低强度脉冲超声调控滑膜巨噬细胞极化抑制关节滑膜炎
|
摘要
目的探讨低强度脉冲超声(low-intensity pulsed ultrasound,LIPUS)抑制关节滑膜炎的作用和可能机制。方法采用小鼠内侧半月板不稳定(destabilization of medial meniscus,DMM)关节炎模型。21只小鼠按随机数字表法分为3组:对照组、DMM组和DMM+LIPUS组。组织学分析各组膝关节滑膜病理改变,统计滑膜中iNOS或CD206阳性细胞占F4/80阳性巨噬细胞的比例。将体外培养的THP-1细胞系分为对照组、LIPUS组、LPS(lipopolysaccharide)组和LPS+LIPUS组,将Raw 264.7细胞分为LPS组和LPS+LIPUS组。采用荧光定量PCR技术检测IL-1β、TNF-α、IL-10和精氨酸酶1(Arg1)等巨噬细胞极化相关基因的表达,Western blot检测p-JNK和p-NF-κB p65蛋白表达水平;激光共聚焦显微镜观察THP-1细胞NF-κBp65核转位状态。结果在DMM关节炎模型中,LIPUS处理后的小鼠滑膜的厚度比未处理组变薄(P<0.05),滑膜中M1型巨噬细胞比例减少(P<0.05),M2型巨噬细胞比例增加(P<0.05);在LPS诱导的炎症条件下,LIPUS处理抑制THP-1和Raw 264.7细胞的M1型巨噬细胞相关基因的表达,同时促进M2型巨噬细胞相关基因的表达,抑制了p-JNK和p-NF-κB p65的蛋白表达水平(P<0.05),LIPUS抑制THP-1细胞NF-κB p65的核转位(P<0.05)。结论 LIPUS可以缓解关节滑膜炎,该作用可能与JNK和NF-κB通路介导的滑膜巨噬细胞极化有关。
Objective To investigate the therapeutic effect of low-intensity pulsed ultrasound(LIPUS) on synovitis in a mouse model of osteoarthritis and explore the possible mechanism. Methods We assessed the therapeutic effect of LIPUS on synovitis in a mouse model of destabilization of medial meniscus(DMM). The histopathology of the synovium of the knee joint was examined in 7 mice with DMM(model group), 7 normal mice(control group) and 7 mice with DMM treated with LIPUS. The proportion of iNOS-and CD206-positive cells in F4/80-positive macrophages in the synovium was detected. Cultured THP-1 macrophages were divided into control group, LIPUS group, lipopolysaccharide(LPS) group and LPS+LIPUS group, and Raw 264.7 cells were divided into LPS group and LPS+LIPUS group with corresponding treatments. The mRNA levels of polarization-related indexes(IL-1β, TNF-α, IL-10 and Arg1) were detected using real-time quantitative PCR after the treatments, and the protein levels of p-JNK and p-NF-κB p65 were detected using Western blotting. Confocal laser scanning microscopy was used to observe the nuclear translocation of nuclear factor-κB(NF-κB) in THP-1 cells after LPS or LPS+LIPUS treatment. Results In the mouse models of DMM, treatment with LIPUS significantly reduced the thicknesses of the synovium(P<0.05), decreased the proportion of M1 macrophages and upregulated the proportion of M2 macrophages in the synovium(P<0.05). In THP-1 and Raw 264.7 macrophages stimulated with LPS, LIPUS treatment significantly lowered IL-1β and TNF-α levels but increased IL-10 and Arg1 levels(P<0.05). LIPUS also decreased the protein levels of p-JNK and p-NF-κB p65 in LPS-stimulated macrophages(P<0.05) and suppressed LPS-induced nuclear translocation of NF-κB(P<0.05). Conclusion LIPUS produces anti-inflammatory effects on the synovium in the mouse model of osteoarthritis possibly by modulating the polarization of synovial macrophages through the JNK and NF-κB signaling pathways.
