1. Introduction {#sec1}
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Cisplatin is a potent chemotherapeutic agent for the treatment of multiple human malignancies \[[@B1], [@B2]\]. It accumulates in all segments of nephron but is predominantly taken up by the proximal tubule cells, which then provokes severe damage \[[@B3]\]. The efficacy of cisplatin is dose dependent, but the side effect in kidney limits the use of higher doses to improve its chemotherapeutic effects \[[@B4], [@B5]\]. The toxic effects of cisplatin mainly occur via oxidative stress and DNA damage \[[@B6], [@B7]\], ultimately leading to apoptotic pathways in tumour cells \[[@B8]\] and also in renal cells \[[@B4], [@B9], [@B10]\].

For centuries, many natural products have been identified for the prevention and/or treatment of kidney diseases because they are believed to have nephroprotective effects. They are widely used in clinical practice in many parts of the world. For example,*Silybum marianum* was found to attenuate nephrotoxicity induced by gentamicin in dogs \[[@B11]\]. A water extract of*Kalanchoe pinnata* leaves protected rat kidneys from gentamicin-induced nephrotoxicity \[[@B12]\]. Salviae Radix extract exerted a protective effect against cisplatin-induced renal cell injury, and its effect might be mediated by its antioxidant effect \[[@B13]\].

*Artemisia asiatica* Nakai is a traditional oriental medicine and it has been used for the treatment of several inflammatory disorders. Recent studies revealed that*A. asiatica* has antioxidative and anti-inflammatory effects contributing to its protective effects against various pathophysiological conditions including gastric damage \[[@B14]\], liver damage \[[@B15]\], experimental pancreatitis \[[@B16]\], and tumor promotion \[[@B17]\].

Stillen is a commercially available extract from*A. asiatica*. Eupatilin ([Figure 1(a)](#fig1){ref-type="fig"}), an active compound isolated from*A. asiatica*, has been reported to treat peptic ulcers and gastritis. It has antioxidative and anti-inflammatory effects against gastric mucosal injury \[[@B18], [@B19]\]. Various inflammatory mediators such as cytokines and oxidative stress that can affect gastric mucosal injury are thought to be involved in its action mechanism \[[@B20], [@B21]\]. Eupatilin was also reported to have therapeutic potential for the treatment of gastric cancer \[[@B22], [@B23]\].

Although cisplatin-induced nephrotoxicity has been well documented, the effects of*A. asiatica* and eupatilin on apoptosis in kidney cells after cisplatin exposure remain under active investigation.

2. Materials and Methods {#sec2}
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2.1. Chemicals and Reagents {#sec2.1}
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An ethanolic extract of*A. asiatica* and its active compound eupatilin were prepared as reported previously \[[@B17], [@B18]\]. Cisplatin and 1,1-diphenyl-2-picryl-hydrazyl (DPPH) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The stock solution of chemicals was prepared in 100% dimethylsulfoxide (DMSO) and stored at −20°C until use. Antibodies for p38, p-p38, JNK, p-JNK, ERK, p-ERK, cleaved caspase-3, and GAPDH were purchased from Cell Signaling (Boston, MA, USA).

2.2. Protective Effect against Cisplatin-Induced Nephrotoxicity in Cells {#sec2.2}
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Possible renoprotective effects against cisplatin-induced damage were evaluated in LLC-PK1 cells as reported previously \[[@B24]\]. In brief, LLC-PK1 cells were seeded in 96-well culture plates at 1 × 10^4^ cells per well and the test sample and/or radical donor, 25 *μ*M cisplatin, were added to the culture medium. Twenty-four hours later, the cell viability was measured by using a microplate reader (PowerWave XS; Bio-Tek Instruments, Winooski, VT, USA).

2.3. DPPH Radical Scavenging Assay {#sec2.3}
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The radical scavenging activity of*A. asiatica* and eupatilin against DPPH was determined spectrophotometrically. In microwells, 100 *μ*L of an aqueous solution of the completely dissolved sample (control: 100 *μ*L DW) was added to an ethanolic solution of DPPH (100 *μ*L, 60 *μ*M) according to the reported method \[[@B25]\]. The final concentrations of the tested samples in the assayed solution were 10, 25, 50, and 100 *μ*g/mL. Vitamin C was used as the standard for comparison. The ability to scavenge DPPH radicals was calculated in terms of percentage of inhibition according to the following equation: % inhibition = \[(*A* ~0~ − *A* ~1~)/*A* ~0~ × 100\], where *A* ~0~ is the absorbance of the control (without extract) and *A* ~1~ is the absorbance in the presence of the extract.

