NLRP3/caspase-1/IL-1β信号通路在HK-2细胞高糖缺氧复氧损伤中的作用
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摘要
目的探讨Nod样受体蛋3炎性体(nod-like receptor protein-3,NLRP3)/半胱天冬酶-1(caspase-1)/白介素-1β(interleukin-1β,IL-1β)信号通路介导的高糖缺氧复氧诱导人肾小管上皮细胞(human renal proximal tubular cells,HK-2)的损伤。方法采用数字表法将人肾小管上皮细胞(HK-2)随机分为5组(n=5),即低糖组(NG组)、低糖缺氧复氧组(NHR组)、高糖组(HG组)、高糖缺氧复氧组(HHR组)和高糖缺氧复氧+NLRP3抑制剂BAY11-7082(5μmol/L)组(HHR-BAY组)。采用高糖(30mmol/L)刺激72h建立高糖模型,缺氧4h复氧2h建立缺氧复氧模型。CCK-8检测细胞存活率;酶标仪测定超氧化物歧化酶(superoxide dismutase,SOD)活性;荧光探针DCFH-DA法检测细胞内活性氧自由基(reactive oxygen species,ROS)含量; ELISA法检测IL-1β含量和caspase-1活性;免疫印迹法和免疫荧光法检测细胞NLRP3蛋白的表达。结果与NG组比较,NHR组与HG组ROS和IL-1β含量,caspase-1活性,NLRP3表达升高,细胞存活率,SOD活性降低(P均<0. 05);分别与HG组和NHR组比较,HHR组ROS和IL-1β含量,caspase-1活性,NLRP3表达升高,细胞存活率,SOD活性降低(P均<0. 05);而NLRP3抑制剂BAY11-7082预处理可显著抑制细胞损伤和氧化应激水平,下调NLRP3蛋白水平,caspase-1活性和IL-1β含量(P均<0. 05)。结论 NLRP3/caspase-1/IL-1β信号通路参与了高糖缺氧复氧诱导的HK-2细胞损伤过程。
Objective To evaluate whether NLRP3/caspase-1/IL-1β signaling pathway mediates high glucose and hypoxia/reoxygenation induced injury in human renal proximal tubular cells( HK-2). Methods HK-2 cells were randomly divided into five groups( n = 5) : normal glucose group( group NG),normal glucose and hypoxia/reoxygenation group( group NHR),high glucose group( group HG),high glucose and hypoxia/reoxygenation group( group HHR),high glucose and hypoxia/reoxygenation + BAY11-7082( NLRP3 inhibitor)( 5μmol/L) group( group HHR-BAY). The high glucose and hypoxia/reoxygenation model was established by incubated in30 mmol/L glucose for 72 h,then exposed to hypoxia 4 h and reoxygenation 2 h. The cell viability was examined by CCK-8,the superoxide dismutase( SOD) activity in the cell culture supernatants was measured by microplate reader and the generation of reactive oxygen species( ROS) was determined by DCFH-DA staining. IL-1β content and caspase-1 activity were detected by ELISA. The expression of NLRP3 was detected by Western blot and immunofluorescence. Results Compared with group NG,ROS and IL-1β content,caspase-1 activity and NLRP3 level were increased,cell viability and SOD activity were decreased( all P < 0. 05) in group NHR and group HG;compared with group HG and group NHR respectively,ROS and IL-1β content,caspase-1 activity and NLRP3 level were increased,cell viability and SOD activity were decreased( all P < 0. 05) in group HHR. However,pretreatment with the inhibitor of NLRP3 BAY11-7082 significantly inhibited cell injury and oxidative stress,decreases in the expression of NLRP3,caspase-1 activity and IL-1β content( all P < 0. 05). Conclusion NLRP3/caspase-1/IL-1β signaling pathway is involved in the high glucose and hypoxia/reoxygenation induced injury in HK-2 cell.
