紫苏叶水提物对阿霉素致HK-2细胞氧化损伤的保护及机制
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摘要
目的:探讨阿霉素(ADR)诱导人肾小管上皮细胞(HK-2)发生氧化损伤后,紫苏叶水提取物(PFAE)对其细胞活性、氧化损伤标记物及细胞凋亡等关键因子的影响。方法:ADR刺激HK-2细胞建立损伤模型,使用N-乙酰半胱氨酸(NAC)或不同浓度PFAE(5,15,45 g·L~(-1))干预后,采用细胞增殖/毒性检测(CCK-8)法检测细胞存活率,结合光镜下细胞形态变化,筛选出PFAE保护细胞的最佳浓度。后续实验分为6组:空白组,ADR(0.05 g·L~(-1))组,PFAE(15 g·L~(-1))组,ADR+PFAE(0.05+15)g·L~(-1)组,NAC(0.81 g·L~(-1))组,ADR+NAC(0.05+81)g·L~(-1)组。检测细胞匀浆中的丙二醛(MDA),超氧化物歧化酶(SOD)和细胞总抗氧化能力,2',7'-二氯荧光黄双乙酸盐(DCFH-DA)荧光探针检测细胞内活性氧(ROS)水平,流式细胞术及脱氧核糖核苷酸末端转移酶(TUNEL)染色法检测细胞凋亡率,蛋白免疫印迹法(Western blot)检测细胞线粒体凋亡相关蛋白B淋巴细胞瘤-2基因(Bcl-2),Bcl-2相关X蛋白(Bax),半胱氨酸天冬氨酸蛋白酶9和3(Caspase-9,Caspase-3),聚腺苷二磷酸-核糖聚合酶(PARP)包括其剪切体的表达,及丝裂原活化蛋白激酶(MAPK)信号转导通路中p38丝裂素活化蛋白激酶(p38 MAPK),细胞外信号调节激酶(ERK),c-Jun氨基端激酶(JNK)及其磷酸化蛋白的表达。结果:与空白组比较,ADR组的细胞活性显著降低(P<0.01),与ADR组比较,5,15 g·L~(-1)的PFAE和NAC能促进细胞的增殖(P<0.01)。与空白组比较,ADR组抗氧化能力和SOD水平显著降低(P<0.01),MDA和ROS的水平显著增高(P<0.01),与ADR组比较,ADR+PFAE组和ADR+NAC组抗氧化能力和SOD水平显著升高(P<0.01),MDA和ROS的水平显著下降(P<0.01)。与空白组比较,ADR组细胞凋亡率上升(P<0.01),凋亡相关蛋白Bax/Bcl-2,cleaved Caspase-9/Caspase-9,cleaved Caspase-3/Caspase-3,cleaved PARP/PARP水平显著上升(P<0.01),MAPKs通路中的p38 MAPK,ERK和JNK的磷酸化蛋白表达明显增高(P<0.05,P<0.01);与ADR组比较,PFAE或NAC干预后减轻了细胞凋亡率,降低了凋亡蛋白的相对比值,并且抑制了MAPK信号通路中p38 MAPK,ERK蛋白的磷酸化(P<0.01),但对磷酸化的JNK蛋白表达无影响。结论:PFAE可以减轻ADR诱导的HK-2细胞氧化损伤,并发挥抗氧化作用,通过线粒体凋亡途径和ERK/p38 MAPK信号通路来抑制细胞凋亡。
Objective: To investigate the protective effect of Perillae Folium with aqueous extract( PFAE on some key factors of Adriamycin( ADR)-induced oxidative injury in human renal tubular epithelial cells( HK-2),including the survival rate,oxidative injury indexes and cell apoptosis,in order to define the underlying mechanism. Method: A model of ADR-induced HK-2 cells oxidative injury was established in vitro,then cell viability was detected by cell counting kit-8( CCK-8) after intervention with positive reference N-acetylcysteine( NAC) or PFAE( 5,15,45 g·L~(-1)) at different concentrations. According to the morphological changes under microscopy,the optimum concentration of PFAE was screened out for the follow-up experiments. Then,the experiments were divided into six groups: blank group,ADR( 0. 05 g·L~(-1)) group,PFAE( 15 g·L~(-1)) group,ADR + PFAE( 0. 05 + 15) g·L~(-1) group,NAC( 0. 81 g·L~(-1)) group,and ADR + NAC( 0. 05 + 81) g·L~(-1) group.After that,malondialdehyde( MDA),superoxide dismutase( SOD),total antioxidant capacity( TAC) were measured in the cell homogenate after 24 h administration. The level of reactive oxygen species( ROS) was detected by 2',7'-dichloroflurescin diacetate( DCFH-DA) fluorescence probe. Flow cytometry and Td T-mediated dUTP Nick-End Labeling( TUNEL) were used to monitor the cell apoptosis. Western blot was used to observed the expressions of mitochondrial apoptosis-associated proteins,like B lymphocyte tumor-2 gene( Bcl-2),Bcl-2 related X protein( Bax),cysteine aspartate protease-9( Caspase-9),cysteine aspartate protease-3( Caspase-3) and poly ADP-ribose polymerase( PARP), as well as their shear bodies. In addition, the phosphorylation protein expressions of p38 mitogen-activated protein kinase( p38 MAPK),extracellular signal-regulated kinase( ERK),c-Jun amino-terminal kinase( JNK) in mitogen-activated protein kinase( MAPK) signaling transduction pathway were detected by Western blot. Result: Compared with blank group,ADR group showed a decreased cell viability( P < 0. 01),and lower SOD level( P < 0. 01),but higher expressions of MDA and ROS( P < 0. 01),and an increased apoptotic rate( P < 0. 01). The ADR group also increased in rate of Bax/Bcl-2,cleaved Caspase-9/Caspase-9,cleaved Caspase-3/Caspase-3,and cleaved PARP/PARP( P < 0. 01),as well as the phosphorylation protein expressions of p38 MAPK,ERK and JNK( P < 0. 05,P < 0. 01). Compared with the ADR group,both ADR + PFAE groups and ADR + NAC group had higher cell proliferation rates( P < 0. 