2.4. Western Blot Analysis {#sec2.4}
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Proteins (whole cell extracts, 30 *μ*g/lane) were separated by electrophoresis in a precast 4--15% Mini-PROTEAN TGX gel (Bio-Rad, CA, USA) blotted onto PVDF transfer membranes as reported previously \[[@B26]\]. Bound antibodies were visualized using ECL Advance Western Blotting Detection Reagents (GE Healthcare, UK) and a LAS 4000 imaging system (Fujifilm, Japan).

2.5. Image-Based Cytometric Assay {#sec2.5}
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To determine the portion of the population that had become apoptotic, cells were stained with annexin V-Alexa Fluor 488 conjugate using a Tali image-based cytometer (Invitrogen, CA, USA) \[[@B27]\]. Propidium iodide (PI) was used to differentiate dead cells (annexin V-positive/PI positive or annexin V-negative/PI positive) from those that were apoptotic (annexin V-positive/PI negative). The percentages of the population reported as viable, apoptotic, and dead by the Tali cytometer were comparable with data from the same samples independently run on a flow cytometer.

2.6. Statistical Analysis {#sec2.6}
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Statistical significance was determined through analysis of variance (ANOVA). *p* values of less than 0.05 were considered statistically significant.

3. Results and Discussion {#sec3}
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3.1. Effects of*A. asiatica* Extract and Eupatilin on Cisplatin-Induced Nephrotoxicity in LLC-PK1 Cells {#sec3.1}
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The antioxidant effects of*A. asiatica* and eupatilin were tested using DPPH, a stable free radical. DPPH decolorizes in the presence of antioxidants. The scavenging ability of*A. asiatica* and eupatilin was represented by a line diagram and compared with vitamin C ([Figure 1(b)](#fig1){ref-type="fig"}). This result suggests that eupatilin is the antioxidant and active component of*A. asiatica*. As shown in [Figure 1](#fig1){ref-type="fig"}, the cell viability was decreased significantly to about 60% of that of untreated control cells (*p* = 0.0004) after 25 *μ*M cisplatin treatment. However, pretreatment with the*A. asiatica* extract and eupatilin markedly restored cell viability to 80 and 82%, respectively, in a dose-dependent manner (Figures [1(c)](#fig1){ref-type="fig"} and [1(d)](#fig1){ref-type="fig"}).

3.2. Involvement of MAPKs-Caspase-3 Signaling Pathway in the Protective Effect of*A. asiatica* Extract and Eupatilin against Cytotoxicity in Cultured LLC-PK1 Cells {#sec3.2}
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[Figure 2](#fig2){ref-type="fig"} shows the protein expressions of p38, p-p38, JNK, p-JNK, ERK, p-ERK, and cleaved caspase-3 after*A. asiatica* (250 *μ*g/mL) and eupatilin (10 and 50 *μ*g/mL) treatment. As shown in [Figure 2](#fig2){ref-type="fig"}, the phosphorylation of p38 and JNK was decreased in LLC-PK1 cells by*A. asiatica* and eupatilin treatments. In addition, the elevated protein expression of cleaved caspase-3 was also markedly reduced by*A. asiatica* and eupatilin treatments. ERK protein expression in the LLC-PK1 cells was slightly decreased by cisplatin treatment and increased by*A. asiatica* and eupatilin treatment; however, the differences were not of significant effect.

3.3. Effects of*A. asiatica* Extract and Eupatilin on Apoptosis in LLC-PK1 Cells {#sec3.3}
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[Figure 3](#fig3){ref-type="fig"} shows the effects of the*A. asiatica* extract and eupatilin on apoptosis in LLC-PK1 cells. As shown in [Figure 3(a)](#fig3){ref-type="fig"}, the number of dead and apoptotic cells, which were stained with red or green colors, was increased by cisplatin treatment, whereas it was decreased after cotreatment with the*A. asiatica* extract (*p* = 0.008) and more significantly eupatilin (*p* = 0.003). The elevated percentage of apoptotic cells after cisplatin treatment was markedly decreased after cotreatment with the*A. asiatica* extract and eupatilin ([Figure 3(b)](#fig3){ref-type="fig"}).

