雷帕霉素对大肠杆菌诱发的大鼠乳腺上皮细胞炎症反应的影响
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摘要
[目的]阐明雷帕霉素(Rapa)对大肠杆菌诱发的乳腺感染的保护作用机制,为临床上寻找乳腺炎防控新方法提供理论依据。[方法]原代培养大鼠乳腺上皮细胞,待细胞贴壁后,试验组采用100 nmol·L~(-1)的Rapa处理4 h后,细胞经感染比(MOI)为10的大肠杆菌处理2 h后用细胞刮刀刮取细胞,收集细胞上清液,用于Toll样受体4(TLR-4)及髓样分化因子(MyD88)蛋白、促炎性细胞因子表达及活性氧(ROS)释放检测。[结果]大肠杆菌刺激原代培养的乳腺上皮细胞后,TLR-4和MyD88蛋白表达显著升高;而Rapa预处理可降低MyD88蛋白表达;同时,大肠杆菌刺激后,乳腺上皮细胞中促炎性细胞因子,如肿瘤坏死因子α(TNF-α)、白介素1β(IL-1β)的表达及ROS的释放均显著升高。雷帕霉素能下调促炎性细胞因子的释放,显著降低ROS的释放量。[结论]Rapa抑制MyD88信号通路后可减轻大肠杆菌诱导的大鼠乳腺炎症反应。
[Objectives]The present study aimed to investigate the effect of rapamycin(Rapa)on inflammatory response in Escherichia coli(E.coli)induced rat mastitis model. [Methods]Primary mammary epithelial cells were separated and when cells fused to 80%,the monolayers in treated group were treated with 100 nmol·L~(-1) Rapa for 4 h. Then cells were infected with E.coli in logarithmic phase at a multiplicity of infection(MOI)of 10 for 2 h at 37 ℃. Cells and the supernatant were collected. [Results]The protein level of Toll like receptor 4(TLR-4)and Myeloid differentiation factor 88(MyD88)significantly increased in E.coli stimulated primary rat mammary epithelial cells while the elevated expression of MyD88 was significantly suppressed by Rapa pretreatment compared with the control. The pro-inflammatory cytokines level,such as TNF-α,IL-1β expression was all significantly up-regulated by E.coli stimulation. Besides that,ROS releasing and the level of MDA also increased and the anti-oxidant indices of SOD and T-AOC increased by E.coli stimulation. However,Rapa treatment effectively attenuated E.coli-induced inflammatory responses by suppressing the over-expression of pro-inflammatory cytokines,decreasing the release of ROS and strengthening the anti-oxidant system. [Conclusions]The treatment of Rapa abolished E.coli-induced inflammatory response by inactivating the MyD88 signaling pathway.
[Objectives]The present study aimed to investigate the effect of rapamycin(Rapa)on inflammatory response in Escherichia coli(E.coli)induced rat mastitis model. [Methods]Primary mammary epithelial cells were separated and when cells fused to 80%,the monolayers in treated group were treated with 100 nmol·L~(-1) Rapa for 4 h. Then cells were infected with E.coli in logarithmic phase at a multiplicity of infection(MOI)of 10 for 2 h at 37 ℃. Cells and the supernatant were collected. [Results]The protein level of Toll like receptor 4(TLR-4)and Myeloid differentiation factor 88(MyD88)significantly increased in E.coli stimulated primary rat mammary epithelial cells while the elevated expression of MyD88 was significantly suppressed by Rapa pretreatment compared with the control. The pro-inflammatory cytokines level,such as TNF-α,IL-1β expression was all significantly up-regulated by E.coli stimulation. Besides that,ROS releasing and the level of MDA also increased and the anti-oxidant indices of SOD and T-AOC increased by E.coli stimulation. However,Rapa treatment effectively attenuated E.coli-induced inflammatory responses by suppressing the over-expression of pro-inflammatory cytokines,decreasing the release of ROS and strengthening the anti-oxidant system. [Conclusions]The treatment of Rapa abolished E.coli-induced inflammatory response by inactivating the MyD88 signaling pathway.
引文
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[22] Higashimoto T,Panopoulos A,Hsieh CL,et al.TNF-α induces chromosomal abnormalities independent of ROS through IKK,JNK,p38 and caspase pathways[J].Cytokine,2006,34:39-50.
[2] Li J,Kim S G,Blenis J.Rapamycin:one drug,many effects[J].Cell Metabolism,2014,19:373-379.
[3] Wang Y,Chen J,Tang W Q,et al.Rapamycin inhibits the proliferation of endothelial cells in hemangioma by blocking the mTOR-FABP4 pathway[J].Biomedicine & Pharmacotherapy,2017,85:272-279.
