Evaluation of the Liver Cancer Prevention of Anthocyanin Extracts from Mulberry ( Morus alba L . ) Variety PR-01

This study aims to evaluate the preventive effects of anthocyanins extracts (MAEs) from mulberry variety PR-01 against N-nitrosodiethylamine (NDEA)induced hepatocarcinogenesis in rats. It was found that 150 mg·kg MAEs treatment significantly reduced the NDEA-induced hepatic nodules incidence and hepatocellular carcinoma incidence by 58.30% and 41.70% compared to the model group. Meanwhile, MAEs significantly restored the elevated the liver function enzymes, inhibited the tumor necrosis factor alpha and interleukin-6 levels, elevated the serum interleukin-10 and interferon-γ and increased hepatic glutathione-S-transferase and UDP-glucuronosyltransferase 2B1 enzyme activity. Moreover, 150 mg·kg MAEs supplement enhanced glutathione content and the activities of superoxide dismutase, catalase, glutathione peroxidase activities but reduced the malondialdehyde and thiobarbituric acid-reactive substances content by 37.90% and 44.52%. Furthermore, MAEs pretreatment maintained nuclear factor erythroid 2-related factor 2 (Nrf2), Kelch-like ECH-associated protein 1, heme oxygenase-1, and NAD(P)H: quinine oxidoreductase1 stimulation and inhibited the expression of TNF-α, nuclear factor-kappaB (NF-κB), and cyclooxygenase-2 (COX-2), indicating that MAEs exhibit effectively prevention effects against liver cancer via decreased lipid peroxidation, induced Nrf2-mediated antioxidant enzymes and attenuating the inflammatory mediators COX-2 through NF-κB pathway. Thus, MAEs of mulberry variety PR-01 may be used as a good functional dietary supplement against liver cancer.


Introduction
According to the histological classification, primary liver cancer can be divided into hepatocellular carcinoma (HCC), cholangiocarcinoma and mixed liver cancer, among which HCC is the most common [1] [2].Despite significant improvements in the clinical diagnosis and treatment of early HCC, the advanced HCC is a highly aggressive tumor with poor or no response to conventional treatment [3] [4].Therefore, it is urgent to find effective ways or measures to prevent liver cancer.
Mulberry (Morus alba L.) fruit has been traditionally used in Chinese medicines for its pharmacological effects including antioxidant activity, anti-inflammatory, hepatoprotective effect, and anti-diabetic properties, etc. [13].Mulberry is rich in anthocyanins (The anthocyanins content was 11.20 mg•100g −1 -193.00 mg•100g −1 in mulberry varieties), among which, the C3G and cyaniding-3-rutinoside (C3R) have been demonstrated to dose-dependently inhibit the migration and invasion of A549 human lung carcinoma cells [14].Yan et al. [15] showed that mulberry anthocyanin extract ameliorates oxidative damage in HepG2 Cells and prolongs the lifespan of Caenorhabditis elegans through mitogen-activated protein kinase and nuclear factor erythroid 2-related factor 2 (Nrf2) pathways.
Li, et al. [10] reported that mulberry anthocyanin has a strong protective effect on the carbon tetrachloride-induced liver fibrosis.Furthermore, it was recently demonstrated that the mulberry extract can increase the protein expression of liver antioxidant, accelerate the metabolism and excretion of nitrophenol, and detoxify the toxicity of nonyl phenol-induced rats [6].Results of the above studies suggested that mulberry anthocyanins may have potential effects in reducing the risk of cancers by anti-inflammatory, detoxication and chemoprotective properties.However, the anti-cancer activity and the potential anti-cancer mechanisms of mulberry anthocyanins in vivo have not been well elucidated.
In our preliminary work, we have found that black mulberry fruit is abundant in anthocyanins, especially the new cultivars, mulberry variety PR-01, which the Advances in Bioscience and Biotechnology anthocyanins content is 193.00 mg•100g −1 , 10.16-fold that of the common variety (19.00 mg•100g −1 ) [16].Therefore, the present study is to determine the mulberry anthocyanin extracts (MAEs) composition of mulberry variety PR-01 fruits and investigate its prevention effects against the N-nitrosodiethylamine (NDEA)induced hepatocarcinogenesis in rats and its possible mechanisms.

