miRNA-136-5p对急性脊髓损伤模型大鼠炎症因子的作用
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摘要
背景:miRNA-136-5p对脊髓损伤后病理改变、炎症反应和再生修复产生重要的调节作用。目的:观察miR NA-136-5p对大鼠脊髓损伤后血清细胞炎性因子、脊髓组织NF-κB蛋白的影响,探讨其作用分子机制。方法:SPF级雄性SD大鼠36只,购买于广西医科大学动物实验中心。制备慢病毒载体系统转染脊髓损伤大鼠。改良Allen’s法成功建立36只大鼠脊髓损伤模型,对大鼠进行Basso Beattie Brenham(BBB)评分,随机分成正常对照组、模型组(LV-ctrl+脊髓损伤组)、过表达组(脊髓损伤+LV-miRNA-136-5p)、抑制组(脊髓损伤+LV-sponge抑制组),每组9只。手术前7 d及手术当天过表达组、抑制组在损伤区连续注射慢病毒悬液,正常对照组、模型组注射等量生理盐水。各组在第1,3,7天分别处死3只大鼠,取血、脊髓组织。ELISA测定大鼠血清白细胞介素1β,白细胞介素6和干扰素α表达水平;Western Blot和双重免疫荧光检测NF-κB蛋白的表达。结果与结论:①术前BBB评分结果无显著差异(P> 0.05);脊髓损伤模型组大鼠活动表现为俯卧位缓慢行走,出现不同程度的尿潴留,双后肢的运动、感觉功能完全丧失,肌肉力量为0,出现脊髓休克;②与正常对照组相比,其他3组炎症因子水平均有升高(P <0.05),过表达组各种炎症因子水平升高更加明显;各组炎症因子水平分别按过表达组、模型组、抑制组顺序逐渐递减;③Western Blot、双重免疫荧光检测NF-κB蛋白表达水平,模型组、过表达组、抑制组均高于正常对照组(P <0.05),其中过表达组表达水平最高;④结果提示,miRNA-136-5p对急性脊髓损伤大鼠炎症因子、NF-κB可产生影响。
BACKGROUND: miRNA-136-5 p plays a crucial regulatory role in pathological changes, inflammatory response and regeneration after spinal cord injury. OBJECTIVE: To investigate the effect of miRNA-136-5 p on the expression of cytokines in serum and NF-κB protein in spinal cord in rats with spinal cord injury and to explore the molecular mechanism. METHODS: Thirty-six male Sprague-Dawley rats, of SPF grade were provided by Laboratory Animal Center of Guangxi Medical University. The lentiviral vector system was prepared and transfected into spinal cord injured rats. Thirty-six rat models of spinal cord injury were established by modified Allen's method. Basso Beattie Bresnahan scores were performed. Rats were randomly divided into normal control, modeling(LV-ctrl plus spinal cord injury), overexpression(spinal cord injury plus LV-miRNA-136-5 p), and inhibition(spinal cord injury plus LV-sponge) groups(n=9/group). Seven days before surgery and the day of surgery, the overexpression and inhibition groups were continuously injected with the lentivirus suspension into the injured area, and the normal control and modeling groups were injected with the same amount of normal saline. Three rats were sacrificed at 1, 3 and 7 days, and blood and spinal cord tissues were taken. The levels of interleukin-1β, interleukion-6 and interferon-α in rat serum were determined by ELISA. The expression of NF-κB protein was detected by western blot assay and double immunofluorescence. RESULTS AND CONCLUSION:(1) There was no significant difference in preoperative Basso Beattie Bresnahan scores(P > 0.05). In the modeling group, the rats showed prone walking, vary degrees of urinary retention, and spinal shock, with complete loss of function of both hind limbs and muscle strength of 0.(2) Compared with the normal control group, the levels of inflammatory factors in the other groups were increased significantly(P < 0.05). The expression levels of inflammatory factors were highest in the overexpression group, followed by modeling group, and lowest in the inhibition group.(3) Results of western blot assay and double immunofluorescence showed that the expression level of NF-κB protein in the modeling, overexpression and inhibition groups was significantly higher than that in the normal control group(P < 0.05), and the level was highest in the overexpression group.(4) In summary, miRNA-136-5 p can affect inflammatory factors and NF-κB in rats with acute spinal cord injury.