4. Discussion {#sec4}
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The protective effect of the*A. asiatica* extract and eupatilin against cisplatin-induced nephrotoxicity was tested using LLC-PK1 cells, which are the most vulnerable renal tubular cells to oxidative stress \[[@B24], [@B28]\]. It is reported that reactive oxygen species (ROS) play vital biological roles in cellular homeostasis whereas the increased ROS levels are associated with apoptosis in cells \[[@B29]--[@B31]\]. Our result suggests that eupatilin is the antioxidant and active component of*A. asiatica*. In addition, pretreatment with the*A. asiatica* extract and eupatilin markedly ameliorated reduced LLC-PK1 cell viability by cisplatin in a dose-dependent manner.

It has been reported that ROS act as a second messenger initiating signal transduction cascades, including the MAPKs signaling pathway \[[@B32], [@B33]\]. The MAPKs are important mediators for apoptosis induction in response to anticancer drugs, in particular cisplatin \[[@B34], [@B35]\]. In the present study, the increased protein expressions of phosphorylated JNK and p38 by cisplatin in LLC-PK1 cells were markedly ameliorated after*A. asiatica* extract or eupatilin cotreatment. To further investigate the ability of*A. asiatica* and eupatilin to prevent apoptosis, we measured the expression levels of cleaved caspase-3, known as an index of apoptosis, in the kidney. As shown in the results,*A. asiatica* and eupatilin significantly reduced the expression of cleaved caspase-3. These results suggest that cisplatin-induced increases in the ROS level activate MAPKs in LLC-PK1 cells, whereas the*A. asiatica* extract and eupatilin inhibit the activation of p38 and JNK.

Cisplatin-induced renal cell damage is dependent on apoptosis induced by DNA damage \[[@B36], [@B37]\]. Apoptosis in renal tubular cells has been observed in several kinds of renal disorders \[[@B38]\]. The elevated protein level of cleaved caspase-3 decreased after treatment with*A. asiatica* extract and eupatilin.

In conclusion, our results suggest that the*A. asiatica* extract can ameliorate nephrotoxicity in LLC-PK1 cells. Eupatilin serves as one of the major components by blocking the MAPKs-caspase-3 signaling cascade.

This research was funded by the Gangneung Asan Hospital Biomedical Research Center Promotion Fund. This work was also supported by a grant from Korea Institute of Science and Technology, Republic of Korea (2Z04371).

Conflict of Interests
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The authors declare no conflict of interests in this work.

Authors\' Contribution
======================

Jun Yeon Park and Dahae Lee contributed equally to the content of this paper.

![Effects of*A. asiatica* extract and eupatilin on cisplatin-induced nephrotoxicity in LLC-PK1 cells. (a) Structure of eupatilin. (b) Comparison of DPPH radical scavenging effects of*A. asiatica* extract, eupatilin, and vitamin C. (c) Dose-dependent protective effect of*A. asiatica* extract against cisplatin-induced nephrotoxicity in cells. (d) Dose-dependent protective effect of eupatilin against cisplatin-induced nephrotoxicity in cells.](ECAM2015-483980.001){#fig1}

![Involvement of the MAPKs-caspase-3 signaling pathway in the protective effect of*A. asiatica* extract and eupatilin against cytotoxicity in cultured LLC-PK1 cells. Results of the Western blot show the levels of p-p38, p38, p-JNK, JNK, p-ERK, ERK, and cleaved caspase-3 in LLC-PK1 cells treated with*A. asiatica* extract and eupatilin and/or cisplatin at different concentrations for 24 h. Whole cell lysates (20 *μ*g) were separated by SDS-PAGE, transferred onto PVDF transfer membranes, and probed with the indicated antibodies. Proteins were visualized using an ECL detection system.](ECAM2015-483980.002){#fig2}

![Effects of*A. asiatica* extract and eupatilin on apoptosis in LLC-PK1 cells. (a) Representative images of apoptosis detection. (b) Percentage of annexin V-positive-stained apoptotic cells. Dead and apoptotic cells were stained red and green, respectively. Apoptosis was determined by a Tali image-based cytometer.](ECAM2015-483980.003){#fig3}

[^1]: Academic Editor: Avni Sali
