Objective:

2

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:

2

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 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:

2

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:

2

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.