TSP1对高糖诱导的肾小管上皮细胞损伤的影响研究
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摘要
目的探讨血小板反应蛋白1(TSP1)对高糖诱导的肾小管上皮细胞损伤及炎症因子分泌的影响。方法以肾小管上皮细胞为研究对象,通过转染TSP-1 shRNA(shTSP-1),沉默TSP-1基因,探讨下调TSP1对高糖条件下肾小管上皮细胞损伤的影响。肾小管上皮细胞依次分为:对照组(以5.6 mmol/L葡萄糖培养)、高糖组(以30 mmol/L葡萄糖培养)、NC+高糖组(对照慢病毒转染,以30 mmol/L葡萄糖培养)、干扰+高糖组(TSP1 shRNA慢病毒转染以30mmol/L葡萄糖培养)。Realtime PCR和Western Blot检测高糖对细胞中TSP1表达影响,同时检测TSP1 shRNA干扰效果。DCFH-DA法检测细胞中活性氧(ROS)水平,硫代巴比妥酸法检测培养液中丙二醛(MDA)含量,ELISA法检测培养液上清中肿瘤坏死因子-α(TNF-α)、白细胞介素-8(IL-8)含量,Annexin V-FITC/PI双染法检测细胞凋亡,Western Blot法检测细胞中活化的Caspase-3(c-caspase-3)蛋白水平。两组均数差异比较采用独立样本t检验,多组均数间差异比较采用单因素方差分析,组间两两比较采用SNK-q检验。结果高糖组细胞中TSP1mRNA和蛋白水平高于对照组(P <0.05)。干扰+高糖组细胞中TSP1 mRNA和蛋白水平低于高糖组(P <0.05)。与对照组比较,高糖组细胞中ROS水平升高,培养液中MDA、TNF-α、IL-8含量升高[(2.36±0.21)nmol/ml、(45.91±2.87)ng/ml、(25.42±3.26) ng/ml:(1.05±0.13)nmol/ml、(20.14±1.36)ng/ml、(12.98±1.63)ng/ml],差异有统计学意义(F=18.595,F=43.825,F=21.155,P <0.05);细胞凋亡率升高(P <0.05),细胞中c-caspase-3蛋白水平升高(P <0.05)。与高糖组和NC+高糖组比较,干扰+高糖组细胞中ROS水平降低,培养液中MDA、TNF-α、IL-8含量降低[(1.63±0.10)nmol/ml、(34.20±2.06)ng/ml、(18.75±1.62)ng/ml:(2.36±0.21) nmol/ml、(45.91±2.87)ng/ml、(25.42±3.26)ng/ml和(2.30±0.42)nmol/ml、(46.32±5.24) ng/ml、(26.91±2.74)ng/ml],差异具有统计学意义(F=18.595,F=43.825,F=21.155,P <0.05);细胞凋亡率降低,细胞中c-caspase-3蛋白水平降低(P <0.05)。结论高糖诱导肾小管上皮细胞中TSP1表达,下调其表达可以减少细胞分泌炎症因子,抑制高糖诱导的肾小管上皮细胞损伤。
Objective To investigate the effects of TSP1 on renal tubular epithelial cell injury and secretion of inflammatory factors induced by high glucose. Methods TSP-1 shRNA(shTSP-1)was transfected into renal tubular epithelial cells to silence TSP-1 gene. The renal tubular epithelial cells were divided into control group(treated with 5.6 mmol/L glucose), high glucose group(treated with 30 mmol/L glucose), NC + high glucose group(treated with 30 mmol/L glucose, transfected with lentivirus control), interference + high glucose group(treated with 30 mmol/L glucose culture, transfected with TSP1 shRNA lentivirus). Realtime PCR and Western Blot were used to detect the effect of high glucose on the expression of TSP1 and the effect of TSP1 shRNA on the expression of TSP1. The level of ROS in cells was detected by DCFH-DA. The content of MDA in culture medium was detected by thiobarbituric acid. ELISA was used to detect TNF-α and IL-8 in the supernatant of the culture medium. Annexin V-FITC/PI double staining was used to detect apoptosis. Western Blot assay was used to detect c-caspase-3 in cells. Independent sample t test was used to compare the mean difference between the two groups, single factor analysis of variance was used to compare the difference among multiple groups, and SNK-q test was used to compare the two groups. Results The levels of TSP1 mRNA and protein in the high glucose group were higher than those in the control group(P < 0.05). The levels of TSP1 mRNA and protein in the interference + high glucose group were lower than those in the high glucose group(P < 0.05). Compared with the control group, the level of ROS in the high glucose group increased, the contents of MDA, TNF-αand IL-8 increased in culture medium, the rate of apoptosis increased, the level of c-caspase-3 protein in the cells increased [(2.36±0.21)nmol/ml,(45.91±2.87)ng/ml,(25.42±3.26)ng/ml vs(1.05±0.13)nmol/ml,(20.14±1.36)ng/ml,(12.98±1.63)ng/ml], the difference has statistical significance(P < 0.05). Compared with high glucose group and NC + high glucose group, the level of ROS in the interference + high glucose group decreased, the contents of MDA, TNF-α and IL-8 in culture medium decreased, the apoptosis rate decreased, the level of c-caspase-3 decreased [(1.63±0.10) nmol/ml,(34.20±2.06)ng/ml,(18.75±1.62)ng/ml vs(2.36±0.21) nmol/ml,(45.91±2.87) ng/ml,(25.42±3.26)ng/ml 和(2.30±0.42)nmol/ml,(46.32±5.24)ng/ml,(26.91±2.74)ng/ml], the difference has statistical significance(P < 0.05). Conclusion High glucose induces TSP1 expression in renal tubular epithelial cells, down-regulation of its expression can inhibit high glucose-induced renal tubular epithelial cell injury and reduce cell secretion of inflammatory factors.
