SIRT1减轻缺氧复氧肾小管上皮细胞炎性损伤的实验研究
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摘要
目的:研究沉默信息调节因子1(SIRT1)对缺氧复氧肾小管上皮细胞炎性损伤的影响。方法:构建缺氧复氧肾小管上皮细胞损伤模型,用qRT-PCR和Western blot方法检测细胞中SIRT1的表达变化。在肾小管上皮细胞中转染pc DNA3. 1-SIRT1,qRT-PCR和Western blot检测过表达效率。ELISA法检测细胞分泌肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)水平。Western blot检测CCAAT/增强子结合蛋白同源蛋白(CHOP)、Caspase-11蛋白水平。全自动生化分析仪检测培养液中乳酸脱氢酶(LDH)含量。结果:缺氧复氧肾小管上皮细胞中SIRT1 mRNA和蛋白水平均降低。pc DNA3. 1-SIRT1转染后的肾小管上皮细胞中SIRT1 mRNA和蛋白水平均明显升高。缺氧复氧肾小管上皮细胞分泌的TNF-α、IL-1β水平升高,细胞中CHOP、Caspase-11蛋白水平升高,细胞培养液中LDH水平升高。过表达SIRT1的肾小管上皮细胞经缺氧复氧处理以后,细胞分泌的TNF-α、IL-1β水平降低,细胞中CHOP、Caspase-11蛋白水平降低,细胞培养液中LDH水平降低。结论:SIRT1具有减轻缺氧复氧肾小管上皮细胞炎性损伤的作用,其可减少细胞分泌炎性因子,作用机制与内质网应激介导的Caspase-11途径有关。
Objective: To study the effects of SIRT1 on inflammatory injury of renal tubular epithelial cells after hypoxia reoxygenation. Methods: Construction of hypoxia reoxygenation injury model of renal tubular epithelial cells. The expression of SIRT1 in cells was detected by qRT-PCR and Western blot. Transfection pc DNA3. 1-SIRT1 into renal tubular epithelial cells. qRT-PCR and Western blot were used to detect the efficiency of overexpression. ELISA assay was used to detect the secretion of TNF-α and IL-1β in cells. The level of CHOP and Caspase-11 protein was detected by Western blot. The content of LDH in culture medium was detected by automatic biochemical analyzer. Results: The levels of SIRT1 mRNA and protein were decreased in anoxia reoxygenation renal tubular epithelial cells. After transfection,the levels of SIRT1 mRNA and protein in pc DNA3. 1-SIRT1 cells increased significantly. The levels of TNF-α and IL-1β secreted by anoxia reoxygenation renal tubular epithelial cells increased,the levels of CHOP and Caspase-11 protein in the cells increased. The level of LDH increased in the cell culture medium. After overexpression of SIRT1,renal tubular epithelial cells were treated with anoxia and reoxygenation. The levels of TNF-α and IL-1β secreted by the cells decreased,the level of CHOP and Caspase-11 protein in the cells decreased,the level of LDH in cell culture medium decreased. Conclusion: SIRT1 can alleviate inflammatory injury of renal tubular epithelial cells after hypoxia reoxygenation,it can reduce the secretion of inflammatory factors in cells,the mechanism is related to endoplasmic reticulum stress mediated Caspase-11 pathway.
Objective: To study the effects of SIRT1 on inflammatory injury of renal tubular epithelial cells after hypoxia reoxygenation. Methods: Construction of hypoxia reoxygenation injury model of renal tubular epithelial cells. The expression of SIRT1 in cells was detected by qRT-PCR and Western blot. Transfection pc DNA3. 1-SIRT1 into renal tubular epithelial cells. qRT-PCR and Western blot were used to detect the efficiency of overexpression. ELISA assay was used to detect the secretion of TNF-α and IL-1β in cells. The level of CHOP and Caspase-11 protein was detected by Western blot. The content of LDH in culture medium was detected by automatic biochemical analyzer. Results: The levels of SIRT1 mRNA and protein were decreased in anoxia reoxygenation renal tubular epithelial cells. After transfection,the levels of SIRT1 mRNA and protein in pc DNA3. 1-SIRT1 cells increased significantly. The levels of TNF-α and IL-1β secreted by anoxia reoxygenation renal tubular epithelial cells increased,the levels of CHOP and Caspase-11 protein in the cells increased. The level of LDH increased in the cell culture medium. After overexpression of SIRT1,renal tubular epithelial cells were treated with anoxia and reoxygenation. The levels of TNF-α and IL-1β secreted by the cells decreased,the level of CHOP and Caspase-11 protein in the cells decreased,the level of LDH in cell culture medium decreased. Conclusion: SIRT1 can alleviate inflammatory injury of renal tubular epithelial cells after hypoxia reoxygenation,it can reduce the secretion of inflammatory factors in cells,the mechanism is related to endoplasmic reticulum stress mediated Caspase-11 pathway.
