miRNA-146a-5p对癫痫大鼠海马神经元NF-κB信号转导通路的影响
|
摘要
目的观察miRNA-146a-5p(miR-146a-5p)对癫痫大鼠海马神经元核因子κB(NF-κB)信号转导通路的影响。方法体外培养新生SD大鼠海马神经元,随后制备癫痫海马神经元模型,随机分为三组,空白对照组(control组):转染时添加等体积opti-MEM培养基;阴性对照组(NC组):转染80 n M浓度NC至癫痫大鼠海马神经元; miR-146a-5p组:转染80 n M浓度miR-146a-5p mimics至癫痫大鼠海马神经元。转染后培养24 h,再用200 ng/ml脂多糖(LPS)诱导6 h,采用免疫荧光双染法鉴定体外培养海马神经元,采用膜片钳技术检测正常大鼠海马神经元和癫痫大鼠海马神经元自发性放电频率,采用逆转录-聚合酶链反应(RT-PCR)检测未经LPS诱导各组海马神经元miR-146a-5p表达水平及经LPS诱导后各组海马神经元核因子κB抑制蛋白α(IκBα)、磷酸化IκBα(p IκBα)、NF-κB P65、IKB激酶-β(IKKβ) mRNA表达水平,采用Western blot法检测经LPS诱导后各组海马神经元IκBα、p IκBα、NF-κB P65、IKKβ蛋白表达水平,采用酶联免疫吸附(ELISA)法检测经LPS诱导后各组细胞培养液中肿瘤坏死因子α(TNF-α)、白细胞介素1(IL-1)、白细胞介素6(IL-6)、细胞间黏附分子1(ICAM-1)表达水平。结果体外培养7 d的癫痫大鼠海马神经元与正常大鼠海马神经元相比较形态未见明显异常;癫痫大鼠海马神经元自发性放电频率明显较正常大鼠海马神经元明显增加(P <0. 05); miR-146a-5p组海马神经元miR-146a-5p表达水平明显高于control组和NC组(P <0. 05);经LPS诱导后miR-146a-5p组海马神经元p IκBα、NF-κB P65、IKKβmRNA及蛋白表达水平明显低于control组和NC组(P <0. 05),而IκBαmRNA及蛋白表达水平明显高于control组和NC组(P <0. 05);经LPS诱导后miR-146a-5p组细胞培养液中TNF-α、IL-1、IL-6、ICAM-1表达水平明显低于control组和NC组(P <0. 05)。结论miR-146a-5p可抑制LPS诱导的癫痫大鼠海马神经元NF-κB信号转导通路中p IκBα、NF-κB P65、IKKβmRNA及蛋白表达,促进IκBαmRNA及蛋白表达,减少TNF-α、IL-1、IL-6、ICAM-1等炎症因子的释放。
Objective To observe the effect of miRNA-146 a-5 p on the signal transduction pathway of NF-κB in hippocampal neurons of epileptic rats. Methods The hippocampal neurons of neonatal SD rats were cultured in vitro,and then the epileptic hippocampal neuron model was prepared. The rats were randomly divided into three groups,the blank control group( control group) : the same volume opti-MEM medium was added at the time of transfection,and negative control group( NC group) : 80 nM concentration NC was transfected into hippocampal neurons of epileptic rats,and miR-146 a-5 p group: 80 nM miR-146 a-5 p mimics was transfected into hippocampal neurons of epileptic rats. After transfection,they were cultured for 24 hours,and then induced by 200 ng/ml LPS for 6 hours. The cultured hippocampal neurons were identified by immunofluorescence double staining. The spontaneous firing frequency of hippocampal neurons in normal rats and epileptic rats was detected by patch clamp technique. The expression levels of miR-146 a-5 p in the hippocampal neurons in each group without LPS induction and the expression levels of IκBα,pIκBα,NF-κB P65,IKKβ mRNA in hippocampal neurons of each group after LPS induction were detected by reverse transcription-polymerase chain reaction( RT-PCR). The expression levels of IκBα,pIκBα,NF-κB P65,IKKβ protein in hippocampal neurons of each group after LPS induction were detected by Western blot. The expression levels of TNF-α,IL-1,IL-6,ICAM-1 in LPS-induced cell cultures were determined by ELISA. Results There was no obvious abnormality between the morphology of hippocampal neurons of epileptic rats cultured in vitro for 7 days and that of normal rats. The spontaneous discharge frequency of hippocampal neurons of epileptic rats significantly increased than that of normal rats( P < 0. 05). The expression level of miR-146 a-5 p in hippocampal neurons of miR-146 a-5 p group was significantly higher than that of control group and NC group( P < 0. 05). The expression levels of pIκBα,NF-κB P65,IKKβ mRNA and protein in the hippocampal neurons of themiR-146 a-5 p group induced by LPS were significantly lower than those of the control group and NC group( P < 0. 05),while the expression levels of IκBα mRNA and protein were significantly higher than those of the control group and NC group( P< 0. 05). The expression levels of TNF-α,IL-1,IL-6,ICAM-1 in the cell culture medium of the miR-146 a-5 p group induced by LPS were significantly higher than those of the control group and NC group( P < 0. 05). Conclusion MiR-146 a-5 p can inhibit the expression of pIκBα,NF-κB P65,IKKβ mRNA and protein in the NF-κB signal transduction pathway in hippocampal neurons of epileptic rats induced by LPS,promote the expression of IκBα mRNA and protein,and reduce the release of inflammatory factors such as TNF-α,IL-1,IL-6 and ICAM-1.
