双膦酸盐纳米乳在大鼠实验性自身免疫性神经炎中的应用
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摘要
目的:探讨双膦酸盐(bisphosphonate nanoemulsion,BP)纳米乳对实验性自身免疫性神经炎(experimental autoimmune neuritis,EAN)的治疗作用。方法:BP纳米乳体外处理巨噬细胞系RAW264.7,实时定量PCR(real-time PCR,RT-PCR)检测炎症因子白介素(interleukin,IL)-1β、IL-6、诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)及抑炎因子IL-10、巨噬细胞M2型标记物CD206的表达。构建EAN大鼠模型,给予BP纳米乳处理,测大鼠体重,分析机械疼痛阈值及临床评分,劳克坚牢蓝(luxol fast blue,LFB)染色检测坐骨神经髓鞘病变,免疫组化观察其炎性细胞浸润及病理改变;RT-PCR检测EAN大鼠坐骨神经IL-1β、IL-17、i NOS及基质金属蛋白酶9(matrix metalloproteinase 9,MMP-9)等炎症因子表达。结果:BP与BP纳米乳体外处理巨噬细胞,均可降低IL-1β、iNOS,同时提高IL-10和CD206的表达,促进M1型巨噬细胞向M2型转化。BP纳米乳干预的EAN大鼠体重下降减轻、临床评分降低,机械性疼痛程度缓解,病程明显缩短。同时坐骨神经炎性细胞浸润及髓鞘病变显著减轻,IL-1β、IL-17、i NOS、MMP-9表达降低。结论:BP纳米乳可能通过减轻大鼠坐骨神经炎性细胞浸润及抑制促炎因子表达,来缓解EAN的临床病理变化,提示BP纳米乳可作为神经炎性病变的潜在治疗药物。
Objective:To test potential therapeutic effects of bisphosphonate nanoemulsion(BP nanoemulsion)in the experimental autoimmune neuritis(EAN). Methods:In vitro BP nanoemulsion treatment was performed in macrophage cell line RAW264.7,realtime PCR(RT-PCR)was used to evaluate the expression of inflammatory factors interleukin(IL)-1β,IL-6,inducible nitric oxide synthase(iNOS),anti-inflammatory factor IL-10 and macrophage M2 marker CD206. After successfully establishing EAN rats model,BP nanoemulsion treatment was given,weight change of rats was assessed. The mechanical pain threshold and clinical score were analyzed. Luxol fast blue(LFB)staining was applied to show sciatic myelinopathy. Inflammatory cell infiltration and pathological changes in the sciatic nerve were evaluated by immunohistochemistry. The expressions of inflammatory cytokines IL-1β,IL-17,iNOS and matrix metalloproteinase 9(MMP-9)m RNA level in sciatic nerve were detected by RT-PCR. Results:BP and BP nanoemulsion treatments inhibited IL-1β and iNOS,increased the expressions of IL-10 and CD206,and induced phenotypic switch of macrophage polarization from M1 to M2 subtype in cell culture. In EAN rats:BP nanoemulsion ameliorated body weight loss and neurological signs,ameliorated mechanical allodynia,shortened disease duration,reduced myelin lesions. Meanwhile,it inhibited accumulation of macrophages and other immune cells,and decreased expressions of inflammatory cytokines IL-1β,IL-17,iNOS,and MMP-9 mRNA level in sciatic nerves of EAN rats. Conclusion:BP nanoemulsion can reduce accumulation of immune cells and attenuated inflammatory cytokines in sciatic nerves,and improve the clinical pathological changes of EAN rats. BP nanoemulsion may therefore be considered a potential therapeutic option for neuroinflammatory diseases.