01). In addition,the protective effect of PFAE on cells was the most obvious at the concentration of 15 g·L~(-1). The ATC and SOD levels were increased in ADR + PFAE group and ADR + NAC group( P < 0. 01),while their content of MDA and ROS,cell apoptosis,relative ratio of apoptotic protein expression,and phosphorylation protein expressions of p38 MAPK and ERK were all decreased( P < 0. 01). However,there was no effect on the expression of phosphorylated JNK protein. Conclusion: PFAE could alleviate the oxidative injury of HK-2 cells induced by ADR,and have an antioxidant effect,which inhibited cell apoptosis through mitochondrial apoptotic pathway and ERK/p38 MAPK signaling pathway.
Objective: To investigate the protective effect of Perillae Folium with aqueous extract( PFAE on some key factors of Adriamycin( ADR)-induced oxidative injury in human renal tubular epithelial cells( HK-2),including the survival rate,oxidative injury indexes and cell apoptosis,in order to define the underlying mechanism. Method: A model of ADR-induced HK-2 cells oxidative injury was established in vitro,then cell viability was detected by cell counting kit-8( CCK-8) after intervention with positive reference N-acetylcysteine( NAC) or PFAE( 5,15,45 g·L~(-1)) at different concentrations. According to the morphological changes under microscopy,the optimum concentration of PFAE was screened out for the follow-up experiments. Then,the experiments were divided into six groups: blank group,ADR( 0. 05 g·L~(-1)) group,PFAE( 15 g·L~(-1)) group,ADR + PFAE( 0. 05 + 15) g·L~(-1) group,NAC( 0. 81 g·L~(-1)) group,and ADR + NAC( 0. 05 + 81) g·L~(-1) group.After that,malondialdehyde( MDA),superoxide dismutase( SOD),total antioxidant capacity( TAC) were measured in the cell homogenate after 24 h administration. The level of reactive oxygen species( ROS) was detected by 2',7'-dichloroflurescin diacetate( DCFH-DA) fluorescence probe. Flow cytometry and Td T-mediated dUTP Nick-End Labeling( TUNEL) were used to monitor the cell apoptosis. Western blot was used to observed the expressions of mitochondrial apoptosis-associated proteins,like B lymphocyte tumor-2 gene( Bcl-2),Bcl-2 related X protein( Bax),cysteine aspartate protease-9( Caspase-9),cysteine aspartate protease-3( Caspase-3) and poly ADP-ribose polymerase( PARP), as well as their shear bodies. In addition, the phosphorylation protein expressions of p38 mitogen-activated protein kinase( p38 MAPK),extracellular signal-regulated kinase( ERK),c-Jun amino-terminal kinase( JNK) in mitogen-activated protein kinase( MAPK) signaling transduction pathway were detected by Western blot. Result: Compared with blank group,ADR group showed a decreased cell viability( P < 0. 01),and lower SOD level( P < 0. 01),but higher expressions of MDA and ROS( P < 0. 01),and an increased apoptotic rate( P < 0. 01). The ADR group also increased in rate of Bax/Bcl-2,cleaved Caspase-9/Caspase-9,cleaved Caspase-3/Caspase-3,and cleaved PARP/PARP( P < 0. 01),as well as the phosphorylation protein expressions of p38 MAPK,ERK and JNK( P < 0. 05,P < 0. 01). Compared with the ADR group,both ADR + PFAE groups and ADR + NAC group had higher cell proliferation rates( P < 0. 01). In addition,the protective effect of PFAE on cells was the most obvious at the concentration of 15 g·L~(-1). The ATC and SOD levels were increased in ADR + PFAE group and ADR + NAC group( P < 0. 01),while their content of MDA and ROS,cell apoptosis,relative ratio of apoptotic protein expression,and phosphorylation protein expressions of p38 MAPK and ERK were all decreased( P < 0. 01). However,there was no effect on the expression of phosphorylated JNK protein. Conclusion: PFAE could alleviate the oxidative injury of HK-2 cells induced by ADR,and have an antioxidant effect,which inhibited cell apoptosis through mitochondrial apoptotic pathway and ERK/p38 MAPK signaling pathway.
