黄芪甲苷对高糖诱导肾小管上皮细胞凋亡及线粒体自噬相关蛋白表达的影响
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摘要
【目的】探讨黄芪甲苷对高糖诱导肾小管上皮细胞凋亡及线粒体自噬相关蛋白表达的影响。【方法】将大鼠近端肾小管上皮细胞NRK-52E分为3组:正常对照组、高糖刺激组(给予葡萄糖30 mmol/L,作用48 h)及黄芪甲苷干预组(给予葡萄糖30 mmol/L刺激的同时加用黄芪甲苷100μmol/L,48 h)。采用细胞计数试剂盒8(CCK-8)检测细胞增殖能力;采用Westernblot法检测促凋亡蛋白cleaved-caspase-3及同源性磷酸酶张力蛋白诱导的激酶1(PINK1)/帕金蛋白(Parkin)介导的线粒体自噬相关蛋白PINK1、Parkin、LC3-Ⅱ、LC3-Ⅰ的表达。【结果】黄芪甲苷可有效缓解高糖对细胞增殖的抑制作用(P <0.01)。高糖可上调NRK-52E细胞促凋亡蛋白cleaved-caspase-3,线粒体自噬相关蛋白PINK1、Parkin的表达水平及LC3-Ⅱ/LC3-Ⅰ比值(均P <0.01);而黄芪甲苷干预可显著下调上述促凋亡蛋白及线粒体自噬相关蛋白的表达(P <0.05或P <0.01)。【结论】黄芪甲苷可抑制高糖诱导的肾小管上皮细胞NRK-52E的凋亡,减轻其由PINK1/Parkin介导的线粒体自噬。
Objective To explore the effect of astragaloside Ⅳ(AS-Ⅳ)on apoptosis-and mitophagy-related proteins in high glucose(HG)-induced renal tubular epithelial cells.Methods The proximal renal tubular epithelial cells NRK-52E were divided into 3 groups,namely normal control group,HG stimulating group(at the dosage of 30 mmol/L for 48h),and AS-Ⅳintervention group(treated with HG 30 mmol/L and AS-Ⅳ100μmol/L for 48 h).The activity of cell proliferation was tested by cell counting kit 8(CCK-8).The expression levels of apoptosis-related protein cleaved-caspase-3 and proteins including PINK1,Parkin,LC3-Ⅱ,LC3-Ⅰassociated with mitophagy mediated by phosphatase and tensin homolog deleted on chromosome ten induced kinase 1(PINK1)/Parkin were detected by Western blotting method.Results The inhibition of HG-induced cell proliferation was effectively lightened by AS-Ⅳ(P<0.01).The expression levels of pro-apoptotic protein cleavedcaspase-3 and mitophagy-associated proteins PINK1 and Parkin as well as the ratio of LC3-Ⅱ/LC3-Ⅰin NRK-52E cells were up-regulated by HG(P<0.01),and then were significantly down-regulated by AS-Ⅳ(P<0.05 or P<0.01).Conclusion AS-Ⅳcould inhibit the apoptosis of HG-induced renal tubular epithelial cells NRK-52E,and alleviate PINK1/Parkin-mediated mitophagy.
Objective To explore the effect of astragaloside Ⅳ(AS-Ⅳ)on apoptosis-and mitophagy-related proteins in high glucose(HG)-induced renal tubular epithelial cells.Methods The proximal renal tubular epithelial cells NRK-52E were divided into 3 groups,namely normal control group,HG stimulating group(at the dosage of 30 mmol/L for 48h),and AS-Ⅳintervention group(treated with HG 30 mmol/L and AS-Ⅳ100μmol/L for 48 h).The activity of cell proliferation was tested by cell counting kit 8(CCK-8).The expression levels of apoptosis-related protein cleaved-caspase-3 and proteins including PINK1,Parkin,LC3-Ⅱ,LC3-Ⅰassociated with mitophagy mediated by phosphatase and tensin homolog deleted on chromosome ten induced kinase 1(PINK1)/Parkin were detected by Western blotting method.Results The inhibition of HG-induced cell proliferation was effectively lightened by AS-Ⅳ(P<0.01).The expression levels of pro-apoptotic protein cleavedcaspase-3 and mitophagy-associated proteins PINK1 and Parkin as well as the ratio of LC3-Ⅱ/LC3-Ⅰin NRK-52E cells were up-regulated by HG(P<0.01),and then were significantly down-regulated by AS-Ⅳ(P<0.05 or P<0.01).Conclusion AS-Ⅳcould inhibit the apoptosis of HG-induced renal tubular epithelial cells NRK-52E,and alleviate PINK1/Parkin-mediated mitophagy.
引文
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[12]李素娟,李敛敏,汪洋,等.氧化应激和P53参与波动高糖诱导的肾小管上皮细胞凋亡[J].中国病理生理杂志,2011,27(12):2302.
[13]徐维佳,牟姗,王琴,等.黄芪甲苷对高糖诱导的肾小管上皮细胞损伤的保护作用[J].中国中西医结合肾病杂志,2012,13(9):765.
[14] Higgins G C, Coughlan M T. Mitochondrial dysfunction and mitophagy:the beginning and end to diabetic nephropathy[J].Brit J Pharmacol,2014,171(8):1917.