Objective To evaluate whether NLRP3/caspase-1/IL-1β signaling pathway mediates high glucose and hypoxia/reoxygenation induced injury in human renal proximal tubular cells( HK-2). Methods HK-2 cells were randomly divided into five groups( n = 5) : normal glucose group( group NG),normal glucose and hypoxia/reoxygenation group( group NHR),high glucose group( group HG),high glucose and hypoxia/reoxygenation group( group HHR),high glucose and hypoxia/reoxygenation + BAY11-7082( NLRP3 inhibitor)( 5μmol/L) group( group HHR-BAY). The high glucose and hypoxia/reoxygenation model was established by incubated in30 mmol/L glucose for 72 h,then exposed to hypoxia 4 h and reoxygenation 2 h. The cell viability was examined by CCK-8,the superoxide dismutase( SOD) activity in the cell culture supernatants was measured by microplate reader and the generation of reactive oxygen species( ROS) was determined by DCFH-DA staining. IL-1β content and caspase-1 activity were detected by ELISA. The expression of NLRP3 was detected by Western blot and immunofluorescence. Results Compared with group NG,ROS and IL-1β content,caspase-1 activity and NLRP3 level were increased,cell viability and SOD activity were decreased( all P < 0. 05) in group NHR and group HG;compared with group HG and group NHR respectively,ROS and IL-1β content,caspase-1 activity and NLRP3 level were increased,cell viability and SOD activity were decreased( all P < 0. 05) in group HHR. However,pretreatment with the inhibitor of NLRP3 BAY11-7082 significantly inhibited cell injury and oxidative stress,decreases in the expression of NLRP3,caspase-1 activity and IL-1β content( all P < 0. 05). Conclusion NLRP3/caspase-1/IL-1β signaling pathway is involved in the high glucose and hypoxia/reoxygenation induced injury in HK-2 cell.
引文
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17 Parekh J,Roll GR,Feng S,et al.Peri-operative hyperglycemia is associated with delayed graft function in deceased donor renal transplantation[J].Clin Transplant,2013,27(4):E424-E430
18 Shen ZY,Sun Q,Xia ZY,et al.Overexpression of DJ-1 reduces oxidative stress and attenuates hypoxia/reoxygenation injury in NRK-52E cells exposed to high glucose[J].Int J Mol Med,2016,38(3):729-736
19 Jiang M,Liu K,Luo J,et al.Autophagy is a renoprotective mechanism during in vitro hypoxia and in vivo ischemia-reperfusion injury[J].Am J Pathol,2010,176(3):1181-1192
20 De Nardo D,Latz E.NLRP3 inflammasomes link inflammation and metabolic disease[J].Trends Immunol,2011,32(8):373-379
21 Pulskens WP,Butter LM,Teske GJ,et al.Nlrp3 prevents early renal interstitial edema and vascular permeability in unilateral ureteral obstruction[J].PLo S One,2014,9(1):e85775
22 Abderrazak A,Syrovets T,Couchie D,et al.NLRP3 inflammasome:from a danger signal sensor to a regulatory node of oxidative stress and inflammatory diseases[J].Redox Biol,2015,4:296-307
23 Bivol LM,Iversen BM,Hultstrom M,et al.Unilateral renal ischemia in rats induces a rapid secretion of inflammatory markers to renal lymph and increased peritubular capillary permeability[J].J Physiol,2016,594(6):1709-1726
24 Liu Y,Lian K,Zhang L,et al.TXNIP mediates NLRP3 inflammasome activation in cardiac microvascular endothelial cells as a novel mechanism in myocardial ischemia/reperfusion injury[J].Basic Res Cardiol,2014,109(5):415
2 Thakar CV,Christianson A,Himmelfarb J,et al.Acute kidney injury episodes and chronic kidney disease risk in diabetes mellitus[J].Clin J Am Soc Nephrol,2011,6(11):2567-2572
3 Hao JL,Li YF,Li RS.A novel mechanism of NALP3 inducing ischemia reperfusion injury by activating MAPK pathway in acute renal failure[J].Med Hypotheses,2013,80(4):463-465