BACKGROUND: miRNA-136-5 p plays a crucial regulatory role in pathological changes, inflammatory response and regeneration after spinal cord injury. OBJECTIVE: To investigate the effect of miRNA-136-5 p on the expression of cytokines in serum and NF-κB protein in spinal cord in rats with spinal cord injury and to explore the molecular mechanism. METHODS: Thirty-six male Sprague-Dawley rats, of SPF grade were provided by Laboratory Animal Center of Guangxi Medical University. The lentiviral vector system was prepared and transfected into spinal cord injured rats. Thirty-six rat models of spinal cord injury were established by modified Allen's method. Basso Beattie Bresnahan scores were performed. Rats were randomly divided into normal control, modeling(LV-ctrl plus spinal cord injury), overexpression(spinal cord injury plus LV-miRNA-136-5 p), and inhibition(spinal cord injury plus LV-sponge) groups(n=9/group). Seven days before surgery and the day of surgery, the overexpression and inhibition groups were continuously injected with the lentivirus suspension into the injured area, and the normal control and modeling groups were injected with the same amount of normal saline. Three rats were sacrificed at 1, 3 and 7 days, and blood and spinal cord tissues were taken. The levels of interleukin-1β, interleukion-6 and interferon-α in rat serum were determined by ELISA. The expression of NF-κB protein was detected by western blot assay and double immunofluorescence. RESULTS AND CONCLUSION:(1) There was no significant difference in preoperative Basso Beattie Bresnahan scores(P > 0.05). In the modeling group, the rats showed prone walking, vary degrees of urinary retention, and spinal shock, with complete loss of function of both hind limbs and muscle strength of 0.(2) Compared with the normal control group, the levels of inflammatory factors in the other groups were increased significantly(P < 0.05). The expression levels of inflammatory factors were highest in the overexpression group, followed by modeling group, and lowest in the inhibition group.(3) Results of western blot assay and double immunofluorescence showed that the expression level of NF-κB protein in the modeling, overexpression and inhibition groups was significantly higher than that in the normal control group(P < 0.05), and the level was highest in the overexpression group.(4) In summary, miRNA-136-5 p can affect inflammatory factors and NF-κB in rats with acute spinal cord injury.
引文
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[18]程素利,徐家淳,王剑歌,等.急性脊髓损伤大鼠Allen's造模方法的改良及行为学评价[J].四川中医,2015,33(10):43-45.
[19]Kozomara A,Griffiths-Jones S.mi RBase:integrating micro RNA annotation and deep-sequencing data.Nucleic Acids Res.2011;39(Database issue):D152-157.
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[21]Bak M,Silahtaroglu A,M?ller M,et al.MicroRNA expression in the adult mouse central nervous system.RNA(New York,N.Y.).2008;14(3):432-444.
[22]Bartel DP.MicroRNAs:genomics,biogenesis,mechanism,and function.Cell.2004;116(2):281-297.
[23]Pauley KM,Cha S,Chan EK.MicroRNA in autoimmunity and autoimmune diseases.(New York,N.Y.)J Autoimmun.2009;32(3-4):189-194.
[24]杨俊娜,何丽,徐陶,等.miR-124在神经系统发育、损伤及修复中作用的研究进展[J].山东医药,2018,58(20):100-103.
[25]田密,丁宇,侯德仁,等.实时荧光定量PCR检测转基因阿尔茨海默病小鼠脑组织中miRNAs的差异表达[J].南方医科大学学报,2013,33(2):262-266.
[26]孔程程,杨文平,华建,等.血清miRNA-181a在帕金森病中神经保护作用及机制分析[J].南京医科大学学报(自然科学版),2017,37(9):1081-1085.
[27]Shi XZ,Zong SH,He JC,et al.miR-136-5p effect on A20expression in interleukin-17-stimulated astrocytes.Zhongguo Zuzhi Gongcheng Yanjiu.2017;21(16):2587-2592.
[28]Bortolotto V,Grilli M.Novel insights into the role of NF-κB p50in astrocyte-mediated fate specification of adult neural progenitor cells.Neural Regen Res.2017;12(3):354-357.
[29]Strickland ER,Hook MA,Balaraman S,et al.MicroRNAdysregulation following spinal cord contusion:implications for neural plasticity and repair.Neuroscience.2011;186:146-160.