[4] Zheng L H,Xu Y Y,Lu J Y,et al.Variant innate immune responses of mammary epithelial cells to challenge by Staphylococcus aureus,Escherichia coli and the regulating effect of taurine on these bioprocesses[J].Free Radical Biology & Medicine,2016,96:166-180.
[5] Liu S,Shi X,Bauer I,et al.Lingual antimicrobial peptide and IL-8 expression are oppositely regulated by the antagonistic effects of NF-κB p65 and C/EBPβ in mammary epithelial cells[J].Microbiology and Immunology,2011,48:895-908.
[6] Strandberg Y,Gary C,Vuocolo T,et al.Lipopolysaccharide and lipoteichoic acid induce different innate immune responses in bovine mammary epithelial cells[J].Cytokine,2005,31:72-86.
[7] Gu B B,Miao J F,Fa Y M,et al.Retinoid attenuates lipopolysaccharide-induced inflammatory responses by suppressing TLR4/NF-κB expression in rat mammary tissue[J].Int Immunopharmacol,2010,10(7):799-805.
[8] Miao J,Zheng L,Zhang J,et al.The effect of taurine on the toll-like receptors/nuclear factor kappa B(TLRs/NF-κB)signaling pathway in Streptococcus uberis-induced mastitis in rats[J].Int Immunopharmacol,2011,11:1740-1746.
[9] Fu H,Gao R,Gao Y,et al.Curcumin attenuates inflammatory responses by suppressing TLR4-mediated NF-κB signaling pathway in lipopolysaccharide-induced mastitis in mice[J].Int Immunopharmacol,2014,20:54-58.
[10] Lorraine M S,Katie L S.Mamammary gland immunity and mastitis susceptibility[J].J Mammary Gland Biol,2002,7(2):135-146.
[11] Bradley A J.Bovine mastitis:an evolving disease[J].Vet J,2002,164:116-128.
[12] Rinaldi M,Li R W,Capuco A V.Mastitis associated transcriptomic disruptions in cattle[J].Vet Immunol Immunopathol,2010,138:267-279.
[13] 贺鹏.PM2.5对大鼠变应性鼻炎鼻黏膜上皮中TLR2/4及mTOR信号通路表达的影响[D].长春:吉林大学,2017.He P.Effect of PM2.5 on TLR2/4 and mTOR signaling pathway expression in nasal epithelium of rats with allergic rhinitis[D].Changchun:Jilin University,2017(in Chinese with English abstract).
[14] Zhao J,Benakanakere M R,Hosur K B,et al.Mammalian target of rapamycin(mTOR)regulates TLR3 induced cytokines in human oral keratinocytes[J].Molecular Immunology,2010,48(1/2/3):294-304.
[15] Lorne E,Zhao X,Zmijewski J W,et al.Participation of mammalian target of rapamycin complex 1 in Toll-like receptor 2- and 4-induced neutrophil activation and acute lung injury[J].American Journal of Respiratory Cell & Molecular Biology,2009,41(2):237-245.
[16] Sun R,Zhang Y,Ma S,et al.Down-regulation of mitogen-activated protein jinases and nuclear-κB signaling is involved in rapamycin suppression of TLR2-induced inflammatory response in monocytic THP-1 cells[J].Microbiol Immunol,2015,59(10):614-622.
[17] Schmitz F,Heit A,Dreher S,et al.Mammalian target of rapamycin(mTOR)orchestrates the defense program of innate immune cells[J].European Journal of Immunology,2008,38(1):2981-2992.
[18] Wakkad A E I,Nel-M H,Sibaii H,et al.Proinflammatory,anti-inflammatory cytokines and adiponkines in students with central obesity[J].Cytokine,2013,61(2):682-687.
[19] Atwell D M,Grichnik K P,Newman M F,et al.Balance of proinflammatory and antiinflammatory cytokines at thoracic cancer operation:an important conceot in modern anesthesia[J].The Annals of Thoracic Surgery,1998,68(4):1134-1150.
[20] Oguma K.In vitro effect of recombinat human tumor necrosis factor on canine neutrophil apoptosis[J].Research in Veterinary Science,2006,80(2):162-166.
[21] Ho W Y,Wong C K,Lam C W K.Tumor necrosis factor-α up-regulates the expression of CCL2 and adhesion molecules of human proximal tubular epithelial cells through MAPK signaling pathways[J].Immunobiology,2008,213:533-544.
[22] Higashimoto T,Panopoulos A,Hsieh CL,et al.TNF-α induces chromosomal abnormalities independent of ROS through IKK,JNK,p38 and caspase pathways[J].Cytokine,2006,34:39-50.