三七皂苷对TGF-β_1诱导的大鼠肾小管上皮细胞EMT的影响
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摘要
目的:研究三七皂苷(PNS)对转化生长因子-β_1(TGF-β_1)诱导的大鼠肾小管细胞上皮-间充质转化(EMT)的干预作用,并从沉默信息调节因子2相关酶1(SIRT1)/TGF-β_1/Smad通路分析其可能机制。方法:在DMEM培养基中,用10%胎牛血清培养大鼠肾小管上皮细胞(NRK-52E),分为正常组,TGF-β_1组(5μg·L~(-1)),白藜芦醇组(50 mg·L~(-1)),SIRT1阻断剂EX527组(10μmol·L~(-1)),PNS组(100 mg·L~(-1)),EX527+PNS组(EX527 10μmol·L~(-1),PNS 100 mg·L~(-1)),48 h后收集细胞;采用实时荧光定量聚合酶链式反应(Real-time PCR)检测SIRT1,α-平滑肌肌动蛋白(α-SMA),E-钙黏附蛋白(E-cadherin),TGF-β_1,Smad3,Smad4 mRNA表达;蛋白免疫印迹法(Western blot)检测SIRT1,α-SMA,E-cadherin,TGF-β_1蛋白表达。结果:与正常组比较,TGF-β_1组α-SMA mRNA及蛋白表达明显增多(P <0. 05),E-cadherin表达显著减少(P <0. 01);与TGF-β_1组比较,PNS,白藜芦醇组,E-cadherin mRNA及蛋白表达均显著增加(P <0. 01),α-SMA表达显著减少(P <0. 01),EMT发生被抑制,同时,SIRT1表达显著增加(P <0. 01),TGF-β_1,Smad3,Smad4表达显著降低(P <0. 01)。在SIRT1阻断剂EX527的干预下,PNS则不能发挥明显抑制作用。结论:PNS可以阻止TGF-β_1诱导的肾小管上皮细胞EMT的发生,其机制可能是通过激活SIRT1进而抑制TGF-β_1/Smad通路实现的。
Objective: To investigate the intervention effect of panax notoginseng saponins( PNS) on epithelial-mesenchymal transition( EMT) of rat renal tubular epithelial cells( NRK-52 E) induced by transforming growth factor-β_1( TGF-β_1),and analyze the mechanism based on the silent information regulation 2 homolog 1( SIRT1)/TGF-β_1/Smad signaling pathway. Method: NRK-52 E were cultured in DMEM medium with 10% fetal bovine serum,and divided into normal control group,TGF-β_1 group( 5 μg·L~(-1)),resveratrol( RSV) group( 50 mg·L~(-1)),EX527 group( 10 μmol·L~(-1)),Panax notoginseng saponins( PNS) group( 100 mg·L~(-1)),and EX527 + PNS group( 10 μmol·L~(-1)+ 100 mg·L~(-1)). Then cells were collected after drug intervention for 48 h. The expressions of α-SMA,E-cadherin,SIRT1,TGF-β_1,Smad3,Smad4 mRNA in each group were detected by Real-time PCR. The protein expressions of α-SMA,E-cadherin,SIRT1 and TGF-β_1 were detected by Western blot. Result: Compared with normal group,mRNA and protein expressions of α-SMA increased obviously( P <0. 05),but E-cadherin decreased significantly( P < 0. 01) in TGF-β_1 group. Compared with TGF-β_1 group,mRNA and protein expressions of α-SMA decreased significantly( P < 0. 01),while E-cadherin increased( P <0. 01) in resveratrol and PNS groups,and EMT was inhibited. Meanwhile,mRNA and protein expressions of SIRT1 increased significantly( P < 0. 01),while mRNA expressions of TGF-β_1,Smad3,and Smad4 decreased( P < 0. 01). Under the intervention of SIRT1 blocker EX527,PNS could not play a significant inhibitory effect on the cells. Conclusion: PNS can prevent the occurrence of EMT of renal tubular epithelial cells induced by TGF-β_1,and the mechanism may be related to active SIRT1 to inhibit TGF-β_1/Smad pathway.