引文
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[12] Liu XJ,Tan Y,Geng YQ,et al. Proximal tubule toll-like receptor4 expression linked to inflammation and apoptosis following hypoxia/reoxygenation injury[J]. Am J Nephrol,2014,39(4):337-347.
[13] Cao Y,Jiang X,Ma H,et al. SIRT1 and insulin resistance[J]. J Diabetes Complications,2016,5(7):178-183.
[14] Gao J,Zhou R,You X,et al. Salidroside suppresses inflammation in a D-galactose-induced rat model of Alzheimer's disease via SIRT1/NF-κB pathway[J]. Metab Brain Dis,2016,31(4):771-778.
[15] Ye Z,Fang J,Dai S,et al. MicroRNA-34a induces a senescencelike change via the down-regulation of SIRT1 and up-regulation of p53 protein in human esophageal squamous cancer cells with a wild-type p53 gene background[J]. Cancer Lett,2016,370(2):216-221.
[16] Xu F,Lin B,Zheng X,et al. GLP-1 receptor agonist promotes brown remodelling in mouse white adipose tissue through SIRT1[J]. Diabetologia,2016,59(5):1059-1069.
[17]曹荣,张晓燕,康继宏,等. Sirt1在肾脏疾病中的作用[J].生理科学进展,2013,44(4):305-309.Cao R,Zhang XY,Kang JH,et al. The role of Sirt1 in renal diseases[J]. Prog Physiol Sci,2013,44(4):305-309.
[18]邵滢,吕川,吴灿,等. Mir-217通过Sirt1/HIF-1α信号通路介导高糖诱导的大鼠肾小球系膜细胞炎症反应及纤维化[J].中华内分泌代谢杂志,2016,32(7):556-563.Shao Y,Lv C,Wu C,et al. Mir-217 promotes inflammation and fibrosis induced by high glucose in rat glomerular mesangial cells via Sirt1/HIF-1αsignal pathway[J]. Chin J Endocrinol Metab,2016,32(7):556-563.
[19] Zhou L,Xu DY,Sha WG,et al. High glucose induces renal tubular epithelial injury via Sirt1/NF-kappaB/microR-29/Keap1signal pathway[J]. J Transl Med,2015,13(1):352.
[20] Hasegawa K,Wakino SK,Tatematsu S,et al. Sirt1 protects against oxidative stress-induced renal tubular cell apoptosis by the bidirectional regulation of catalase expression[J]. Biochem Biophys Res Commun,2008,372(1):51-56.
[21]曲柳,仇丽鸿. SIRT1抑制炎症反应的研究进展[J].中国实用口腔科杂志,2013,6(9):564-568.Qu L,Qiu LH. Research progress of SIRT1 in suppressing inflammatory response[J]. Chin J Pract Stomatol,2013,6(9):564-568.
[22] Carloni S,Favrais G,Saliba E,et al. Melatonin modulates neonatal brain inflammation through ER stress,autophagy and miR-34a/SIRT1 pathway[J]. J Pineal Res,2016,61(3):370-380.
[23] Morishima N,Nakanishi K,Takenouchi H,et al. An endoplasmic reticulum stress-specific caspase cascade in apoptosis. Cytochrome c-independent activation of caspase-9 by caspase-12[J]. J Biol Chem,2002,277(37):34287-34294.
[24] Shenderov K,Riteau N,Yip R,et al. Cutting edge:Endoplasmic reticulum stress licenses macrophages to produce mature IL-1βin response to TLR4 stimulation through a caspase-8-and TRIFdependent pathway[J]. J Immunol,2014,192(5):2029-2033.