Objective To investigate the effects of TSP1 on renal tubular epithelial cell injury and secretion of inflammatory factors induced by high glucose. Methods TSP-1 shRNA(shTSP-1)was transfected into renal tubular epithelial cells to silence TSP-1 gene. The renal tubular epithelial cells were divided into control group(treated with 5.6 mmol/L glucose), high glucose group(treated with 30 mmol/L glucose), NC + high glucose group(treated with 30 mmol/L glucose, transfected with lentivirus control), interference + high glucose group(treated with 30 mmol/L glucose culture, transfected with TSP1 shRNA lentivirus). Realtime PCR and Western Blot were used to detect the effect of high glucose on the expression of TSP1 and the effect of TSP1 shRNA on the expression of TSP1. The level of ROS in cells was detected by DCFH-DA. The content of MDA in culture medium was detected by thiobarbituric acid. ELISA was used to detect TNF-α and IL-8 in the supernatant of the culture medium. Annexin V-FITC/PI double staining was used to detect apoptosis. Western Blot assay was used to detect c-caspase-3 in cells. Independent sample t test was used to compare the mean difference between the two groups, single factor analysis of variance was used to compare the difference among multiple groups, and SNK-q test was used to compare the two groups. Results The levels of TSP1 mRNA and protein in the high glucose group were higher than those in the control group(P < 0.05). The levels of TSP1 mRNA and protein in the interference + high glucose group were lower than those in the high glucose group(P < 0.05). Compared with the control group, the level of ROS in the high glucose group increased, the contents of MDA, TNF-αand IL-8 increased in culture medium, the rate of apoptosis increased, the level of c-caspase-3 protein in the cells increased [(2.36±0.21)nmol/ml,(45.91±2.87)ng/ml,(25.42±3.26)ng/ml vs(1.05±0.13)nmol/ml,(20.14±1.36)ng/ml,(12.98±1.63)ng/ml], the difference has statistical significance(P < 0.05). Compared with high glucose group and NC + high glucose group, the level of ROS in the interference + high glucose group decreased, the contents of MDA, TNF-α and IL-8 in culture medium decreased, the apoptosis rate decreased, the level of c-caspase-3 decreased [(1.63±0.10) nmol/ml,(34.20±2.06)ng/ml,(18.75±1.62)ng/ml vs(2.36±0.21) nmol/ml,(45.91±2.87) ng/ml,(25.42±3.26)ng/ml 和(2.30±0.42)nmol/ml,(46.32±5.24)ng/ml,(26.91±2.74)ng/ml], the difference has statistical significance(P < 0.05). Conclusion High glucose induces TSP1 expression in renal tubular epithelial cells, down-regulation of its expression can inhibit high glucose-induced renal tubular epithelial cell injury and reduce cell secretion of inflammatory factors.
引文
1 Kim DY,Kang MK,Park SH,et al.Eucalyptol ameliorates Snail1/beta-catenin-dependent diabetic disjunction of renal tubular epithelial cells and tubulointerstitial fibrosis[J].Oncotarget,2017,8(63):106190-106205.
2 Gou R,Chen J,Sheng S,et al.KIM-1 mediates high Glucose-Induced autophagy and apoptosis in renal tubular epithelial cells[J].Cell Physiol Biochem,2016,38(6):2479-2488.
