还原型辅酶Ⅱ氧化酶抑制剂Apocynin对急性坏死性胰腺炎大鼠肾损伤的保护作用
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摘要
目的探讨还原型辅酶Ⅱ氧化酶(NADPH oxidase,NOX2)抑制剂夹竹桃麻素(Apocynin)对急性坏死性胰腺炎(acute necrotic pancreatitis,ANP)模型大鼠肾损伤的保护作用。方法 30只Wistar大鼠随机分为3组:假手术组(SO组)、急性坏死性胰腺炎组(ANP组)、Apocynin治疗组(APO组),每组各10只大鼠。ANP组采用逆行胆胰管注射牛磺胆酸钠溶液1 ml/kg(STC)制备ANP模型。APO组在造模前30 min股静脉注射Apocynin(50 mg/kg),其余同ANP组。SO组开腹后仅翻动十二指肠和胰腺。造模后12 h处死大鼠,下腔静脉采血,留取各组大鼠肾组织以4%多聚甲醛固定。全自动生化仪检测血清淀粉酶(AMY)、脂肪酶(LIPA)、肌酐(Cr)、尿素(BUN)水平,肾脏组织固定行HE染色,光学显微镜下观察肾脏病理改变,免疫组化及免疫荧光法检测肾脏组织半胱天冬酶-3(Caspase-3)、核转录因子-κB(NF-κB)、肿瘤坏死因子-α(TNF-α)、肠黏膜髓过氧化酶(MPO)水平。结果 (1)ANP组大鼠血清AMY、LIP、Cr、BUN水平及组织病理改变较SO组明显升高(P<0.05),应用Apocynin后降低(P<0.05)。(2)ANP组大鼠Caspase-3、NF-κB、TNF-α表达水平较SO组升高(P<0.05),应用Apocynin后有所降低(P<0.05)。(3)ANP组大鼠髓过氧化酶(MPO)表达水平较SO组升高(P<0.05),应用Apocynin后有所降低(P<0.05)。结论 NOX2抑制剂Apocynin可减轻ANP模型大鼠肾脏损伤。
[Objective]To investigate the protective effect of Apocynin, the inhibitor of NADPH oxidase(NOX), on renal injury induced by acute necrotic pancreatitis(ANP) in rats. [Methods] A total of 30 Wistar rats were randomly divided into 3 groups:sham operation group(SO group, n=10), acute necrotizing pancreatitis group(ANP group, n=10), and Apocynin treated group(APO group, n=10). ANP models were induced by retrograde injection of 1 ml/kg sodium Taurocholate(STC) in biliopancreatic duct in ANP group. Apocynin was injected 30 mins before the induction of ANP models in APO group, the rest were the same as ANP group. Rats of SO group were just flipped the pancreas and duodenum. All the rats were sacrificed at 12 h after modeling. Blood samples were collected by inferior vena cava puncture, and serum activity of AMY, LIP, Cr, BUN were measured by autochemistry analyzer. The pathological changes of kidney were observed under optical microscope with HE staining.Immunohistochemistry and immunofluorescence were used to determine Caspase-3, NF-κB, TNF-α and MPO levels.[Results](1)Compared with SO group, the serum activity of AMY, LIP, Cr, BUN and histopathological changes were increased in ANP group(P<0.05) and decreased in APO group(P<0.05).(2)Compared with SO group, the expression level of Caspase-3, NF-κB and TNF-α were significantly increased in ANP group(P<0.05) and decreased in APO group(P<0.05).(3)Compared with SO group, the expression level of MPO was increased in ANP group( P < 0. 05) and decreased in APO group(P<0.05).[Conclusion]Apocynin can ameliorate renal injury of ANP model rats by inhibiting the activity of NOX2.