Materials and Chemicals
Fresh mature mulberry variety PR-01 fruits was obtained in May 2016 from the Institute of Agricultural Product Quality, Fujian Agriculture and Forestry University (Fujian, China) and stored at −80˚C on the same day.

Preparation of Anthocyanin Rich-Extract of Mulberry
The PR-01 mulberry anthocyanin rich-extract was extracted as previously described [17].Freeze mulberry fruits were dried in an ALPHA 1-2LD PLUS lyophilizer (Beijing, China), then ground and sifted for homogenization and stored at −80˚C.The finedly ground mulberries (3 kg) were extracted with 300 L of methanol-hydrochloric acid (999:1) at 37˚C under the condition of ultrasonic power 200 w and frequency 40 kHz for 30 min.This process was repeated three consecutive times.The extracts is then mixed and evaporated into syrup using RE-2000E rotatory evaporator (Xi'an Taikang Biotechnology Co., Ltd., Xi'an, China).The syrup was dissolved in 1000 mL of 0.01 mol/L HCl, and was defatted with 1200 mL of ethyl acetate.The water solution was subjected to column chromatography of Amberlite XAD-7 orderly eluted with water and methanol-formic acid (9:1) [18] [19].The methanol-formic acid elution solution was evaporated into syrup and freeze-dried to yield 403 g of MAEs.The MAEs stored at −80˚C for further use.

pH Difference Method Analysis of Total Anthocyanins Content
Reference to previous study [20], the MAEs was diluted 10-fold with hydrochloric acid-sodium chloride of pH 1.0 and pH 4.5, respectively.The absorbance of MAEs diluents was measured at wavelengths of 520 nm and 700 nm by 755b UV-Vis spectrophotometer (Xi'an Heb Biotechnology Co., Ltd, Xi'an, China), respectively.The absorbance (A) was calculated by the Equation (1): Advances in Bioscience and Biotechnology A = (A520 nm − A700 nm) pH 1.0 − (A520 nm − A700 nm) pH 4.5 (1) The total anthocyanin content of MAEs was calculated according to the formula derived by Wrolstad et al. [21]: Total anthocyanin content (mg•g −1 ) = 1000 1 where MW is the molecular weight of cyclamine-3-glucoside (449.2),DF is the dilution, ε is the molar absorptivity (29,600), l is the optical path (1.0 cm), V is the total volume of MAEs diluents (1.0 ml), and M (0.01 g) is the weight of the MAEs.blood samples and blood samples were centrifuged at 3000 rpm at 4˚C for 10 min to obtain serum.Livers were fully excised from the animal and accurately weighed [6].The size and number of liver nodules were measured as described earlier [3].

Electrospray Ionization
Liver tissue was collected for biochemical and histopathological analyses.
Precisely 1 gram of liver was taken, rinsed with cold PBS, pH 7.4, dried with paper wipe, and placed in a 10-mL centrifuge tube.Then the tissue homogenate was centrifuged at 3500 rpm for 15 min.The supernatant was collected in a 10-mL centrifuge tube (namely 10% liver homogenate), immediately frozen in liquid nitrogen and stored at −20˚C until use [6].The contents of malondialdehyde (MDA), glutathione (GSH), and the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) were assayed using standard commercially available kits (Nanjing Jiancheng Bioengineering Engineering Institute, Nanjing, China) according to the manufacturer's instructions.

Histological Examination
Hepatic tissues from each group were washed with cold physiological saline and rapidly excised after the rats were sacrificed.The tissue samples were immediately fixed in 4% phosphate-buffered paraformaldehyde for 48 h, then dehydrated in a graded ethanol series, cleared in xylene and embedded in paraffin.
Sections (4 μm thick) were stained with hematoxylin and eosin (H&E) according to published methods [22].The samples were also sectioned and Masson's trichome staining was performed as previous studies [23].For histological analysis, the tissue sections were photographed with a microscope (Nikon E200, Nikon Corp., Japan).