3 Wu Y,Zhang M,Liu R,et al.Oxidative Stress-Activated NHE1 is involved in high Glucose-Induced apoptosis in renal tubular epithelial cells[J].Yonsei Med J,2016,57(5):1252-1259.
4 Li K,Wu Q,Sun X,et al.Tsp1 promotes alveolar stem cell proliferation and its downregulation relates to lung inflammation in intralobar pulmonary sequestration[J].Oncotarget,2017,8(39):64867-64877.
5 Fleitas T,Martinez-Sales V,Vila V,et al.VEGF and TSP1 levels correlate with prognosis in advanced non-small cell lung cancer[J].Clin Transl Oncol,2013,15(11):897-902.
6杨小燕,曾嵘,王俭勤,等.急性肾衰大鼠肾小管上皮细胞TSP1表达及凋亡的意义[J].按摩与康复医学:中旬刊,2011,2(5):10-11.
7付玲,代甜,刘建社.吡格列酮对糖尿病大鼠肾组织TSP1表达的影响[J].华中科技大学学报(医学版),2009,38(6):725-728.
8 De Kort H,Heutinck KM,Ruben JM,et al.Primary human RenalDerived tubular epithelial cells fail to recognize and suppress BK virus infection[J].Transplantation,2017,101(8):1820-1829.
9 Pena C,Hernandez-Fonseca JP,Pedreanez AA,et al.Renal oxidative stress and renal CD8(+)T-cell infiltration in mercuric chloride-induced nephropathy in rats:role of angiotensin II[J].J Immunotoxicol,2016,13(3):324-334.
10 Zhou C,Yool AJ,Byard RW.The etiology of basal vacuolizations in renal tubular epithelial cells evaluated in an isolated perfused kidney model[J].J Forensic Sci,2017,62(4):915-920.
11 Yan Q,Luo H,Wang BY,et al.Correlation between PKB/Akt,GSK-3beta expression and tubular epithelial-mesenchymal transition in renal allografts with chronic active antibody-mediated rejection[J].Exp Ther Med,2017,13(5):2217-2224.
12 Lopez-Ramirez M A,Fonseca G,Zeineddine H A,et al.Thrombospondin1(TSP1)replacement prevents cerebral cavernous malformations[J].J Exp Med,2017,214(11):3331-3346.
13 Suades R,PadróT,Alonso R,et al.High levels of TSP1+/CD142+platelet-derived microparticles characterise young patients with high cardiovascular risk and subclinical atherosclerosis[J].Thromb Haemost,2015,114(6):1310-1321.
14 Labrousse-Arias D,Castillo-Gonzalez R,Rogers NM,et al.HIF-2alpha-mediated induction of pulmonary thrombospondin-1 contributes to hypoxia-driven vascular remodelling and vasoconstriction[J].Cardiovasc Res,2016,109(1):115-130.
15 Gao P,Li L,Ji L,et al.Nrf2 ameliorates diabetic nephropathy progression by transcriptional repression of TGF beta 1 through interactions with c-Jun and SP1[J].Biochim Biophys Acta,2014,1839(11):1110-1120.
16 Li X,Zeng L,Cao C,et al.Long noncoding RNA MALAT1 regulates renal tubular epithelial pyroptosis by modulated miR-23c targeting of ELAVL1 in diabetic nephropathy[J].Exp Cell Res,2017,350(2):327-335.
17 Xiang L,Wu TT,Chen J,et al.Elevated expression levels of serum IGF-1,TNF-αand VEGF 165,May exacerbate type 2 diabetic nephropathy[J].J Diabetes Investig,2016,8(1):108-114.
18 Yang HL,Wu SK.Ligustrazine attenuates renal damage by inhibiting endoplasmic reticulum stress in diabetic nephropathy by inactivating MAPK pathways[J].RSC Adv,2018,8(39):21816-21822.
19 Contrerasruiz L,Ryan DS,Sia RK,et al.Polymorphism in THBS1gene is associated with post-refractive surgery chronic ocular surface inflammation[J].Ophthalmology,2014,121(7):1389-1397.
20 Vives-Bauza C,Gonzalo R,Manfredi G,et al.Enhanced ROSproduction and antioxidant defenses in cybrids harbouring mutations in mtDNA[J].Neurosci Lett,2006,391(3):136-141.
21 Yang CP,Zhang ZH,Zhang LH,et al.Neuroprotective role of MicroRNA-22 in a 6-Hydroxydopamine-Induced cell model of parkinson’s disease via regulation of its target gene TRPM7[J].J Mol Neurosci,2016,60(4):445-452.
2 Gou R,Chen J,Sheng S,et al.KIM-1 mediates high Glucose-Induced autophagy and apoptosis in renal tubular epithelial cells[J].Cell Physiol Biochem,2016,38(6):2479-2488.
