抑制免疫抑制因子COX-2对视网膜神经节细胞凋亡的影响及p38MAPK信号的调控作用
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摘要
目的:探讨环氧合酶-2(COX-2)抑制剂NS-398对H_2O_2诱导的视网膜神经节RGC-5细胞活力和凋亡及p38MAPK信号通路的调控作用。方法:200、300、400、600、800μmol/L的H_2O_2刺激视网膜神经节RGC-5细胞建立H_2O_2损伤模型,根据半数抑制浓度选择H_2O_2的浓度; COX-2抑制剂NS-398处理H_2O_2诱导的RGC-5细胞7 h,SB203580作为p38MAPK信号通路抑制剂,通过MTT法及流式细胞术分别检测细胞的活力和凋亡率; Western blot检测细胞增殖核抗原(PCNA)、p53、p38丝裂原活化蛋白激酶(p38MAPK)、p-p38的蛋白表达。结果:不同浓度的H_2O_2均可抑制RGC-5细胞活力,且随H_2O_2浓度升高细胞活力降低,由于400μmol/L的H_2O_2可抑制一半的细胞活力,选择作为研究对象;与空白对照组比较,H_2O_2组细胞活力及PCNA蛋白表达均显著降低,细胞凋亡率及p53和p-p38的蛋白表达均显著升高,与H_2O_2组比较,H_2O_2+NS-398组细胞活力及PCNA蛋白表达均显著升高,细胞凋亡率及p53和p-p38的蛋白表达均显著降低(P<0. 05);与H_2O_2+NS-398组比较,H_2O_2+NS-398+SB203580组细胞活力及PCNA蛋白表达均显著升高,细胞凋亡率及p53和p-p38的蛋白表达均显著降低(P<0. 05)。结论:抑制免疫抑制因子COX-2表达可通过调控p38MAPK信号通路提高视网膜神经节细胞活力和抑制细胞凋亡。
Objective: To investigate the effect of COX-2 inhibitor bevacizumab on the activity and apoptosis of retinal ganglion RGC-5 cells induced by H_2O_2 and the regulation of p38 MAPK signaling pathway. Methods: Retinal ganglion RGC-5 cells was stimulated using H_2O_2( 200,300,400,600,800 μmol/L) to establish a H_2O_2 damage model,H_2O_2 concentration was selected based on half inhibitory concentration,COX-2 inhibitor bevacizumab treated RGC-5 cell induced by H_2O_2 for 7 h,SB203580 as a p38 MAPK signaling pathway inhibitor,cell viability and apoptosis rate were detected by MTT method and flow cytometry,respectively; the expression of PCNA,p53,p38 and p-p38 protein were detected by Western blot. Results: Different concentrations of H_2O_2 could inhibit the viability of RGC-5 cells,and the cell viability decreased with the increase of H_2O_2 concentration,because 400 μmol/L H_2O_2 inhibited half of the cell viability,it was selected as an object of study. Compared with the control group,the cell viability and the expression of PCNA were decreased significantly in H_2O_2 group,the apoptosis rate and the expression of p53 and p-p38 protein was increased significantly; compared with H_2O_2 group,the cell viability and PCNA expression were increased significantly in the H_2O_2+bevacizumab group,the apoptosis rate and the expression of p53 and p-p38 protein were decreased significantly( P < 0. 05); compared with H_2O_2+ bevacizumab group,the cell viability and PCNA expression were increased significantly in H_2O_2+bevacizumab+SB203580 group,the apoptosis rate and the expression of p53 and p-p38 protein were decreased significantly( P<0. 05). Conclusion: Inhibition of immunosuppressive factor COX-2 expression can improve the activity of retinal ganglion cells and inhibit apoptosis by regulating the p38 MAPK signaling pathway.
