Protective Effect of Panaxynol Isolated from Panax vietnamensis against Cisplatin-Induced Renal Damage: In Vitro and In Vivo Studies

Polyacetylenic compounds isolated from Panax species are comprised of non-polar C17 compounds, exhibiting anti-inflammatory, antitumor, and antifungal activities. Panaxynol represents the major component of the essential oils of ginseng. We investigated whether panaxynol isolated from Panax vietnamensis (Vietnamese ginseng, VG) could prevent cisplatin-induced renal damage induced in vitro and in vivo. Cisplatin-induced apoptotic cell death was observed by staining with annexin V conjugated with Alexa Fluor 488, and western blotting evaluated the molecular mechanism. Panaxynol at concentrations above 0.25 μM prevented cisplatin-induced LLC-PK1 porcine renal proximal tubular cell death. LLC-PK1 cells treated with cisplatin demonstrated an increase in apoptotic cell death, whereas pretreatment with 2 and 4 μM panaxynol decreased this effect. Cisplatin demonstrated a marked increase in the phosphorylation of c-Jun N-terminal kinase (JNK), P38, and cleaved caspase-3. However, pretreatment with 2 and 4 μM panaxynol reversed the upregulated phosphorylation of JNK, P38, and the expression of cleaved caspase-3. We confirmed that the protective effect of panaxynol isolated from P. vietnamensis in LLC-PK1 cells was at least partially mediated by reducing the cisplatin-induced apoptotic damage. In the animal study, panaxynol treatment ameliorated body weight loss and blood renal function markers and downregulated the mRNA expression of inflammatory mediators.


Introduction
The kidney is the main organ responsible for maintaining homeostasis in the body by regulating the volume and chemical composition of body fluids [1]. Thus, the kidney accumulates more toxic chemicals than other organs [2]. Studies have reported that 60% of all clinical nephrotoxicity cases are primarily attributed to anticancer drugs, particularly cisplatin [3]. Therefore, the incidence of nephrotoxicity may increase with the incidence of cancer, a leading cause of death with 18.1 million

Extraction and Isolation
VG dried powder (300 g) was extracted with 1000 mL of MeOH by sonication six times. The filtered MeOH extract was evaporated in vacuo to yield the MeOH total extract (120 g), which was suspended with water and partitioned between diethyl ether (Et 2 O) and water. The Et 2 O fraction was then evaporated in vacuo to yield the Et 2 O extract (4.2 g). The Et 2 O fraction was chromatographed on a silica gel column using a stepwise gradient elution with a mixture of n-hexane and ethyl acetate (30:1→15:1→8:1→4:1→1:1) to yield five sub-fractions (Et1-Et5). Sub-fraction Et3 (1.2 g) was further purified using a silica gel column and elution with n-hexane and ethyl acetate (8:1) to yield panaxynol (600 mg) as a yellow liquid. The structure of panaxynol was confirmed through comparison with published data of 1 H and 13 C NMR [30].

Cell Culture
The porcine renal proximal tubular cell line LLC-PK1 was purchased from the American Type Culture Collection (Rockville, MD, USA). Cells were cultured in Dulbecco s modified Eagle medium (DMEM) (Cellgro, Manassas, VA, USA) containing 10% FBS, 1% penicillin/streptomycin (Invitrogen Co., Grand Island, NY, USA), and 4 mM l-glutamine. To maintain cell growth, cells were maintained in 5% CO 2 in humidified air and at 37 • C.

Cell Viability Assay
To detect the cytotoxicity of cisplatin (Sigma, St. Louis, MO, USA) and evaluate the protective effect of panaxynol in cisplatin-induced cytotoxicity in LLC-PK1 cells, the cells were seeded in 96-well culture plates (10,000 cells per well) and incubated for 24 h. The cells were pretreated with panaxynol at a wide range of concentrations (0.0625, 0.125, 0.25, 0.5, 1, 2, 4 µM) for 2 h. Next, 25 µM cisplatin was added to each well, except for the control (0.5% DMSO) wells. After incubation for 24 h, cell viability was determined using the Ez-Cytox (Daeil Lab Services; Seoul, Korea) assay in accordance with the manufacturer's instructions. The mean optical density (OD, absorbance) of each well at 450 nm was measured using a microplate reader (PowerWave XS; Bio-Tek Instruments, Winooski, VT, USA) and used to calculate the percentage of cell viability (percentage of cell viability = (A treatment − A blank )/ (A control − A blank ) × 100% (where, A = absorbance)).