Objective:To test potential therapeutic effects of bisphosphonate nanoemulsion(BP nanoemulsion)in the experimental autoimmune neuritis(EAN). Methods:In vitro BP nanoemulsion treatment was performed in macrophage cell line RAW264.7,realtime PCR(RT-PCR)was used to evaluate the expression of inflammatory factors interleukin(IL)-1β,IL-6,inducible nitric oxide synthase(iNOS),anti-inflammatory factor IL-10 and macrophage M2 marker CD206. After successfully establishing EAN rats model,BP nanoemulsion treatment was given,weight change of rats was assessed. The mechanical pain threshold and clinical score were analyzed. Luxol fast blue(LFB)staining was applied to show sciatic myelinopathy. Inflammatory cell infiltration and pathological changes in the sciatic nerve were evaluated by immunohistochemistry. The expressions of inflammatory cytokines IL-1β,IL-17,iNOS and matrix metalloproteinase 9(MMP-9)m RNA level in sciatic nerve were detected by RT-PCR. Results:BP and BP nanoemulsion treatments inhibited IL-1β and iNOS,increased the expressions of IL-10 and CD206,and induced phenotypic switch of macrophage polarization from M1 to M2 subtype in cell culture. In EAN rats:BP nanoemulsion ameliorated body weight loss and neurological signs,ameliorated mechanical allodynia,shortened disease duration,reduced myelin lesions. Meanwhile,it inhibited accumulation of macrophages and other immune cells,and decreased expressions of inflammatory cytokines IL-1β,IL-17,iNOS,and MMP-9 mRNA level in sciatic nerves of EAN rats. Conclusion:BP nanoemulsion can reduce accumulation of immune cells and attenuated inflammatory cytokines in sciatic nerves,and improve the clinical pathological changes of EAN rats. BP nanoemulsion may therefore be considered a potential therapeutic option for neuroinflammatory diseases.
引文
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[19] Liu X. Bone site-specific delivery of siRNA[J]. Journal ofBiomedical Research,2016,30(4):264-271
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[23] Sambataro G,Sambataro D,Polosa R. Emerging potentialfor bisphosphonates in the treatment of axial spondyloar-thritis[J]. Ther Adv Chronic Dis,2017,8(6-7):97-99
[24] Palmieri B,Rottigni V,Iannitti T. Preliminary study of highly cross-linked hyaluronic acid-based combination therapy for management of knee osteoarthritis-related pain[J]. Drug Des Devel Ther,2013,7:7-12
[25] Kim JH,Kang HM,Yu SB,et al. Cytoprotective effect of flavonoid-induced autophagy on bisphosphonate mediated cell death in osteoblast[J]. 2018,119(7):5571-5580
[26] Niebel W,Walkenbach K,Beduneau A,et al. Nanoparticle-based clodronate delivery mitigates murine experimental colitis[J]. J Control Release,2012,160(3):659-665
[27] Li C,Ding XY,Xiang DM,et al. Enhanced M1 and Impaired M2 macrophage polarization and reduced mitochondrial biogenesis via inhibition of AMP kinase in chronic kidney disease[J]. Cell Physiol Biochem,2015,36(1):358-372
[28] Zhang ZY,Zhang Z,Schluesener HJ. FTY720 attenuates lesional interleukin-17(+)cell accumulation in rat experimental autoimmune neuritis[J]. Neuropathol Appl Neurobiol,2009,35(5):487-495
[29] Yi C,Zhang Z,Wang W,et al. Doxycycline attenuates peripheral inflammation in rat experimental autoimmune neuritis[J]. Neurochem Res,2011,36(11):1984-1990
[2] Ding Y,Han R,Jiang W,et al. Programmed death ligand1 plays a neuroprotective role in experimental autoim-mune neuritis by controlling peripheral nervous system In-flammation of rats[J]. J Immunol,2016,197(10):3831-3840
[3] Soliven B. Animal models of autoimmune neuropathy[J].ILAR J,2014,54(3):282-290
[4] Shen D,Chu F,Lang Y,et al. Beneficial or harmful roleof macrophages in Guillain-Barre syndrome and experi-mental autoimmune neuritis[J]. Mediators Inflamm,2018,2018:4286364
[5] Iannitti T,Rosini S,Lodi D,et al. Bisphosphonates:focuson inflammation and bone loss[J]. Am J Ther,2012,19(3):228-246
[6] D’Antonio C,Passaro A,Gori B,et al. Bone and brain me-tastasis in lung cancer:recent advances in therapeuticstrategies[J]. Ther Adv Med Oncol,2014,6(3):101-114