3 Wu Y,Zhang M,Liu R,et al.Oxidative Stress-Activated NHE1 is involved in high Glucose-Induced apoptosis in renal tubular epithelial cells[J].Yonsei Med J,2016,57(5):1252-1259.
4 Li K,Wu Q,Sun X,et al.Tsp1 promotes alveolar stem cell proliferation and its downregulation relates to lung inflammation in intralobar pulmonary sequestration[J].Oncotarget,2017,8(39):64867-64877.
5 Fleitas T,Martinez-Sales V,Vila V,et al.VEGF and TSP1 levels correlate with prognosis in advanced non-small cell lung cancer[J].Clin Transl Oncol,2013,15(11):897-902.
6杨小燕,曾嵘,王俭勤,等.急性肾衰大鼠肾小管上皮细胞TSP1表达及凋亡的意义[J].按摩与康复医学:中旬刊,2011,2(5):10-11.
7付玲,代甜,刘建社.吡格列酮对糖尿病大鼠肾组织TSP1表达的影响[J].华中科技大学学报(医学版),2009,38(6):725-728.
8 De Kort H,Heutinck KM,Ruben JM,et al.Primary human RenalDerived tubular epithelial cells fail to recognize and suppress BK virus infection[J].Transplantation,2017,101(8):1820-1829.
9 Pena C,Hernandez-Fonseca JP,Pedreanez AA,et al.Renal oxidative stress and renal CD8(+)T-cell infiltration in mercuric chloride-induced nephropathy in rats:role of angiotensin II[J].J Immunotoxicol,2016,13(3):324-334.
10 Zhou C,Yool AJ,Byard RW.The etiology of basal vacuolizations in renal tubular epithelial cells evaluated in an isolated perfused kidney model[J].J Forensic Sci,2017,62(4):915-920.
11 Yan Q,Luo H,Wang BY,et al.Correlation between PKB/Akt,GSK-3beta expression and tubular epithelial-mesenchymal transition in renal allografts with chronic active antibody-mediated rejection[J].Exp Ther Med,2017,13(5):2217-2224.
12 Lopez-Ramirez M A,Fonseca G,Zeineddine H A,et al.Thrombospondin1(TSP1)replacement prevents cerebral cavernous malformations[J].J Exp Med,2017,214(11):3331-3346.
13 Suades R,PadróT,Alonso R,et al.High levels of TSP1+/CD142+platelet-derived microparticles characterise young patients with high cardiovascular risk and subclinical atherosclerosis[J].Thromb Haemost,2015,114(6):1310-1321.
14 Labrousse-Arias D,Castillo-Gonzalez R,Rogers NM,et al.HIF-2alpha-mediated induction of pulmonary thrombospondin-1 contributes to hypoxia-driven vascular remodelling and vasoconstriction[J].Cardiovasc Res,2016,109(1):115-130.
15 Gao P,Li L,Ji L,et al.Nrf2 ameliorates diabetic nephropathy progression by transcriptional repression of TGF beta 1 through interactions with c-Jun and SP1[J].Biochim Biophys Acta,2014,1839(11):1110-1120.
16 Li X,Zeng L,Cao C,et al.Long noncoding RNA MALAT1 regulates renal tubular epithelial pyroptosis by modulated miR-23c targeting of ELAVL1 in diabetic nephropathy[J].Exp Cell Res,2017,350(2):327-335.
17 Xiang L,Wu TT,Chen J,et al.Elevated expression levels of serum IGF-1,TNF-αand VEGF 165,May exacerbate type 2 diabetic nephropathy[J].J Diabetes Investig,2016,8(1):108-114.
18 Yang HL,Wu SK.Ligustrazine attenuates renal damage by inhibiting endoplasmic reticulum stress in diabetic nephropathy by inactivating MAPK pathways[J].RSC Adv,2018,8(39):21816-21822.
19 Contrerasruiz L,Ryan DS,Sia RK,et al.Polymorphism in THBS1gene is associated with post-refractive surgery chronic ocular surface inflammation[J].Ophthalmology,2014,121(7):1389-1397.
20 Vives-Bauza C,Gonzalo R,Manfredi G,et al.Enhanced ROSproduction and antioxidant defenses in cybrids harbouring mutations in mtDNA[J].Neurosci Lett,2006,391(3):136-141.
21 Yang CP,Zhang ZH,Zhang LH,et al.Neuroprotective role of MicroRNA-22 in a 6-Hydroxydopamine-Induced cell model of parkinson’s disease via regulation of its target gene TRPM7[J].J Mol Neurosci,2016,60(4):445-452.