Objective To observe the effect of miRNA-146 a-5 p on the signal transduction pathway of NF-κB in hippocampal neurons of epileptic rats. Methods The hippocampal neurons of neonatal SD rats were cultured in vitro,and then the epileptic hippocampal neuron model was prepared. The rats were randomly divided into three groups,the blank control group( control group) : the same volume opti-MEM medium was added at the time of transfection,and negative control group( NC group) : 80 nM concentration NC was transfected into hippocampal neurons of epileptic rats,and miR-146 a-5 p group: 80 nM miR-146 a-5 p mimics was transfected into hippocampal neurons of epileptic rats. After transfection,they were cultured for 24 hours,and then induced by 200 ng/ml LPS for 6 hours. The cultured hippocampal neurons were identified by immunofluorescence double staining. The spontaneous firing frequency of hippocampal neurons in normal rats and epileptic rats was detected by patch clamp technique. The expression levels of miR-146 a-5 p in the hippocampal neurons in each group without LPS induction and the expression levels of IκBα,pIκBα,NF-κB P65,IKKβ mRNA in hippocampal neurons of each group after LPS induction were detected by reverse transcription-polymerase chain reaction( RT-PCR). The expression levels of IκBα,pIκBα,NF-κB P65,IKKβ protein in hippocampal neurons of each group after LPS induction were detected by Western blot. The expression levels of TNF-α,IL-1,IL-6,ICAM-1 in LPS-induced cell cultures were determined by ELISA. Results There was no obvious abnormality between the morphology of hippocampal neurons of epileptic rats cultured in vitro for 7 days and that of normal rats. The spontaneous discharge frequency of hippocampal neurons of epileptic rats significantly increased than that of normal rats( P < 0. 05). The expression level of miR-146 a-5 p in hippocampal neurons of miR-146 a-5 p group was significantly higher than that of control group and NC group( P < 0. 05). The expression levels of pIκBα,NF-κB P65,IKKβ mRNA and protein in the hippocampal neurons of themiR-146 a-5 p group induced by LPS were significantly lower than those of the control group and NC group( P < 0. 05),while the expression levels of IκBα mRNA and protein were significantly higher than those of the control group and NC group( P< 0. 05). The expression levels of TNF-α,IL-1,IL-6,ICAM-1 in the cell culture medium of the miR-146 a-5 p group induced by LPS were significantly higher than those of the control group and NC group( P < 0. 05). Conclusion MiR-146 a-5 p can inhibit the expression of pIκBα,NF-κB P65,IKKβ mRNA and protein in the NF-κB signal transduction pathway in hippocampal neurons of epileptic rats induced by LPS,promote the expression of IκBα mRNA and protein,and reduce the release of inflammatory factors such as TNF-α,IL-1,IL-6 and ICAM-1.
引文
[1] Varadkar S,Cross JH. Rasmussen Syndrome and Other Inflammatory Epilepsies[J]. Semin Neurol,2015,35(3):259-268.
[2] de Vries EE,van den Munckhof B,Braun KP,et al. Inflammatory mediators in human epilepsy:A systematic review and meta-analysis[J]. Neurosci Biobehav Rev,2016,63:177-190.
[3] Gasparini C,Feldmann M. NF-κB as a target for modulating inflammatory responses[J]. Curr Pharm Des,2012,18(35):5735-5745.
[4] Kontomanolis EN,Koukourakis MI. MicroRNA:The Potential Regulator of Endometrial Carcinogenesis[J]. Microrna,2015,4(1):18-25.
[5] Brostoff T,Pesavento PA,Barker CM,et al. MicroRNA reduction of neuronal West Nile virus replication attenuates and affords a protective immune response in mice[J]. Vaccine,2016,34(44):5366-5375.
[6] Hébert SS,Wang WX,Zhu Q,et al. A study of small RNAs from cerebral neocortex of pathology-verified Alzheimer's disease,dementia with lewy bodies,hippocampal sclerosis,frontotemporal lobar dementia,and non-demented human controls[J]. J Alzheimers Dis,2013,35(2):335-348.