[25] Yang JR,Yao FH,Zhang JG,et al. Ischemia-reperfusion induces renal tubule pyroptosis via the CHOP-caspase-11 pathway[J].Am J Physiol Renal Physiol,2014,306(1):75-84.
[26]谢静,杜敏,史栋梁. SIRT1过表达可抑制衣霉素诱导软骨细胞内质网应激[J].基础医学与临床,2016,36(1):89-93.Xie J,Du M,Shi DL. SIRT1 overexpression inhibits tunicamycininduced endoplasmic reticulum stress in chondrocytes[J]. Basic Clin Med,2016,36(1):89-93.
[27] Hong EH,Lee SJ,Kim JS,et al. Ionizing radiation induces cellular senescence of articular chondrocytes via negative regulation of SIRT1 by p38 kinase[J]. J Biol Chem 2010,285(2):1283-1295.
[28] Li Y,Xu S,Giles A,et al. Hepatic overexpression of SIRT1 in mice attenuates endoplasmic reticulum stress and insulin resistance in the liver[J]. FASEB J,2011,25(5):1664-1679.
[2] Emma F,Montini G,Parikh SM,et al. Mitochondrial dysfunction in inherited renal disease and acute kidney injury[J]. Nat Rev Nephrol,2016,12(5):267-280.
[3] Ueki M,Asaga T,Chujo K,et al. D-allose protects against endotoxemic acute renal injury[J]. J Biosci Bioeng,2008,105(5):481-485.
[4]陈厚早,张祝琴,韦玉生,等.去乙酰化酶SIRT1的研究进展[J].中国医学科学院学报,2007,29(3):441-447.Chen HZ,Zhang ZQ,Wei YS,et al. Research progression of deacetylase(SIRT1)[J]. Acta Acad Med Sinicae,2007,29(3):441-447.
[5] Xie J,Zhang X,Zhang L. Negative regulation of inflammation by SIRT1[J]. Pharmacol Res,2013,67(1):60-67.
[6] Julien C,Tremblay C,mond V,et al. SIRT1 decrease parallels the accumulation of tau in alzheimer disease[J]. J Neuropathol Exp Neurol,2009,68(1):48-58.
[7] Hasegawa K,Wakino S,Yoshioka K,et al. Kidney-specific overexpression of Sirt1 protects against acute kidney injury by retaining peroxisome function[J]. J Biol Chem,2010,285(17):13045-13056.
[8] Fan H,Yang HC,You L,et al. The histone deacetylase,SIRT1,contributes to the resistance of young mice to ischemia/reperfusioninduced acute kidney injury[J]. Kidney Int,2013,83(3):404-413.
[9] Chen H,Huang RS,Yu XX,et al. Emodin protects against oxidative stress and apoptosis in HK-2 renal tubular epithelial cells after hypoxia/reoxygenation[J]. Exp Ther Med,2017,14(1):447-452.
[10]公伟,刘丹,岳会敏,等.冬虫夏草菌丝体提取物抑制顺铂诱导的肾小管上皮细胞损伤[J].中国免疫学杂志,2016,32(5):669-672.Gong W,Liu D,Yue HM,et al. Effect of hirsutella sinensis mycelium on cis-dia minodichloroplatin-induced injury of renal tubular epithelial cell[J]. Chin J Immun,2016,32(5):669-672.
[11] Franquesa M,Riera M,Herrero-Fresneda I,et al. Tubular epithelial cells transfected with hHGF counteracts monocyte chemotactic protein-1 up-regulation after hypoxia/reoxygenation insult[J]. Transplant Proc,2009,41(6):2069-2072.
[12] Liu XJ,Tan Y,Geng YQ,et al. Proximal tubule toll-like receptor4 expression linked to inflammation and apoptosis following hypoxia/reoxygenation injury[J]. Am J Nephrol,2014,39(4):337-347.
[13] Cao Y,Jiang X,Ma H,et al. SIRT1 and insulin resistance[J]. J Diabetes Complications,2016,5(7):178-183.
