白细胞介素-27在顺铂所致急性肾损伤中的高表达及对细胞凋亡的抑制作用
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摘要
目的·探讨白细胞介素-27(interleukin-27,IL-27)在顺铂所致急性肾损伤(acute kidney injury,AKI)中的表达及在AKI中对细胞调亡的抑制作用。方法·将C57BL/6小鼠随机分为对照组、顺铂组,于注射顺铂后24、48、72 h处死小鼠;取血清样本,检测血尿素氮和血清肌酐水平;实时荧光定量PCR检测肾脏组织中IL-27亚单位p28和EBI3在mRNA水平的表达变化。将人近端肾小管上皮细胞系HK-2分为对照组、IL-27组、顺铂组及顺铂+IL-27组,实时荧光定量PCR分析顺铂处理0.5、1、6、12和24 h后IL-27受体GP130和WSX-1在mRNA水平的表达;CCK-8检测细胞活性;Hoechst染色观察细胞核形态;Western blotting检测凋亡相关蛋白Bax、Bcl-2和多聚ADP核糖聚合酶剪切体(cleaved poly ADP-ribose polymerase,cleaved PARP)的表达。结果·顺铂组小鼠血尿素氮、血清肌酐的水平呈时间依赖性升高,肾脏组织中p28和Ebi3 mRNA的表达水平增加并在顺铂注射48 h时出现峰值。顺铂处理1h内HK-2细胞GP130和WSX-1的表达增高,处理6、12、24 h后GP130和WSX-1的表达无明显变化。与顺铂组比较,顺铂+IL-27组细胞活力显著提升,细胞核浓缩、破裂减少,Bcl-2表达增加,Bax及cleaved PARP表达下降。结论·IL-27在顺铂所致AKI中高表达且可能通过抑制细胞凋亡发挥保护作用。
Objective · To investigate the expression of interleukin-27(IL-27) in cisplatin-induced acute kidney injury(AKI) and explore its inhibitory effect on apoptosis in AKI. Methods · C57 BL/6 mice were randomly divided to control group and cisplatin group, in which mice were sacrificed at 24,48 and 72 h after cisplatin administration. Blood urea nitrogen(BUN) and serum creatinine(Scr) levels were estimated. And the levels of mRNA for the IL-27 subunits, i.e. p28 and EBI3 in kidneys were determined by real-time PCR. As for in vitro experiments, after cisplatin incubation for 0.5, 1, 6, 12 and 24 h, HK-2 cells, a line of proximal tubular epithelial cells, were collected to assess the expression of IL-27 receptors(GP130 and WSX-1) at mRNA level.After that, HK-2 cells were treated with phosphate buffer saline or recombinant human IL-27 protein after cisplatin treatment, cell viability was detected by CCK-8, nuclear morphology was observed by Hoechst staining, and apoptosis related proteins including Bax, Bcl-2, and cleaved poly ADP-ribose polymerase(PARP) were estimated by Western blotting. Results · Compared with control group, BUN and Scr in cisplatin group significantly increased in a time-dependent manner after cisplatin injection. The mRNA levels of p28 and Ebi3 grew in injured kidneys, and their highest expressions were exhibited at 48 h after cisplatin injection. In HK-2 cells, GP130 and WSX-1 mRNA significantly increased at 1 h after cisplatin treatment but reduced to the basal level at 6, 12 and 24 h. IL-27 treatment significantly up-regulated cell viability and alleviated apoptosis and necrosis in cisplatin-treated HK-2 cells. In addition, IL-27 treatment inhibited the cleaved PARP and pro-apoptotic protein Bax, and upregulated antiapoptotic protein Bcl-2 in cisplatin-treated HK-2 cells. Conclusion · The expression of IL-27 increases in cisplatin-induced AKI, and it may protect against AKI by reducing cell apoptosis.
Objective · To investigate the expression of interleukin-27(IL-27) in cisplatin-induced acute kidney injury(AKI) and explore its inhibitory effect on apoptosis in AKI. Methods · C57 BL/6 mice were randomly divided to control group and cisplatin group, in which mice were sacrificed at 24,48 and 72 h after cisplatin administration. Blood urea nitrogen(BUN) and serum creatinine(Scr) levels were estimated. And the levels of mRNA for the IL-27 subunits, i.e. p28 and EBI3 in kidneys were determined by real-time PCR. As for in vitro experiments, after cisplatin incubation for 0.5, 1, 6, 12 and 24 h, HK-2 cells, a line of proximal tubular epithelial cells, were collected to assess the expression of IL-27 receptors(GP130 and WSX-1) at mRNA level.After that, HK-2 cells were treated with phosphate buffer saline or recombinant human IL-27 protein after cisplatin treatment, cell viability was detected by CCK-8, nuclear morphology was observed by Hoechst staining, and apoptosis related proteins including Bax, Bcl-2, and cleaved poly ADP-ribose polymerase(PARP) were estimated by Western blotting. Results · Compared with control group, BUN and Scr in cisplatin group significantly increased in a time-dependent manner after cisplatin injection. The mRNA levels of p28 and Ebi3 grew in injured kidneys, and their highest expressions were exhibited at 48 h after cisplatin injection. In HK-2 cells, GP130 and WSX-1 mRNA significantly increased at 1 h after cisplatin treatment but reduced to the basal level at 6, 12 and 24 h. IL-27 treatment significantly up-regulated cell viability and alleviated apoptosis and necrosis in cisplatin-treated HK-2 cells. In addition, IL-27 treatment inhibited the cleaved PARP and pro-apoptotic protein Bax, and upregulated antiapoptotic protein Bcl-2 in cisplatin-treated HK-2 cells. Conclusion · The expression of IL-27 increases in cisplatin-induced AKI, and it may protect against AKI by reducing cell apoptosis.