Immunohistochemical Analysis
The proteins expression of inflammatory markers in liver tissue was detected by the SABC immunohistochemical method.The main steps are as follows: paraffin section (4 μm thick) was de-waxed and incubated with sodium citrate buffer (pH terstained with hematoxylin and the brown color signifying the presence of antigen bound to antibody was detected by light microscopy.For the negative control, TBS was used instead of a primary antibody.From ten randomly selected sections of each slide, 500 cells were counted.The percentage of positive cells for each group was calculated using the Image-Pro Plus 6.0 image analysis system.

Western Blot Analysis
Liver tissue was lysated in RIPA lysis buffer containing protease inhibitor cocktail protease inhibitors and phosphatase inhibitors according to the M-PER (R)

Real-Time PCR Analysis
Total RNA was extracted from liver tissue using a TRIzol reagent (Invitrogen, Thermo Fisher Scientific Inc., Beijing, China) according to the manufacturer's instructions.Real-time PCR was performed using the SYBR Green Kit (Takara Biomedical Technology Co., Ltd., Beijing, China) on the ABI Step One RT-PCR system.Primers designed with Primer Premier 5 and Beacon Designer 8.1 was listed in Table 2.

Statistical Analysis
Data was presented by mean standard deviation (SD).Data from study was dealed with SPSS 21.0 statistical package.Statistical analysis was performed by one-way analysis of variance (ANOVA) with Tukey's multiple comparisons or Student's t-test.A P-value of <0.05 was considered statistically significant.The GraphPad Prism 6.01 was used for the graphical evaluations.

Alterations in Body Weight, Food Intake, and Liver Index
As shown in Table 3, the body weight of rats in NDEA group was significantly reduced subsequent to NDEA compared with that in control group (P < 0.01), while the weight gains of animals treated with MAEs and C3G-150 were similar(P > 0.05).With regard to average daily feed consumption, the NDEA group consumed a lower amount of the feed than the control group (P < 0.01).However, after treatment with MAEs and C3G, the daily feed intakes were significantly increased than the NDEA group (P < 0.05).Compared to NDEA group, the relative liver weight was remarkably decreased in the MAEs-75 and MAEs-150 groups by 23.64% and 28.37%, respectively after MAEs treatment (P < 0.01).

Changes in Liver Function Biomarkers
NDEA causes liver damage and consequently releases liver enzymes in its selected dose [25].The past research suggested that mulberry water extracts may serve as liver protective agents [4] [25].In this study, the elevated activity of ALT, AST, TBiL, ALP, and GGT are indicative of poor hepatic function in the NDEA alone treated animals compared to the control animals (Table 4), as reported by Latief et al. [26].After treated with MAEs, the levels of the elevated liver function enzymes above mentioned were significantly restored in MAEs-75 and MAEs-150 group animals, suggesting that MAEs has an efficacy function on liver protection.Pretreatment with the higher dose of MAEs (150 mg•kg −1 ) also reversed the NDEA-induced increase in serum BUN and creatinine to normal levels (Table 4).In addition, the serum CEA and AFP levels were obviously higher in NDEA group than that of the control group (P < 0.05), while 75  liver damage in rats.

