Cisplatin is one of the most effective and potent antineoplastic agent, 25~30% of patients receiving cisplatin experience acute kidney injury formally called acute renal failure (ARF) [1,2]. Moreover, cisplatin use can also result in a variety of clinical renal complications such as hypomagnesemia, hypocalcemia, distal renal tubular acidosis, thrombotic microangiopathy [3].

The etiology of ARF is associated to the accumulation of cisplatin in renal proximal tubular cells and cisplatin is well-known to permeate proximal tubular cells through apical and basolateral organic cation transporters [4,5]. Especially, the global burden of ARF is calculated to be 13.3 million cases per year, 11.3 million of which are in low-income countries. According to the UK’s National Institute for Health and Care Excellence (NICE), adequate care of ARF could avoid 42,000 deaths every year [6]. The various efforts which include intravenous volume expansion, avoidance of other potential nephrotoxins, renal replacement therapy have been tried to prevent nephrotoxicity. However, none of them have demonstrated definite positive clinical results. Ultimately, the clinical use of cisplatin has been limited because of this side effect [3] and is also greatly demanded the development of new therapeutic approaches to support and improve renal function.

Panax ginseng (P. ginseng) has been used as traditional herbal medicine in Korea, China, and Japan and especially roots have been provided a health food or various remedies. P. ginseng has been reported to possess multiple biological activities including antioxidant, anti-inflammatory, anti-tumor, antiaging, antidiabetic, anti-fatigue effects [7-9]. Ginsenosides, saponin molecules that are characteristic of Panax species, are commonly considered to be the major active pharmacological components of P. ginseng. The bioactive components in ginseng root include more than 60 ginsenosides such as Rg1, Re, Rb1, Rg2(s), Rg2(r), Rc, Rb3, Rd, Rg6, Rg3(s), Rg3(r), Rk1, Rg5, etc. [10,11].

Fresh ginseng is easily degraded at room temperature and consequentially decreases its efficacy. Therefore, fresh ginseng is processed into red ginseng or black ginseng through the process of steaming and drying or processed into white ginseng by a simple drying process [12]. These processing methods increase their pharmacological effects and decrease their cytotoxicity through the chemical alteration of components and the content change [13]. Thus, recent many studies have examined the enhancement of active components and physiological activites by fermenting ginseng. Moreover, fermentation-processed ginseng was absorbed and digested more easily [14,15] and also improves their pharmacological efficacy [16].

However, virtually no studies have investigated the changes in chemical and biological activities according to fermentation of P. ginseng, which is undergone 9 times steaming and drying repeatedly, was called generally ‘Black Ginseng’ in Korea. Therefore, we separated the major ginsenosides and analyzed their contents by high-performance liquid chromatographic (HPLC) and compared it with white ginseng by a simple drying process. Moreover, we evaluated the antioxidant and anti-inflammatory effects of fermented black ginseng on Cisplatin-induced nephrotoxicity in mice.

Materials

Cisplatin (cis-diamminedichloroplatinum(II) (CDDP)), phenylmethylsulfonyl fluoride (PMSF), dithiothreitol (DTT), N-1- naphthylenediamine dihydrochloride, sulphanilamide, phosphoric acid, diethylenetriaminepenta-acetic acid (DTPA), and dihydrorhodamine 123 (DHR 123) were obtained from Sigma Chemical Co. (St Louis, MO, USA). The protease inhibitor mixture and ethylenediaminetetraacetic acid (EDTA) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). 2’, 7’-Dichlorofluorescein diacetate (DCF-DA) was obtained from Molecular Probes (Eugene, OR, USA). The pierce bicinchoninic acid (BCA) protein assay kit was obtained from Thermo Scientific (Rockford, IL, USA). Goat polyclonal antibodies against tumor necrosis factor-α (TNF-α; 1:1,000) and interleukin-6 (IL-6; 1:1,000); rabbit polyclonal antibodies against nuclear factor-κB (NF- κB; 1:1,000), superoxide dismutase (SOD; 1:1,000), catalase (1:1,000), glutathione peroxidase (GPx, 1:1,000); mouse monoclonal antibodies against phosphor- inhibitor of nuclear factor kappa B (p-IκBα; 1:1,000), cyclooxygenase-2 (COX-2, 1:1,000) and inducible nitric oxide synthase (iNOS, 1:1,000), β-actin (1:1,000), and histone (1:1,000) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Rabbit anti-goat and goat anti-mouse immunoglobulin G (IgG) horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5,000) were acquired from Santa Cruz Biotechnology, Inc. ECL Western Blotting Detection Reagents were supplied by GE Healthcare (Piscataway, NJ, USA). The all agent were used for the cultivation of the microorganisms were purchased from BD Difco (Detroit, MI. USA) and Streptococcus thermophilus obtained from prof. Lee CY ( Microbiology and Biotechnology, Daejeon University, Korea). Complex enzyme liquid was manufactured using Cellulclast, HTec2, and Amyloglucosidase and used when black ginseng (BG) was fermented. Here, each ratio of three agents is 1:1:1. Complex enzyme liquid obtained from the Korea Research Institute of Chemical Technology (Daejeon, Korea).