Objective: To investigate the effect of COX-2 inhibitor bevacizumab on the activity and apoptosis of retinal ganglion RGC-5 cells induced by H_2O_2 and the regulation of p38 MAPK signaling pathway. Methods: Retinal ganglion RGC-5 cells was stimulated using H_2O_2( 200,300,400,600,800 μmol/L) to establish a H_2O_2 damage model,H_2O_2 concentration was selected based on half inhibitory concentration,COX-2 inhibitor bevacizumab treated RGC-5 cell induced by H_2O_2 for 7 h,SB203580 as a p38 MAPK signaling pathway inhibitor,cell viability and apoptosis rate were detected by MTT method and flow cytometry,respectively; the expression of PCNA,p53,p38 and p-p38 protein were detected by Western blot. Results: Different concentrations of H_2O_2 could inhibit the viability of RGC-5 cells,and the cell viability decreased with the increase of H_2O_2 concentration,because 400 μmol/L H_2O_2 inhibited half of the cell viability,it was selected as an object of study. Compared with the control group,the cell viability and the expression of PCNA were decreased significantly in H_2O_2 group,the apoptosis rate and the expression of p53 and p-p38 protein was increased significantly; compared with H_2O_2 group,the cell viability and PCNA expression were increased significantly in the H_2O_2+bevacizumab group,the apoptosis rate and the expression of p53 and p-p38 protein were decreased significantly( P < 0. 05); compared with H_2O_2+ bevacizumab group,the cell viability and PCNA expression were increased significantly in H_2O_2+bevacizumab+SB203580 group,the apoptosis rate and the expression of p53 and p-p38 protein were decreased significantly( P<0. 05). Conclusion: Inhibition of immunosuppressive factor COX-2 expression can improve the activity of retinal ganglion cells and inhibit apoptosis by regulating the p38 MAPK signaling pathway.
引文
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[25]Yang M,Zhai X,Xia B,et al.Long noncoding RNA CCHE1 promotes cervical cancer cell proliferation via upregulating PCNA[J].Tumor Biol,2015,36(10):7615-7622.
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[2]Katome T,Namekata K,Guo X,et al.Inhibition of ASK1-p38 pathway prevents neural cell death following optic nerve injury[J].Cell Death Differentiation,2013,20(2):270-280.
[3]Li Y,Andereggen L,Yuki K,et al.Mobile zinc increases rapidly in the retina after optic nerve injury and regulates ganglion cell survival and optic nerve regeneration[J].Proc Nat Acad Sci,2017,114(2):E209-E218.
[4]Adamiec-Mroczek J,Zajac-Pytrus H,Misiuk-Hojlo M.Caspase-dependent apoptosis of retinal ganglion cells during the development of diabetic retinopathy[J].Adv Clin Exp Med,2015,24(3):531-535.
[5]Ohlemacher SK,Sridhar A,Xiao Y,et al.Stepwise differentiation of retinal ganglion cells from human pluripotent stem cells enables analysis of glaucomatous neurodegeneration[J].Stem Cells,2016,34(6):1553-1562.
[6]Giurdanella G,Anfuso CD,Olivieri M,et al.Aflibercept,bevacizumab and ranibizumab prevent glucose-induced damage in human retinal pericytes in vitro,through a PLA 2/COX-2/VEGF-Apathway[J].Biochem Pharmacol,2015,96(3):278-287.
[7]Gui D,Li Y,Chen X,et al.HIF 1 signaling pathway involving i NOS,COX 2 and caspase 9 mediates the neuroprotection provided by erythropoietin in the retina of chronic ocular hypertension rats[J].Mole Med Rep,2015,11(2):1490-1496.
[8]Hu J,Li T,Du S,et al.The MAPK signaling pathway mediates the GPR91-dependent release of VEGF from RGC-5 cells[J].Int JMol Med,2015,36(1):130-138.
[9]Fleischman A,Oron Y,Geyer O.COX-2 Inhibition Improves Retinal Function in Rats'Ischemic Eyes[J].J Ocular Pharmacol Therapeutics,2014,30(8):634-641.
[10]黄晶,宋方,龙向淑,等.IFN-α通过IFI16抑制人脑血管外膜成纤维细胞增殖并促进细胞凋亡[J].中国免疫学杂志,2017,33(4):494-497.Huang J,Song F,Long XS,et al.Interferon alpha suppresses proliferation and induces apoptosis of human brain vascular adventitial fibroblasts via IFI16[J].Chin J Immunol,2017,33(4):494-497.
[11]Wilson AM,Morquette B,Abdouh M,et al.ASPP1/2 regulate p53-dependent death of retinal ganglion cells through PUMA and Fas/CD95 activation in vivo[J].J Neurosci,2013,33(5):2205-2216.
