Protective Effect of Phenolic Compounds Isolated from Mugwort (Artemisia argyi) against Contrast-Induced Apoptosis in Kidney Epithelium Cell Line LLC-PK1

We investigated whether 14 phenolic compounds isolated from Artemisia argyi could prevent the apoptotic damage caused by iodixanol, an iodinated contrast agent, on LLC-PK1 cells. Iodixanol was used to induce cytotoxicity in LLC-PK1 cells. Apoptotic cell death was observed as the fluorescence intensity emitted by annexin V and Hoechst 33342 stains. Western blotting was used to detect specific proteins. Seven phenolic compounds protected against iodixanol-induced LLC-PK1 cell death in a concentration-dependent manner. Among them, methyl caffeate exerted the strongest protective effect, and co-treatment with 50 and 100 μM methyl caffeate decreased intracellular reactive oxygen species elevated by 25 mg/mL iodixanol. In addition, the treatment of LLC-PK1 cells with iodixanol resulted in an increase in apoptotic cell death, which decreased by co-treatment with methyl caffeate. Iodixanol caused a cytotoxicity-related increase in the phosphorylation of extracellular-signal-regulated kinase, c-Jun N-terminal kinase, and P38; and a similar increase in the expression levels of kidney injury molecule-1 and cleaved caspase-3. However, the up-regulation of these proteins was reversed by co-treatment with methyl caffeate. These findings suggest that phenolic compounds isolated from A. argyi play an important role in protecting kidney epithelium cells against apoptotic damage caused by iodixanol.


Introduction
Contrast agents are widely used to improve the visibility of blood vessels and internal organs, including the tissues and urinary tract in patients undergoing elective coronary procedures [1]. Based on osmolality, contrast agents are classified into three distinct groups-nonionic low-osmolar iopromide, ionic ioxitalamate, and isoosmolar iodixanol [1,2]. Although newer and safer contrast agents have been developed, several recent studies have shown that the incidence of renal dysfunction persists after contrast agents use [3][4][5].
Contrast agent-induced toxicity in renal proximal tubular cells has been implicated in the pathogenesis of acute kidney injury [4]. The cytotoxicity of contrast agents towards renal proximal tubular cells was indicated in in vitro and in vivo researches as well as in clinical trials [6,7]. The major reasons for cytotoxicity are the reduction in medullary blood flow and direct renal proximal tubular cell damage. The latter is related to the hypoxia-mediated formation of reactive oxygen species (ROS) [2,8,9]. Therefore, the prevention of the formation of ROS can be beneficial in protecting against contrast agent-induced renal proximal tubular cell damage. Many studies have demonstrated that N-acetylcysteine (NAC), a known antioxidant, prevents contrast agent-induced nephrotoxicity in the human embryonic kidney cell line, the porcine renal proximal tubular cell line, and the canine Madin-Darby distal tubular renal cell line [1,8,10]. Vitamin E also protects against contrast agent-induced nephrotoxicity in patients undergoing elective coronary procedures [11,12].
Artemisia argyi, an aromatic herb belonging to the Asteraceae family, has been mainly used in traditional oriental medicine for treating various diseases, such as menstrual disorders, infertility, uterine bleeding, and inflammatory diseases [13][14][15][16][17]. In addition, the extract of A. argyi is known to reduce ethanol-induced gastrointestinal damage [18], carbon tetrachloride (CCl 4 )-induced hepatic damage [19], and cerulein-induced pancreatic damage [20] in rats. A. argyi contains phytochemicals such as sesquiterpenoids [21][22][23], triterpenoids [24,25], phenolics, and flavonoids [15,[26][27][28]. These phytochemicals, isolated from A. argyi, exhibit various biological and pharmaceutical activities, including anti-oxidant, anti-inflammatory and anti-apoptosis activities [15,22,23,28]. In our previous study, we showed that artemetin, a flavonoid isolated from A. argyi, can protect against contrast agent-induced cytotoxicity in renal proximal tubular cells through the inhibition of ROS generation and apoptosis [28]. Since A. argyi contains a large number of compounds that possess biological activity [13], we continued our efforts to find more active compounds from A. argyi that exert protective effects against contrast agent-induced toxicity in renal proximal tubular cells and hypothesized that flavonoids may attenuate contrast agent-induced cytotoxicity in renal proximal tubular LLC-PK1 cells and focused on elucidating the molecular mechanism involved.