[7] Xu Y,He Z,Li Z,et al. Irgm1 is required for the inflam-matory function of M1 macrophage in early experimentalautoimmune encephalomyelitis[J]. J Leukoc Biol,2017,101(2):507-517
[8] Katzav A,Bina H,Aronovich R,et al. Treatment for exper-imental autoimmune neuritis with clodronate(Bonefos)[J]. Immunol Res,2013,56(2-3):334-340
[9] Ganta S,Deshpande D,Korde A,et al. A review of multi-functional nanoemulsion systems to overcome oral andCNS drug delivery barriers[J]. Mol Membr Biol,2010,27(7):260-273
[10] Yang P,Bo S,Qin G,et al. Caspase-1 inhibition attenu-ates activation of BV2 microglia induced by LPS-treatedRAW264.7 macrophages[J]. J Biomed Res,2016,30(3):225-233
[11] Zhang Z,Zhang ZY,Schluesener HJ. Compound A,aplant origin ligand of glucocorticoid receptors,increasesregulatory T cells and M2 macrophages to attenuate exper-imental autoimmune neuritis with reduced side effects[J]. J Immunol,2009,183(5):3081-3091
[12] Oaklander AL,Lunn MP,Hughes RA,et al. Treatmentsfor chronic inflammatory demyelinating polyradiculoneu-ropathy(CIDP):an overview of systematic reviews[J]. Co-chrane Database Syst Rev,2017,1:Cd010369
[13]Leger JM,Guimaraes-Costa R,Muntean C. Immunothera-py in peripheral neuropathies[J]. Neurotherapeutics,2016,13(1):96-107
[14] Liu Y,Luo B,Han F,et al. Erythropoietin-derived nonery-thropoietic peptide ameliorates experimental autoimmuneneuritis by inflammation suppression and tissue protection[J]. PLoS One,2014,9(3):e90942
[15] Kuwabara S,Yuki N. Axonal Guillain-Barre syndrome:concepts and controversies[J]. Lancet Neurol,2013,12(12):1180-1188
[16] Pitarokoili K,Ambrosius B,Meyer D,et al. Dimethyl fu-marate ameliorates lewis rat experimental autoimmuneneuritis and mediates axonal protection[J]. PLoS One,2015,10(11):e0143416
[17] Maurer M,Toyka KV,Gold R. Cellular immunity in in-flammatory autoimmune neuropathies[J]. Rev Neurol(Paris),2002,158(12 Pt 2):S7-15
[18] Han R,Xiao J,Zhai H,et al. Dimethyl fumarate attenu-ates experimental autoimmune neuritis through the nucle-ar factor erythroid-derived 2-related factor 2/hemoxygen-ase-1 pathway by altering the balance of M1/M2 macro-phages[J]. J Neuroinflammation,2016,13(1):97
[19] Liu X. Bone site-specific delivery of siRNA[J]. Journal ofBiomedical Research,2016,30(4):264-271
[20]Allavena P,Mantovani A. Immunology in the clinic reviewseries;focus on cancer:tumour-associated macrophages:undisputed stars of the inflammatory tumour microenviron-ment[J]. Clin Exp Immunol,2012,167(2):195-205
[21] Zaslona Z,Przybranowski S,Wilke C,et al. Resident alve-olar macrophages suppress,whereas recruited monocytespromote,allergic lung inflammation in murine models ofasthma[J]. J Immunol,2014,193(8):4245-4253
[22]Perez-Rial S,del Puerto-Nevado L,Terron-Exposito R,etal. Role of recently migrated monocytes in cigarette smoke-induced lung inflammation in different strain of mice[J].PLoS One,2013,8(9):e72975
[23] Sambataro G,Sambataro D,Polosa R. Emerging potentialfor bisphosphonates in the treatment of axial spondyloar-thritis[J]. Ther Adv Chronic Dis,2017,8(6-7):97-99
[24] Palmieri B,Rottigni V,Iannitti T. Preliminary study of highly cross-linked hyaluronic acid-based combination therapy for management of knee osteoarthritis-related pain[J]. Drug Des Devel Ther,2013,7:7-12
[25] Kim JH,Kang HM,Yu SB,et al. Cytoprotective effect of flavonoid-induced autophagy on bisphosphonate mediated cell death in osteoblast[J]. 2018,119(7):5571-5580
[26] Niebel W,Walkenbach K,Beduneau A,et al. Nanoparticle-based clodronate delivery mitigates murine experimental colitis[J]. J Control Release,2012,160(3):659-665
[27] Li C,Ding XY,Xiang DM,et al. Enhanced M1 and Impaired M2 macrophage polarization and reduced mitochondrial biogenesis via inhibition of AMP kinase in chronic kidney disease[J]. Cell Physiol Biochem,2015,36(1):358-372
[28] Zhang ZY,Zhang Z,Schluesener HJ. FTY720 attenuates lesional interleukin-17(+)cell accumulation in rat experimental autoimmune neuritis[J]. Neuropathol Appl Neurobiol,2009,35(5):487-495
[29] Yi C,Zhang Z,Wang W,et al. Doxycycline attenuates peripheral inflammation in rat experimental autoimmune neuritis[J]. Neurochem Res,2011,36(11):1984-1990