Quantification of Apoptosis with Image-Based Cytometric Assay
An image-based cytometric assay was performed to observe the effect of panaxynol on cisplatin-induced apoptosis in LLC-PK1 cells. The cells were seeded in 6-well culture plates (200,000 cells per well) and incubated for 24 h. The cells were pretreated with panaxynol at 2 and 4 µM for 2 h, and then 25 µM cisplatin was added to each well, except for the control (0.5% DMSO) wells. After 24 h, harvested cells were suspended at 500,000 cells/100 µL in binding buffer. The cell suspension in 100 µL aliquot was incubated with 5 µL of annexin V Alexa Fluor 488 for 30 min in the dark at room temperature. Annexin V-positive-stained cells were measured using a Tali image-based cytometer (Invitrogen, Temecula, CA, USA).

Western Blot Analysis
The expression levels of c-Jun N-terminal kinase (JNK), P38, and cleaved caspase-3 were detected using the western blotting method. Cells were treated as described in the above image-based cytometric assay and harvested on ice. According to the manufacturer's instructions, cells were lysed using the radioimmunoprecipitation assay buffer (RIPA buffer) (Cell Signaling Technology Inc., Beverly, MA, USA) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF). The protein concentrations were quantified using the Pierce BCA protein assay kit. An equal amount of protein (20 µg) per lane was separated by electrophoresis in a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred onto PVDF transfer membranes. Specific proteins on the membrane were detected by epitope-specific primary antibodies to JNK, P-JNK, P38, P-P38, cleaved caspase-3, GAPDH, and horseradish peroxidase (HRP)-conjugated anti-rabbit antibodies, followed by enhanced chemiluminescence (ECL) advance western blotting detection reagents and visualized using a FUSION Solo chemiluminescence system (PEQLAB Biotechnologie GmbH, Erlangen, Germany).

Cisplatin-Induced Nephrotoxicity in Mice
The animal experiment was approved by the Animal Care and Use Committee of Gachon University (GIACUC-R2019026). Seven-week-old male C57BL/6 mice were used to evaluate cisplatin-induced nephrotoxicity. The animals were housed at 23 ± 2 • C, 55 ± 5% humidity, with a 12 h light/dark cycle, and free access to water and food. Acute kidney injury was induced by a single intraperitoneal injection of cisplatin at a dose of 16 mg/kg in saline (day 0). N-acetyl-l-cysteine (NAC, 1000 mg/kg) and panaxynol (10 and 50 mg/kg) were orally administered once daily for 5 days. NAC (Sigma, St. Louis, MO, USA), a well known antioxidant, was used as a reference drug because there are many comparative experimental data using the NAC against cisplatin-induced nephrotoxicity using both in vitro and in vivo experimental models [31][32][33]. Five mice were allotted to the normal group and eight mice were allotted to each of the cisplatin-injected groups (cisplatin, NAC, panaxynol). Panaxynol and NAC were dissolved in 0.5% methylcellulose solution, and oral administration began 1 day before the cisplatin injection. The body weight was measured daily for 4 days (day 0-3). Three days after the cisplatin injection (day 3), blood samples were collected by performing a cardiac puncture under light anesthesia using diethyl ether, followed by the dissection and isolation of kidneys.

Determination of Serum Creatinine and Blood Urea Nitrogen
The serum was obtained from whole blood by centrifugation at 1200× g for 20 min. Serum level of creatinine and blood urea nitrogen (BUN) were measured using a commercial kit (YD diagnostics, Yongin, Korea) according to the manufacturer's instructions.