Effect of Methyl Caffeate on Iodixanol-Induced Apoptosis and ROS Generation in LLC-PK1 Cells
We tested whether methyl caffeate could reduce iodixanol-induced apoptosis in LLC-PK1 cells. Treatment of 25 mg/mL iodixanol increased the fluorescence intensity of Hoechst 33342 in cells. In contrast, the treatment of 50 and 100 µM methyl caffeate and 10 mM NAC significantly reduced the iodixanol-induced increase in fluorescence intensity of Hoechst 33342 ( Figure 3A). Similarly, the percentage of apoptotic cells with annexin V conjugated with V Alexa Fluor 488 (green fluorescence) increased significantly by 50.33 ± 4.16% by treatment with 25 mg/mL iodixanol, whereas the corresponding fluorescence was decreased by the treatments of 50 and 100 µM methyl caffeate and 10 mM NAC to 36.66 ± 4.50%, 19.33 ± 2.51% and 17.33 ± 2.51%, respectively ( Figure 3B). We then explored whether methyl caffeate could decrease iodixanol-induced ROS generation in LLC-PK1 cells. Fluorescence intensity of 2 ,7 -dichlorodihydrofluorescein (DCF) (in terms of fold increase) was significantly increased by 5.00 ± 0.32-fold by treatment with 25 mg/mL iodixanol, whereas it decreased by 2.25 ± 0.05-, 1.315 ± 0.16-, and 1.15 ± 0.27-fold by treatment with 50, 100 µM methyl caffeate and 10 mM NAC, respectively ( Figure 3C).

Effect of Methyl Caffeate on Expression Levels of MAP Kinase (JNK, ERK and P38), Kidney Injury Molecule-1 (KIM-1) and Cleaved Caspase-3 on Iodixanol-Treated LLC-PK1 Cells
To explore the molecular mechanism of the protective effect of methyl caffeate, cells were exposed to 25 mg/mL iodixanol in the presence or absence of compound 14 and NAC, followed by Western blot analysis to evaluate the phosphorylation of ERK, JNK, and P38 and the expression levels of KIM-1 and cleaved caspase-3. Exposure of LLC-PK1 cells to 25 mg/mL iodixanol resulted in changes in the phosphorylation of MAPKs (ERK, JNK, and P38), whereas methyl caffeate significantly decreased the iodixanol-induced phosphorylation of MAPKs to control levels ( Figure 4A). In addition, the elevated protein expression levels of KIM-1 and cleaved caspase-3 were decreased after treatment with 100 µM methyl caffeate ( Figure 4A). This inhibitory effect of methyl caffeate on expressions of apoptosis-related proteins was similar to that of the 10 mM NAC, a positive control group ( Figure 4B). To explore the molecular mechanism of the protective effect of methyl caffeate, cells were exposed to 25 mg/mL iodixanol in the presence or absence of compound 14 and NAC, followed by Western blot analysis to evaluate the phosphorylation of ERK, JNK, and P38 and the expression levels of KIM-1 and cleaved caspase-3. Exposure of LLC-PK1 cells to 25 mg/mL iodixanol resulted in changes in the phosphorylation of MAPKs (ERK, JNK, and P38), whereas methyl caffeate significantly decreased the iodixanol-induced phosphorylation of MAPKs to control levels ( Figure  4A). In addition, the elevated protein expression levels of KIM-1 and cleaved caspase-3 were decreased after treatment with 100 μM methyl caffeate ( Figure 4A). This inhibitory effect of methyl caffeate on expressions of apoptosis-related proteins was similar to that of the 10 mM NAC, a positive control group ( Figure 4B).