Objective: To investigate the intervention effect of panax notoginseng saponins( PNS) on epithelial-mesenchymal transition( EMT) of rat renal tubular epithelial cells( NRK-52 E) induced by transforming growth factor-β_1( TGF-β_1),and analyze the mechanism based on the silent information regulation 2 homolog 1( SIRT1)/TGF-β_1/Smad signaling pathway. Method: NRK-52 E were cultured in DMEM medium with 10% fetal bovine serum,and divided into normal control group,TGF-β_1 group( 5 μg·L~(-1)),resveratrol( RSV) group( 50 mg·L~(-1)),EX527 group( 10 μmol·L~(-1)),Panax notoginseng saponins( PNS) group( 100 mg·L~(-1)),and EX527 + PNS group( 10 μmol·L~(-1)+ 100 mg·L~(-1)). Then cells were collected after drug intervention for 48 h. The expressions of α-SMA,E-cadherin,SIRT1,TGF-β_1,Smad3,Smad4 mRNA in each group were detected by Real-time PCR. The protein expressions of α-SMA,E-cadherin,SIRT1 and TGF-β_1 were detected by Western blot. Result: Compared with normal group,mRNA and protein expressions of α-SMA increased obviously( P <0. 05),but E-cadherin decreased significantly( P < 0. 01) in TGF-β_1 group. Compared with TGF-β_1 group,mRNA and protein expressions of α-SMA decreased significantly( P < 0. 01),while E-cadherin increased( P <0. 01) in resveratrol and PNS groups,and EMT was inhibited. Meanwhile,mRNA and protein expressions of SIRT1 increased significantly( P < 0. 01),while mRNA expressions of TGF-β_1,Smad3,and Smad4 decreased( P < 0. 01). Under the intervention of SIRT1 blocker EX527,PNS could not play a significant inhibitory effect on the cells. Conclusion: PNS can prevent the occurrence of EMT of renal tubular epithelial cells induced by TGF-β_1,and the mechanism may be related to active SIRT1 to inhibit TGF-β_1/Smad pathway.
引文
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[13]杜月光,章科娜,姜雪儿,等.三七皂苷对高糖诱导的大鼠肾小球系膜细胞保护作用及其机制研究[J].浙江中医药大学学报,2015,39(9):647-653.
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[18]Vasko R,Xavier S,CHEN J,et al.Endothelial sirtuin 1deficiency perpetrates nephrosclerosis through downregulation of matrix metalloproteinase-14:relevance to fibrosis of vascular senescence[J].J Am Soc Nephrol,2014,25(2):276-291.
[19]Chávez E,Reyes-Gordillo K,Segovia J,et al.Resveratrol prevents fibrosis,NF-κB activation and TGF-βincreases induced by chronic CCl4treatment in rats[J].J Appl Toxicol,2008,28(1):35-43.
[20]吴海江,邓新娜,史永红,等.白藜芦醇对高糖刺激下人肾小管上皮细胞发生EMT的作用[J].中国细胞生物学学报,2015,37(11):1522-1527.
[21]LI Z,WANG F,ZHA S,et al.SIRT1 inhibits TGF-β-induced endothelial-mesenchymal transition in human endothelial cells with Smad4 deacetylation[J].J Cell Physiol,2018,233(11):9007-9014.
[22]贾宁,郑晶,王汉.注射用血栓通治疗临床糖尿病肾病的临床观察[J].中国实用医药,2011,6(26):17-18.
[23]杜月光,柴可夫,杨明华,等.三七皂苷对糖尿病大鼠肾脏骨形成蛋白7表达的影响[J].中华中医药学刊,2009,27(2):329-332.
[24]杜月光,柴可夫,杨明华,等.三七皂苷对糖尿病大鼠肾脏保护作用的实验研究[J].中国中医药科技,2010,17(1):40-41.
[2]Leask A,Abraham D J.TGF-beta signaling and the fibrotic response[J].FASEB J,2004,18(7):816-827.
[3]李峥,王灿,宋丹青,等.小檗碱类似物Y53在STZ诱导的糖尿病C57BL/6J小鼠中抗糖尿病肾病的作用研究[J].中国药理学通报,2016,32(9):1236-1242.
[4]Hubbard B P,Sinclair D A.Small molecule SIRT1activators for the treatment of aging and age-related diseases[J].Trends Pharmacol Sci,2014,35(3):146-154.