[7] Jimenez-Mateos EM,Henshall DC. Epilepsy and microRNA[J].Neuroscience,2013,238:218-229.
[8] Huntzinger E,Izaurralde E. Gene silencing by microRNAs:contributions of translational repression and mRNA decay[J]. Nat Rev Genet,2011,12(2):99-110.
[9] Alemdehy MF,Erkeland SJ. MicroRNAs:key players of normal and malignant myelopoiesis[J]. Curr Opin Hematol,2012,19(4):261-267.
[10] Xia Li,Zheng Ji,Si Li,et al. miR-146a-5p Antagonized AGEsand P. g-LPS-Induced ABCA1 and ABCG1 Dysregulation in Macrophages via IRAK-1 Downregulation[J]. Inflammation,2015,38(5):1761-1768.
[11]徐祖才,刘华,李雨芹.新生大鼠海马神经元癫痫样放电模型的初步研究[J].重庆医科大学学报,2008,33(6):697-699.
[12] Genemaras AA,Ennis H,Kaplan L,et al. Inflammatory cytokines induce specific time-and concentration‐dependent MicroRNA release by chondrocytes,synoviocytes,and meniscus cells[J]. J Orthop Res,2016,34(5):779-790.
[13] Maes T,Cobos FA,Schleich F,et al. Asthma inflammatory phenotypes show differential microRNA expression in sputum[J]. J Allergy Clin Immunol,2016,137(5):1433-1446.
[14] Vezzzani A,Ravizza T,Baiosso S,et al. Glia as a source of cytokines:implications for neuronal excitability and survival[J]. Epilepsia,2008,49(2):24-32.
[15] Bhatt K,Lanting LL,Jia Y,et al. Anti-Inflammatory Role of MicroRNA-146a in the Pathogenesis of Diabetic Nephropathy[J]. J Am Soc Nephrol,2016,27(8):2277-2288.
[16] Ahmed O,Jing P,Ciliu Z,et al. Interleukin-1b and microRNA-146a in an immature rat model and children with mesial temporal lobe epilepsy[J]. Epliepsia,2012,53(7):1215-1224.
[17] Aronica E,Fluiter K,Lyer A,et al. Expression pattern of miR-146a,an inflammation-associated microRNA,in experimental and human temporal lobe epilepsy[J]. Eur J Neurosci,2010,31(6):1100-1107.
[18] Zhong Y,Liang Y,Chen J,et al. Propofol inhibits proliferation and induces neuroapoptosis of hippocampal neurons in vitro via downregulation of NF-κB p65 and Bcl-2 and upregulation of caspase-3[J]. Cell Biochem Funct,2014,32(8):720-729.
[19] Grau A,Tabib A,Grau I,et al. Apoptotic Cells Induce NF-κB and Inflammasome Negative Signaling[J]. PLo S One,2015,10(3):e0122440.
[20]杨国庆,刘德军,程保育,等.七氟醚预处理对心肌缺血再灌注损伤大鼠血清NF-κB、TNF-α、IL-6表达的影响[J].临床和实验医学杂志,2018,17(17):1801-1805.
[21] Chuang YC,Chen SD,Lin TK,et al. Transcriptional upregulation of nitric oxide synthase II by nuclear factor-kappa B promotes apoptotic neuronal cell death in the hippocampus following experimental status epilepticus[J]. J Neurosci Res,2010,88(9):1898-1907.
[22] Herrington FD,Carmody RJ,Goodyear CS. Modulation of NF-κB Signaling as a Therapeutic Target in Autoimmunity[J]. J Biomol Screen,2016,21(3):223-242.
[23] Ridder DA,Schwaninger M. NF-kappaB signaling in cerebral ischemia[J]. Neuroscience,2009,158(3):995-1006.
[24] Hess S,Methe H,Kim JQ,et al. NF-kappaB activity in endothelial cells is modulated by cell substratum interactions and influences chemokine-mediated adhesion of natural killer cells[J]. Cell Transplant,2009,18(3):261-273.
[25] Rummel C,Gerstberger R,Rhth J,et al. Parthenolide attenuates LPS-induced fever,circulating cytokines and markers of brain inflammation in rats[J]. Cytokine,2011,56(3):739-748.
[2] de Vries EE,van den Munckhof B,Braun KP,et al. Inflammatory mediators in human epilepsy:A systematic review and meta-analysis[J]. Neurosci Biobehav Rev,2016,63:177-190.
[3] Gasparini C,Feldmann M. NF-κB as a target for modulating inflammatory responses[J]. Curr Pharm Des,2012,18(35):5735-5745.
[4] Kontomanolis EN,Koukourakis MI. MicroRNA:The Potential Regulator of Endometrial Carcinogenesis[J]. Microrna,2015,4(1):18-25.