Discussion
In this study, we investigated the effects of 14 phenolic compounds isolated from A. argyi on iodixanol-induced cytotoxicity in LLC-PK1 cells. LLC-PK1 cells were chosen as the renal epithelial model system because previous studies found direct evidence for the toxicity of contrast agents during in vitro experiments using renal proximal tubular cells. This avoids any confounding variables, such as a variety of systemic physiological factors, that an in vivo system is subject to [2,5,29]. Moreover, contrast agents are concentrated in the renal proximal tubules and their concentration by water tubular reabsorption is much higher than in the plasma [1,2,30].
Among the 14 phenolic compounds isolated from A. argyi, 4-hydroxyacetophenone (5), benzoic acid (7), vanillic acid (13), and methyl caffeate (14) exhibited potent protective effects on iodixanol-induced LLC-PK1 cell death. The protective effects of these compounds were stronger than NAC, which is well-known an effective antioxidant. In previous studies on these four compounds, 4-hydroxyacetophenone isolated from Tagetes mendocina [31], vanillic acid isolated from Origanum vulgare [32], and methyl caffeate isolated from rice bran exhibited a scavenging activity on the DPPH radical [33]. Benzoic acid isolated from Triticum aestivum exhibited improving activities of superoxide dismutase (SOD) [34]. In addition, the intracellular ROS level in Madin Darby distal nonhuman tubular epithelial cells was increased by treatment with iodixanol (50, 100, 200 mg/mL) in a concentration-dependent manner, whereas it was decreased by pretreatment with 100 mM NAC [5]. In the present study, among the tested phenolic compounds, methyl caffeate proved to be the most effective phenolic compound of A. argyi and exhibited intracellular ROS scavenging activity, reflected by the decrease in oxidation of DCFH2 to DCF by intracellular ROS caused by iodixanol. In addition, methyl caffeate reduced the level apoptotic cell death caused by iodixanol. These results suggest that methyl caffeate may protect LLC-PK1 cells against oxidative damage and apoptotic cell death caused by iodixanol.
Previous studies suggest that contrast agent-induced renal damage is caused by molecular mechanisms related to oxidative stress, MAPKs and apoptosis [2,4,5,35]. MAPKs (JNK, P38 and ERK) were activated by the increased production of ROS, which then activated caspase-9 and caspase-3, leading to DNA fragmentation [2,5]. The phosphorylation of JNK and the expression of caspase-3 were increased in epithelial tubular cells collected from patients receiving 1 mL/kg iodixanol per hour. In an in vitro experiment, 100 mM NAC decreased the phosphorylation of P38 and JNK and the expression of caspase-3 in Madin Darby distal nonhuman tubular epithelial cells treated with 200 mg/mL iodixanol [5]. In addition, 100 mM NAC and 0.2 µg statins (known as HMG-CoA reductase inhibitors) decreased the phosphorylation of JNK and the expression of caspase-3 that were increased in both human embryonic proximal tubules cells and Madin Darby distal nonhuman tubular epithelial cells treated with 200 mg/mL iodixanol [35]. In previous clinical trials, KIM-1 was also an important molecule in contrast agent-induced renal damage and was activated in renal proximal tubules during ischemia and renal injury. The levels of urinary KIM-1 increased in urine collected from patients with contrast agent-induced nephropathy [36][37][38]. However, there is very limited information on KIM-1 expression in renal proximal tubular cells exposed to contrast agents. Thus, we evaluated the change in KIM-1 expression in renal proximal tubular cells exposed to contrast media, as well as the effect of antioxidants on KIM-1 expression.
In this study, treatment with iodixanol significantly increased the phosphorylation of JNK, ERK and P38 and the expression levels of cleaved caspase-3 and KIM-1; these effects were reversed by treatment with methyl caffeate. These results suggest that the antioxidative effect of methyl caffeate may inhibit the phosphorylation of JNK, ERK and P38, which are activated in response to oxidative stress [39,40]; and inhibit, as well, the expression of caspase-3, which is activated both by extrinsic and intrinsic apoptosis pathways [41] and KIM-1.

Chemicals and Reagents
Ez-Cytox cell viability assay kit was purchased form Dail Lab Service Co. (Seoul, Korea)

Plant Material
The dried leaves of Artemisia argyi, H. Lév., and Vaniot were obtained in April 2013 from the Gyeongdong herbal medicine market located in Seoul (Republic of Korea), and was identified by Dr. Je-Hyun Lee (College of Oriental Medicine, Dongguk University, Gyeongju, Republic of Korea). A voucher specimen of A. argyi (AA1-103-130429) was deposited at the Department of Biosystems and Biotechnology, Korea University (Seoul, Republic of Korea).

