青蒿琥酯通过诱导HO-1产生对结核分枝杆菌诱导的THP-1细胞TNF-α分泌的影响
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摘要
目的探讨青蒿琥酯(ASN)诱导HO-1产生对结核分枝杆菌(Mtb)诱导的THP-1细胞分泌细胞因子肿瘤坏死因子α(TNF-α)的影响并探讨其可能的作用机制。方法体外培养THP-1细胞,将细胞分为对照组、ASN组、Mtb组、ASN预处理组及Zn PP预处理组。ASN预处理组为浓度0～20μg/ml ASN预处理THP-1细胞4 h,随后加入Mtb(感染复数MOI=10)刺激24 h。锌原卟啉(Zn PP)预处理组为10μmol/L Zn PP抑制剂与THP-1预处理4 h,随后加入Mtb(MOI=10)刺激24 h。MTT法检测ASN对细胞的活性影响。RT-q PCR检测细胞因子TNF-α、核因子E2相关因子2(Nrf2)及血红素氧合酶1(HO-1) mRNA表达; ELISA检测TNF-α蛋白分泌。结果 MTT法检测ASN浓度在0～20μg/ml范围内对细胞无毒性;与对照组相比,Mtb显著增加TNF-αmRNA及蛋白表达,而5～20μg/ml ASN组呈浓度依赖性抑制TNF-αmRNA及蛋白表达(P <0. 05)。此外,ASN能上调Nrf2及HO-1mRNA表达,HO-1竞争性抑制剂Zn PP能增加TNF-α蛋白分泌。结论 ASN通过诱导HO-1产生抑制Mtb诱导的炎症反应。
Objective To investigate the effect of artesunate( ASN) on the secretion of cytokine TNF-α in THP-1 cells induced by Mtb and to investigate its possible mechanism. Methods THP-1 cells were cultured in vitro. The cells were divided into five groups. THP-1 cells were pretreated with 0 ～ 20 μg/ml ASN for 4 h and then stimulated with Mtb( MOI = 10) for 24 h as ASN pretreatment group. THP-1 cells were pretreated with 10 μmol/L ZnPP inhibitor for 4 h and then stimulated with Mtb( MOI = 10) for 24 h as Zn PP pretreatment group. MTT assay was used to detect the effect of ASN on the activity of cells. RT-qPCR was used to detect the mRNA expression of TNF-α,NF-E2-related factor 2( Nrf2) and Heme oxygenase-1( HO-1). ELISA was used to detect the secretion of TNF-α protein. Results There was no cytotoxicity to cells dealt with the concentrations of 0 ～ 20 μg/ml of ASN detected by MTT. Compared with the control group,Mtb significantly increased mRNA and protein expression of TNF-α. ASN could inhibit the secretion of TNF-α at mRNA and protein level in a dose-dependent manner( P < 0. 05). In addition,ASN could up-regulate the mRNA expression of Nrf2 and HO-1. Conclusion ASN can inhibit the inflammatory responses induced by Mtb through up-regulation of HO-1 expression.
Objective To investigate the effect of artesunate( ASN) on the secretion of cytokine TNF-α in THP-1 cells induced by Mtb and to investigate its possible mechanism. Methods THP-1 cells were cultured in vitro. The cells were divided into five groups. THP-1 cells were pretreated with 0 ～ 20 μg/ml ASN for 4 h and then stimulated with Mtb( MOI = 10) for 24 h as ASN pretreatment group. THP-1 cells were pretreated with 10 μmol/L ZnPP inhibitor for 4 h and then stimulated with Mtb( MOI = 10) for 24 h as Zn PP pretreatment group. MTT assay was used to detect the effect of ASN on the activity of cells. RT-qPCR was used to detect the mRNA expression of TNF-α,NF-E2-related factor 2( Nrf2) and Heme oxygenase-1( HO-1). ELISA was used to detect the secretion of TNF-α protein. Results There was no cytotoxicity to cells dealt with the concentrations of 0 ～ 20 μg/ml of ASN detected by MTT. Compared with the control group,Mtb significantly increased mRNA and protein expression of TNF-α. ASN could inhibit the secretion of TNF-α at mRNA and protein level in a dose-dependent manner( P < 0. 05). In addition,ASN could up-regulate the mRNA expression of Nrf2 and HO-1. Conclusion ASN can inhibit the inflammatory responses induced by Mtb through up-regulation of HO-1 expression.
