Protective Effect and Mechanism of Aqueous Extract of Perillae Folium on Adriamycin-induced Oxidative Injury in HK-2 Cell

Yi-ye ZHU; En-chao ZHOU; Kun GAO; Guo-shun HUANG; Wei LI; Ping XIA

doi:10.13422/j.cnki.syfjx.20191238

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Protective Effect and Mechanism of Aqueous Extract of Perillae Folium on Adriamycin-induced Oxidative Injury in HK-2 Cell

Pharmacology | 更新时间：2021-02-09

- Protective Effect and Mechanism of Aqueous Extract of Perillae Folium on Adriamycin-induced Oxidative Injury in HK-2 Cell
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 25, Issue 12, Pages: 50-57(2019)
- 作者机构：
  
  南京中医药大学　附属医院，南京　 210029
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20191238
  CLC： R285; R730.2; R364.5; R978.7
- Received：01 May 2019，
  
  Published Online：05 March 2019，
  
  Published：20 June 2019
- 稿件说明：
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Yi-ye ZHU, En-chao ZHOU, Kun GAO, et al. Protective Effect and Mechanism of Aqueous Extract of Perillae Folium on Adriamycin-induced Oxidative Injury in HK-2 Cell[J]. Chinese journal of experimental traditional medical formulae, 2019, 25(12): 50-57.
DOI：

Yi-ye ZHU, En-chao ZHOU, Kun GAO, et al. Protective Effect and Mechanism of Aqueous Extract of Perillae Folium on Adriamycin-induced Oxidative Injury in HK-2 Cell[J]. Chinese journal of experimental traditional medical formulae, 2019, 25(12): 50-57. DOI： 10.13422/j.cnki.syfjx.20191238.

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

-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(

0.01)，与ADR组比较，5，15 g·L

－1

的PFAE和NAC能促进细胞的增殖(

0.01)。与空白组比较，ADR组抗氧化能力和SOD水平显著降低(

0.01)，MDA和ROS的水平显著增高(

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(

0.01)，MDA和ROS的水平显著下降(

0.01)。与空白组比较，ADR组细胞凋亡率上升(

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(

0.01)，MAPKs通路中的p38 MAPK，ERK和JNK的磷酸化蛋白表达明显增高(

0.05，

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(

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。

Abstract

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

-acetylcysteine (NAC) or PFAE (5

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 TdT-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 (

0.01)

and lower SOD level (

0.01)

but higher expressions of MDA and ROS (

0.01)

and an increased apoptotic rate (

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 (

0.01)

as well as the phosphorylation protein expressions of p38 MAPK

ERK and JNK (

0.05

0.01). Compared with the ADR group

both ADR+ PFAE groups and ADR+ NAC group had higher cell proliferation rates (

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 (

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 (

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.

关键词

Keywords

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