Identification of Renoprotective Phytosterols from Mulberry (Morus alba) Fruit against Cisplatin-Induced Cytotoxicity in LLC-PK1 Kidney Cells

The aim of this study was to explore the protective effects of bioactive compounds from the fruit of the mulberry tree (Morus alba L.) against cisplatin-induced apoptosis in LLC-PK1 pig kidney epithelial cells. Morus alba fruit is a well-known edible fruit commonly used in traditional folk medicine. Chemical investigation of M. alba fruit resulted in the isolation and identification of six phytosterols (1–6). Their structures were determined as 7-ketositosterol (1), stigmast-4-en-3β-ol-6-one (2), (3β,6α)-stigmast-4-ene-3,6-diol (3), stigmast-4-ene-3β,6β-diol (4), 7β-hydroxysitosterol 3-O-β-d-glucoside (5), and 7α-hydroxysitosterol 3-O-β-d-glucoside (6) by analyzing their physical and spectroscopic data as well as liquid chromatography/mass spectrometry data. All compounds displayed protective effects against cisplatin-induced LLC-PK1 cell damage, improving cisplatin-induced cytotoxicity to more than 80% of the control value. Compound 1 displayed the best effect at a relatively low concentration by inhibiting the percentage of apoptotic cells following cisplatin treatment. Its molecular mechanisms were identified using Western blot assays. Treatment of LLC-PK1 cells with compound 1 decreased the upregulated phosphorylation of p38 and c-Jun N-terminal kinase (JNK) following cisplatin treatment. In addition, compound 1 significantly suppressed cleaved caspase-3 in cisplatin-induced LLC-PK1 cells. Taken together, these findings indicated that cisplatin-induced apoptosis was significantly inhibited by compound 1 in LLC-PK1 cells, thereby supporting the potential of 7-ketositosterol (1) as an adjuvant candidate for treating cisplatin-induced nephrotoxicity.


Introduction
Cis-diamminedichloroplatinum II (cisplatin) is one of the most common platinum chemotherapeutic agents used for the treatment of many types of solid tumors [1]. In more than 30% of patients taking cisplatin, a variety of side effects, including allergic reactions, ototoxicity, myelotoxicity, nephrotoxicity, and gastrotoxicity, have been reported [2]. Of these side effects, nephrotoxicity is a dose-limiting one that makes patients unable to continue cisplatin treatment [3]. Cisplatin can seriously damage the S3 segment of the proximal tubules, causing kidney dysfunction [4]. Forced diuresis using mannitol, magnesium supplementation, and kidney-protective therapeutic approaches using enzymes and compounds that can help treat or prevent cisplatin-induced nephrotoxicity was reported [5].
In addition, the effects of plant extracts and plant-derived natural products on cisplatininduced nephrotoxicity were studied [6]. However, the detailed molecular mechanisms underlying their protective effects remain unclear. In previous studies using kidney cells, treatment with cisplatin (16-300 µM) induced cell death and activated cellular signaling pathways, including p53, mitogen-activated protein kinases (MAPKs), and caspases [7,8], which can be molecular targets for the mechanism of nephroprotection.
The mulberry tree (Morus alba L.), also known as white mulberry, belongs to the family Moraceae. Morus alba fruit is a well-known edible fruit commonly used in traditional folk medicine to improve diabetes and eyesight [9]. Its leaves are also consumed as a fodder for silkworms (Bombyx mori L.) and used in health products such as tea and beverages [10]. In previous studies on M. alba, extracts from its fruit have exhibited pharmacological activities, including anti-microbial [11], anti-inflammatory [12], anti-obesity [13,14], anticancer [15], and anti-oxidant activities [12,16,17]. Previous phytochemical investigations of M. alba fruit have reported a variety of bioactive secondary metabolites such as chlorogenic acid, ferulic acid, protocatechuic acid, apigenin, quercetin, and rutin [18]. In our ongoing endeavor to find bioactive products from diverse natural resources [19][20][21][22], we have carried out chemical investigations of many natural materials to identify bioactive compounds exhibiting protective effects against cisplatin-induced nephrotoxicity. As a result, we have identified several kidney-protective phytochemicals, such as ginsenoside Rb1 from Panax ginseng [23], ergosterols from the fruiting bodies of the mushroom Pleurotus cornucopiae [24], and flavonoids from peat moss Sphagnum palustre [25]. Recently, we also identified butyl pyroglutamate, a renoprotective compound, from M. alba fruit [26]. Its renoprotection was mediated by inhibition of MAPK protein expression and cleaved caspase-3 protein expression [26].
To extend our previous studies, we further investigated an ethanol extract of M. alba fruit to identify potential renoprotective compounds in the present study. Phytochemical analysis of the M. alba fruit extract led to the isolation of six phytosterols (1)(2)(3)(4)(5)(6). Their structures were determined by detailed analyses of their nuclear magnetic resonance (NMR) spectroscopic and physical data as well as mass spectrometry (MS) data from liquid chromatography (LC)/MS analyses. Herein, we report the isolation and structural characterization of these six compounds along with their protective effects against cisplatininduced cell death and their underlying mechanism of action in LLC-PK1 cells.