[12]Wang L,Ma Q.Toll-like receptors promote apoptosis of retinal ganglion cells in diabetic retinopathy via regulating Caspase-3 and Bcl-xL[J].Int J Clin Exp Pathol,2016,9(1):73-80.
[13]柏凌,李鹏,陈凌,等.MicroRNA在氧化应激诱导晶状体上皮细胞凋亡中作用的初步研究[J].国际眼科杂志,2014,14(10):1770-1772.Bai L,Li P,Chen L,et al.MicroRNA profiling on oxidative stress induced apoptosis of human lens epithelium[J].Int Eye Sci,2014,14(10):1770-1772.
[14]周希彬,刘瑞,吴志鸿.氧化应激在青光眼中作用的研究进展[J].中华眼科杂志,2014,50(8):634-637.Zhou XB,Liu R,Wu ZH.Recent progress in research on oxidative stress in glaucoma[J].Chin J Ophthalmol,2014,50(8):634-637.
[15]Backman A,Makdoumi K.Riboflavin/UVA-mediated Elimination of Pathogenic Bacteria with and without Antibiotic Resistance U-sing an In Vitro Model of Keratitis Treatment[J].Invest Ophthalmol Visual Sci,2014,55(13):6273-6273.
[16]Jia WC,Liu G,Zhang CD,et al.Formononetin attenuates hydrogen peroxide(H2O2)-induced apoptosis and NF-κB activation in RGC-5 cells[J].Eur Rev Med Pharmacol Sci,2014,18(15):2191-2197.
[17]于艳华,石卓,李艳杰,等.人参皂甙Rd抑制LPS刺激的RAW264.7细胞中i NOS及COX-2表达的研究[J].中国免疫学杂志,2014,30(2):209-212.Yu YH,Shi Z,Li YJ,et al.Inhibitory effect of ginsenoside Rd on i NOS and COX-2 expression in LPS-stimulated RAW264.7 cells[J].Chin J Immunol,2014,30(2):209-212.
[18]Wang J,Liu YT,Xiao L,et al.Anti-inflammatory effects of apigenin in lipopolysaccharide-induced inflammatory in acute lung injury by suppressing COX-2 and NF-kB pathway[J].Inflammation,2014,37(6):2085-2090.
[19]Vessieres E,Guihot AL,Toutain B,et al.COX-2-derived prostanoids and oxidative stress additionally reduce endotheliummediated relaxation in old type 2 diabetic rats[J].PLoS One,2013,8(7):e68217.
[20]Zhang R,Liu Z,Zhang H,et al.The COX-2-selective antagonist(NS-398)inhibits choroidal neovascularization and subretinal fibrosis[J].PLoS One,2016,11(1):e0146808.
[21]Liu N,Wu Q,Wang Y,et al.Helicobacter pylori promotes VEGFexpression via the p38 MAPK mediated COX 2 PGE2 pathway in MKN45 cells[J].Mol Med Rep,2014,10(4):2123-2129.
[22]丘桂华,张雪莹,曹焕玲,等.Smad4基因条件性敲除对T细胞发育的影响[J].中国免疫学杂志,2016,32(5):640-643.Qiu GH,Zhang XY,Cao HL,et al.Effects of conditional deletion of Smad4 gene on T cell development[J].Chin J Immunol,2016,32(5):640-643.
[23]Zhang X,Lai D,Bao S,et al.Triamcinolone acetonide inhibits p38MAPK activation and neuronal apoptosis in early diabetic retinopathy[J].Curr Mole Med,2013,13(6):946-958.
[24]Zhou RM,Shen Y,Yao J,et al.Nmnat 1:a security guard of retinal ganglion cells(RGCs)in response to high glucose stress[J].Cell Physiol Biochem,2016,38(6):2207-2218.
[25]Yang M,Zhai X,Xia B,et al.Long noncoding RNA CCHE1 promotes cervical cancer cell proliferation via upregulating PCNA[J].Tumor Biol,2015,36(10):7615-7622.
[26]Gohari M,Neámatzadeh H,Jafari MA,et al.Association between the p53 codon 72 polymorphism and primary open-angle glaucoma risk:Meta-analysis based on 11 case-control studies[J].Indian JOphthalmol,2016,64(10):756-761.