[30]Aoki E,Yano R,Yokoyama H,et al.Role of nuclear transcription factor kappa B(NF-kappaB)for MPTP-induced apoptosis in nigral neurons of mice.Experimental and Molecular Pathlogy2009;86:57-64.
[31]Ahn KS,Sethi G,Aggarwal BB:Nuclear factor-kappa B:from clone to clinic.Current Mol Med 2007;7:619-637
[32]Sohn KH,Jo JM,Cho WJ,et al.Bojesodok-eum,a herbal prescription,ameliorates acute inflammation in association with the inhibition of NF-κB-mediated nitric oxide and proinflammatory cytokine production.Evid-Based Complement Alternat Med.2012:457370.
[33]Cortez M,Carmo LS,Rogero MM,et al.A high-fat diet increases IL-1,IL-6,and TNF-αproduction by increasing NF-κB and attenuating PPAR-γexpression in bone marrow mesenchymal stem cells.Inflammation.2013;36:379-386.
[34]Jobin C,Sartor RB.NF-κB signaling proteins as therapeutic targets for inflammatory bowel diseases.Inflamm Bowel Dis.2000;6:206-213.
[35]He J,Zhao J,Peng X,et al.Molecular Mechanism of MiR-136-5p Targeting NF-κB/A20 in the IL-17-Mediated Inflammatory Response after Spinal Cord Injury.Cell Physiol Biochem.2017;44(3):1224-1241.
[2]B?thig R,Fiebag K,Thietje R,et al.Morbidity of urinary tract infection after urodynamic examination of hospitalized SCIpatients:the impact of bladder management.Spinal cord.2013;51(1):70-73.
[3]梁锦前,沈建雄,邱贵兴.干细胞移植修复脊髓损伤的研究进展[J].中国脊柱脊髓杂志,2008,18(6):475478.
[4]Kwon BK,Dyomk MF,Fisher CG,et al.Spinal cord injury regene rative strategies and obstacles.Curr 0pin 0rthop.2004;15(3):196-201.
[5]Zhao T,Zhang ZN,Rong Z,et al.Immunogenicity of induced pluripotent stem cells.Nature.2011;474(7350):212-215.
[6]van Solingen C,Seghers L,Bijkerk R,et al.Antagomirmediated silencing of endothelial cell specific microRNA-126impairs ischemia-induced angiogenesis.J Cell Mol Med.2009;13(8A):1577-1585.
[7]Ma X,Zhou J,Zhong Y,et al.Expression,regulation and function of microRNAs in multiple sclerosis.Int J Med Sci.2014;11(8):810-818.
[8]Ksiazekwiniarek DJ,Kacperska MJ,Glabinski A.MicroRNAs as Novel Regulators of Neuroinflammation.Mediators Inflamm 2013;2013:519-530.
[9]Lee HK,Bier A,Cazacu S,et al.MicroRNA-145 is downregulated in glial tumorsand and regulates glioma cell migration by targeting connective tissue growth factor.Plos One.2013;8:e54652.
[10]Wang XH,Wang TL.MicroRNAs of microglia:Wrestling with central nervous system disease.Neural Regen Res.2018;13(12):2067-2072.
[11]Nishida N,Nagahara M,Sato T,et al.Microarray analysis of colorectal cancer stromal tissue reveals upregulation of two oncogenic miRNA clusters.Clin Cancer Res.2012;18(11):3054-3070.
[12]Visone R,Russo L,Pallante P,et al.MicroRNAs miR-221 and miR-222,both overexpressed in human thyroid papillary carcinomas,regulate p27Kip1 protein levels and cell cycle.Endocrine-Related Cancer.2007;14(3):791-798.
[13]Chen HY,Lin YM,Chung HC,et al.miR-103/107 promote metastasis of colorectal cancer by targeting the metastasis suppressors DAPK and KLF4.Cancer Res.2012;72(14):3631-3641.
[14]Schulte LN,Westermann AJ,Vogel J.Differential activation and functional specialization of miR-146 and miR-155 in innate immune sensing.Nucleic Acids Res.2013;41(1):542-553.