Statistical Analysis
Statistical significance was determined using one-way analysis of variance (ANOVA), and multiple comparisons were performed using a Bonferroni correction. P-values of less than 0.05 indicated statistical significance. All analyses were performed using SPSS Statistics ver. 19.0 (SPSS Inc., Chicago, IL, USA).

Protective Effect of Panaxynol Isolated from Panax vietnamensis on Cisplatin-Induced LLC-PK1 Cell Death
In the present study we isolated panaynol from VG, and it was analyzed with retention times of 22.3 min (Figure 1). To determine the non-toxic dose ranges of panaxynol, we assessed the cytotoxic effect of various concentrations of panaxynol on LLC-PK1 cells. As shown in Figure S1, panaxynol at 1, 2 and 4 µM show no toxic effects. To evaluate the protective effect of panaxynol (Figure 2A), LLC-PK1 cells were exposed to 25 µM cisplatin in the presence or absence of panaxynol for 24 h. As shown in Figure 2B, no protective effect on LLC-PK1 cells was observed at panaxynol concentrations below 0.125 µM. However, the cisplatin-induced decrease in cell viability to 59.94 ± 2.72% was increased to 75.95 ± 3.73%, 77.24 ± 2.93%, 77.47 ± 2.24%, 81.40 ± 3.60%, and 82.63 ± 0.35%, after pretreatment with 0.25, 0.5, 1, 2, and 4 µM of panaxynol, respectively. The cell morphology was confirmed using inverted phase-contrast microscopy under a 20× objective. Figure 2C revealed that the morphological shapes of the cisplatin-treated cells were detached from the surface and characterized by bubbling, cellular shrinkage, and deformation of cell bodies, indicating cell death. These changes were concentration-dependently suppressed by panaxynol. Thus, 2 and 4 µM of panaxynol were chosen as the highest optimal dose for further study.

Effect of Panaxynol Isolated from Panax vietnamensis on Cisplatin-Induced Apoptosis in LLC-PK1 Cells
To explore whether panaxynol could decrease cisplatin-induced apoptosis, LLC-PK1 cells were exposed to 25 µM cisplatin in the presence or absence of 2 and 4 µM of panaxynol for 24 h and stained with annexin V conjugated with Alexa Fluor 488 to identify apoptotic cells and propidium iodide to identify necrotic cells. As shown in Figure 3A, cisplatin-treated cells displayed a highly increased green fluorescence indicating apoptotic cells stained with annexin V conjugated with Alexa Fluor 488 compared to control cells. However, pretreatment with 2 and 4 µM of panaxynol reduced the green fluorescence. As shown in Figure 3B, the percentage of apoptotic cells increased by 39.96 ± 1.76% after treatment with 25 µM cisplatin and decreased following treatment with 2 and 4 µM panaxynol to 26.23 ± 1.51% and 15.96 ± 1.53%, respectively. These results suggest that panaxynol prevented cisplatin-induced apoptotic cell death through its anti-apoptotic activity.

Effect of Panaxynol Isolated from Panax vietnamensis on Expression Levels of JNK, P38, and Cleaved Caspase-3 in Cisplatin-Treated LLC-PK1 Cells
Western blot analysis was performed to evaluate the molecular mechanism, focusing on JNK, P38, and cleaved caspase-3. As shown in Figure 3, treatment with 25 µM cisplatin increased the phosphorylation of JNK, P38, and the expression level of cleaved caspase-3. However, pretreatment with 2 and 4 µM panaxynol reversed the upregulated phosphorylation of JNK, P38, and expression of cleaved caspase-3 ( Figure 4).