Quantification of Cellular Viability with Ez-Cytox Assay
We implemented methods used to study protective effects on iodixanol-induced cytotoxicity in kidney cells [28,55]. The cells were seeded at 1 × 10 4 cells per well in 96-well culture plates and incubated for 24 h to promote adhesion. Then, the cells were either treated with 0.5% DMSO (control), or the indicated concentrations of flavonoid compounds. NAC treatment was used as a positive control (10 mM). After incubation for further 2 h, iodixanol (25 mg/mL) was added to each well and incubated for 3 h. The experiments were performed in triplicate and readings of the cell viability using the the Ez-Cytox assay kit according to the manufacturer's instructions, with the absorbance value at 450 nm measured using a microplate reader (PowerWave XS; Bio-Tek Instruments, Winooski, VT, USA).

Quantification of DNA Fragmentation by Hoechst 33342 Staining
LLC-PK1 cells seeded at 4 × 10 5 cells per well in 6-well plates were treated with control (0.5% DMSO), or compound 14 and NAC as the positive control compound (10 mM). After incubation for 2 h, iodixanol (25 mg/mL) was then added to each well and incubated for 3 h, followed by the addition of 2 µL of Hoechst 33342 solution and incubation for 10 min at 37 • C [56]. The stained cells were observed under an IX50 fluorescent microscope equipped with a CCD camera (Olympus, Tokyo, Japan).

Quantification of ROS Level by DCFH-DA Staining
LLC-PK1 cells seeded at 4 × 10 5 cells per well in 6-well plates were treated with 0.5% DMSO (control), or compound 14, and 10 mM NAC (positive control compound). After incubation for 2 h, iodixanol (25 mg/mL) was added to each well and incubated for 3 h. Next, cells were incubated with 10 µM DCFH-DA for 30 min at 37 • C and then washed with phosphate-buffered saline (PBS). The stained cells were observed under an IX50 fluorescent microscope equipped with a CCD camera (Olympus, Tokyo, Japan) and the fluorescence intensity of DCF was measured at 495/517 nm (ex/em) using a SPARK 10M fluorescent mAcroplate reader (Tecan, Männedorf, Switzerland) [57].

Quantification of Apoptosis with Image-Based Cytometric Assay
LLC-PK1 cells seeded at 4 × 10 5 cells per well in 6-well plates were treated with 0.5% DMSO (control), or compound 14, and 10 mM NAC (positive control compound). After incubation for 2 h, iodixanol (25 mg/mL) was added to each well and incubated for further 3 h. Next, cells were harvested and washed with PBS. The cells were resuspended at 5 × 10 5 cell/100 µL in binding buffer and an aliquot of 100 µL was incubated with 5 µL of annexin V Alexa Fluor 488 for 30 min in the dark at room temperature. The Annexin V-positive-stained cells were measured by a Tali image-based cytometer (Invitrogen, Temecula, CA, USA) [58].

Statistical Analysis
Statistical analysis was conducted using analysis of variance (ANOVA) followed by a multiple comparison test with a Bonferroni adjustment. The analysis was carried out using SPSS ver. 19.0 (SPSS Inc., Chicago, IL, USA). All the assays were done in triplicate for each assay and were repeated at least three times. The data with P values of less than 0.05 were considered as a statistically significant effect.

Conclusions
Our findings indicate that methyl caffeate, a phenolic compound isolated from A. argyi, protects against iodixanol-induced LLC-PK1 cell death, an action that we showed to be due to the combination of antioxidant and antiapoptotic effects resulting from the inhibition of the activities of MAPKs (JNK, P38 and ERK), caspase-3, and KIM-1. These findings suggest the potential of methyl caffeate as a new and efficient therapeutic approach to prevent contrast agent-induced cytotoxicity and preserve renal function during the pathogenesis of an acute kidney injury.

Conflicts of Interest:
The authors declare no conflict of interest.