Objective To investigate the therapeutic effect of low-intensity pulsed ultrasound(LIPUS) on synovitis in a mouse model of osteoarthritis and explore the possible mechanism. Methods We assessed the therapeutic effect of LIPUS on synovitis in a mouse model of destabilization of medial meniscus(DMM). The histopathology of the synovium of the knee joint was examined in 7 mice with DMM(model group), 7 normal mice(control group) and 7 mice with DMM treated with LIPUS. The proportion of iNOS-and CD206-positive cells in F4/80-positive macrophages in the synovium was detected. Cultured THP-1 macrophages were divided into control group, LIPUS group, lipopolysaccharide(LPS) group and LPS+LIPUS group, and Raw 264.7 cells were divided into LPS group and LPS+LIPUS group with corresponding treatments. The mRNA levels of polarization-related indexes(IL-1β, TNF-α, IL-10 and Arg1) were detected using real-time quantitative PCR after the treatments, and the protein levels of p-JNK and p-NF-κB p65 were detected using Western blotting. Confocal laser scanning microscopy was used to observe the nuclear translocation of nuclear factor-κB(NF-κB) in THP-1 cells after LPS or LPS+LIPUS treatment. Results In the mouse models of DMM, treatment with LIPUS significantly reduced the thicknesses of the synovium(P<0.05), decreased the proportion of M1 macrophages and upregulated the proportion of M2 macrophages in the synovium(P<0.05). In THP-1 and Raw 264.7 macrophages stimulated with LPS, LIPUS treatment significantly lowered IL-1β and TNF-α levels but increased IL-10 and Arg1 levels(P<0.05). LIPUS also decreased the protein levels of p-JNK and p-NF-κB p65 in LPS-stimulated macrophages(P<0.05) and suppressed LPS-induced nuclear translocation of NF-κB(P<0.05). Conclusion LIPUS produces anti-inflammatory effects on the synovium in the mouse model of osteoarthritis possibly by modulating the polarization of synovial macrophages through the JNK and NF-κB signaling pathways.
引文
[1] GLYN-JONES S,PALMER A J,AGRICOLA R,et al.Osteoarthritis[J].Lancet,2015,386(9991):376-387.DOI:10.1016/S0140-6736(14)60802-3.
[2] HUNTER D J.Osteoarthritis[J].Ann Int Med,2007,147(3):8-16.
[3] BAY-JENSEN A C,THUDIUM C S,GUALILLO O,et al.Biochemical marker discovery,testing and evaluation for facilitating OA drug discovery and development[J].Drug Discov Today,2018,23(2):349-358.DOI:10.1016/j.drudis.2017.10.008.
[4] PRICE A J,ALVAND A,TROELSEN A,et al.Knee replacement[J].Lancet,2018,392(10158):1672-1682.DOI:10.1016/s0140-6736(18)32344-4.
[5] VAN DE GRAAF V A,NOORDUYN J C A,WILLIGENBURG N W,et al.Effect of early surgery vs physical therapy on knee function among patients with nonobstructive meniscal tears[J].JAMA,2018,320(13):1328.DOI:10.1001/jama.2018.13308.
[6] RUBIN C,BOLANDER M,RYABY J P,et al.The use of low-intensity ultrasound to accelerate the healing of fractures[J].J Bone Joint Surg Am,2001,83-A(2):259-270.
[7] ROTHENBERG J B,JAYARAM P,NAQVI U,et al.The role of low-intensity pulsed ultrasound on cartilage healing in knee osteoarthritis:A review[J].PM&R,2017,9(12):1268-1277.DOI:10.1016/j.pmrj.2017.05.008.
[8] ZHOU X Y,ZHANG XX,YU G Y,et al.Effects of low-intensity pulsed ultrasound on knee osteoarthritis:A meta-analysis of randomized clinical trials[J].Bio Med Res Int,2018,2018:1-7.DOI:10.1155/ 2018/7469197.
[9] CHUNG J I,BARUA S,CHOI B H,et al.Anti-inflammatory effect of low intensity ultrasound (LIUS) on complete Freund’s adjuvant-induced arthritis synovium[J].Osteoarthr Cartilage,2012,20(4):314-322.DOI:10.1016/j.joca.2012.01.005.
[10] NAKAMURA T,FUJIHARA S,YAMAMOTO-NAGATA K,et al.Low-intensity pulsed ultrasound reduces the inflammatory activity of synovitis[J].Ann Biomed Eng,2011,39(12):2964-2971.DOI:10.1007/s10439-011-0408-0.
[11] ZHANG X,HU B,SUN J C,et al.Inhibitory effect of low-intensity pulsed ultrasound on the expression of lipopolysaccharide-induced inflammatory factors in U937 cells[J].J Ultrasound Med,2017,36(12):2419-2429.DOI:10.1002/jum.14239.