[14] Gao J,Zhou R,You X,et al. Salidroside suppresses inflammation in a D-galactose-induced rat model of Alzheimer's disease via SIRT1/NF-κB pathway[J]. Metab Brain Dis,2016,31(4):771-778.
[15] Ye Z,Fang J,Dai S,et al. MicroRNA-34a induces a senescencelike change via the down-regulation of SIRT1 and up-regulation of p53 protein in human esophageal squamous cancer cells with a wild-type p53 gene background[J]. Cancer Lett,2016,370(2):216-221.
[16] Xu F,Lin B,Zheng X,et al. GLP-1 receptor agonist promotes brown remodelling in mouse white adipose tissue through SIRT1[J]. Diabetologia,2016,59(5):1059-1069.
[17]曹荣,张晓燕,康继宏,等. Sirt1在肾脏疾病中的作用[J].生理科学进展,2013,44(4):305-309.Cao R,Zhang XY,Kang JH,et al. The role of Sirt1 in renal diseases[J]. Prog Physiol Sci,2013,44(4):305-309.
[18]邵滢,吕川,吴灿,等. Mir-217通过Sirt1/HIF-1α信号通路介导高糖诱导的大鼠肾小球系膜细胞炎症反应及纤维化[J].中华内分泌代谢杂志,2016,32(7):556-563.Shao Y,Lv C,Wu C,et al. Mir-217 promotes inflammation and fibrosis induced by high glucose in rat glomerular mesangial cells via Sirt1/HIF-1αsignal pathway[J]. Chin J Endocrinol Metab,2016,32(7):556-563.
[19] Zhou L,Xu DY,Sha WG,et al. High glucose induces renal tubular epithelial injury via Sirt1/NF-kappaB/microR-29/Keap1signal pathway[J]. J Transl Med,2015,13(1):352.
[20] Hasegawa K,Wakino SK,Tatematsu S,et al. Sirt1 protects against oxidative stress-induced renal tubular cell apoptosis by the bidirectional regulation of catalase expression[J]. Biochem Biophys Res Commun,2008,372(1):51-56.
[21]曲柳,仇丽鸿. SIRT1抑制炎症反应的研究进展[J].中国实用口腔科杂志,2013,6(9):564-568.Qu L,Qiu LH. Research progress of SIRT1 in suppressing inflammatory response[J]. Chin J Pract Stomatol,2013,6(9):564-568.
[22] Carloni S,Favrais G,Saliba E,et al. Melatonin modulates neonatal brain inflammation through ER stress,autophagy and miR-34a/SIRT1 pathway[J]. J Pineal Res,2016,61(3):370-380.
[23] Morishima N,Nakanishi K,Takenouchi H,et al. An endoplasmic reticulum stress-specific caspase cascade in apoptosis. Cytochrome c-independent activation of caspase-9 by caspase-12[J]. J Biol Chem,2002,277(37):34287-34294.
[24] Shenderov K,Riteau N,Yip R,et al. Cutting edge:Endoplasmic reticulum stress licenses macrophages to produce mature IL-1βin response to TLR4 stimulation through a caspase-8-and TRIFdependent pathway[J]. J Immunol,2014,192(5):2029-2033.
[25] Yang JR,Yao FH,Zhang JG,et al. Ischemia-reperfusion induces renal tubule pyroptosis via the CHOP-caspase-11 pathway[J].Am J Physiol Renal Physiol,2014,306(1):75-84.
[26]谢静,杜敏,史栋梁. SIRT1过表达可抑制衣霉素诱导软骨细胞内质网应激[J].基础医学与临床,2016,36(1):89-93.Xie J,Du M,Shi DL. SIRT1 overexpression inhibits tunicamycininduced endoplasmic reticulum stress in chondrocytes[J]. Basic Clin Med,2016,36(1):89-93.
[27] Hong EH,Lee SJ,Kim JS,et al. Ionizing radiation induces cellular senescence of articular chondrocytes via negative regulation of SIRT1 by p38 kinase[J]. J Biol Chem 2010,285(2):1283-1295.
[28] Li Y,Xu S,Giles A,et al. Hepatic overexpression of SIRT1 in mice attenuates endoplasmic reticulum stress and insulin resistance in the liver[J]. FASEB J,2011,25(5):1664-1679.