龙.星形胶质细胞与老龄脑内中枢神经炎症反应[J].北京医学,2015,37(8):777-779.
[5]张瀚元,张秀英,施路一.疾病的炎症本质及其中药干预[J].西北农业学报,2017,26(1):1-13.
[6]张昕,黄李法,寿迪文,等.天麻钩藤饮对轻型颅脑损伤患者脑保护作用的临床研究[J].浙江医学,2018,40(6):557-559.
[7]黄华,丁伯平.钩藤生物碱对中枢神经系统的药理作用研究进展[J].现代药物与临床,2013,28(5):806-810.
[8]吴二兵,黄燮南,石京山,等.钩藤碱对脑缺血再灌注损伤的保护作用及其机制的实验研究[J].四川生理科学杂志,2001,23(3):121
[9]石京山,Kenneth H.钩藤碱对DA诱导NT2细胞凋亡的保护作用[J].中国药理学会通讯,2000,17(2):18.
[10]武良玉,鲍秀琦,孙华,等.星形胶质细胞上的清道夫受体与神经炎症的关系[J].中国医学科学院学报,2014,36(3):330-335.
[11]张永全,谭文澜,陆晖,等.天麻钩藤饮合美多巴治疗帕金森病62例[J].陕西中医,2008,29(6):666-667
[12]王宏献.天麻钩藤饮治疗高血压病的临床研究[J].中华中医药学刊,2008,26(2):338-340
[13]李根龙.辩证分型治疗老年痴呆120例临床观察[J].实用中医内科杂志,2013,27(4):9-11.
[14] Ramberg P,Sekino S,Uzel NG,et al.Bacterial colonization during de novo plaque formation[J].J Clin Periodontol.2003,30(11):990-995.
[15]周武庆,郑家润.一氧化氮在炎症和免疫调节中的作用[J].国外医学(皮肤性病学分册),1999,25(4):233-235.
[16]王冬梅,王久敏,刘桂芝.IL-23/IL-17炎性通路的研究进展[J].现代中西医结合杂志,2013,22(6):674-677.
[17]张伟洁,郑宏.IL-6介导免疫炎性反应作用及其与疾病关系的研究进展[J].细胞与分子免疫学杂志,2017,33(5):699-703.
[18]刘利群,毛定安,薄涛,等.川芎嗪对幼鼠惊厥性脑损伤脂质过氧化及抗氧化水平的影响[J].中国医院药学杂志,2008,28(4):253-256.
[19]付俊鲜,杨光路.醒脑静注射液联合高压氧治疗病毒性脑炎疗效及对血清SOD活性、MDA含量及脑神经损害的影响[J].现代中西医结合杂志,2016,25(32):3606-3608.
[20]李申,湛先保,许国铭.胃黏膜损伤与保护[M].基础与临床:上海,上海科学技术出版社,2004:300-309.
[21] Weichhart T.Mammalian target of rapamycin:a signaling kinase for every aspect of cellular life[J].Methods Mol Biol,2012,821:1-14.
[22] Dziennis S,Alkayed N J.Role of signal transducer and activator of transcription 3in neuronal survival and regeneration[J].Rev Neurosci,2008,19(4-5):341-361.
[23] PARK S Y,BAIK Y H,CHO J H,et al.Inhibition of lipopolysaccharide-induced nitric oxide synthesisby nicotine through S6K1-p42/44MAPK pathway and STAT3(Ser 727)phosphorylation in Raw 264.7cells[J].Cytokine,2008,44(1):126-134.
[24]吕婧.mTOR信号通路在慢性痛形成和维持中的作用及机制[D].广州:广州医科大学,2015.