Discussion
Although many studies have reported the protective effects of ginsenosides in cisplatin-induced renal damage, the protective effect of panaxynol remains unknown. Previous studies have suggested that cisplatin damages the proximal tubular cells using molecular mechanisms related to the MAPK signaling and apoptotic pathways. In the MAPK signaling pathway, JNK and P38 are the main mediators of apoptotic cell death in the proximal tubular cells in response to cisplatin [34]. Cleaved caspase-3 with JNK and P38 are regarded as important therapeutic targets against cisplatin-induced damage in the proximal tubular cells [35]. Several studies have demonstrated that ginsenosides can attenuate the cisplatin-induced upregulation of phosphorylation of JNK, P38, and the expression levels of cleaved caspase-3 in proximal tubule cells. Ginsenosides Rb1 [7], Rh2 [8], Rh3 [14], and Rg3 [15,36] provide protection in cisplatin-induced tubular cell damage by suppressing the activation of caspase-3 that activates the extrinsic and intrinsic apoptotic pathways. In addition, ginsenosides Rh3 [14] and Rg3 [15] attenuate cisplatin-induced tubular cell damage by inhibiting phosphorylation of JNK and P38. In this study, we investigated the effects of panaxynol isolated from P. vietnamensis on cisplatin-induced cytotoxicity in LLC-PK1 cells. In the present study, cisplatin-induced LLC-PK1 cell death was suppressed by panaxynol. To further investigate the detailed mechanism, staining with annexin V conjugated with V Alexa Fluor 488 was performed. Cisplatin exposure increased the apoptotic cell death in LLC-PK1 cells. In contrast, panaxynol protected LLC-PK 1 cells from the cisplatin-induced apoptosis. Although various proteins were involved in cisplatin-induced apoptosis, we confirmed the protective effect of panaxynol in apoptotic cell death in LLC-PK1 cells using JNK, P38, and cleaved caspase-3. Cisplatin increased the phosphorylation of JNK, P38, and the expression level of cleaved caspase-3, and pretreatment with panaxynol effectively suppressed these observed upregulations. Hence, panaxynol inhibited apoptosis by downregulating the phosphorylation of JNK, P38, and the expression levels of cleaved caspase-3 in cisplatin-treated cells, contributing to the protective effect of panaxynol in cisplatin-induced LLC-PK1 cell death.
In the in vivo study, the oral administration of panaxynol ameliorated the cisplatin-induced kidney injury. Serum creatinine and BUN are the most commonly used biomarkers of renal dysfunction. Cisplatin is known to demonstrate toxic effects on the renal tubule, vasculature, and glomeruli. It results in structural damage to the kidney and consequently increases creatinine and urea in blood [37][38][39]. Panaxynol treatment reduced the serum creatinine and BUN in mice with cisplatin-induced kidney injury. Several reports have indicated that various inflammatory factors such as TNF-α, IL-6, MCP-1, and COX-2 are upregulated in cisplatin-induced nephrotoxicity in both humans and rodents [40,41]. The panaxynol-treated group exhibited significantly decreased COX-2 mRNA expression and slightly decreased MCP-1 mRNA expression in the kidneys compared to the cisplatin alone group. Collectively, these results indicate that panaxynol possesses a protective role in cisplatin-induced renal toxicity in mice and LLC-PK1 cells.
Panaxynol is a polyacetylene compound, which demonstrated renoprotective effects at low doses. The triple bonds in the structure could explain the ROS scavenging effects of panaxynol. Therefore, the kidney cells treated with this polyacetylene could possibly alleviate the oxidative stress induced by cisplatin. Additionally, panaxynol previously reported a strong antiproliferative effect [19,42]. The synergetic effect of the nephroprotective and antiproliferative effect of panaxynol could be an exciting combination in cancer patients, reducing the tumor as well as protecting the kidneys from cisplatin-induced injury. However, the potential clinical value of a panaxynol to protect against the cisplatin-induced nephrotoxicity will depend on its drug solubility, oral absorption, intestinal membrane permeability, and metabolism. Thus, further in vivo bioavailability studies will be needed.

Conclusions
This study, for the first time, confirmed the effect of panaxynol isolated from P. vietnamensis in cisplatin-induced renal damage in vitro and in vivo. Panaxynol reported a protective effect against cisplatin-induced LLC-PK1 cell death. We elucidated that this action was due to the antiapoptotic effects mediated through the downregulation of JNK, P38, and cleaved caspase-3, providing a better understanding of the pharmacological mechanisms of panaxynol in the treatment of cisplatin-induced nephrotoxicity.

Conflicts of Interest:
The authors declare no competing financial interest.