[Objective]To investigate the protective effect of Apocynin, the inhibitor of NADPH oxidase(NOX), on renal injury induced by acute necrotic pancreatitis(ANP) in rats. [Methods] A total of 30 Wistar rats were randomly divided into 3 groups:sham operation group(SO group, n=10), acute necrotizing pancreatitis group(ANP group, n=10), and Apocynin treated group(APO group, n=10). ANP models were induced by retrograde injection of 1 ml/kg sodium Taurocholate(STC) in biliopancreatic duct in ANP group. Apocynin was injected 30 mins before the induction of ANP models in APO group, the rest were the same as ANP group. Rats of SO group were just flipped the pancreas and duodenum. All the rats were sacrificed at 12 h after modeling. Blood samples were collected by inferior vena cava puncture, and serum activity of AMY, LIP, Cr, BUN were measured by autochemistry analyzer. The pathological changes of kidney were observed under optical microscope with HE staining.Immunohistochemistry and immunofluorescence were used to determine Caspase-3, NF-κB, TNF-α and MPO levels.[Results](1)Compared with SO group, the serum activity of AMY, LIP, Cr, BUN and histopathological changes were increased in ANP group(P<0.05) and decreased in APO group(P<0.05).(2)Compared with SO group, the expression level of Caspase-3, NF-κB and TNF-α were significantly increased in ANP group(P<0.05) and decreased in APO group(P<0.05).(3)Compared with SO group, the expression level of MPO was increased in ANP group( P < 0. 05) and decreased in APO group(P<0.05).[Conclusion]Apocynin can ameliorate renal injury of ANP model rats by inhibiting the activity of NOX2.
引文
[1]WALLING A,FREELOVE R. Pancreatitis and Pancreatic Cancer[J].Prim Care,2017,44(4):609-620.
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[4]孙海涛,陈辰,邓文宏,等.塞来昔布调节ORP-150对重症急性胰腺炎大鼠保护作用的最佳剂量[J].职业与健康,2011,27(18):2044-2047.
[5]古升,陈家诚,周贞学,等.胰淀粉酶水平变化与急性胰腺炎病情严重程度的相关性分析[J].中国临床研究,2016,29(5):629-631.
[6]PAVLIDIS P,CRICHTON S,LEMMICH SMITH J,et al. Improved Outcome of Severe Acute Pancreatitis in the Intensive Care Unit[J/OL].Critical Care Research and Practice,2013,2013:1-5.[2018-08-16].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3594930.DOI:10.1155/2013/897107.
[7]HALONEN KI,PETTILA V,LEPPANIEMI AK,et al. Multiple organ dysfunction associated with severe acute pancreatitis[J]. Crit Care Med,2002,30(6):1274-1279.
[8]CHENG G,CAO Z,XU X,et al. Homologs of gp91phox:cloning and tissue expression of Nox3,Nox4,and Nox5[J]. Gene,2001,269(1-2):131-140.
[9]LAMBETH JD. NOX enzymes and the biology of reactive oxygen[J].Nat Rev Immunol,2004,4(3):181-189.
[10]SUH YA,ARNOLD RS,LASSEGUE B,et al. Cell transformation by the superoxide-generating oxidase Mox1[J]. Nature,1999,401(6748):79-82.
[11] BEDARD K,KRAUSE KH. The NOX family of ROS-generating NADPH oxidases:physiology and pathophysiology[J]. Physiol Rev,2007,87(1):245-313.
[12]LIU PG,HE SQ,ZHANG YH,et al. Protective effects of apocynin and allopurinol on ischemia/reperfusion-induced liver injury in mice[J].World J Gastroenterol,2008,14(18):2832-2837.
[13] BARNES MA,MCMULLEN MR,ROYCHOWDHURY S,et al.Macrophage migration inhibitory factor contributes to ethanol-induced liver injury by mediating cell injury,steatohepatitis,and steatosis[J].Hepatology,2013,57(5):1980-1991.
[14] SIDARALA V,KOWLURU A. The regulatory roles of mitogen-activated protein kinase(MAPK)pathways in health and diabetes:Lessons learned from the pancreatic beta-Cell[J]. Recent Pat Endocr Metab Immune Drug Discov,2017,10(2):76-84.
[15] WITTE D,BARTSCHT T,KAUFMANN R,et al. TGF-beta1-induced cell migration in pancreatic carcinoma cells is RAC1 and NOX4-dependent and requires RAC1 and NOX4-dependent activation of p38 MAPK[J]. Oncol Rep,2017,38(6):3693-3701.