引文
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[13]Lou H,Kaur K,Sharma A K,et al. Adriamycininduced oxidative stress,activation of MAP kinases and apoptosis in isolated cardiomyocytes[J].Pathophysiology,2006,13(2):103-109.
[14]Ki Y W,Park J H,Lee J E,et al. JNK and p38MAPK regulate oxidative stress and the inflammatory response in chlorpyrifos-induced apoptosis[J]. Toxicol Lett,2013,218(3):235-245.
[15]Luanpitpong S,Chanvorachote P,Nimmannit U,et al.Mitochondrial superoxide mediates doxorubicin-induced keratinocyte apoptosis through oxidative modification of ERK and Bcl-2 ubiquitination[J]. Biochem Pharmacol,2012,83(12):1643-1654.
[16]马习习,周恩超.浅析周恩超教授益肾法治疗慢性肾病[J].浙江中医药大学学报,2017,41(3):226-227,231.
[17]谭美莲,严明芳,汪磊,等.国内外紫苏研究进展概述[J].中国油料作物学报,2012,34(2):225-231.
[2]Lee V W,Harris D C. Adriamycin nephropathy:a model of focal segmental glomerulosclerosis[J]. Nephrology,2010,16(1):30-38.
[3]SAI Y P,SUN Y C,CHEN X X,et al. Protective effect of astragalosides from Radix Astragali on adriamycininduced podocyteinjury[J]. Exp Ther Med,2018,15(5):4485-4490.
[4]Khan T H,Ganaie M A, Alharthy K M, et al.Naringenin prevents doxorubicin-induced toxicity in kidney tissues by regulating the oxidative and inflammatory insult in Wistar rats[J]. Arch Physiol Biochem, 2018, doi:10. 1080/13813455.2018. 1529799.
[5]TIAN T,LI J,WANG M Y,et al. Protective effect of 20-hydroxyeicosatetraenoic acid(20-HETE)on adriamycininduced toxicity of human renal tubular epithelial cell(HK-2)[J]. Eur J Pharmacol,2012,683(1/3):246-251.
[6]霍立娜,王威,刘洋,等.紫苏叶化学成分研究[J].中草药,2016,47(1):26-31.
[7]马丽娜,黄纯绚,莫晓晖.紫苏叶总黄酮提取物对过氧化氢所致人肾小管上皮细胞HK-2的氧化损伤保护作用[J].华夏医学,2016,29(3):14-17.
[8]王震,张绒,宋清,等.荷叶、绞股蓝及紫苏叶提取物复方制剂的减肥降脂作用[J].中国实验方剂学杂志,2015,21(13):144-147.
[9]杜艳,殷丽君,朱巧梅,等.紫苏油的药理活性及其缓释技术研究进展[J].河南工业大学学报:自然科学版,2016,37(4):114-117,123.
[10]Shalom S T,Mina K,Seung J L,et al. Antiobesity effects of purple perilla(perillafrutescens var acuta)on adipocyte differentiation and mice fed a high-fat diet[J]. J Food Sci,2018,83(9):2384-2393.
[11]HU H,Batteux F, Chéreau C, et al. Clopidogrel protects from cell apoptosis and oxidative damage in a mouse model of renal ischaemia-reperfusion injury[J].J Pathol,2011,225(2):265-275.
[12]LI W N, HAN H, ZI Y J, et al. Mitochondrial oxidative damage and apoptosis induced by high glucose through Rho kinase signal pathway in renal tubular epithelial cells[J]. Asian Pac J Trop Med,2018,11(6):399-404.
[13]Lou H,Kaur K,Sharma A K,et al. Adriamycininduced oxidative stress,activation of MAP kinases and apoptosis in isolated cardiomyocytes[J].Pathophysiology,2006,13(2):103-109.
[14]Ki Y W,Park J H,Lee J E,et al. JNK and p38MAPK regulate oxidative stress and the inflammatory response in chlorpyrifos-induced apoptosis[J]. Toxicol Lett,2013,218(3):235-245.
[15]Luanpitpong S,Chanvorachote P,Nimmannit U,et al.Mitochondrial superoxide mediates doxorubicin-induced keratinocyte apoptosis through oxidative modification of ERK and Bcl-2 ubiquitination[J]. Biochem Pharmacol,2012,83(12):1643-1654.
[16]马习习,周恩超.浅析周恩超教授益肾法治疗慢性肾病[J].浙江中医药大学学报,2017,41(3):226-227,231.
[17]谭美莲,严明芳,汪磊,等.国内外紫苏研究进展概述[J].中国油料作物学报,2012,34(2):225-231.
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