[12] ZHOU D X,HUANG C,LIN Z,et al.Macrophage polarization and function with emphasis on the evolving roles of coordinated regulation of cellular signaling pathways[J].Cell Signal,2014,26(2):192-197.DOI:10.1016/j.cellsig.2013.11.004.
[13] PORTA C,RIMOLDI M,RAES G,et al.Tolerance and M2 (alternative) macrophage polarization are related processes orchestrated by p50 nuclear factor B[J].Proc Nat Acad Sci U S A,2009,106(35):14978-14983.DOI:10.1073/pnas.0809784106.
[14] GLASSON SS,BLANCHET T J,MORRIS E A.The surgical destabilization of the medial meniscus (DMM) model of osteoarthritis in the 129/SvEv mouse[J].Osteoarthr Cartilage,2007,15(9):1061-1069.DOI:10.1016/j.joca.2007.03.006.
[15] CHEVALIER X,EYMARD F,RICHETTE P.Biologic agents in osteoarthritis:hopes and disappointments[J].Nat Rev Rheumatol,2013,9(7):400-410.DOI:10.1038/ nrrheum.2013.44.
[16] O’NEILL T W,PARKES M J,MARICAR N,et al.Synovial tissue volume:A treatment target in knee osteoarthritis (OA)[J].Ann Rheum Dis,2016,75(1):84-90.DOI:10.1136/ annrheumdis-2014-206927.
[17] ROEMER F W,KASSIM JAVAID M,GUERMAZI A,et al.Anatomical distribution of synovitis in knee osteoarthritis and its association with joint effusion assessed on non-enhanced and contrast-enhanced MRI[J].Osteoarthr Cartilage,2010,18(10):1269-1274.DOI:10.1016/j.joca.2010.07.008.
[18] KRAUS V B,MCDANIEL G,HUEBNER J L,et al.Directin vivo evidence of activated macrophages in human osteoarthritis[J].Osteoarthr Cartil,2016,24(9):1613-1621.DOI:10.1016/j.joca.2016.04.010.
[19] BONDESON J,BLOM A B,WAINWRIGHT S,et al.The role of synovial macrophages and macrophage-produced mediators in driving inflammatory and destructive responses inosteoarthritis[J].Arthritis Rheum,2010,62(3):647-657.DOI:10.1002/art.27290.
[20] LAWRENCE T,NATOLI G.Transcriptional regulation of macrophage polarization:enabling diversity withidentity[J].Nat Rev Immunol,2011,11(11):750-761.DOI:10.1038/ nri3088.
[21] ZHANG H Y,LIN C X,ZENG C,et al.Synovial macrophage M1 polarisation exacerbates experimental osteoarthritis partially through R-spondin-2[J].Ann Rheum Dis,2018,77(10):annrheumdis-2018-213450.DOI:10.1136/annrheumdis-2018-213450.
[22] LIU B L,ZHANG M Q,ZHAO J M,et al.Imbalance of M1/M2 macrophages is linked to severity level of kneeosteoarthritis[J].Exp Ther Med,2018,16(6):5009-5014.DOI:10.3892/etm.2018.6852.
[23] MANFERDINI C,PAOLELLA F,GABUSI E,et al.Adipose stromal cells mediated switching of the pro-inflammatory profile of M1-like macrophages is facilitated by PGE2:in vitro evaluation[J].Osteoarthr Cartilage,2017,25(7):1161-1171.DOI:10.1016/j.joca.2017.01.011.
[24] DA SILVA JUNIOR E M,MESQUITA-FERRARI R A,FRAN?A C M,et al.Modulating effect of low intensity pulsed ultrasound on the phenotype of inflammatory cells[J].Biomed Pharmacother,2017,96:1147-1153.DOI:10.1016/j.biopha.2017.11.108.
[25] ZHU L N,ZHAO Q J,YANG T,et al.Cellular metabolism and macrophage functional polarization[J].Int Rev Immunol,2015,34(1):82-100.DOI:10.3109/ 08830185.2014.969421.
[26] GAO S,LI C W,ZHU Y J,et al.PEDF mediates pathological neovascularization by regulating macrophage recruitment and polarization in the mouse model of oxygen-induced retinopathy[J].Sci Rep,2017,7:42846.DOI:10.1038/ srep42846.