Histopathological Study of Liver Samples
As one of the most important environmental hepatotoxin and carcinogen, NDEA can cause severe liver damage and lead to severe alterations in the lobular architecture and hampers liver functioning [26].During the experiment, 2 rats died of severe hepatic tumor pathogenesis at 16 th and 18 th week in the NDEA group.No death was visible in other group animals.The results of this study shown that low-dose multiple injection of NDEA (25 mg•kg −1 of NDEA twice a week) significantly increased the nodule incidence and the apparent HCC incidence (100.0%,50.0%, respectively) and only a low mortality rate (16.7%) for 18 weeks in the NDEA group, indicating successful induction of liver cancer model.
However, there was significant decrease in hepatic nodules incidence, maximum diameter, average number in the MAEs and C3G-150 group rats as compared with NDEA group animals (Table 5, P < 0.05).In particularly, treatment of 150 mg•kg −1 MAEs prior to C3G showed a significant reduction of the nodule incidence (41.7%) compared to the NDEA group animals (100%).
As shown in Table 5, treatment of 150 mg•kg −1 MAEs prior to C3G showed a significant reduction of the apparent HCC incidence (8.3%) compared to the NDEA group animals (50.0%).The liver phenotype of rats in the control group was not significantly changed, while the liver size and structure of NDEA group rats were significantly changed (Figure 2).Apparently the liver structure of injured rats was characterized by cells necrosis, hemorrhage, scars and hepatic nodule like structure, abundant eosinophilic cells, basophilic cells and egg cells hyperplasia obviously accompanied by inflammatory cells infiltration [26], which were observed in the liver of NDEA group rats.However, MAEs (75 mg•kg −1 or 150 mg•kg −1 ) and 150 mg•kg −1 C3G administration regress these changes significantly (Figure 2(B)).The results of masson-stained sections showed that rats developed liver fibrosis after NDEA administration.However, the degree of   These results suggested that MAEs is an effective chemopreventive agent for preventing or delaying NDEA-induced hepatocarcinogenesis in rats, among which the high dose of MAEs has the best effect and is superior to C3G.Advances in Bioscience and Biotechnology simultaneous decrease in the Phase II detoxifying enzyme [6] [28].GST and UGT2b1 play an important role in the detoxification and excretion of toxins, carcinogens, which may actively modulated by Nrf2 and the antioxidant response element [29].In this study, administration of NDEA, the significant reduction in UGT2b1 and GST activity in livers of NDEA group were observed (Figure 1(A), P < 0.05).Whereas the levels of GST and UGT2b1 in livers of MAEs-75 and MAEs-150 group rats were remarkably increased as compared to the NDEA group.What's more, the hepatic GST and UGT2b1 levels of MAEs-150 group were obviously higher than that of C3G-150 group (P < 0.05), and closed to the normal level.The result of the qRT-PCR also NDEA induced GST and UGT2b1 mRNA expressions and MAEs repressed these alterations (Figure 3(B)).These results suggested MAEs can inhibit tumor development by stimulating the activity of phase II detoxification enzyme.

MAEs Activated the Nrf2 Signaling Pathway and Its Downstream Detoxification Enzymes
Nrf2-mediated antioxidant, detoxification enzymes and anti-inflammatory signaling are through Nrf2-ARE pathways to protect organisms against cellular damage caused by oxidative stress [28].Yan et al. [15] reported that consumption of mulberry anthocyanin extract maintained Nrf2, HO-1, and p38 MAPK stimulation that were involved in oxidative stress modulation.The results of this study shown that Nrf2 activation in livers of rats was induced by 150 mg•kg −1 MAEs, accompanied by the stimulation the mRNA expressions of NQO-1, Keap1, and HO-1 (Figure 3(B), P < 0.05).Similar findings were observed in the measurements of Nrf2, Keap1, HO-1, and NQO-1 protein levels.Compared with the control and NDEA groups, treatment of 150 mg•kg −1 MAEs caused significant changes in the protein expressions of Nrf2, Keap1, HO-1, and NQO-1 in the MAEs-150 group (P < 0.05), but less obvious changes were found in MAEs-75 group (Figure 3(C)).Combining these results, it was concluded that MAEs may activate the Nrf2 signaling pathway and induce Nrf2-mediated antioxidant enzymes, further activate the expression of downstream phase II detoxifying GST and UGT2b1, which then accelerated the elimination of the metabolites of NDEA, thereby achieving the goal of detoxification.
6.0) at 37˚C for 5 min (twice), then washed with 0.1 M PBS for 5 min (3 times), incubated with 3% hydrogen peroxide at 37˚C for 10 min, washed with PBS for 5 min (3 times) and placed in a blocking solution at room temperature for 1.5 -3 h.The sections were incubated with rabbit polyclonal antibodies, NF-κB, COX-2, and TNF-α (Cell Signaling, Danvers, MA, USA) with TBS and Tween 20 overnight at 4˚C.Then sections were washed with PBS and incubated with HRP-labeled sheep-anti-rabbit secondary antibody at 37˚C for 1 -2 h followed by streptavidin-biotin-peroxidase at room temperature for 30 min.The slides were washed and the immunoprecipitation was visualized by treating with 3, 3'-diaminobenzidine for color development for 25 min.Then slides were coun- Protein Extraction Reagent (Thermo Scientific, Fair Lawn, NJ, USA).The protein extraction was collected and the concentrations were quantified using a BCA kit (Biotechnology Development Co., Ltd., Beijing, China).The same amount of proteins was separated using 10% SDS-polyacrylamide gel and transferred to polyvinylidence fluoride (PVDF) membranes.After blocked with 5% skim milk for 1 h at room temperature, membranes were incubated with various antibodies against Nrf2 (1:2000), NQO1 (1:1000), HO-1 (1:2000), Keap1(1:500), NF-κB (1:4000), COX-2 (1:500), TNF-α (1:1000), and β-actin (1:2000) which purchased from Cell Signaling Technology (Danvers, MA, USA) or Santa S. F. Liao et al.DOI: 10.4236/abb.2018.99030429 Advances in Bioscience and Biotechnology Cruz Biotechnology (Santa Cruz, CA, USA) overnight at 4˚C.Then membranes were washed and exposed to HRP-conjugated secondary antibodies (1:10,000) at room temperature for 1 h.Immunoreactive bands were detected by enhanced cheiluminescence solution (CUSABIO Biotech Co., Ltd., Wuhan, China) and exposed to X-ray film using the Bio-Rad Chemi Doc XRS imaging System.The immunoreactive bands were visualized and quantified using Quantity One software and normalized to β-actin.