[15]Psathas JN,Doonan PJ,Raman P,et al.The Myc-miR-1792axis amplifies B-cell receptor signaling via inhibition of ITIMproteins:a novel lymphomagenic feed-forward loop.Blood.2013;122(26):4220-4229.
[16]Zardo G,Ciolfi A,Vian L,et al.Polycombs and microRNA-223regulate human granulopoiesis by transcriptional control of target gene expression.Blood.2012;119(17):4034-4046.
[17]Siddle KJ,Deschamps M,Tailleux L,et al.A genomic portrait of the genetic architecture and regulatory impact of microRNAexpression in response to infection.Genome research.2014;24(5):850-859.
[18]程素利,徐家淳,王剑歌,等.急性脊髓损伤大鼠Allen's造模方法的改良及行为学评价[J].四川中医,2015,33(10):43-45.
[19]Kozomara A,Griffiths-Jones S.mi RBase:integrating micro RNA annotation and deep-sequencing data.Nucleic Acids Res.2011;39(Database issue):D152-157.
[20]Zhou X,Jeker LT,Fife BT.Selective mi RNA disruption in Treg cells leads to uncontrolled autoimmunity.J Exp Med.2008;205(9):1983-1991.
[21]Bak M,Silahtaroglu A,M?ller M,et al.MicroRNA expression in the adult mouse central nervous system.RNA(New York,N.Y.).2008;14(3):432-444.
[22]Bartel DP.MicroRNAs:genomics,biogenesis,mechanism,and function.Cell.2004;116(2):281-297.
[23]Pauley KM,Cha S,Chan EK.MicroRNA in autoimmunity and autoimmune diseases.(New York,N.Y.)J Autoimmun.2009;32(3-4):189-194.
[24]杨俊娜,何丽,徐陶,等.miR-124在神经系统发育、损伤及修复中作用的研究进展[J].山东医药,2018,58(20):100-103.
[25]田密,丁宇,侯德仁,等.实时荧光定量PCR检测转基因阿尔茨海默病小鼠脑组织中miRNAs的差异表达[J].南方医科大学学报,2013,33(2):262-266.
[26]孔程程,杨文平,华建,等.血清miRNA-181a在帕金森病中神经保护作用及机制分析[J].南京医科大学学报(自然科学版),2017,37(9):1081-1085.
[27]Shi XZ,Zong SH,He JC,et al.miR-136-5p effect on A20expression in interleukin-17-stimulated astrocytes.Zhongguo Zuzhi Gongcheng Yanjiu.2017;21(16):2587-2592.
[28]Bortolotto V,Grilli M.Novel insights into the role of NF-κB p50in astrocyte-mediated fate specification of adult neural progenitor cells.Neural Regen Res.2017;12(3):354-357.
[29]Strickland ER,Hook MA,Balaraman S,et al.MicroRNAdysregulation following spinal cord contusion:implications for neural plasticity and repair.Neuroscience.2011;186:146-160.
[30]Aoki E,Yano R,Yokoyama H,et al.Role of nuclear transcription factor kappa B(NF-kappaB)for MPTP-induced apoptosis in nigral neurons of mice.Experimental and Molecular Pathlogy2009;86:57-64.
[31]Ahn KS,Sethi G,Aggarwal BB:Nuclear factor-kappa B:from clone to clinic.Current Mol Med 2007;7:619-637
[32]Sohn KH,Jo JM,Cho WJ,et al.Bojesodok-eum,a herbal prescription,ameliorates acute inflammation in association with the inhibition of NF-κB-mediated nitric oxide and proinflammatory cytokine production.Evid-Based Complement Alternat Med.2012:457370.
[33]Cortez M,Carmo LS,Rogero MM,et al.A high-fat diet increases IL-1,IL-6,and TNF-αproduction by increasing NF-κB and attenuating PPAR-γexpression in bone marrow mesenchymal stem cells.Inflammation.2013;36:379-386.
[34]Jobin C,Sartor RB.NF-κB signaling proteins as therapeutic targets for inflammatory bowel diseases.Inflamm Bowel Dis.2000;6:206-213.
[35]He J,Zhao J,Peng X,et al.Molecular Mechanism of MiR-136-5p Targeting NF-κB/A20 in the IL-17-Mediated Inflammatory Response after Spinal Cord Injury.Cell Physiol Biochem.2017;44(3):1224-1241.