[27] SICA A,MANTOVANI A.Macrophage plasticity and polarization:in vivo veritas[J].J Clin Invest,2012,122(3):787-795.DOI:10.1172/JCI59643.
[28] MAYU N G,TANABE N,MANAKA S,et al.LIPUS suppressed LPS-induced IL-1α through the inhibition of NF-κB nuclear translocation via AT1-PLCβ pathway in MC3T3-E1 cells[J].J Cell Physiol,2017,232(12):3337-3346.DOI:10.1002/jcp.25777.
[29] ENGELMANN J,VITTO M F,CESCONETTO P A,et al.Pulsed ultrasound and dimethylsulfoxide gel treatment reduces the expression of pro-inflammatory molecules in an animal model of muscle injury[J].Ultrasound Med Biol,2012,38(8):1470-1475.DOI:10.1016/j.ultrasmedbio.2012.03.020.
[2] HUNTER D J.Osteoarthritis[J].Ann Int Med,2007,147(3):8-16.
[3] BAY-JENSEN A C,THUDIUM C S,GUALILLO O,et al.Biochemical marker discovery,testing and evaluation for facilitating OA drug discovery and development[J].Drug Discov Today,2018,23(2):349-358.DOI:10.1016/j.drudis.2017.10.008.
[4] PRICE A J,ALVAND A,TROELSEN A,et al.Knee replacement[J].Lancet,2018,392(10158):1672-1682.DOI:10.1016/s0140-6736(18)32344-4.
[5] VAN DE GRAAF V A,NOORDUYN J C A,WILLIGENBURG N W,et al.Effect of early surgery vs physical therapy on knee function among patients with nonobstructive meniscal tears[J].JAMA,2018,320(13):1328.DOI:10.1001/jama.2018.13308.
[6] RUBIN C,BOLANDER M,RYABY J P,et al.The use of low-intensity ultrasound to accelerate the healing of fractures[J].J Bone Joint Surg Am,2001,83-A(2):259-270.
[7] ROTHENBERG J B,JAYARAM P,NAQVI U,et al.The role of low-intensity pulsed ultrasound on cartilage healing in knee osteoarthritis:A review[J].PM&R,2017,9(12):1268-1277.DOI:10.1016/j.pmrj.2017.05.008.
[8] ZHOU X Y,ZHANG XX,YU G Y,et al.Effects of low-intensity pulsed ultrasound on knee osteoarthritis:A meta-analysis of randomized clinical trials[J].Bio Med Res Int,2018,2018:1-7.DOI:10.1155/ 2018/7469197.
[9] CHUNG J I,BARUA S,CHOI B H,et al.Anti-inflammatory effect of low intensity ultrasound (LIUS) on complete Freund’s adjuvant-induced arthritis synovium[J].Osteoarthr Cartilage,2012,20(4):314-322.DOI:10.1016/j.joca.2012.01.005.
[10] NAKAMURA T,FUJIHARA S,YAMAMOTO-NAGATA K,et al.Low-intensity pulsed ultrasound reduces the inflammatory activity of synovitis[J].Ann Biomed Eng,2011,39(12):2964-2971.DOI:10.1007/s10439-011-0408-0.
[11] ZHANG X,HU B,SUN J C,et al.Inhibitory effect of low-intensity pulsed ultrasound on the expression of lipopolysaccharide-induced inflammatory factors in U937 cells[J].J Ultrasound Med,2017,36(12):2419-2429.DOI:10.1002/jum.14239.
[12] ZHOU D X,HUANG C,LIN Z,et al.Macrophage polarization and function with emphasis on the evolving roles of coordinated regulation of cellular signaling pathways[J].Cell Signal,2014,26(2):192-197.DOI:10.1016/j.cellsig.2013.11.004.
[13] PORTA C,RIMOLDI M,RAES G,et al.Tolerance and M2 (alternative) macrophage polarization are related processes orchestrated by p50 nuclear factor B[J].Proc Nat Acad Sci U S A,2009,106(35):14978-14983.DOI:10.1073/pnas.0809784106.
[14] GLASSON SS,BLANCHET T J,MORRIS E A.The surgical destabilization of the medial meniscus (DMM) model of osteoarthritis in the 129/SvEv mouse[J].Osteoarthr Cartilage,2007,15(9):1061-1069.DOI:10.1016/j.joca.2007.03.006.