Cell Culture and Cell Viability Assay
LLC-PK1 cells and kidney epithelial cells from pigs were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). These cells were grown at 37 • C in a humidified atmosphere incubator with 5% CO 2 in air using Dulbecco's modified eagle medium (ATCC) supplemented with 1% penicillin/streptomycin, 10% fetal bovine serum (Invitrogen, Grand Island, NY, USA), and 4 mM l-glutamine. These cells were seeded into 96-well culture plates at a density of 1 × 10 4 cells/mL. After 24 h, cells were pretreated with 2.5, 5, 10, 25, and 50 µM of test samples for 2 h at 37 • C. Next, 25 µM cisplatin was added to cells. After incubation for 24 h at 37 • C, cell viability was measured using an EZ-Cytox assay kit (Daeillab Service, Seoul, South Korea) according to the method described in a previous study [26].

Image-Based Cytometric Assay
Annexin V Alexa Fluor 488 staining was performed to determine the percentage of apoptotic cells. Briefly, cells were seeded in six-well plates at a density of 4 × 10 5 cells/mL. After 24 h, cells were pretreated with 2.5 and 5 µM compound 1 for 2 h at 37 • C. Next, 25 µM cisplatin was added to cells. After incubation for 24 h at 37 • C, cells were stained with Annexin V Alexa Fluor 488 (Invitrogen, Temecula, CA, USA). The percentage of apoptotic cells was analyzed using a Tali image-based cytometer (Invitrogen, Temecula, CA, USA) according to the method described in a previous study [26].

Statistical Analysis
All data, including cell viability, percentage of apoptotic cells, and protein expression, are presented as average value and standard deviation (SD). All assays were performed in triplicate and repeated at least thrice. In this study, only a small number of repetitions for each cell experiment were included. Thus, a non-parametric analysis method was adopted for the statistical analysis. The Kruskal-Wallis test was used for the statistical analysis of each variable. The SPSS statistical package (IBM SPSS Statistics version 21, Boston, MA, USA) was used for all analyses. Statistical significance was considered at p < 0.05.

Compound 1 Inhibits Cisplatin-Induced Apoptosis in LLC-PK1 Cells
We evaluated the effects of compound 1 on cisplatin-induced apoptotic cell death using Annexin V Alexa Fluor 488 staining. As shown in Figure 3A, apoptotic cells were stained with Annexin V Alexa Fluor 488 (green fluorescence). The percentage of apoptotic cells was increased by 25 μM cisplatin from 2.13% ± 0.19% to 46.41% ± 3.21%, whereas it was decreased by 13.74% ± 1.31% and 4.86% ± 0.49% when cells were pretreated with 10 μM and 25 μM of compound 1, respectively ( Figure 3B).

Compound 1 Inhibits Cisplatin-Induced Apoptosis in LLC-PK1 Cells
We evaluated the effects of compound 1 on cisplatin-induced apoptotic cell death using Annexin V Alexa Fluor 488 staining. As shown in Figure 3A, apoptotic cells were stained with Annexin V Alexa Fluor 488 (green fluorescence). The percentage of apoptotic cells was increased by 25 µM cisplatin from 2.13% ± 0.19% to 46.41% ± 3.21%, whereas it was decreased by 13.74% ± 1.31% and 4.86% ± 0.49% when cells were pretreated with 10 µM and 25 µM of compound 1, respectively ( Figure 3B).