[15] Picard M, Juster R P, McEwen B S. Mitochondrial allostatic load puts the‘gluc’back in glucocorticoids[J]. Nat Rev Endocrinol,2014,10(5):303.
[16] Czajka A, Ajaz S, Gnudi L, et al. Altered mitochondrial function, mitochondrial DNA and reduced metabolic flexibility in patients with diabetic nephropathy[J]. EBioMedicine,2015,2(6):499.
[17] Sharma K,Karl B,Mathew A V,et al. Metabolomics reveals signature of mitochondrial dysfunction in diabetic kidney disease[J]. J Am Soc Nephrol,2013,24(11):1901.
[18] Supale S, Li N, Brun T, et al. Mitochondrial dysfunction in pancreaticβcells[J]. Trends Endocrin Met, 2012, 23(9):477.
[19] Eiyama A, Okamoto K. PINK1/Parkin-mediated mitophagy in mammalian cells[J]. Curr Opin Cell Biol,2015,33:95.
[20] Smith M A,Covington M D,Schnellmann R G. Loss of calpain 10causes mitochondrial dysfunction during chronic hyperglycemia[J].Arch Biochem Biophys,2012,523(2):161.
[21] Zhan M,Usman I M,Sun L,et al. Disruption of renal tubular mitochondrial quality control by myo-inositol oxygenase in diabetic kidney disease[J]. J Am Soc Nephrol,2015,26(6):1304.
[2] Tang S C W,Lai K N. The pathogenic role of the renal proximal tubular cell in diabetic nephropathy[J]. Nephrol Dial Transpl,2012,27(8):3049.
[3] Isermann B, Vinnikov I A, Madhusudhan T, et al. Activated protein C protects against diabetic nephropathy by inhibiting endothelial and podocyte apoptosis[J]. Nat Med,2007,13(11):1349.
[4] Tan A L,Sourris K C,Harcourt B E,et al. Disparate effects on renal and oxidative parameters following RAGE deletion, AGE accumulation inhibition, or dietary AGE control in experimental diabetic nephropathy[J]. Am J Physiol Renal Physiol,2010,298(3):F763.
[5] Fischer F,Hamann A,Osiewacz H D. Mitochondrial quality control:an integrated network of pathways[J]. Trends Biochem Sci,2012,37(7):284.
[6]王盛发,唐劭,曹克光.糖尿病肾病中医治疗用药规律分析[J].天津中医药,2009,26(2):167.
[7]段贤春,方朝晖,姚先梅,等.中药黄芪治疗糖尿病及糖尿病肾病研究进展[J].安徽医药,2013,17(9):1592.
[8]孙豪栋,庞晓斌,李继扬.黄芪甲苷生物活性研究进展[J].中国药房,2011,22(7):657.
[9]郭维文,黎帅,陈玲玲,等.黄芪甲苷对糖尿病肾病大鼠肾脏的保护作用及其机制[J].中国病理生理杂志,2014,30(2):351.
[10]尤良震,林逸轩,方朝晖,等.黄芪甲苷治疗糖尿病及其并发症药理作用研究进展[J].中国中药杂志,2017,42(24):4700.
[11] Liu X,Wang W,Song G,et al. Astragaloside IV ameliorates diabetic nephropathy by modulating the mitochondrial quality control network[J]. PLoS One,2017,12(8):e0182558.
[12]李素娟,李敛敏,汪洋,等.氧化应激和P53参与波动高糖诱导的肾小管上皮细胞凋亡[J].中国病理生理杂志,2011,27(12):2302.
[13]徐维佳,牟姗,王琴,等.黄芪甲苷对高糖诱导的肾小管上皮细胞损伤的保护作用[J].中国中西医结合肾病杂志,2012,13(9):765.
[14] Higgins G C, Coughlan M T. Mitochondrial dysfunction and mitophagy:the beginning and end to diabetic nephropathy[J].Brit J Pharmacol,2014,171(8):1917.
[15] Picard M, Juster R P, McEwen B S. Mitochondrial allostatic load puts the‘gluc’back in glucocorticoids[J]. Nat Rev Endocrinol,2014,10(5):303.
[16] Czajka A, Ajaz S, Gnudi L, et al. Altered mitochondrial function, mitochondrial DNA and reduced metabolic flexibility in patients with diabetic nephropathy[J]. EBioMedicine,2015,2(6):499.
[17] Sharma K,Karl B,Mathew A V,et al. Metabolomics reveals signature of mitochondrial dysfunction in diabetic kidney disease[J]. J Am Soc Nephrol,2013,24(11):1901.
[18] Supale S, Li N, Brun T, et al. Mitochondrial dysfunction in pancreaticβcells[J]. Trends Endocrin Met, 2012, 23(9):477.
[19] Eiyama A, Okamoto K. PINK1/Parkin-mediated mitophagy in mammalian cells[J]. Curr Opin Cell Biol,2015,33:95.
[20] Smith M A,Covington M D,Schnellmann R G. Loss of calpain 10causes mitochondrial dysfunction during chronic hyperglycemia[J].Arch Biochem Biophys,2012,523(2):161.
[21] Zhan M,Usman I M,Sun L,et al. Disruption of renal tubular mitochondrial quality control by myo-inositol oxygenase in diabetic kidney disease[J]. J Am Soc Nephrol,2015,26(6):1304.