Plant materials and Strain used for fermentation

The fresh ginseng roots used standardized 6-yr-old Korean ginseng harvested in 2013 (Yeoju-si, Gyeonggi-do, South Korea). A voucher herbarium specimen has been deposited at the Herbarium of Daejon University and was identified by Prof. Y.B. Seo, the herbarium leader of Daejon University. White ginseng (WG) was experienced a simple drying process (drying temperature; 50°C, water content; less than or equal to 15%) without steaming and fermentation. Black gingeng (BG) was undergoed 9 times steaming (steaming temperature; 90ºC, steaming time; 4 h) using non-pneumatic steam steaming machine (Ace groundbreaking, Seoul, Korea) and drying repeatedly, thereafter was fermented by Streptococcus thermophilus. This fermented BG was called FBG hereafter. WG and BG are showed Figure 1A and 1B. First, mix BG 50 g and distilled water (250 mL) (20% w/v) in air-tight containers. Second, reacts complex enzyme liquid (2 mL) for 24 h. Third, inoculates Streptococcus thermophilus (2 mL) and incubates at 35°C. Fourth, inoculates the strain for the solid fermentation (about 10⁷~10⁸ cells/mL) and measure pH at 0 day, 4 days, 7 days. Fifth, FBG 5 g fermented for 6 days was inserted 10 times distilled water and repeatedly extracted 2 times in 100°C for 3 h. Six, filtered using Whatman No.2 and evaporated on a rotary evaporator (Buchi, flawil, Switzerland). Next, dried using a freeze drier in vacuo (Eyela FDU-540, Tokyo, Japan) 120 min at -30ºC, 120 min at -15°C, 240 min at 0 ºC, 120 min at 15°C, 120 min at 30ºC, in regular sequence. The extract powder was stored at -20°C.

Figure 1: Morphology change from WG to BG. WG (A) BG (B) WG, White ginseng; BG, 9 times steaming and drying repeatedly. This figure is showed that WG is undergoed 0, 1, 3, 6, 9 times processing and is changed in its morphology.

pH Measurement

The pH values were measured using Oakton 35634-40 pH Spear (Varnon hills, IL, USA) at 0 day, 4 days, 7 days according to fermentation days. The change of pH is showed in Figure 2A-C.

Figure 2: HPLC chromatograms of WG (A), BG (B), and FBG (C) at a detection wavelength of UV 203 nm. (1) Rg1, (2) Re, (3) Rb1, (4) Rg2(s), (5) Rg2(r), (6) Rc, (7) Rb3, (8) Rd, (9) Rg6, (10) F4+Rk3, (11) Rh4, (12) Rg3(s), (13) Rg3(r), (14) Rk1, (15) Rg5. WG, White ginseng; BG, 9 times steaming and drying repeatedly; FBG, BG fermented by Streptococcus thermophiles.