引文
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[11]Cao J,Xu F,Lin S,et al.IL-27 controls sepsis-induced impairment of lung antibacterial host defence[J].Thorax,2014,69(10):926-937.
[12]Yang B,Suwanpradid J,Sanchez-Lagunes R,et al.IL-27 facilitates skin wound healing through induction of epidermal proliferation and host defense[J].JInvest Dermatol,2017,137(5):1166-1175.
[13]Jones SA,Jenkins BJ.Recent insights into targeting the IL-6 cytokine family in inflammatory diseases and cancer[J].Nat Rev Immunol,2018,18(12):773-789.
[14]Qi W,Niu J,Qin Q.Glycated albumin triggers fibrosis and apoptosis via an NADPH oxidase/Nox4-MAPK pathway-dependent mechanism in renal proximal tubular cells[J].Mol Cell Endocrinol,2015,405:74-83.
[15]Ishibashi Y,Matsui T,Ohta K,et al.PEDF inhibits AGE-induced podocyte apoptosis via PPAR-γactivation[J].Microvasc Res,2013,85:54-58.
[16]Yang SD,Bai ZL,Zhang F,et al.Levofloxacin increases the effect of serum deprivation on anoikis of rat nucleus pulposus cells via Bax/Bcl-2/caspase-3pathway[J].Toxicol Mech Methods,2014,24(9):688-696.
[17]Shukla P,Mansoori MN,Kakaji M,et al.IL-27 alleviates bone loss in estrogen deficient conditions by induction of early growth response-2 gene[J].J Biol Chem,2017,292(11):4686-4699.
[2]de Mendon?a A,Vincent JL,Suter PM,et al.Acute renal failure in the ICU:risk factors and outcome evaluated by the SOFA score[J].Intensive Care Med,2000,26(7):915-921.
[3]Hoste EA,Clermont G,Kersten A,et al.RIFLE criteria for acute kidney injury are associated with hospital mortality in critically ill patients:a cohort analysis[J].Crit Care,2006,10(73):496-504.
[4]Lebwohl D,Canetta R.Clinical development of platinum complexes in cancer therapy:an historical perspective and an update[J].Eur J Cancer,1998,34(10):1522-1534.
[5]Pflanz S,Timans JC,Cheung J,et al.IL-27,a heterodimeric cytokine composed of EBI3 and p28 protein,induces proliferation of naive CD4+T cells[J].Immunity,2002,16(6):779-790.
[6]Basset L,Chevalier S,Danger Y,et al.Interleukin-27 and IFNγregulate the expression of CXCL9,CXCL10,and CXCL11 in hepatitis[J].J Mol Med(Berl),2015,93(12):1355-1367.
[7]Pickens SR,Chamberlain ND,Volin MV,et al.Local expression of interleukin-27 ameliorates collagen-induced arthritis[J].Arthritis Rheum,2011,63(8):2289-2298.
[8]Su Y,Yao H,Wang H,et al.IL-27 enhances innate immunity of human pulmonary fibroblasts and epithelial cells through upregulation of TLR4expression[J].Am J Physiol Lung Cell Mol Physiol,2015,310(2):133-141.
[9]Tanida S,Yoshitomi H,Ishikawa M,et al.IL-27-producing CD14+cells infiltrate inflamed joints of rheumatoid arthritis and regulate inflammation and chemotactic migration[J].Cytokine,2011,55(2):237-244.
[10]Ma MC,Wang BW,Yeh TP,et al.Interleukin-27,a novel cytokine induced by ischemia-reperfusion injury in rat hearts,mediates cardioprotective effects via the gp130/STAT3 pathway[J].Basic Res Cardiol,2015,110(3):22-34.
[11]Cao J,Xu F,Lin S,et al.IL-27 controls sepsis-induced impairment of lung antibacterial host defence[J].Thorax,2014,69(10):926-937.
[12]Yang B,Suwanpradid J,Sanchez-Lagunes R,et al.IL-27 facilitates skin wound healing through induction of epidermal proliferation and host defense[J].JInvest Dermatol,2017,137(5):1166-1175.
[13]Jones SA,Jenkins BJ.Recent insights into targeting the IL-6 cytokine family in inflammatory diseases and cancer[J].Nat Rev Immunol,2018,18(12):773-789.
[14]Qi W,Niu J,Qin Q.Glycated albumin triggers fibrosis and apoptosis via an NADPH oxidase/Nox4-MAPK pathway-dependent mechanism in renal proximal tubular cells[J].Mol Cell Endocrinol,2015,405:74-83.
[15]Ishibashi Y,Matsui T,Ohta K,et al.PEDF inhibits AGE-induced podocyte apoptosis via PPAR-γactivation[J].Microvasc Res,2013,85:54-58.
[16]Yang SD,Bai ZL,Zhang F,et al.Levofloxacin increases the effect of serum deprivation on anoikis of rat nucleus pulposus cells via Bax/Bcl-2/caspase-3pathway[J].Toxicol Mech Methods,2014,24(9):688-696.
[17]Shukla P,Mansoori MN,Kakaji M,et al.IL-27 alleviates bone loss in estrogen deficient conditions by induction of early growth response-2 gene[J].J Biol Chem,2017,292(11):4686-4699.
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