Rnd3在中枢神经系统对IL-8的调控作用及其机制
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摘要
目的:探讨小GTP酶蛋白家族成员Rnd3对白细胞介素-8(IL-8)的调控作用,及可能的调控位点及机制。方法:通过免疫荧光染色、q-PCR、免疫印迹等方法检测Rnd3-/-及Rnd3+/+小鼠脑组织内IL-8表达量改变;通过转染siCtrl,siRnd3,myc,myc-Rnd3至PC12细胞,构建体外Rnd3低表达/高表达细胞模型,通过q-PCR、荧光素酶检测、免疫印迹检测PC12细胞内IL-8表达活性及表达量的改变。结果:Rnd3-/-小鼠脑组织内IL-8表达量较Rnd3+/+小鼠增高。在转染Rnd3小干扰片段至PC12细胞致Rnd3低表达后,IL-8的mRNA和蛋白水平较siCtrl组明显升高(P<0. 05)。较myc组,myc-Rnd3组细胞内IL-8的mRNA和蛋白水平明显降低(P<0. 05)。提示Rnd3可能通过转录前水平调控IL-8的表达量。结论:Rnd3在神经细胞内与IL-8表达量呈负相关,可能在mRNA或蛋白水平对IL-8进行调控,是一个新的IL-8调控位点。
Objective: To explore the relationship between interleukin - 8(IL - 8) and Rnd3, a member of small GTPase family, in central nervous system.Methods: Immunofluorescence, q-PCR and Western blot were performed to explore the expression of IL-8 in mice brain tissue. Then, siCtrl, siRnd3,myc, myc-Rnd3 were transfected to PC12 cells by liposome, respectively, to construct Rnd3 knock-down or overexpression cell models. The mRNA and protein levels of Rnd3 and IL-8 were detected by q-PCR and Western Blot, respectively. Meanwhile, luciferase was also performed.Results: The expression of IL-8 was increased in Rnd3-/-mice brain tissue as compared with Rnd3+/+mice. When Rnd3 was knocked down, the mRNA and protein level of IL-8 was increased as compared with siCtrl group(P<0. 05), while, when Rnd3 was over-expressed, the mRNA and protein level of IL-8 was decreased(P<0. 05).Conclusion: The results showed that the expression of Rnd3 was negatively re-lated to IL-8 in PC12 cell line. Rnd3 probably regulates IL-8 on both mRNA and protein level, and Rnd3 might be a new regulatory site for IL-8.
Objective: To explore the relationship between interleukin - 8(IL - 8) and Rnd3, a member of small GTPase family, in central nervous system.Methods: Immunofluorescence, q-PCR and Western blot were performed to explore the expression of IL-8 in mice brain tissue. Then, siCtrl, siRnd3,myc, myc-Rnd3 were transfected to PC12 cells by liposome, respectively, to construct Rnd3 knock-down or overexpression cell models. The mRNA and protein levels of Rnd3 and IL-8 were detected by q-PCR and Western Blot, respectively. Meanwhile, luciferase was also performed.Results: The expression of IL-8 was increased in Rnd3-/-mice brain tissue as compared with Rnd3+/+mice. When Rnd3 was knocked down, the mRNA and protein level of IL-8 was increased as compared with siCtrl group(P<0. 05), while, when Rnd3 was over-expressed, the mRNA and protein level of IL-8 was decreased(P<0. 05).Conclusion: The results showed that the expression of Rnd3 was negatively re-lated to IL-8 in PC12 cell line. Rnd3 probably regulates IL-8 on both mRNA and protein level, and Rnd3 might be a new regulatory site for IL-8.
引文
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[16] Zhao AM, Qiu WR, Mao LJ, et al. The efficacy and safety of Jiedu Tongluo granules for treating post-stroke depression with qi deficiency and blood stasis syndrome:study protocol for a randomized controlled trial[J]. Tri-als, 2018,19(1):275.
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[21] Lin X, Liu B, Yang X, et al. Genetic deletion of Rnd3results in aqueductal stenosis leading to hydrocephalus through up-regulation of Notch signaling[J]. Proc Natl Acad Sci U S A, 2013,110(20):8 236-8 241.
[22] Dong H, Lin X, Li Y, et al. Genetic deletion of Rnd3in neural stem cells promotes proliferation via upregulation of Notch signaling[J]. Oncotarget, 2017, 8(53):91 112-91 122.
[2] Brenner P, Granqvist M, Konigsson J, et al. Depression and fatigue in multiple sclerosis:Relation to exposure to violence and cerebrospinal fluid immunomarkers[J]. Psychoneuroendocrinology, 2018,89:53-58.