HPLC analysis

We injected 20 μL of WG or FBG into a reverse-phase high-performance liquid chromatography (HPLC) using a XbridgeTM RP18 (4.6 × 250 mm, 5-μm pore size), with a column temperature of 40°C. Mobile phase component A=water and B=acetonitrile. The gradient conditions were as follows: 0-42 min, 18% B; 42-46 min, 24% B; 46-79 min, 40% B; 79-115 min, 65% B; 115-135 min, 85% B; 150-151 min, 18% B. The flow rate was 1.0 mL/min. The UV absorbance from 203 nm was monitored using PDA (Waters 1525, detector 2998, USA). All peaks were assigned by carrying out co-injection tests with authentic samples and comparing them with the UV spectral data. The components of major compounds (Rg1, Re, Rb1, Rc, Rb3, Rd, Rg3, Rk1, and Rg) were detected from WG, BG, and FBG. The measurement was repeated three times for each sample. Representative HPLC results are illustrated in Figure 3.

Figure 3: pH change according to fermented days. WG, White ginseng; BG, 9 times steaming and drying repeatedly. pH level was measured at fermentation 1 day, 4 days, and 7 days.

Animals and experimental design

Six-week-old ICR mice were purchased from Orient Bio (Gyeonggi-do, Korea). The animals were maintained under a 12 h light/dark cycle, and housed with a controlled temperature (24°C) and humidity (55 ± 5%). All animal experimental protocols were approved by the Institutional Animal Care and Use Committee of the Daegu Haany University (Ethical approval number: 2015-050). For the main study, 36 mice were randomly divided into four groups. The group 1 (Normal group, n = 9) received water; group 2 (Control group, n=9) received only a single injection of cisplatin (20 mg/kg) intraperitoneally; group 3 (WG group, n = 9) received WG (200 mg/kg/day) orally for 4 successive days thereafter injected cisplatin; group 4 (FBG group, n = 9) received FBG (200 mg/kg/day) orally for 4 successive days thereafter injected cisplatin. At 24 h after cisplatin challenge, blood samples were collected by cardiac puncture from anesthetized mice. The serum was immediately separated from the blood samples by centrifugation. Subsequently, the kidney was perfused through the artery with ice-cold physiological saline (0.9% NaCl, pH 7.4), removed, quickly frozen, and kept at -80ºC until analysis.

Measurement of renal functional parameters

Renal functional parameters, blood urea nitrogen (BUN) and Creatinine assay kits were conducted spectrophotometrically using commercially available kits from Asan Pharm. Co., Ltd., (Seoul, Korea).

Measurement of ROS and GSH in serum and kidney

ROS level was measured employing the method of Ali et al. [17]. DCF-DA was added to serum or kidney tissue. After incubation for 30 min, the changes in fluorescence values were determined at an excitation wavelength of 486 nm and emission wavelength of 530. GSH assay was carried out by employing the method of Hissin and Hilf [18]. Renal tissues were homogenized on ice with 1 mM EDTA-50 mM sodium phosphate buffer (pH 7.4). Then, 25% metaphosphoric acid was added for protein precipitation. The homogenate was centrifuged at 4°C for 30 min to obtain the supernatant for the assay of GSH. To assay, 1 mM EDTA-50 mM sodium phosphate buffer (pH 7.4) was added to the supernatant, followed by o-phthalaldehyde. After 20 min at room temperature, fluorescence was estimated at an excitation wavelength at 360 nm and emission wavelength of 460 nm. Protein assay was carried out using a Bio-Rad protein kit (Bio- Rad Laboratories, Hercules, CA, USA).

Preparation of cytosol and nuclear fractions

Protein extraction was performed according to the method of Komatsu with minor modifications [18,19]. Esophageal tissues for cytosol fraction were homogenized with ice-cold lysis buffer A (250 mL) containing 10 mM HEPES (pH 7.8), 10 mM KCl, 2 mM MgCl₂, 1 mM DTT, 0.1 mM EDTA, 0.1 mM PMSF, and 1,250 μL protease inhibitor mixture solution. The homogenate incubated at 4°C for 20 min. And then 10% NP-40 was added and mixed well. After centrifugation (13,400 × g for 2 min at 4°C) using Eppendorf 5415R (Hamburg, Germany), the supernatant liquid (cytosol fraction) was seperated new e-tube. The left pellets were washed twice by buffer A and discard the supernatant. Next, the pellets were suspended with lysis buffer C (20 mL) containing 50 mM HEPES (pH 7.8), 50 mM KCl, 300 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 0.1 mM PMSF, 1% (v/v) glycerol, and 100 μL protease inhibitor mixture solution suspended and incubated at 4°C for 30 min. After centrifugation (13,400 × g for 10 min at 4°C), the nuclear fraction was prepared to collect the supernatant. Both cytosol and nuclear fractions were kept at -80°C before the analysis.