[5] Brostoff T,Pesavento PA,Barker CM,et al. MicroRNA reduction of neuronal West Nile virus replication attenuates and affords a protective immune response in mice[J]. Vaccine,2016,34(44):5366-5375.
[6] Hébert SS,Wang WX,Zhu Q,et al. A study of small RNAs from cerebral neocortex of pathology-verified Alzheimer's disease,dementia with lewy bodies,hippocampal sclerosis,frontotemporal lobar dementia,and non-demented human controls[J]. J Alzheimers Dis,2013,35(2):335-348.
[7] Jimenez-Mateos EM,Henshall DC. Epilepsy and microRNA[J].Neuroscience,2013,238:218-229.
[8] Huntzinger E,Izaurralde E. Gene silencing by microRNAs:contributions of translational repression and mRNA decay[J]. Nat Rev Genet,2011,12(2):99-110.
[9] Alemdehy MF,Erkeland SJ. MicroRNAs:key players of normal and malignant myelopoiesis[J]. Curr Opin Hematol,2012,19(4):261-267.
[10] Xia Li,Zheng Ji,Si Li,et al. miR-146a-5p Antagonized AGEsand P. g-LPS-Induced ABCA1 and ABCG1 Dysregulation in Macrophages via IRAK-1 Downregulation[J]. Inflammation,2015,38(5):1761-1768.
[11]徐祖才,刘华,李雨芹.新生大鼠海马神经元癫痫样放电模型的初步研究[J].重庆医科大学学报,2008,33(6):697-699.
[12] Genemaras AA,Ennis H,Kaplan L,et al. Inflammatory cytokines induce specific time-and concentration‐dependent MicroRNA release by chondrocytes,synoviocytes,and meniscus cells[J]. J Orthop Res,2016,34(5):779-790.
[13] Maes T,Cobos FA,Schleich F,et al. Asthma inflammatory phenotypes show differential microRNA expression in sputum[J]. J Allergy Clin Immunol,2016,137(5):1433-1446.
[14] Vezzzani A,Ravizza T,Baiosso S,et al. Glia as a source of cytokines:implications for neuronal excitability and survival[J]. Epilepsia,2008,49(2):24-32.
[15] Bhatt K,Lanting LL,Jia Y,et al. Anti-Inflammatory Role of MicroRNA-146a in the Pathogenesis of Diabetic Nephropathy[J]. J Am Soc Nephrol,2016,27(8):2277-2288.
[16] Ahmed O,Jing P,Ciliu Z,et al. Interleukin-1b and microRNA-146a in an immature rat model and children with mesial temporal lobe epilepsy[J]. Epliepsia,2012,53(7):1215-1224.
[17] Aronica E,Fluiter K,Lyer A,et al. Expression pattern of miR-146a,an inflammation-associated microRNA,in experimental and human temporal lobe epilepsy[J]. Eur J Neurosci,2010,31(6):1100-1107.
[18] Zhong Y,Liang Y,Chen J,et al. Propofol inhibits proliferation and induces neuroapoptosis of hippocampal neurons in vitro via downregulation of NF-κB p65 and Bcl-2 and upregulation of caspase-3[J]. Cell Biochem Funct,2014,32(8):720-729.
[19] Grau A,Tabib A,Grau I,et al. Apoptotic Cells Induce NF-κB and Inflammasome Negative Signaling[J]. PLo S One,2015,10(3):e0122440.
[20]杨国庆,刘德军,程保育,等.七氟醚预处理对心肌缺血再灌注损伤大鼠血清NF-κB、TNF-α、IL-6表达的影响[J].临床和实验医学杂志,2018,17(17):1801-1805.
[21] Chuang YC,Chen SD,Lin TK,et al. Transcriptional upregulation of nitric oxide synthase II by nuclear factor-kappa B promotes apoptotic neuronal cell death in the hippocampus following experimental status epilepticus[J]. J Neurosci Res,2010,88(9):1898-1907.
[22] Herrington FD,Carmody RJ,Goodyear CS. Modulation of NF-κB Signaling as a Therapeutic Target in Autoimmunity[J]. J Biomol Screen,2016,21(3):223-242.
[23] Ridder DA,Schwaninger M. NF-kappaB signaling in cerebral ischemia[J]. Neuroscience,2009,158(3):995-1006.
[24] Hess S,Methe H,Kim JQ,et al. NF-kappaB activity in endothelial cells is modulated by cell substratum interactions and influences chemokine-mediated adhesion of natural killer cells[J]. Cell Transplant,2009,18(3):261-273.
[25] Rummel C,Gerstberger R,Rhth J,et al. Parthenolide attenuates LPS-induced fever,circulating cytokines and markers of brain inflammation in rats[J]. Cytokine,2011,56(3):739-748.