Compound 1 Inhibits Expression Levels of p38, JNK, and Cleaved Caspase-3 in Cisplatin-Treated LLC-PK1 Cells
We also evaluated the possible molecular mechanisms of compound 1, focusing on p38, JNK, and cleaved caspase-3 using a Western blot analysis. Treatment with 25 μM cisplatin increased the expression levels of phosphorylated p38, phosphorylated JNK, and cleaved caspase-3. However, the expression levels of all these proteins in LLC-PK1 cells were decreased by treatment with 2.5 and 5 μM compound 1 in a dose-dependent manner ( Figure 4A). Bar graphs show the expression levels of phosphorylated p38, phosphorylated JNK, and cleaved caspase-3 normalized to glyceraldehyde 3-phosphate dehydro-

Compound 1 Inhibits Expression Levels of p38, JNK, and Cleaved Caspase-3 in Cisplatin-Treated LLC-PK1 Cells
We also evaluated the possible molecular mechanisms of compound 1, focusing on p38, JNK, and cleaved caspase-3 using a Western blot analysis. Treatment with 25 µM cisplatin increased the expression levels of phosphorylated p38, phosphorylated JNK, and cleaved caspase-3. However, the expression levels of all these proteins in LLC-PK1 cells were decreased by treatment with 2.5 and 5 µM compound 1 in a dose-dependent manner ( Figure 4A). Bar graphs show the expression levels of phosphorylated p38, phosphorylated JNK, and cleaved caspase-3 normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Figure 4B-D).

Discussion
Many drugs, including antifungal agents, anti-retroviral drugs, aminoglycoside antibiotics, and anticancer drugs, are known to cause nephrotoxicity [32]. Various assays have been used to assess the protective effects of plant extracts and plant-derived natural products against drug-induced cytotoxicity in kidney cells. The primary assay to identify an effective substance is based on measurement of cell viability. In the present study, we identified cell-protective compounds from M. alba fruit using the EZ-Cytox assay to measure the metabolic activities of cells in the presence of cisplatin. All compounds displayed protective effects against cisplatin-induced LLC-PK1 cell damage, improving cisplatininduced cytotoxicity to more than 80% of the control value. Compound 1 displayed the best effect at a relatively low concentration. The LLC-PK1 cell viability that was reduced by 25 μM cisplatin to 60% increased to nearly 100% after co-treatment with 5 μM compound 1. In our previous study, 10 μM butyl pyroglutamate isolated from M. alba fruit improved the cell viability by 83%, which was more effective than N-acetylcysteine [33]. N-acetylcysteine has been used as a positive control in cisplatin-induced renal toxicity studies [34,35].
Oxidative stress, apoptosis, and inflammation are three major mechanisms underlying cisplatin-induced cytotoxicity. Among these, the most well-known mechanism is the apoptosis pathway [35]. It is known that cisplatin-induced apoptotic cell death in renal tubular cells is associated with both mitochondrial-mediated and death-receptor-mediated pathways [36]. Both these pathways ultimately induce apoptosis through caspase-3 activation [37]. Additionally, it has been shown that JNK and p38 regulate tumor necrosis factor-α (TNF-α), which plays an important role in cisplatin-induced apoptosis [38,39]. In the present study, compound 1 had a protective effect against apoptotic cell death. This result is consistent with the improved cell viability of compound-1-treated cells. The protective effect of compound 1 on LLC-PK1 cells might be partly due to inhibition of apop-