Immunoblotting analyses

For the estimation of NF-κBp65, 10 μg of protein from each nuclear fraction was electrophoresed through an 8% sodium dodecylsulfate polyacrylamide gel (SDS-PAGE). Separated proteins were transferred to a nitrocellulose membrane, blocked with 5% (w/v) skim milk solution for 1 h, and then incubated with primary antibodies to NF-Bp65 and histone, respectively, overnight at 4°C. After the blots were washed, they were incubated with anti-goat or anti-mouse IgG HRP-conjugated secondary antibody for 1 h at room temperature. Also, 10-15 μg of protein of each cytosol fraction of SOD, catalase, GPx, p-IκBα, COX-2, iNOS, TNF-α, and IL-6 was electrophoresed through 8-15% SDS-PAGE. Each antigen-antibody complex was visualized using ECL Western Blotting Detection Reagents and detected by chemiluminescence with Sensi-Q2000 Chemidoc (Lugen Sci Co., Ltd., Gyeonggi-do, Korea). Band densities were measured using ATTO Densitograph Software (ATTO Corporation, Tokyo, Japan) and quantified as the ratio to histone and/ or β-actin. The protein levels of groups are expressed relative to those of normal mice (represented as 1).

Statistical analysis

The data are expressed as the mean ± SEM. Significance was assessed by one-way analysis of variance (ANOVA) followed by least-significant differences (LSD) test (SPSS 22.0 for Windows, SPSS Inc., Chicago, IL, USA). Values of p<0.05 were considered significant.

HPLC analysis for WG and FBG and pH change

Traditional herbal medicines have been processed to enhance their therapeutic effects, remove or reduce toxicity and side effects, and make storage higher. Heat-processed Korean ginseng (Panax ginseng) is one of the representative examples of a processed herbal medicine [20]. Besides, the lactic acid fermentation is considered as one of the appropriate tool to enhance the functional potential or bioactive compounds [21]. Especially, Streptococcus thermophilus, a gram-positive facultative anaerobe, is one of the crucial lactic acid bacteria in the manufacture of yogurt widely. In addition, it is considered as the most important industrial dairy starter during fermentation processes [22].

The major 15 ginsenoides such as Rg1, Re, Rb1, Rg2(s), Rg2(r), Rc, Rb3, Rd, Rg6, Rg3(s), Rg3(r), Rk1, and Rg5 and total ginsenoides contents were determined after comparison with standards Figure 2. In this study, we investigated the heat-processing and fernentation-induced chemical changes of Panax ginseng. As shown in Table 1, WG was high Rg1, Re, Rb1, Rc, and Rb3 levels, however, BG was high Rd, Rg3(s), Rg3(r), Rk1, and Rg5 levels. When BG was fermented, Rd, Rg3(s), Rg3(r), Rk1, and Rg5 levels increases much higher than those of BG. Above all, the Rd level showed the noticeable rise (5.7 fold compared with BG, and 11.7 fold compared with WG). Total ginsenoids content of WG, BG, and FBG was 4.360, 7.857, and 9.252, respectively. These results suggest that fermentation of BG could exert the potent pharmacological activities than WG or BG. As shown in Figure 3, pH level according to fermentation days of BG is reduced. pH of WG is 5.37 and pH of BG is 3.93 at fermentation 7 days. FBG (pH 3.93) was used in the current study.

Table 1: Comparison of composition content of WG, BG, and FBG.