4 Anders HJ,Muruve DA.The inflammasomes in kidney disease[J].JAm Soc Nephrol,2011,22(6):1007-1018
5 Bakker PJ,Butter LM,Claessen N,et al.A tissue-specific role for Nlrp3 in tubular epithelial repair after renal ischemia/reperfusion[J].Am J Pathol,2014,184(7):2013-2022
6张锦华,张建国,杨聚荣,等.NALP3表达与糖尿病肾病肾间质炎症的关系[J].第三军医大学学报,2012,34(10):959-962
7 Gao P,Meng XF,Su H,et al.Thioredoxin-interacting protein mediates NALP3 inflammasome activation in podocytes during diabetic nephropathy[J].Biochim Biophys Acta,2014,1843(11):2448-2460
8 Qiu Z,Lei S,Zhao B,et al.NLRP3 inflammasome activation-mediated pyroptosis aggravates myocardial ischemia/reperfusion injury in diabetic rats[J].Oxid Med Cell Longev,2017,2017:9743280
9 Bonventre JV,Yang L.Cellular pathophysiology of ischemic acute kidney injury[J].J Clin Invest,2011,121(11):4210-4221
10 Basile DP,Anderson MD,Sutton TA.Pathophysiology of acute kidney injury[J].Compr Physiol,2012,2(2):1303-1353
11 Sancho-Martinez SM,Lopez-Novoa JM,Lopez-Hernandez FJ.Pathophysiological role of different tubular epithelial cell death modes in acute kidney injury[J].Clin Kidney J,2015,8(5):548-559
12 Rochette L,Zeller M,Cottin Y,et al.Diabetes,oxidative stress and therapeutic strategies[J].Biochim Biophys Acta,2014,1840(9):2709-2729
13 Wei J,Shi Y,Hou Y,et al.Knockdown of thioredoxin-interacting protein ameliorates high glucose-induced epithelial to mesenchymal transition in renal tubular epithelial cells[J].Cell Signal,2013,25(12):2788-2796
14 Lee MJ,Feliers D,Sataranatarajan K,et al.Resveratrol ameliorates high glucose-induced protein synthesis in glomerular epithelial cells[J].Cell Signal,2010,22(1):65-70
15 Altunkaynak HO,Ozcelikay AT.Cardioprotective effect of postconditioning against ischemia-reperfusion injury is lost in heart of 8-week diabetic rat[J].Gen Physiol Biophys,2016,35(1):63-69
16 Kim HS,Cho JE,Hwang KC,et al.Diabetes mellitus mitigates cardioprotective effects of remifentanil preconditioning in ischemiareperfused rat heart in association with anti-apoptotic pathways of survival[J].Eur J Pharmacol,2010,628(1-3):132-139
17 Parekh J,Roll GR,Feng S,et al.Peri-operative hyperglycemia is associated with delayed graft function in deceased donor renal transplantation[J].Clin Transplant,2013,27(4):E424-E430
18 Shen ZY,Sun Q,Xia ZY,et al.Overexpression of DJ-1 reduces oxidative stress and attenuates hypoxia/reoxygenation injury in NRK-52E cells exposed to high glucose[J].Int J Mol Med,2016,38(3):729-736
19 Jiang M,Liu K,Luo J,et al.Autophagy is a renoprotective mechanism during in vitro hypoxia and in vivo ischemia-reperfusion injury[J].Am J Pathol,2010,176(3):1181-1192
20 De Nardo D,Latz E.NLRP3 inflammasomes link inflammation and metabolic disease[J].Trends Immunol,2011,32(8):373-379
21 Pulskens WP,Butter LM,Teske GJ,et al.Nlrp3 prevents early renal interstitial edema and vascular permeability in unilateral ureteral obstruction[J].PLo S One,2014,9(1):e85775
22 Abderrazak A,Syrovets T,Couchie D,et al.NLRP3 inflammasome:from a danger signal sensor to a regulatory node of oxidative stress and inflammatory diseases[J].Redox Biol,2015,4:296-307
23 Bivol LM,Iversen BM,Hultstrom M,et al.Unilateral renal ischemia in rats induces a rapid secretion of inflammatory markers to renal lymph and increased peritubular capillary permeability[J].J Physiol,2016,594(6):1709-1726
24 Liu Y,Lian K,Zhang L,et al.TXNIP mediates NLRP3 inflammasome activation in cardiac microvascular endothelial cells as a novel mechanism in myocardial ischemia/reperfusion injury[J].Basic Res Cardiol,2014,109(5):415