[5]HUANG X Z,WEN D,ZHANG M,et al.Sirt1 activation ameliorates renal fibrosis by inhibiting the TGF-β/Smad3pathway[J].J Cell Biochem,2014,115(5):996-1005.
[6]Simic P,Williams E O,Bell E L,et al.SIRT1suppresses the epithelial-to-mesenchymal transition in cancer metastasis and organ fibrosis[J].Cell Rep,2013,3(4):1175-1186.
[7]李娟,王如锋,杨莉,等.三七皂苷类成分及对心血管作用的研究进展[J].中国中药杂志,2015,40(17):3480-3487.
[8]陈光,刘超,何浩强,等.含三七中成药用药规律研究[J].中国实验方剂学杂志,2017,23(7):191-197.
[9]HUANG G D,LV J Z,LI T Y,et al.Notoginsenoside R1ameliorates podocyte injury in rats with diabetic nephropathy by activating the PI3K/Akt signaling pathway[J].Int J Mol Med,2016,38(4):1179-1189.
[10]HUANG G D,ZOU B,LV J,et al.NotoginsenosideR1attenuates glucose-induced podocyte injury via the inhibition of apoptosis and the activation of autophagy through the PI3K/Akt/m TOR signaling pathway[J].Int J Mol Med,2017,39(3):559-568.
[11]潘晶,章科娜,柴科夫,等.基于SIRT1/TGF-β1/Smad通路研究三七皂苷对高糖诱导的大鼠肾小管上皮细胞EMT的影响[J].浙江中医药大学学报,2018,42(4):259-265,282.
[12]王海亮,赵威,高志卿等.益肾化瘀方含药血清对NRK52E细胞转分化过程中p-Smad2/3、Smad7表达的影响[J].中华中医药杂志,2015,30(12):4482-4485.
[13]杜月光,章科娜,姜雪儿,等.三七皂苷对高糖诱导的大鼠肾小球系膜细胞保护作用及其机制研究[J].浙江中医药大学学报,2015,39(9):647-653.
[14]Lamouille S,XU J,Derynck R,et al.Molecular mechanisms of epithelial-mesenchymal transition[J].Nat Rev Mol Cell Biol,2014,15(3):178-196.
[15]Verrecchia F,Mauviel A.Transforming growth factor-βand fibrosis[J].World J Gastroenterol,2007,13(22):3056-3062.
[16]LI J,QU X,Ricardo S D,et al.Resveratrol inhibits renal fibrosis in the obstructed kidney:potential role in deacetylation of Smad3[J].Am J Pathol,2010,177(3):1065-1071.
[17]KONG X X,WANG R,LIU X J,et al.Function of SIRT1 in physiology[J].Biochemistry:Mosc,2009,74(7):703-708.
[18]Vasko R,Xavier S,CHEN J,et al.Endothelial sirtuin 1deficiency perpetrates nephrosclerosis through downregulation of matrix metalloproteinase-14:relevance to fibrosis of vascular senescence[J].J Am Soc Nephrol,2014,25(2):276-291.
[19]Chávez E,Reyes-Gordillo K,Segovia J,et al.Resveratrol prevents fibrosis,NF-κB activation and TGF-βincreases induced by chronic CCl4treatment in rats[J].J Appl Toxicol,2008,28(1):35-43.
[20]吴海江,邓新娜,史永红,等.白藜芦醇对高糖刺激下人肾小管上皮细胞发生EMT的作用[J].中国细胞生物学学报,2015,37(11):1522-1527.
[21]LI Z,WANG F,ZHA S,et al.SIRT1 inhibits TGF-β-induced endothelial-mesenchymal transition in human endothelial cells with Smad4 deacetylation[J].J Cell Physiol,2018,233(11):9007-9014.
[22]贾宁,郑晶,王汉.注射用血栓通治疗临床糖尿病肾病的临床观察[J].中国实用医药,2011,6(26):17-18.
[23]杜月光,柴可夫,杨明华,等.三七皂苷对糖尿病大鼠肾脏骨形成蛋白7表达的影响[J].中华中医药学刊,2009,27(2):329-332.
[24]杜月光,柴可夫,杨明华,等.三七皂苷对糖尿病大鼠肾脏保护作用的实验研究[J].中国中医药科技,2010,17(1):40-41.