Effect of FBG in Body and Kidney weights and serum parameters

Cisplatin (cis-diammine-dichloroplatinum II, CDDP) is a widely used cancer chemotherapeutic agent to treat various types of solid tumors in the head and neck, lung, ovaries, uterus, testicles, and bladder [23]. The well-known side effects of cisplatin are nephrotoxicity, neurotoxicity, ototoxicity, electrolyte disturbances, nausea and vomiting, myelotoxicity, and hemolytic anemia. Herein, nephrotoxicity is the most common adverse effect and it is a major issue [24]. High concentrations of cisplatin and its toxic metabolites related to the risk of nephrotoxicity, therefore cisplatin has been used limitedly. Cisplatin induced nephrotoxicity consists of change in kidney weight, histological changes in kidney and increase BUN and creatinine in serum [25]. Recent report showed that Ginsenoside-Rd was proved to decrease the severity of renal injury induced by cisplatin [26]. In the present study, we evaluated the kidney weight, BUN and creatinine which are renal function biomarker. Cisplatin-control mice showed a significant decrease in final body weight (33.3 ± 0.25 vs. 35.5±0.45 g) and increase in the kidney weight (12.25 ± 0.52 vs. 5.47 ± 0.41 mg/g b.w.); which were alleviated with WG or FBG pretreatment Table 2. In addition, cisplatin raised both serum BUN and creatinine levels compared to the normal group. However, BUN and creatinine levels both WG and FBG were decreased significantly. Herein, pretreatment with WG and FBG attenuated cisplatin-induced renal dysfunction as demonstrated by normalization of BUN and creatinine level compared to cisplatin-control mice [27-30].

Table 2: Body weight, kidney weight, kidney functional indices in serum.

ROS and GSH levels in Serum and Kidney

Recently, studies in vitro and in vivo have been accumulated that the pathogenesis of nephrotoxicity is closely related to reactive oxygen metabolites [30,31]. Oxidative stress initially triggered by cisplatin treatment directly acts on cell components, including lipids, proteins, and DNA and destroys their structure. The induction of ROS contributes to the development of nephrotoxicy. Most of free radicals rapidly impair biological functions of cellular constituent. Thus, cisplatin-induced nephrotoxicity was protected by radical scavengers [32,33]. GSH is the major antioxidative tripeptide and plays important role in the detoxification of toxicants to disturb cellular homeostasis.

Table 3 shows the effect of WG and FBG on the level of ROS and GSH in serum and kidney. The serum ROS level of cisplatin-control mice was significantly elevated to 3802 ± 352 fluorescence/min/mL in comparison with that of normal mice of 2106 ± 155 fluorescence/min/ mL. However, in WG or FBG-treated mice, it was significantly decreased to 2859 ± 252 or 2902 ± 199 fluorescence/min/mL, respectively. The renal ROS level of cisplatin-control mice was significantly elevated to 3635 ± 61 fluorescence/min/mg protein in comparison with that of normal mice of 3076 ± 77 fluorescence/min/ mg protein. Besides, in WG or FBG-treated mice, it was remarkably decreased to 2950 ± 153 (p<0.001) or 29676 ± 64 (p<0.001) fluorescence/min/ mg protein, respectively. Moreover, the oral administration of WG or FBG significantly up-regulated the GSH level in serum when it compared with cisplatin-control mice. Similary, the renal GSH level by WG or FBG pretreatment was increased higher than it of normal mice. Especially, FBG administration showed a significant increase (p<0.05) [34,35].

Table 3: Biochemical analysis.

Renal oxidative and inflammatory protein expressions

Compared to normal mice, renal expressions of SOD, catalase, and GPx were markedly reduced in cisplatin-control mice [36]. The administration of WG or FBG led to a significant up-regulation of these protein expressions. The SOD level of FBG was superior to it of WG Figure 4A-C.

Figure 4: Renal SOD (A), Catalase (B), and GPx (C) protein expressions. N, normal mice; Con, Cisplatin-treated mice; WG, Cisplatin-treated and WG 200 mg/ kg pretreated mice; FBG, Cisplatin-treated and FBG 200 mg/kg pretreated mice. Significance: *p<0.05, **p<0.01 vs. Con. (n=9).

An early response to cisplatin-induced nephrotoxicity includes the activation of NF-κBsignaling pathway [34]. NF-κB is a major pro-inflammatory transcriptional factor sequestered in the cytoplasm by the inhibitor protein IκBα. Phosphorylation of IκBα leads to the release of NF-κBand then NF-κB translocates to the nucleus and promotes the transcription of target genes including inflammatory mediators and cytokines such as COX-2, iNOS, TNF-α and IL-6 [35,37].