Discussion
Many drugs, including antifungal agents, anti-retroviral drugs, aminoglycoside antibiotics, and anticancer drugs, are known to cause nephrotoxicity [32]. Various assays have been used to assess the protective effects of plant extracts and plant-derived natural products against drug-induced cytotoxicity in kidney cells. The primary assay to identify an effective substance is based on measurement of cell viability. In the present study, we identified cell-protective compounds from M. alba fruit using the EZ-Cytox assay to measure the metabolic activities of cells in the presence of cisplatin. All compounds displayed protective effects against cisplatin-induced LLC-PK1 cell damage, improving cisplatin-induced cytotoxicity to more than 80% of the control value. Compound 1 displayed the best effect at a relatively low concentration. The LLC-PK1 cell viability that was reduced by 25 µM cisplatin to 60% increased to nearly 100% after co-treatment with 5 µM compound 1. In our previous study, 10 µM butyl pyroglutamate isolated from M. alba fruit improved the cell viability by 83%, which was more effective than N-acetylcysteine [33]. N-acetylcysteine has been used as a positive control in cisplatin-induced renal toxicity studies [34,35].
Oxidative stress, apoptosis, and inflammation are three major mechanisms underlying cisplatin-induced cytotoxicity. Among these, the most well-known mechanism is the apoptosis pathway [35]. It is known that cisplatin-induced apoptotic cell death in renal tubular cells is associated with both mitochondrial-mediated and death-receptor-mediated pathways [36]. Both these pathways ultimately induce apoptosis through caspase-3 activation [37]. Additionally, it has been shown that JNK and p38 regulate tumor necrosis factor-α (TNF-α), which plays an important role in cisplatin-induced apoptosis [38,39]. In the present study, compound 1 had a protective effect against apoptotic cell death. This result is consistent with the improved cell viability of compound-1-treated cells. The protective effect of compound 1 on LLC-PK1 cells might be partly due to inhibition of apoptosis by cisplatin. In addition, treatment with cisplatin increased the expression levels of phosphorylated p38, phosphorylated JNK, and cleaved caspase-3, whereas these expression levels were decreased in a dose-dependent manner by treatment of LLC-PK1 cells with compound 1. These observations indicated that compound 1 inhibited apoptosis through the inhibition of phosphorylated JNK and p38 as well as the inhibition of the expression level of cleaved caspase-3 ( Figure 5). Therefore, the anti-apoptotic effect might be responsible for the protective effect of compound 1 against cisplatin-induced cell death.
Plants 2021, 10, x FOR PEER REVIEW 9 of 11 expression levels were decreased in a dose-dependent manner by treatment of LLC-PK1 cells with compound 1. These observations indicated that compound 1 inhibited apoptosis through the inhibition of phosphorylated JNK and p38 as well as the inhibition of the expression level of cleaved caspase-3 ( Figure 5). Therefore, the anti-apoptotic effect might be responsible for the protective effect of compound 1 against cisplatin-induced cell death.

Conclusions
In summary, as part of an ongoing research project to discover bioactive natural products [40][41][42][43][44][45], we identified renoprotective phytosterols from the fruit of the mulberry tree (M. alba) that ameliorated cisplatin-induced cytotoxicity. All compounds displayed protective effects against cisplatin-induced damage in LLC-PK1 cells. Compound 1 displayed the best effect at a relatively low concentration. In addition, we demonstrated that compound 1 blocked cisplatin-induced LLC-PK1 cell apoptosis by inhibiting expression levels of phosphorylated p38, phosphorylated JNK, and cleaved caspase-3. However, additional detailed mechanisms responsible for the renoprotective effects of compound 1 need to be studied to support the potential of 7-ketositosterol (1) as an adjuvant candidate for treating cisplatin-induced nephrotoxicity.

Conclusions
In summary, as part of an ongoing research project to discover bioactive natural products [40][41][42][43][44][45], we identified renoprotective phytosterols from the fruit of the mulberry tree (M. alba) that ameliorated cisplatin-induced cytotoxicity. All compounds displayed protective effects against cisplatin-induced damage in LLC-PK1 cells. Compound 1 displayed the best effect at a relatively low concentration. In addition, we demonstrated that compound 1 blocked cisplatin-induced LLC-PK1 cell apoptosis by inhibiting expression levels of phosphorylated p38, phosphorylated JNK, and cleaved caspase-3. However, additional detailed mechanisms responsible for the renoprotective effects of compound 1 need to be studied to support the potential of 7-ketositosterol (1) as an adjuvant candidate for treating cisplatin-induced nephrotoxicity.

Data Availability Statement:
The data presented in this study are available in article and supplementary material.

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