引文
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[2]Chakraborty P,Kulkarni S,Rajan R,et al.Drug resistant clinical isolates of Mycobacterium tuberculosis from different genotypes exhibit differential host responses in THP-1 cells[J].PLo S One,2013,8(5):e62966.
[3]Li B,Li J,Pan X,et al.Artesunate protects sepsis model mice challenged with Staphylococcus aureus by decreasing TNF-alpha release via inhibition TLR2 and Nod2 mRNA expressions and transcription factor NF-kappa B activation[J].Int Immunopharmacol,2010,10(3):344-350.
[4]Regev D,Surolia R,Karki S,et al.Heme oxygenase-1 promotes granuloma development and protects against dissemination of mycobacteria[J].Lab Invest,2012,92(11):1541-52.
[5]Silva-Gomes S,Appelberg R,Larsen R,et al.Heme catabolism by heme oxygenase-1 confers host resistance to Mycobacterium infection[J].Infect Immun,2013,81(7):2536-45.
[6]Rushworth SA,Mac Ewan DJ,O'Connell MA.Lipopolysaccharide-induced expression of NAD(P)H:quinone oxidoreductase 1 and heme oxygenase-1 protects against excessive inflammatory responses in human monocytes[J].J Immunol,2008,181(10):6730-6737.
[7]Choi WH.Novel Pharmacological Activity of Artesunate and Artemisinin:Their Potential as Anti-Tubercular Agents[J].J Clin Med,2017,6(3).
[8]Sato K,Kawakami K.Recognition of Cryptococcus neoformans by Pattern Recognition Receptors and its Role in Host Defense to This Infection[J].Med Mycol J,2017,58(3):J83-J90.
[9]Posadas I,Romero-Castillo L,El Brahmi N,et al.Neutral highgeneration phosphorus dendrimers inhibit macrophage-mediated inflammatory response in vitro and in vivo[J].Proc Natl Acad Sci U S A,2017,114(37):E7660-E7669.
[10]Zhang B,Wang B,Cao S,et al.Silybin attenuates LPS-induced lung injury in mice by inhibiting NF-κB signaling and NLRP3 activation[J].Int J Mol Med,2017,39(5):1111-1118.
[11]Ma X,You X,Zeng Y,et al.Mycoplasma fermentans MALP-2 induces heme oxygenase-1 expression via mitogen-activated protein kinases and Nrf2 pathways to modulate cyclooxygenase 2 expression in human monocytes[J].Clin Vaccine Immunol,2013,20(6):827-834.
[12]Park SY,Kim YH,Kim EK,et al.Heme oxygenase-1 signals are involved in preferential inhibition of pro-inflammatory cytokine release by surfactin in cells activated with Porphyromonas gingivalis lipopolysaccharide[J].Chem Biol Interact,2010,188(3):437-445.
[13]Li B,Zhang R,Li J,et al.Antimalarial artesunate protects sepsis model mice against heat-killed Escherichia coli challenge by decreasing TLR4,TLR9 mRNA expressions and transcription factor NF-kappa B activation[J].Int Immunopharmacol,2008,8(3):379-389.
[14]Zhao D,Zhang J,Xu G,et al.Artesunate Protects LPS-Induced A-cute Lung Injury by Inhibiting TLR4 Expression and Inducing Nrf2 Activation[J].Inflammation,2017,40(3):798-805.