Figure 5 shows p-IκBα and NF-κBp65 protein expressions. In cisplatin-control mice, the phosphorylation of IκBα was significantly increased compared with the normal group. Pretreatment of WG or FBG significantly suppressed under the level of normal mice Figure 5A. Moreover, cisplatin-control mice showed up-regulation of the nuclear NF-Bp65 protein compared to normal mice. On the other hand, the administration of WG or FBG led to reduce nearly too normal levels Figure 5B.

Figure 5: Renal p-IB (A) and NF-Bp65 (B) protein expressions. N, normal mice; Con, Cisplatin-treated mice; WG, Cisplatin-treated and WG 200 mg/kg pretreated mice; FBG, Cisplatin-treated and FBG 200 mg/kg pretreated mice. Significance: *p<0.05 vs. Con. (n=9).

We quantified renal COX-2, iNOS, TNF-α, and IL-6 protein expressions, whose activations are involved in ROS and related to inflammatory responses, presented in Figure 6A-D, respectively. COX- 2, iNOS, TNF-α, and IL-6 protein expressions in cisplatin-control mice were significantly increased compared to those in normal mice, whereas these increased protein expressions were down-regulated by the administration of WG or FBG. Especially, COX-2, iNOS, and IL-6 levels in the mice receiving FBG were significantly reduced nearly to those of normal mice or below, as shown Figure 6.

Figure 6: Renal COX-2 (A), iNOS (B), TNF- (C), and IL-6 (D) protein expressions. N, normal mice; Con, Cisplatin-treated mice; WG, Cisplatin-treated and WG 200 mg/kg pretreated mice; FBG, Cisplatin-treated and FBG 200 mg/kg pretreated mice. Significance: *p<0.05, **p<0.01, ***p<0.001 vs. Con. (n=9).

Effect of FBG on renal tubular damage

Cisplatin is filtered at the glomerulus and taken up into renal tubular cells mainly by a transport-mediated process. Accordingly, nephrotoxicity by cisplatin is characterized by renal electrolyte disturbances and by acute fall in glomerular filtration rate (GFR) [27,28]. Cisplatin teatment was found to cause diffuse tubular necrosis and desquamation and parenchyma degeneration in the cortex [29]. In the current study, turbular damage was similar to control kidney of a previous reportd study. However, WB or FBG treatment partially was reduced the histological features of renal injury. Cisplatin-control mice Figure 7A and 7B showed a meaningful tubular damage compared to the normal group Figure 7A. Pretreatment with WG or FBG decreased tubular damage, as shown Figure 7C and 7D.

Figure 7: H/E staining of kidney tissue. (A) normal mice, (B) Cisplatin-treated mice (C) WG, Cisplatin-treated and WG 200 mg/kg pretreated mice (D) FBG, Cisplatin-treated and FBG 200 mg/kg pretreated mice. ×200.

Data from previous studies demonstrated key roles for inflammatory and oxidative pathways in nephrotoxicity induced by cisplatin. Based on these reports, cisplatin nephrotoxicity observed in our study can be attributed to the concomitant deterioration in the antioxidant tendency (reduced SOD, catalase, and GPx), elevations in inflammatory mediators (COX-2 and iNOS), and increases in inflammatory cytokines (TNF-α and IL-6) in renal tissues. Herein, pretreatment both WG and FBG reversed significantly these protein expressions. Especially, antioxidative effect of FBG treatment was more superior than those of WG treatment. These results showed that FBG may more effectively develop the renoprotection through the strong suppression of the oxidative stress. FBG could effectively protect more than it of BG Figure 8.

Figure 8: Predicted mechanism in renal tissues on administering FBG against oxidative stress and inflammation.

In this study, the administration of FBG effectively ameliorates nephrotoxicity in Cisplatin-treated mice through the inhibition of NF- κB activation and the up-regulation of antioxidative enzyme, thus, FBG would result in the improvement of renal disorders caused by the ROS system.

This research was supported by the Daejeon University fund (201501140001).