Figure 2 .
Figure 2. Effects of MAEs on hepatic pathological changes in NDEA-induced hepatocarcinogenesis rats.(A) Liver surface; (B) Liver sections with haematoxylin and esion (H & E, 200×).Basophilic cell focus (white arrow) is an altered hepatic focus.At the center of the lobule is the central vein (CV).PV, portal vein.(C) Liver sections with Masson staining (200×).

Figure 3 .
Figure 3. Effects of MAEs on the expression of Nrf2 and its downstream detoxification enzymes.(A) Effects of MAEs on GST and UGT2b1 levels in liver microsomes of rats exposed to NDEA.Values are expressed as concentration of the hepatic microsomal GST and UGT2b1 activity.GST, glutathione-S-transferase; UGT2b1, UDP glucuronosyl-transferase UGT2b1.(B) The mRNA relative expression levels of GST, UGT2b1, Keap1，HO-1, Nrf2, and NQO-1 in control and treatment groups.GAPDH was used as an internal control.(C) The representative immunoblots and protein levels of Keap1, HO-1, Nrf2, and NQO-1 in liver tissues in the rats of each group.Values with different superscript letters (a, b, c, d) within cultivar are significantly different among groups (P < 0.05).

Figure 4 .
Figure 4. Effects of MAEs on markers of inflammation in NDEA-induced hepato-carcinogenesis rats.(A) The markers of pro-and anti-inflammatory cytokines in serum of each group rats.(B) The mRNA expression of TNF-α, NF-κB, and COX-2 in livers of each group rats.GAPDH was used as an internal control; (C) Representative immunoblots of TNF-α, NF-κB, and COX-2 and its protein levels in livers of each group rats.β-actin was used as an internal control.Values expressed as mean ± SD.Values with different superscript letters (a, b, c, d) within cultivar are significantly different.

Figure 5 .
Figure 5. Representative immunohistochemical staining of TNF-α, COX-2, and NF-κB in the liver of control and experimental animals at ×200 original magnification.Bar graph represents average percentage of positive stained cells of TNF-α, COX-2, and NF-κB in the liver of control and experimental groups.Results are expressed as mean ± SD.Values with different superscript letters (a, b, c, d) within cultivar are significantly different.

2.5. Animals and Experimental Design
The animal experiment design is showned in Table1.Body weights were measured weekly.Twenty-four hours after the last MAEs administration, the rats were anesthetized by ip of pentobarbital sodium (40 mg•kg −1 ) for collection of Advances in Bioscience and Biotechnology

Table 1 .
Sequences of primers used for qRT-PCR analysis.

Table 2 .
Experimental design of the study.

Table 3 .
Effects of MAEs on body weight, feed intake, and liver index in NDEA-induced hepatocarcinogenesis rats.

Table 4 .
Effects of MAEs on serum hepatic enzymes in NDEA-induced hepatocarcinogenesis rats.