[16] YUBERO S,RAMUDO L,MANSO MA,et al. Mechanisms of dexamethasone-mediated chemokine down-regulation in mild and severe acute pa ncreatitis[J]. Biochim Biophys Acta,2009,1792(12):1205-1211.
[17] KIM C,SANO Y,TODOROVA K,et al. The kinase p38 alpha serves cell type-specific inflammatory functions in skin injury and coordinates pro-and anti-inflammatory gene expression[J]. Nat Immunol,2008,9(9):1019-1027.
[2]DIMAGNO MJ. Clinical update on fluid therapy and nutritional support in acute pancreatitis[J]. Pancreatology,2015,15(6):583-588.
[3]HALONEN KI,PETTILA V,LEPPANIEMI AK,et al. Multiple organ dysfunction associated with severe acute pancreatitis[J]. Crit Care Med,2002,30(6):1274-1279.
[4]孙海涛,陈辰,邓文宏,等.塞来昔布调节ORP-150对重症急性胰腺炎大鼠保护作用的最佳剂量[J].职业与健康,2011,27(18):2044-2047.
[5]古升,陈家诚,周贞学,等.胰淀粉酶水平变化与急性胰腺炎病情严重程度的相关性分析[J].中国临床研究,2016,29(5):629-631.
[6]PAVLIDIS P,CRICHTON S,LEMMICH SMITH J,et al. Improved Outcome of Severe Acute Pancreatitis in the Intensive Care Unit[J/OL].Critical Care Research and Practice,2013,2013:1-5.[2018-08-16].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3594930.DOI:10.1155/2013/897107.
[7]HALONEN KI,PETTILA V,LEPPANIEMI AK,et al. Multiple organ dysfunction associated with severe acute pancreatitis[J]. Crit Care Med,2002,30(6):1274-1279.
[8]CHENG G,CAO Z,XU X,et al. Homologs of gp91phox:cloning and tissue expression of Nox3,Nox4,and Nox5[J]. Gene,2001,269(1-2):131-140.
[9]LAMBETH JD. NOX enzymes and the biology of reactive oxygen[J].Nat Rev Immunol,2004,4(3):181-189.
[10]SUH YA,ARNOLD RS,LASSEGUE B,et al. Cell transformation by the superoxide-generating oxidase Mox1[J]. Nature,1999,401(6748):79-82.
[11] BEDARD K,KRAUSE KH. The NOX family of ROS-generating NADPH oxidases:physiology and pathophysiology[J]. Physiol Rev,2007,87(1):245-313.
[12]LIU PG,HE SQ,ZHANG YH,et al. Protective effects of apocynin and allopurinol on ischemia/reperfusion-induced liver injury in mice[J].World J Gastroenterol,2008,14(18):2832-2837.
[13] BARNES MA,MCMULLEN MR,ROYCHOWDHURY S,et al.Macrophage migration inhibitory factor contributes to ethanol-induced liver injury by mediating cell injury,steatohepatitis,and steatosis[J].Hepatology,2013,57(5):1980-1991.
[14] SIDARALA V,KOWLURU A. The regulatory roles of mitogen-activated protein kinase(MAPK)pathways in health and diabetes:Lessons learned from the pancreatic beta-Cell[J]. Recent Pat Endocr Metab Immune Drug Discov,2017,10(2):76-84.
[15] WITTE D,BARTSCHT T,KAUFMANN R,et al. TGF-beta1-induced cell migration in pancreatic carcinoma cells is RAC1 and NOX4-dependent and requires RAC1 and NOX4-dependent activation of p38 MAPK[J]. Oncol Rep,2017,38(6):3693-3701.
[16] YUBERO S,RAMUDO L,MANSO MA,et al. Mechanisms of dexamethasone-mediated chemokine down-regulation in mild and severe acute pa ncreatitis[J]. Biochim Biophys Acta,2009,1792(12):1205-1211.
[17] KIM C,SANO Y,TODOROVA K,et al. The kinase p38 alpha serves cell type-specific inflammatory functions in skin injury and coordinates pro-and anti-inflammatory gene expression[J]. Nat Immunol,2008,9(9):1019-1027.