[3] Xu S, Zhong, Bu X, et al. Salvianolic acid B inhibits platelets-mediated inflammatory response in vascular endothelial cells[J]. Thrombosis Research, 2015, 135(1):137-145.
[4] Nwachuku EL, Puccio AM, Adeboye A, et al. Time course of cerebrospinal fluid inflammatory biomarkers and relationship to 6-month neurologic outcome in adult severe traumatic brain injury[J]. Clin Neurol Neuro-surg, 2016,149:1-5.
[5] Liu J, Li S, Cai C, et al. Cerebrospinal fluid chemo-kine patterns in children with enterovirus 71-related encephalitis[J]. Sci Rep, 2018,8(1):1658.
[6] Talens-Visconti R, Peris, Guerri C, et al. RhoE stimu-lates neurite-like outgrowth in PC12 cells through inhibi-tion of the RhoA/ROCK-Ⅰsignalling[J]. J Neuro-chem, 2010,112(4):1 074-1 087.
[7] Jie W, Andrade KC, Lin X, et al. Pathophysiological functions of Rnd3/RhoE[J]. Compr Physiol, 2015,6(1):169-186.
[8] Yang X, Wang, Lin X, et al. Genetic deletion of Rnd3/RhoE results in mouse heart calcium leakage through upregulation of protein kinase A signaling[J].Circ Res, 2015, 116(1):e1-e10.
[9] Ishizu T, Osoegawa M, Mei FJ, et al. Intrathecal acti-vation of the IL-17/IL-8 axis in opticospinal multiple sclerosis[J]. Brain, 2005,128(Pt 5):988-1 002.
[10] G?owińska B, Urban M. Selected cytokines(Il-6, Il-8,Il-10, MCP-1, TNF-alpha)in children and adolescents with atherosclerosis risk factors:obesity, hypertension,diabetes[J]. Wiad Lek, 2003,56(3-4):109-116.
[11] Koper OM, Kamińska J, Sawicki K, et al. Cerebrospi-nal fluid and serum IL-8, CCL2, and ICAM-1 concen-trations in astrocytic brain tumor patients[J]. Ir J Med Sci, 2017,187(3):767-775.
[12] Hesse R, Wahler A, Gummert P, et al. Decreased IL-8 levels in CSF and serum of AD patients and negative correlation of MMSE and IL-1beta[J]. BMC Neurol,2016,16(1):185.
[13] Bielekova B, Komori M, Xu Q, et al. Cerebrospinal fluid IL-12p40, CXCL13 and IL-8 as a combinatorial biomarker of active intrathecal inflammation[J]. PLoS One, 2012,7(11):e48370.
[14] Janelidze S, Lindqvist D, Francardo V, et al. In-creased CSF biomarkers of angiogenesis in Parkinson disease[J]. Neurology, 2015,85(21):1 834-1 842.
[15] Garcia-Esparcia P, Llorens F, Carmona M, et al.Complex deregulation and expression of cytokines and mediators of the immune response in Parkinson′s dis-ease brain is region dependent[J]. Brain Pathol, 2014,24(6):584-598.
[16] Zhao AM, Qiu WR, Mao LJ, et al. The efficacy and safety of Jiedu Tongluo granules for treating post-stroke depression with qi deficiency and blood stasis syndrome:study protocol for a randomized controlled trial[J]. Tri-als, 2018,19(1):275.
[17] Hayashi T, Ago K, Nakamae T, et al. Interleukin(IL)-8 immunoreactivity of injured axons and surrounding oligodendrocytes in traumatic head injury[J]. Forensic Sci Int, 2016,263:48-54.
[18] Mehta VK, Verma R, Garg RK, et al. Study of inter-leukin-6 and interleukin-8 levels in patients with neuro-logical manifestations of dengue[J]. J Postgrad Med,2017,63(1):11-15.
[19] Riento K, Guasch RM, Garg R, et al. RhoE binds to ROCK I and inhibits downstream signaling[J]. Mol Cell Biol, 2003,23(12):4 219-4 229.
[20] Aspenstr?m P, Fransson A, Saras J. Rho GTPases have diverse effects on the organization of the actin fila-ment system[J]. Biochem J, 2004,377(Pt 2):327-337.
[21] Lin X, Liu B, Yang X, et al. Genetic deletion of Rnd3results in aqueductal stenosis leading to hydrocephalus through up-regulation of Notch signaling[J]. Proc Natl Acad Sci U S A, 2013,110(20):8 236-8 241.
[22] Dong H, Lin X, Li Y, et al. Genetic deletion of Rnd3in neural stem cells promotes proliferation via upregulation of Notch signaling[J]. Oncotarget, 2017, 8(53):91 112-91 122.