[15] CHEVALIER X,EYMARD F,RICHETTE P.Biologic agents in osteoarthritis:hopes and disappointments[J].Nat Rev Rheumatol,2013,9(7):400-410.DOI:10.1038/ nrrheum.2013.44.
[16] O’NEILL T W,PARKES M J,MARICAR N,et al.Synovial tissue volume:A treatment target in knee osteoarthritis (OA)[J].Ann Rheum Dis,2016,75(1):84-90.DOI:10.1136/ annrheumdis-2014-206927.
[17] ROEMER F W,KASSIM JAVAID M,GUERMAZI A,et al.Anatomical distribution of synovitis in knee osteoarthritis and its association with joint effusion assessed on non-enhanced and contrast-enhanced MRI[J].Osteoarthr Cartilage,2010,18(10):1269-1274.DOI:10.1016/j.joca.2010.07.008.
[18] KRAUS V B,MCDANIEL G,HUEBNER J L,et al.Directin vivo evidence of activated macrophages in human osteoarthritis[J].Osteoarthr Cartil,2016,24(9):1613-1621.DOI:10.1016/j.joca.2016.04.010.
[19] BONDESON J,BLOM A B,WAINWRIGHT S,et al.The role of synovial macrophages and macrophage-produced mediators in driving inflammatory and destructive responses inosteoarthritis[J].Arthritis Rheum,2010,62(3):647-657.DOI:10.1002/art.27290.
[20] LAWRENCE T,NATOLI G.Transcriptional regulation of macrophage polarization:enabling diversity withidentity[J].Nat Rev Immunol,2011,11(11):750-761.DOI:10.1038/ nri3088.
[21] ZHANG H Y,LIN C X,ZENG C,et al.Synovial macrophage M1 polarisation exacerbates experimental osteoarthritis partially through R-spondin-2[J].Ann Rheum Dis,2018,77(10):annrheumdis-2018-213450.DOI:10.1136/annrheumdis-2018-213450.
[22] LIU B L,ZHANG M Q,ZHAO J M,et al.Imbalance of M1/M2 macrophages is linked to severity level of kneeosteoarthritis[J].Exp Ther Med,2018,16(6):5009-5014.DOI:10.3892/etm.2018.6852.
[23] MANFERDINI C,PAOLELLA F,GABUSI E,et al.Adipose stromal cells mediated switching of the pro-inflammatory profile of M1-like macrophages is facilitated by PGE2:in vitro evaluation[J].Osteoarthr Cartilage,2017,25(7):1161-1171.DOI:10.1016/j.joca.2017.01.011.
[24] DA SILVA JUNIOR E M,MESQUITA-FERRARI R A,FRAN?A C M,et al.Modulating effect of low intensity pulsed ultrasound on the phenotype of inflammatory cells[J].Biomed Pharmacother,2017,96:1147-1153.DOI:10.1016/j.biopha.2017.11.108.
[25] ZHU L N,ZHAO Q J,YANG T,et al.Cellular metabolism and macrophage functional polarization[J].Int Rev Immunol,2015,34(1):82-100.DOI:10.3109/ 08830185.2014.969421.
[26] GAO S,LI C W,ZHU Y J,et al.PEDF mediates pathological neovascularization by regulating macrophage recruitment and polarization in the mouse model of oxygen-induced retinopathy[J].Sci Rep,2017,7:42846.DOI:10.1038/ srep42846.
[27] SICA A,MANTOVANI A.Macrophage plasticity and polarization:in vivo veritas[J].J Clin Invest,2012,122(3):787-795.DOI:10.1172/JCI59643.
[28] MAYU N G,TANABE N,MANAKA S,et al.LIPUS suppressed LPS-induced IL-1α through the inhibition of NF-κB nuclear translocation via AT1-PLCβ pathway in MC3T3-E1 cells[J].J Cell Physiol,2017,232(12):3337-3346.DOI:10.1002/jcp.25777.
[29] ENGELMANN J,VITTO M F,CESCONETTO P A,et al.Pulsed ultrasound and dimethylsulfoxide gel treatment reduces the expression of pro-inflammatory molecules in an animal model of muscle injury[J].Ultrasound Med Biol,2012,38(8):1470-1475.DOI:10.1016/j.ultrasmedbio.2012.03.020.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |