Two purified proteins from royal jelly with in vitro dual anti-hepatic damage potency: Major royal jelly protein 2 and its novel isoform X1

Accepted Manuscript Two purified proteins from royal jelly with in vitro dual antihepatic damage potency: Major royal jelly protein 2 and its novel isoform X1

Marwa M. Abu-Serie, Noha H. Habashy PII: DOI: Reference:

S0141-8130(18)36781-3 https://doi.org/10.1016/j.ijbiomac.2019.01.210 BIOMAC 11643

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

7 December 2018 31 January 2019 31 January 2019

Please cite this article as: M.M. Abu-Serie and N.H. Habashy, Two purified proteins from royal jelly with in vitro dual anti-hepatic damage potency: Major royal jelly protein 2 and its novel isoform X1, International Journal of Biological Macromolecules, https://doi.org/ 10.1016/j.ijbiomac.2019.01.210

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Two purified proteins from royal jelly with in vitro dual antihepatic damage potency: Major royal jelly protein 2 and its novel isoform X1

Department of Medical Biotechnology, Genetic Engineering, and Biotechnology

RI

a

PT

Marwa M. Abu-Serie a*, Noha H. Habashyb

SC

Research Institute, City of Scientific Research and Technological Applications (SRTACity), New Borg EL-Arab 21934, Alexandria, Egypt.

Biochemistry Department, Faculty of Science, Alexandria University, Alexandria

NU

b

*Author

for

MA

21511, Egypt correspondence.

E-mail:

[email protected],

Phone:

PT E

D

+2034593422, Fax: +2034593407

AC

CE

Running title: Dual anti-hepatic damage potency of royal jelly proteins

1

ACCEPTED MANUSCRIPT Abstract Liver diseases are serious life-threating conditions that should be controlled. Here, we identify a protein fraction from royal-jelly (RJ) that represents the most effective composite against CCl4-induced hepatotoxicity and HepG2 cell growth. Two closely related proteins were purified from this fraction by a new simple method and identified

PT

by MALDI-TOF MS as major RJ protein 2 (MRJP2) and its predicted isoform X1. The

RI

in silico assessment (3D structures and functions) of these proteins were performed

SC

using Iterative Threading ASSEmbly Refinement (I-TASSER) analysis and RAMPAGE program. These two purified proteins were able to relieve the necrotic hepatocytes (by

NU

60.4%) via reducing tumor necrosis factor (TNF)-α, mixed lineage kinase domain-like protein (MLKL) and intracellular reactive species. The latter effects associated with

MA

improving hepatocyte functions. Furthermore, they revealed the potent anticancer effect via induction of caspase-dependent apoptosis and controlling the expression of both

D

Bcl-2 and p53 in HepG2 cells. Thus, MRJP2 and its isoform X1 can be a promising

PT E

dual strategy for fighting hepatic injury and cancer in future animal and human studies. Keywords: Major royal-jelly protein (MRJP) 2; MRJP2 isoform X1; Anti-necrotic

AC

CE

effect; Apoptosis-mediated anticancer effect.

2

ACCEPTED MANUSCRIPT 1. Introduction The liver is the largest and the most important organ in the body that is exposed to various diseases including steatosis, fibrosis, cirrhosis, and cancer. Liver diseases are the main cause of mortality and morbidity worldwide. They can be produced from

PT

different conditions such as metabolic syndromes, hepatitis virus infections, alcohol abuse and toxins [1]. These conditions lead initially to inflammation in the liver and

RI

their persistence caused chronic liver diseases, which is multistage development of

SC

necrosis, fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). The sustained hepatocyte death (necrosis) involves in disruption of the liver architecture and the

NU

development of liver fibrosis. Uncontrolled fibrosis can progress to cirrhosis and HCC

MA

[2,3] which is the third cause of cancer mortality [4]. Although most of the liver diseases have some treatment options, many types still incurable and developing drug

D

resistance is prevalent. Therefore, the discovery of novel therapeutic compounds

PT E

especially that are derived from natural sources is essential for promoting healthcare and will gain considerable popularity.

CE

Royal-jelly (RJ), or bee’s milk, is a creamy botanical origin product secreted from mandibular and hypopharyngeal glands of nurse bees (Apis mellifera). It is the specific

AC

food for the development of bee larva, at the age of 5-15 days, into long-living fertile mature queen bees. Components of RJ has a significant impact on the long-life span of queen bee which lives up to 4-5 years in comparison with short-life span (6-8 weeks) of bee workers [5,6]. RJ is an acidic food composed mainly of water (60-70%), proteins (9-18%), carbohydrates (7-18%), and lipids (3-8%) along with few amounts of other compounds (vitamins, minerals, polyphenols, and organic acids) [6,7]. About 80% of the RJ proteins are water-soluble belonging to the major RJ protein (MRJP) family,

3

ACCEPTED MANUSCRIPT which comprises 9 members (MRJP1-MRJP9) with a molecular mass of 49-87 kDa [5,8]. The MRJPs play an essential nutritional role that direct the development of the honey bee queen whose long-life span [6]. These proteins have been reported to be glycoproteins and their carbohydrate moieties are important for their solubility [9], folding, and biological roles [10]. MRJP2 is a monomeric glycoprotein has eight forms

PT

with a molecular mass from 50 to 59 kDa and PI range from 4.92 to 7.02 [5]. This

RI

protein is N-glycosylated at two sites with high-mannose glycan (Man9GlcNAc2) [11].

SC

This unique composition of RJ is responsible for its numerous pharmacological applications [12]. However, insufficient preclinical studies were performed on the

NU

antioxidant, anti-inflammatory [9], the anti-hepatotoxicity [9,13-15] and anticancer [9,16,17] activities of RJ. Moreover, no previous studies achieved complete

MA

fractionation for the RJ to identify the responsible constituents for its anti-hepatic damage effect. Thus, the present work, for the first time, separated RJ into different

D

fractions and a new simple method was established for purification of the two proteins,

PT E

MRJP2 and its isoform X1 (a predicted protein in the NCBI Protein Database). Then the anti-hepatic injury and anticancer activities of these purified proteins were evaluated in

CE

comparison with standard drugs (silymarin ‘SM’ and doxorubicin ‘DOX’). For that, carbon tetrachloride (CCl4)-exposed hepatocyte culture and a human hepatocellular

AC

carcinoma cell line (HepG2) were used to investigate the anti-hepatotoxicity effect and anticancer activity of RJ constituents, respectively. Here, we conducted the commonly used in vitro models because it has become vital to take advantage of cell cultures in supporting the research prior to committing studies on animals and human clinical trials. A combination of the computational and experimental analyses used here for a better explanation of results.

2. Materials and methods 4

ACCEPTED MANUSCRIPT 2.1. Chemicals Collagenase I, CCl4, polyacrylamide, EB, AO, DOX, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT), trypsin modified sequencing grade, carboxymethyl

2ʹ,7ʹ-DCFH-DA,

(CM)-Sephadex,

streptavidin-fluorescein,

and

PT

Coomassie brilliant blue G-250 were obtained from Sigma-Aldrich (St. Louis, MO, USA). SM capsules were purchased from SEDICO Pharmaceuticals Company, Egypt.

RI

Each capsule contains SM 70% (200 mg), acetylcysteine (200 mg), vitamin E (5 IU),

SC

vitamin A (300 IU), vitamin C (30 mg), selenium (18.3 mg), and zinc (3.65 mg). Ammonium sulfate was from Nentech Ltd (NTL, Brixworth, Northants, UK). Roswell

NU

Park Memorial Institute (RPMI)-1640 medium, William’s E medium and fetal bovine

MA

serum (FBS) were obtained from Lonza (USA). Protease inhibitor cocktails and Gene JET RNA purification kit were obtained from Thermo Scientific, USA. Tumor necrosis factor (TNF)-α ELISA kit and mixed lineage kinase domain-like protein (MLKL)

PT E

D

ELISA kit were from RayBiotech, USA and Cloud-clone Corp, USA respectively. Ki67 immunohistochemistry kit was obtained from Eton Bioscience, US. Caspase-Glo 3/7 assay kit was purchased from Promega, USA. ALT, AST, and albumin kits were

CE

purchased from Biosystem, Spain. Other chemicals were obtained with a high grade.

AC

2.2. Cell lines and culture condition Hepatocytes were isolated from the liver of male Albino rats according to the

method of Whitehead and Robinson [18] with some modifications [19]. Rats were obtained from MISR University for Science and Technology with animal welfare (assurance number: A5865-01). All applicable international and/or institutional guidelines for using animals were pursued. Using experimental rats follows the Research Ethical Committee (REC). This committee was published by the National Health and Medical Research Council policies and the recommendations of the Ministry 5

ACCEPTED MANUSCRIPT of Health and Population, High Committee of Medical Specialties, Egypt. This current research was granted permission by the Medical Biotechnology Department (SRATCity) and Faculty of Science, Alexandria University, Egypt. HepG2 cell line was purchased from American Type Culture Collection (ATCC, USA) and cultured in RPMI medium supplemented with 10% FBS and 2 mM L-

PT

glutamine in a 5% CO2 incubator at 37°C.

RI

2.3. Preparation of MRJP2 and its isoform (MRJP2 X1)

SC

2.3.1. Fractionation of RJ

The RJ was obtained freshly from the local market of Egypt and used

NU

immediately. Carbohydrates, lipids, and proteins were separated then the protein

MA

fraction was further fractionated using ammonium sulfate. All fractions were studied for their efficacy against the CCl4-induced hepatotoxicity and HepG2 cell growth. For preparing carbohydrate fraction, 2 g of RJ was dissolved in 40% methanol and

PT E

D

deproteinized using Carrez I (zinc acetate) and Carrez II (potassium hexacyanoferrate II) reagents. Then, lipids were removed by washing the deproteinized RJ two times with dichloromethane. The aqueous layer (carbohydrate fraction) was filtered through a 0.2

CE

µm disposable syringe filter, quantified, lyophilized (Telstar, Terrassa, Spain) and kept

AC

at -80˚C until used [20]. Lipids were prepared from RJ with petroleum ether using a Soxhlet apparatus for 30 min. The organic solvent was evaporated, and then the lipid fraction was weighed and stored at -80˚C. The water-soluble proteins were extracted from RJ using ammonium sulfate crystals according to the previous method [21] with some modifications. In brief, RJ was dissolved in phosphate buffer saline (PBS, 0.1 M, pH 7) containing 1x protease inhibitor cocktails and the solution was centrifuged at 14,500 rpm and 4˚C for 30 min. Then the water-soluble proteins in the supernatant were precipitated by adding crystals of ammonium sulfate until the saturation reached 60%.

6

ACCEPTED MANUSCRIPT Pellet was dissolved in PBS, dialyzed for 24 h, and finally freeze-dried to obtain the powdered RJ crude protein fraction (CPF). The protein content of CPF was quantified using Bradford’s method [22]. The CPF was subjected to ammonium sulfate precipitation using different degrees of saturation [(0-5), (5-10), (10-15), (15-20), (20-25), (25-30), (30-40), (40-50) and (50-

PT

60%)]. The precipitated proteins were obtained by centrifugation at 14,500 rpm (4˚C)

RI

for 30 min and the protein content was examined in each isolated protein fraction (PF)

SC

using Bradford’s method. The PF containing detectable protein content [PF25 (20-25%), PF30 (25-30%), PF40 (30-40%), PF50 (40-50%), and PF60 (50-60%)] was dialyzed for 24

NU

h against PBS, lyophilized, and then the effective one was analyzed with the sodium

MA

dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 2.3.2. Purification of MRJP2 and its isoform (MRJP2 X1) The PF50 was used for the purification of MRJP2 and MRJP2 isoform X1 using

PT E

D

CM-Sephadex ion-exchange column chromatography. The amount of PF50 that obtained from 10 g of RJ (about 300 mg) was dissolved in 20 ml of the binding buffer (20 mM phosphate buffer containing 1x protease inhibitor cocktails, pH 6.7). The protein

CE

solution then applied to a CM-Sephadex column (16 x 2.5 cm) and left for 1 h at 4˚C.

AC

The unbound protein (MRJP2 isoform X1, fraction 1) was obtained by washing the column with about 100 ml of the binding buffer. Elution of the bound protein (MRJP2, fraction 2) was achieved by a one-step gradient of about 50 ml of 0.5 M NaCl in the binding buffer. The detection of protein in the column eluents was done during the purification step using UV absorption at 280 nm. The purified fractions were dialyzed for 24 h against PBS (pH 7) then freeze-dried and quantified by Bradford method [22]. 2.3.3. Identification of MRJP2 and its isoform (MRJP2 X1)

7

ACCEPTED MANUSCRIPT The two purified protein fractions were identified through the determination of their molecular masses by SDS-PAGE and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). In addition, peptide mass fingerprinting (PMF) after tryptic digestion was used for identification of these proteins

PT

using MALDI-TOF MS analysis. 2.3.3.1. SDS-PAGE

RI

The prepared CPF and its precipitated PFs (PF50, MRJP2, and MRJP2 X1) were

SC

analyzed by 12% SDS-PAGE [23]. For each sample, 30 µg of protein were loaded and electrophoresed at 75 V through stacking gel followed by 125 V for approximately 2 h.

NU

The gel was stained with Coomassie brilliant blue R-250 and the molecular masses of

MA

the prepared PFs were analyzed with a gel documentation system (Geldoc-it, UVP, England) using Totallab analysis software (version 10.1).

D

2.3.3.2. MALDI-TOF MS

PT E

The PMF after tryptic digestion for the purified proteins (MRJP2 and its isoform X1) was done using the in-solution digestion protocol. In brief, 200 µg of each

CE

lyophilized PF was dissolved in 100 µl of 50 mM sodium bicarbonate (pH 8.0). Then cysteines were reduced by 5 µl of DTT (45 mM) and alkylated by 5 µl of iodoacetamide

AC

(IAA, 100 mM). Afterward, proteins were subjected to proteolysis by trypsin modified sequencing grade at an (enzyme: substrate) ratio of (1: 50) and incubated at 37˚C for 24 h. The reaction was terminated by addition of 10 µl of 10% trifluoroacetic acid (TFA). One microliter of the tryptic digest was added to the preloaded MALDI plate with 1 µl of the MALDI matrix (saturated solution of α-cyano-4-hydroxycinnamic acid in 2.5% TFA and 50% acetonitrile) and air-dried. The masses of the tryptic peptides were determined using MALDI-TOF MS UltraFlex system (Bruker Daltonics GmbH, Bremen, Germany). The analysis was done in the linear positive ion mode in the 8

ACCEPTED MANUSCRIPT mass/charge (m/z) range of 500-4000 Da using FlexControl software version 3. The generated spectra were compared to the database (fingerprint) using the Bruker Biotyper (version 3.1) and a library of 5,623 entries. 2.3.3.3. In silico assessment of the purified proteins using I-TASSER analysis

PT

The predicted 3D structures of MRJP2 and its isoform X1 was generated by Iterative Threading ASSEmbly Refinement (I-TASSER) protein-modeling server

RI

(http://zhanglab.ccmb.med.umich.edu/I-TASSER/). This analysis depends on alignment

SC

the sequence of the target proteins with structural protein templates from Protein Data Bank (PDB) using the multiple threading alignments approaches. Then the full-length

NU

structure models were constructed via iterative fragment assembly simulations. The I-

MA

TASSER model with the highest C-score value was matched to all the protein structures in the PDB library using TM-align structural alignment program to know its structural analogs. Depending on the obtained I-TASSER predicted structures, the structure-based

PT E

D

function predictions were deduced using COFACTOR and COACH methods. COFACTOR method can deduce the protein function depending on structure comparison and protein-protein networks. While COACH is a meta-server approach

CE

that combines multiple functions annotation results from different servers, including

AC

COFACTOR. Visualization and analysis of the predicted protein structures were performed via the Discovery Studio 2017 R2 program. The validation of these predicted structures was tested by RM plot. 2.4. Determination of safe doses of RJ and its fractions using normal isolated hepatocytes Hepatocytes were isolated from the liver of male Albino rats according to the method of Abu-Serie [19].

The cytotoxicity of the studied compounds on the

hepatocytes was done using the MTT assay [24]. In brief, hepatocytes were seeded in 9

ACCEPTED MANUSCRIPT 96-well cell culture plate and treated separately with serial dilutions of RJ and its fractions as well as the standard drugs for hepatotoxicity and cancer (SM and DOX, respectively). In addition, the untreated cells were included as a negative control. All plates were incubated in the CO2 incubator at 5% CO2, 37˚C, and 90% relative humidity. After 72 h, 20 μl of 5 mg/ml MTT was added for each well and incubated for a further 4

PT

h. Then MTT was replaced by 150 µl of DMSO and the absorbance was read at 570 nm

RI

using an ELISA reader (BMG LabTech, Germany). Cell viability was determined and

SC

the safe concentrations (EC100, 100% cell viability) were calculated. 2.5. Evaluation of the anti-hepatotoxicity effect of RJ and its fractions

RJ fraction against hepatotoxicity

NU

2.5.1. Flow cytometric analysis of PI-stained cells for selecting the most effective

MA

The hepatotoxicity was induced in the isolated rat hepatocytes by exposure to CCl4 according to the previous method [25] with our modifications. Briefly,

D

hepatocytes were incubated with various concentrations (0.13–1.30 mM) of CCl4 for 72

PT E

h. At the end of the incubation period, the cell viability was assessed [24] and the median lethal concentration (LC50) value was calculated by GraphPad Instat software

CE

version 3. About 10% of this value was considered as the optimum concentration of CCl4 that used in the induction of injury in the hepatocytes (CCl4-treated cells). After

AC

induction of hepatic injury by CCl4-exposure to hepatocytes for 24 h, cells were incubated with the safe concentration (EC100) of RJ, its fractions, and standard drugs in 5% CO2 incubator (37˚C) for 72 h. The normal untreated hepatocytes and the CCl 4exposed cells with no further treatments were used as negative and positive control cells, respectively. After trypsinization, the untreated and treated cells were incubated with annexin V/PI for 15 min. Then cells were fixed and incubated with streptavidinfluorescein (5 µg/ml) for 15 min. The selection of the most effective fraction was based

10

ACCEPTED MANUSCRIPT on quantification of the PI-stained necrotic population in the studied cells by the flow cytometry (Partec, Germany) using the phycoerythrin emission signal detector (FL2) against the Fluorescein isothiocyanate (FITC) signal detector (FL1). 2.5.2. Biochemical and morphological investigations of the anti-hepatotoxicity

PT

effect of the selective RJ fractions 2.5.2.1. MTT assay

RI

Briefly, CCl4-exposed hepatocytes were incubated with serial concentrations of the

SC

EC100 of the studied compounds for 72 h in a 5% CO2 incubator at 37ºC. The percentages of healthy hepatocytes in the RJ selected fractions (PF50, MRJP2, and

NU

MRJP2 X1), DOX and SM-treated cells were determined using the MTT assay as

MA

described above [24]. The effective concentrations (EC50) of these compounds that halt CCl4-induced hepatotoxicity (CIH) by 50% were estimated using GraphPad Instat software version 3. Before and after treatment of CCl4-exposed cells with the most

PT E

D

effective RJ fractions (PF50, MRJP2, and MRJP2 X1) and standard drugs, they were examined morphologically, using a phase contrast microscope

CE

2.5.2.2. Biochemical analyses The biochemical parameters including ALT, AST, and albumin were determined

AC

using the specific kits. TNF-α and MLKL (activation markers of necrosis) were determined by following the manufacturer’s instructions using ELISA kits. The nitric oxide (NO) level was assessed by measurement of the nitrite using the Griess reaction, which produced colored azo dye with a maximum absorbance at 490 nm [26]. The effect of the selective RJ fractions on CCl4-induced hepatocyte death was investigated by staining cells with EB and AO dyes (100 µg/ml for each) and then visualized under the fluorescent phase contrast microscope (Olympus, Japan).

11

ACCEPTED MANUSCRIPT The intracellular ROS level was examined by incubation of the untreated and treated cells with 5 μM of DCFH-DA for 30 min at 37°C in the dark. Then cells were trypsinized and suspended in a fresh PBS. The intensity of the fluorescence was analyzed by flow cytometer with an excitation and an emission wavelength at 488 nm

PT

and 530 nm, respectively [27]. 2.6. Evaluation of the anticancer effect of RJ and its fractions

RI

2.6.1. Flow cytometric analysis of annexin-stained cells for selecting the most

SC

effective RJ fraction against hepatic cancer cells

NU

The RJ or each of its isolated fractions at their safe concentrations was incubated for 72 h with HepG2 cell line. The standard drugs for hepatic injury (SM) and

MA

cancer (DOX) were included. After trypsinization, the untreated and treated cells were incubated with annexin V/PI for 15 min. Then cells were fixed and incubated with

D

streptavidin-fluorescein (5 µg/ml) for 15 min. The apoptosis-dependent anticancer

PT E

effect was determined by quantification of annexin-stained apoptotic cells using the FITC signal detector (FL1) against the phycoerythrin emission signal detector (FL2).

CE

2.6.2. Biochemical, morphological and molecular investigations of anti-hepatic

AC

cancer effect of the selective RJ fraction 2.6.2.1. MTT assay After cell seeding and attachment, serial concentrations of each of the tested

compounds were added and incubated for 72 h in a 5% CO2 incubator at 37ºC. The cytotoxic effect of RJ selected fractions (PF50, MRJP2, and MRJP2 X1) in comparison with DOX and SM were investigated against HepG2 cell line using the MTT assay as described above [24]. The concentration of each of the studied compounds that inhibit cancer cell growth by 50% (IC50 values) was determined using GraphPad Instat 12

ACCEPTED MANUSCRIPT software version 3. The selectivity indices (SI) also were calculated by dividing the CC50 values (the cytotoxic concentration that inhibits growth for 50% of the normal viable hepatocytes) by the IC50 values. The morphological changes of untreated and treated HepG2 cells were examined using the phase-contrast microscope

PT

2.6.2.2. Fluorescence microscope investigation of apoptotic cells HepG2 cell line was incubated separately with the selected RJ fractions (PF50,

RI

MRJP2, and MRJP2 X1) and standard drugs (DOX and SM) for 72 h in the CO2

SC

incubator. Then HepG2 apoptosis was investigated by EB/AO double staining as

NU

described previously.

2.6.2.3. Real-time quantitative polymerase chain reaction (qRT-PCR) of Bcl-2 and

MA

p53

The total RNA was extracted from the treated and untreated cells then cDNA

D

was produced using Gene JET RNA Purification Kit and cDNA Synthesis Kit,

PT E

respectively. The primers that used for SYBR green qRT-PCR of Bcl-2 were forward: 5′-TCCGATCAGGAAGGCTAGAGTT-3′

CE

TCGGTCTCCTAAAAGCAGGC-3′.

and

reverse:

5′-

However, primers for p53 were forward: 5′-

TAACAGTTCCTGCATGGGCGGC-3′

and

reverse:

5′-

AC

AGGACAGGCACAAACACGCACC-3′. Besides, primers of the housekeeping gene (β-actin) were forward: 5′-AAGCAGGAGTATGACGAGTCCG-3′ and reverse: 5′GCCTTCATACATCTCAAGTTGG-3′. The qPCR program was progressed as one cycle of enzyme activation for 15 min at 95°C followed by 40 denaturation cycles for 15 seconds at 95°C, annealing for 1 min at 60°C and extension for 30 sec at 72°C. The change in these gene expressions was estimated before and after treatment using (2−ΔΔCT) equation.

13

ACCEPTED MANUSCRIPT 2.6.2.4. Caspase 3/7 activation assay It was quantified in untreated and treated HepG2 cells using the Caspase-Glo 3/7 kit following the manufacturer’s instructions. This kit used a luminogenic substrate that was cleaved by caspases resulting in the generation of the luminescent signal. This

Germany) at 490 nm excitation and 520 nm emission.

RI

2.6.2.5. Immunohistochemical expression of Ki-67

PT

signal was measured by the fluorescence omega microplate reader (BMG LabTech,

SC

After 72 h incubation of HepG2 cell line with each of the RJ fraction or standard drugs (DOX and SM), cells were trypsinized and fixed using 10% formalin. The fixed

NU

cell specimens were dehydrated in ascending grades of alcohol and immersed in xylene for one hour (three times) followed by impregnation in melted paraffin forming solid

MA

paraffin blocks. Then a rotator microtome was used to cut each block into 3-5 μm thick sections that were transferred into positively charged slides. Slides were dried at 60-

PT E

D

70°C for 1-2 h then dewaxed 3 times by immersion in xylene and rehydrated in descending grading ethanol. Afterward, slides were incubated in 3% H2O2 for 10 min, washed in PBS buffer twice for 3 min and placed in 10 mM citrate buffer followed by

CE

heating for 10-20 min. After cooling, slides were separately soaked overnight in

AC

primary Ki-67 antibody and the method was completed following the manual protocol of the Ki-67 immunohistochemistry specific kit. The percentage of immunostained cells was evaluated by cellSens imaging analysis software of the phase contrast microscope. 2.7. Statistical analysis Data were expressed as mean ± SE and was analyzed by SPSS version 16. The mean values were compared using one-way analysis of variance (ANOVA) with Duncan’s test and significance was considered at P < 0.05. LC50, IC50, and EC100 values were calculated by the GraphPad Instat software version 3. 14

ACCEPTED MANUSCRIPT 3. Results 3.1. Purification and identification of MRJP2 and its isoform X1 The results in Table 1 show the three main fractions of RJ and the predominance of RJ CPF (14.3%) in comparison with carbohydrate (9.5%) and lipid (0.17%) fractions. Also, Table 1 represents the percentage yields of each PF from CPF.

PT

As elucidated by SDS-PAGE analysis (Fig. 1A), the RJ-CPF gave fifteen protein bands

RI

with an estimated molecular mass range of 10.81-128.00 kDa. The fractionation of the

detectable protein content (PF5-PF20).

SC

CPF by ammonium sulfate precipitation gave nine PFs (PF5-PF60), four of them with no

NU

The SDS-PAGE analysis (Fig. 1A) demonstrated two protein bands for PF50

MA

with a molecular mass of 45.41 and 50.55 kDa. The separation of the dialyzed PF50 on CM-Sephadex ion exchange column resulted in the collection of two PFs with slightly

D

different yields (Table 1). The identification of these two collected PFs occurred via

PT E

PMF analysis using MALDI-TOF MS. The firstly eluted PF was identified as Apis mellifera predicted MRJP2 isoform X1 (Fig. 1C). This PF showed eight matched peptides from thirty-five tryptic digest (20.8% sequence coverage). However, the

CE

adsorbed PF on the column matrix was identified as Apis mellifera MRJP2 (Fig. 1B).

AC

This PF revealed five matched peptides from nineteen tryptic peptides (11.5% sequence coverage). The calculated molecular masses of the obtained PFs from the sum of the tryptic peptide masses were 32.716 kDa for MRJP2 and 57.115 kDa for MRJP2 isoform X1. In addition, the isoelectric point (PI) values for MRJP2 (PI=7) and its isoform (PI=6.5) were obtained from the MALDI-TOF MS data. Additional analysis of these PFs using SDS-PAGE (Fig. 1A) showed only a single distinctive protein band for each. MRJP2 gave a molecular mass of about 49.95 kDa and 53.12 kDa was observed for its isoform X1. 15

ACCEPTED MANUSCRIPT 3.2. Structure prediction of MRJP2 and its isoform X1 Based on the NCBI database information, MRJP2 and its predicted isoform X1 consist of 452 amino acids. Each of the two proteins is a monomer with three domains as provided from the Vector Alignment Search Tool (VAST). Prediction of the 3D structure of the two purified proteins was performed by I-TASSER [28], the most

PT

ranked server for protein structure and function prediction. The protein sequences in

RI

Federal Acquisition STreamlining Act (FASTA) format was submitted to this online

SC

server to provide the most accurate predictions for their structure via state-of-the-art algorithms. Five models were obtained with different confidence score (C-score) values,

NU

which indicate the quality of the predicted model. Model one represents the highest quality one with a good C-score value of 1.0 (Fig. 2A, E). The quality of the predicted

MA

structures was verified via Ramachandran (RM) plot (Fig. 2B, F). This plot analyzed phi (F) and psi (ψ) torsion angles of the structural backbone and was generated by the

PDB

files

to

the

RAMPAGE

server

D

submitting

PT E

(http://mordred.bioc.cam.ac.uk/~rapper/rampage.php) [29]. The residues of the 3D structure of MRJP2 and its isoform X1 in the most favored region, allowed, and outlier

CE

region were 67.6% and 64.2%, 19.8% and 26.2%, and 12.7% and 9.6%, respectively. These results indicated the reasonably accurate backbone dihedral angles, F and ψ, in

AC

the selected model.

The basic properties of the 3D structure of both purified proteins were detected

using the Discovery Studio 2017 R2 Client.lnk (v17.2.0.16349) program. The data elucidated the close similarity between both structures. Regarding the amphipathic properties, the hydrophilic and hydrophobic residues in MRJP2 and its isoform X1 were 182 & 183 and 160 &159, respectively beside 110 neutral residues in each (Fig. 2C, G). The surface charge of the two 3D structures was illustrated in Fig. (2D, H) and the

16

ACCEPTED MANUSCRIPT calculated formal net charges for MRJP2 and its isoform X1 were zero and (-2), respectively. 3.3. The structure-function relationships The predicted functions of the two purified proteins were performed using COFACTOR [30] and COACH [31] computational methods based on the I-TASSER

PT

structure prediction. The analysis showed the similarity of the studied proteins with

RI

certain templates in PDB such as 3fcuA (PDB: 3FCU), IK3iA (PDB: IK3I), and

SC

1OLZA (PDB: 1OLZ). Therefore, certain cellular, molecular, and biological functions of both proteins were predicted such as the ability to cell-matrix adhesion and mediating

NU

integrin signaling pathway beside their oxidoreductase, hydrolase, and receptor

MA

activities.

3.4. Cytotoxicity of RJ and its isolated fractions against normal hepatocytes The safe doses (EC100) of RJ, its fractions, and the two studied standard drugs (SM and

PT E

D

DOX) are presented in Table 1. The results showed higher values (>1300 µg/ml) for RJcarbohydrates and RJ-PFs and the lower values (˂350 µg/ml) for RJ-lipids and the used standard drugs. This indicated the safest of RJ-carbohydrates and RJ-PFs on

AC

injuries.

CE

hepatocytes comparing to RJ-lipids and standard currently used drugs for hepatic

3.5. Anti-hepatotoxicity activities of RJ and its isolated fractions The flow cytometric analysis was done for selection of the most potent RJ-

fraction that able to reduce the hepatocytes death-induced by CCl4. Fig. 3 (A, B) reveals significant (P < 0.05) elevation of the necrotic cell percentages after exposure to the CCl4 and significant (P < 0.05) decrease in them after RJ-fractions and SM treatments. The most significant decrease in the percentage of necrotic cells was observed after the

17

ACCEPTED MANUSCRIPT treatment with SM, PF50, MRJP2, and MRJP2 X1 with similar effects. Whereas, no improvement had appeared after DOX treatment. 3.5.1. Morphological changes of necrotic cells in the injured hepatocytes after the treatment with the most effective RJ-PFs

PT

Hepatotoxicity was induced in rat hepatocytes by incubating cells for 24 h with CCl4 at the concentration of 0.13 mM (10% of LC50). As evident in Fig. 3C, the

RI

morphological criteria of the hepatotoxicity appeared as cell rounding and cytoplasmic

SC

swelling, which indicate the cellular death by necrosis. On the other hand, results elucidated the apparently undamaged (spindle-shaped cells) appearance of the

NU

hepatocytes after the treatment with RJ-PFs and SM at their EC50. Whereas DOX

MA

showed no improvement in the CCl4-induced hepatotoxicity (Fig. 3C and Table 1). Fig. 4A represents the double staining of the hepatocytes with ethidium bromide/acridine orange (EB/AO) to know the nature and the stage of the cell death.

PT E

D

The fluorescence microscopy showed that the CCl4 induced early (bright greenishyellow swollen nuclei) and late (reddish swollen nuclei) necrosis after 24 h and 72 h, respectively. The treatment with RJ and its PFs revealed a dramatic decrease in the

CE

appearance of early and late necrotic cells and an increase in the number of healthy

AC

hepatocytes (green nuclei). This effect was obviously observed with the MRJP fractions more than PF50. In addition, the potency of PF50 and MRJP fractions was less than SM which revealed almost only viable cells and enormously few early necrotic cells. In contrast, the treatment with DOX showed no viable cells and high numbers of late necrotic cells. 3.5.2. Biochemical study after the treatment of the injured hepatocytes with the most effective RJ-PFs

18

ACCEPTED MANUSCRIPT The biochemical results (Fig. 4B) revealed that CCl4 significantly (P < 0.05) reduced the activities of ALT (97.14%) and AST (88.79%) and the level of albumin (39.27%). In contrast,

MLKL (a marker of the programmed necrosis) and the

inflammatory mediators including, TNF-α and NO (Fig. 4C) were elevated by 136.8%, 56.612%, and 1065.90%, respectively. A significant increase (42-100%) in the liver

PT

function parameters was observed after the treatment with RJ-PFs as compared to the

RI

CCl4-treated cells, but still less than or nearly equal to the control values. Additionally,

SC

the inflammatory mediator’s levels returned to or less than the control untreated cells. The efficiency of the different RJ-PFs in improving most of these biochemical

NU

parameters was slightly lower than or nearly similar to the SM. However, DOX showed no improvement in either liver function parameters or MLKL level. It significantly (P <

MA

0.05) reduced the inflammatory mediators but, with lower potency than RJ-PFs and SM. The intracellular reactive oxygen species (ROS) was detected using the ROS-

PT E

D

sensitive fluorescent probe, dichlorofluorescein diacetate (DCFH-DA). As shown in Fig. 4 (D, E), the incubation of hepatocytes with CCl4 for 72 h caused a dramatic increase (543.41%) in the intensity of the fluorescence (ROS level). This elevated

CE

fluorescence rate was significantly (P < 0.05) reduced (˃ 67%) after the treatment with

AC

RJ-PFs with potency lower than SM (77.38%). MRJP2 is the most effective RJ-PF in lowering the fluorescence intensity. However, the treatment with DOX was significantly elevated (17.46%) the fluorescence intensity as compared with the CCl4treated cells. 3.6. Anticancer activities of RJ and its isolated fractions 3.6.1. The IC50 values, morphological and flow cytometric detection of apoptosis The cytotoxic activities of each of the RJ-PFs and the standard drugs against HepG2 were represented in terms of IC50 values and the SI that was used to determine 19

ACCEPTED MANUSCRIPT the cytotoxic selectivity. As seen in Table 1, the MRJP2 had the lowest IC50 value and the highest SI value followed by MRJP2 X1 and PF50. In comparison with SM and DOX, MRJP2 had similar potency like SM, but less than DOX. However, the selectivity of MRJP2 to the cancer cells was significantly (P < 0.05) higher than that of SM and DOX. Also, the MRJP2 X1 and PF50 had significantly higher SI values than both the

PT

standard drugs. Fig. 5C shows the morphology of the HepG2 cell line under the phase

RI

contrast microscope after treatment with the most effective RJ-PFs and the standard

SC

drugs. After 72 h incubation of the cancer cells with the different treatments, cells appeared as oval or irregular-shaped and shrinkage with condensed cytoplasm and

NU

apoptotic bodies. All of these features are the hallmarks of the apoptosis, which observed obviously with cancer cells-treated with the MRJP2 more than other

MA

treatments.

Annexin V/PI double staining was used for detection of the most active RJ

D

fraction inducer for apoptosis in HepG2 cell line as compared to the standard drugs. As

PT E

shown in Fig. 5, the highest percentage of the apoptotic cell populations was induced by MRJP2 followed by PF50 and MRJP2 X1. Compared to SM and DOX, PF50 and MRJP2

CE

showed a significantly (P < 0.05) higher apoptotic effect while MRJP2 X1 had an upper effect to SM and equal effect like DOX.

AC

3.6.2. Fluorescence microscopy detection of apoptosis, relative expression of Bcl-2 & p53 and caspase activation in the most effective RJ-PFs-treated HepG2 cell line Apoptosis in HepG2 cell line was clearly observed by the EB/AO double fluorescent staining (Fig. 6A) and the percentage values of caspase 3/7 activation (Fig. 6B). From the results of EB/AO double staining, it was clear that HepG2 viable cell (green nuclei) was depleted tremendously with all treatments. The treatment with each of the RJ-PFs and DOX increased the number of the late apoptotic cells (orange-red

20

ACCEPTED MANUSCRIPT nuclei). While the treatment with SM elevated the number of early apoptotic cells (greenish-yellow nuclei) beside some late apoptotic cells. MRJP2 showed the highest number of the late apoptotic cells followed by PF50 then MRJP2 X1. Fig. 6B demonstrates suppression of Bcl-2 (oncogene) and upregulation of p53

PT

(tumor suppressor gene) expressions in all treated HepG2 cells, particularly MRJP2 X1treated HepG2 cells (0.213±0.01 and 5.11±0.1, respectively). The potency of MRJP2

RI

X1 was higher than DOX and SM in controlling expressions of both genes. The effect

SC

of other RJ-PFs (PF50 and MRJP2) in regulating the expression of Bcl-2 and p53 was significantly higher than SM and with the same efficiency as DOX. Fig. 6C

NU

demonstrated the caspase 3/7 activity that was concordant with the fluorescent staining

MA

results. Hence, the highest caspase activation was observed with MRJP2 and the lowest percentage was demonstrated with SM. While, MRJP2 X1 up-regulated caspase

D

activities greater than SM, similar to DOX, but less than PF50.

PT E

3.6.3. Ki-67 expression in HepG2 cells after the treatment with the most effective RJ-PFs

CE

The proliferative activity of the HepG2 cell line has been investigated using a cellular marker for proliferation (Ki-67) expression as an excellent indicator of the

AC

cancer cell proliferation (Fig. 6C, D). Hence its level is directly associated with the cancer cell growth. The results showed a significant (P < 0.05) elevation in the gene expression of this protein in HepG2 cells and the significant inhibitory effect of all the studied RJ-PFs and the standard drugs. The highest inhibitory effect was observed with the MRJP2 (66.89%) and the lowest effect was observed with SM (41.40%). The MRJP2 X1 (55.89%), PF50 (56.83%), and DOX (60.71%) gave similar inhibitory effects.

21

ACCEPTED MANUSCRIPT 4. Discussion RJ has great importance in folk medicine since ancient time and new evidence has performed regarding some of its biological activities that confirm its traditional benefits. We investigated the safety of RJ and its constituents on the normal rat hepatocytes and their ameliorative effect on the hepatocellular injury and cancer. In the present study,

PT

the RJ-PFs and carbohydrates were the safest fractions on the hepatocytes and they were

RI

safer than SM and DOX drugs. RJ-CPF, which contains fifteen proteins with different

SC

molecular masses, was the most effective anti-hepatic damage fraction in the RJ. Therefore, further fractionation was done to this fraction using different concentrations

NU

of ammonium sulfate to know the responsible composite(s). Previously, RJ proteins were precipitated using ammonium sulfate with the concentration of either 30% or 60%

MA

only [32]. However, no previous studies evaluated the fractionation of RJ proteins using different ammonium sulfate concentrations, which is unique to this work. Ammonium

D

sulfate precipitation method is the most common technique for protein purification,

PT E

concentration, and fractionation. The mechanism of this technique based on the release of the bounded water molecules to the protein, leading to its folding, self-association,

CE

and precipitation (salting-out theory) [33]. In the present work, PF50 exhibited the best anti-hepatic damage fraction among

AC

all other RJ-PFs. The SDS-PAGE analysis revealed only two protein bands in this PF with slightly different molecular masses. These proteins were further identified through their PMF using MALDI-TOF MS as MRJP2 (Accession: ACS66837) and MRJP2 isoform X1 (Accession: XP_016770144). The MRJP2 isoform X1 was not identified before and it was added in the NCBI database as a predicted protein. Also, this protein present in the UniProt database under the name of UPI0007D8888F. Whilst, MRJP2 was purified before from RJ but via expensive methods using two columns

22

ACCEPTED MANUSCRIPT chromatographic techniques, ion exchange followed by gel filtration [34] or two successive ion exchange separations [10]. However, no previous studies tried the current simple protocol using the ammonium sulfate precipitation followed by single ion exchange chromatographic separation. Both MRJP2 and its isoform X1 are hydrophilic with nearly the same number of the surface hydrophilic residues (Fig. 2C,

PT

G) so they precipitated as a single fraction (PF50) at the ammonium sulfate saturation of

RI

40-50%. The CM-Sephadex ion exchange chromatography using a binding buffer

SC

(mobile phase) at pH 6.7 was the ideal condition for separation of these two proteins based on their differences in the charge. Selection of the binding buffer pH depends on

NU

the protein PI value to determine the matrix functional group and the protein net charges [35]. In the present study, PI values of the PF50 proteins as obtained from the MALDI-

MA

TOF data were 7 (MRJP2) and 6.5 (MRJP2 X1). The obtained PI value of MRJP2 agrees with Yu et al. [36]. These PI values were confirmed by the predicted formal net

D

charges of the studied proteins 3D structures obtained from the computational analysis.

PT E

Hence, MRJP2 carries zero charges (neutral protein) and its isoform carries two negative charges (neutral to slightly acidic protein). Thus, the selected pH (6.7) of the

CE

binding buffer is the ideal value for perfect separation of these two PFs due to this condition will introduce a net positive charge on MRJP2 and net negative charge on its

AC

isoform. Therefore, MRJP2 was absorbed into the CM-Sephadex matrix and its isoform moved firstly without retaining. Then the use of the one-step gradient of 0.5 M NaCl allowed desorption of MRJP2 from the column matrix due to the higher interaction affinity between the matrix functional group and salt ions. The molecular masses of both MRJP2 and its isoform X1 were determined by SDS-PAGE and confirmed by MALDI-TOF MS. Slightly different values were achieved for MRJP2 X1, but the large difference was observed with MRJP2. Hence,

23

ACCEPTED MANUSCRIPT the MALDI-TOF result gave a smaller value (32.72 kDa) than the SDS-PAGE (49.95 kDa) and at the same time, this protein generated a lower number of tryptic peptides. These results are in accordance with Srisuparbh et al. [34], Tamura et al. [37], and Rosmilah et al. [38]. No doubt, the MALDI-TOF MS is a very sensitive and accurate technique for determining the protein molecular masses [39] while the SDS-PAGE gave

PT

an only rough estimate due to its dependence on the protein shape and size [40].

RI

However, false results may be obtained from MALDI-TOF with the glycoproteins. The

SC

attached carbohydrate moieties to the protein resulted in shielding the proteolytic cleavage sites of trypsin leading to a lower yield of peptides and may affect the value of

NU

the protein molecular mass [39]. Therefore, the lower molecular mass value and tryptic peptides number of MRJP2 that obtained from MALDI-TOF analysis may be owed to

MA

its glycoprotein nature. Hence this protein was reported previously as a highly glycosylated protein contains high-mannose glycans [41].

D

The present study evaluated the role of RJ fractions against CCl4-induced

PT E

hepatotoxicity. CCl4 is the most commonly used hepatic toxins; its metabolism by cytochromes generates reactive metabolites that can interact with and damage the

CE

cellular biomolecules. The trichloromethyl (CCl3) radical is the early formed metabolite that able to react with the oxygen to form the highly reactive metabolites and ROS,

AC

which implicated principally in the CCl4 hepatotoxicity [42]. In the present study, the level of ROS was significantly elevated in the hepatocytes after their exposure to the CCl4 as indicated by the DCFH-DA fluorescent probe (Fig. 4D, E). The crosstalk between the ROS and nuclear factor к-B (NF-кB) pathway was postulated previously. The NF-кB is a transcription factor stimulates the expression of TNF-α, cyclooxygenase-2, inducible nitric oxide synthase (iNOS), and other proteins. The iNOS is an inflammatory enzyme produces a high amount of NO [43]. Consequently, in

24

ACCEPTED MANUSCRIPT this study, the elevation in the TNF-α and NO levels in rat hepatocytes after CCl4 treatment resulted from the CCl4-induced upregulation of ROS level. More importantly, this abnormal increase of TNF-α is an initiator of the programmed necrosis via activation of receptor interacting proteins and later MLKL activation. Then MLKL induced ROS production and disrupted membrane integrity and led to necrotic death

PT

[44]. There is evidence that great production of ROS, TNF-α, and MLKL in the

RI

hepatocytes contributes to their extensive necrosis [2,43-45]. The CCl4-induced necrotic

SC

cell death can be confirmed by the rounded and swollen hepatocytes under the phase contrast microscope (Fig. 3C) and as large red nuclei after staining with the EB/AO

NU

(Fig. 4A). It has been postulated that the hepatocyte necrotic death is associated with the leakage of the aminotransferases (ALT and AST), the hallmarks of liver damage. In

MA

contrast, the activities of these enzymes in this study were depleted after exposure of the hepatocytes to CCl4. In addition, this hepatotoxin decreased the hepatocytes secretion of

D

albumin (Fig. 4B). These results confirmed the extreme damage of the hepatocytes [46]

PT E

and are in line with the previous study by Shaban et al. [47]. We found that the treatment of CCl4-exposed cells with RJ and its fractions and

CE

SM decreased the necrotic cell death and improved hepatocellular functions. The SM showed the most significant enhancement proficiency followed by the RJ-PFs but DOX

AC

had nonsignificant improving effect as compared to the CCl4-treated cells. Under the phase contrast and fluorescence microscopes, SM-, PF50-, MRJP2-, and MRJP2 X1treated cells had the typical morphological appearance and the green nuclear staining. While DOX-treated cells were still dead with orange-red nuclei. As a result, a significant improvement in the liver function parameters was observed in consequence of these treatments except for DOX. This improving effectiveness of RJ-PFs and SM was related to their abilities to obviously decrease the ROS levels and its subsequent

25

ACCEPTED MANUSCRIPT influence on the inflammatory mediators and MLKL. Very few studies were conducted in vitro and in vivo to approve the improving effect of RJ on histochemical and histological hepatic parameters, antioxidant and anti-inflammatory activities of RJ [8, 9, 48]. In agreement with this study, other previous studies were reported the in vivo hepatoprotective and antioxidant roles of RJ against CCl4, oxymetholone, paracetamol,

PT

and fumonisin-induced liver injury, without identifying the responsible components

RI

[13,14,48,49]. Regarding the RJ-proteins, a previous study purified twenty-nine

SC

antioxidative peptides from RJ hydrolysate having antiradical activities due to their phenolic hydroxyl groups [50]. As elucidated by the I-TASSER analysis, MRJP2 and its

NU

isoform X1 are cell-matrix adhesion like proteins, one of their homologous templates is 3fcuA (PDB: 3FCU). This template is the headpiece and ligand-binding site of aIIBb3

MA

integrin able to bind with a specific peptide called Arg-Gly-Asp (RGD) which is known for its anti-hepatic damage effect [51,52]. Similarly, MRJP2 and its isoform X1 may be

D

RGD attachment site-containing proteins and another cause for their hepatic damage

PT E

improving capability may be owed to RGD attachment. SM is a well-known flavonolignan compound has several hepatoprotective roles,

CE

one of its principal mechanisms of action was directed to its antiradical activities. The hepatoprotective effect of SM against CCl4-induced oxidative stress via normalization

AC

the hepatic levels of transaminases and lipid peroxidation has been shown previously [53]. On the other hand, the worsening effect of DOX, the commonly used chemotherapeutic drug may be related to its celebrated toxicity on the hepatocytes and other cells [54]. The anticancer effect of RJ and its purified fractions were evaluated here using HepG2 cell line. The activation of proto-oncogenes (e.g. Bcl-2) and impairment of tumor suppressor genes or apoptosis checkpoints (mostly p53) are the most common

26

ACCEPTED MANUSCRIPT molecular alterations in cancer [55-57]. Accordingly, targeting therapy for cancer has two common approaches, enhancing the proapoptotic molecules or suppressing the antiapoptotic molecules [55]. Therefore, the selection of the most effective anticancer RJ fraction was performed according to evaluating their potency in increasing the apoptotic populations in the cancer cells. Our results, using flow cytometry, revealed the

PT

ability of the RJ and all of its isolated fractions, except PF60, in significant induction of

RI

HepG2 cells apoptosis at variable degrees (Fig. 5A, B). However, the cells-treated with

SC

MRJP2 have the highest frequency of the apoptotic cells followed by PF50 then MRJP2 X1. Compared to SM and DOX, these effective fractions exhibited significantly

NU

superior effectiveness except for the MRJP2 X1, which induced apoptosis by the same percentage as DOX. Therefore, PF50 and its purified PFs were the most potent

MA

anticancer fractions in the RJ. According to the IC50 values (Table 1), the anticancer potency of the SM and DOX was higher than the RJ-PFs. However, the selective

D

cytotoxicity of the RJ-PFs (SI ˃ 2) to HepG2 cells was higher than the standard drugs

PT E

(SI ˂ 2). This means that the RJ-PFs were toxic to HepG2 cells and safe on the normal hepatocytes. Therefore, high concentrations of these proteins can be used safely to

CE

achieve the maximum anticancer efficiencies. In contrast, low concentrations must be used from the studied standard drugs to minimize their toxic effects. This will increase

AC

the importance of the RJ proteins in cancer therapy. The anticancer effect of RJ was investigated previously [5,9] on certain types of tumors like lung metastases and its crude proteins suppress the human breast cancer cell line (MCF-7). In addition, RJ inhibits the activity of N-acetyltransferase, a key enzyme in some tumor cells like bladder, colorectal, and breast cancers [9, 16, 17]. However, no previous studies were conducted on the effect of RJ on HepG2 cell line. Here, the growth inhibitory effect of RJ-PFs on HepG2 cells may be related to oncogene suppression (Bcl-2), tumor

27

ACCEPTED MANUSCRIPT suppressor gene (p53) upregulation and caspase 3, 7 activation. The alteration in the expression of these genes may be the critical approach for fighting different cancer types [57] as well as caspases are crucial for the apoptosis execution [58]. In harmony with the flow cytometric results, the RJ-PFs showed potent apoptosispromoting effect (repression of Bcl-2, induction of p53 and caspase 3) followed by

PT

DOX then SM and the MRJP2 is the most effective one (Fig. 6B,C). The morphological

RI

features of the apoptosis were confirmed by the images of phase contrast and

SC

fluorescence microscopes. Results showed that the number of early and late apoptotic cells increased after cancer treatment with MRJP2 more than the other treatments and

NU

the less effective one was the SM (Fig. 6B). Moreover, our immunohistochemical results revealed that the RJ-PFs had anti-proliferative effect by reducing the expression

MA

of Ki-67 (a proliferation marker protein strongly concomitant with the tumor growth). A great correlation was present between this protein marker and apoptosis. Since the

D

down-regulation of Ki-67 leads to cell cycle arrest and cell apoptosis [59]. Our findings

PT E

agree with this evidence hence the MRJP2 exhibited the most effective RJ fraction in reducing the Ki-67 expression followed by MRJP X1, PF50, and DOX while SM

CE

showed the less potency. Collectively, the ability of the RJ-PFs in reducing the Bcl-2 and Ki-67 expression with the enhancement of p53 and caspase 3/7 indicated their great

AC

potency in suppressing the tumor progression. Also, DOX like other chemotherapeutic agents were able to kill cancer cells by apoptosis through elevation of ROS level [60] and inhibition of Ki-67 expression [61]. Our results confirmed the ability of DOX to elevate the ROS level after its addition to the CCl4-treated hepatocytes (Fig. 4D, E). In fact, the ROS actions depend on its level since its moderate levels trigger cell proliferation and the high levels induced cell death [62]. At high ROS level, the cellular redox state will be decreased leading to activation of the intrinsic pathway of apoptosis

28

ACCEPTED MANUSCRIPT [63]. On the other hand, SM was reported as an anticancer agent through different mechanisms including the induction of apoptosis and inhibition of the cancer cell proliferation [64].

5. Conclusions

PT

The present study evaluated the in vitro anti-hepatic damage (hepatic necrosis and

RI

tumor) effect of RJ ingredients to identify the responsible components in control with

SC

two standard drugs of these two damages. The purified MRJP2 and its isoform X1 proved high efficiency in the amelioration of the CCl4-induced hepatotoxicity and in the

NU

inhibition of HepG2 cell growth. The potency of these proteins was compared with SM and DOX standard drugs at their EC100. The purified RJ proteins demonstrated double

MA

therapeutic potentials toward both the chemically induced hepatotoxicity and the cancer cell proliferation. SM showed these double therapeutic actions, but it had less influence

D

as an anticancer agent as well as its lower safety comparing to the purified RJ proteins.

PT E

Whilst, DOX was highly toxic to the hepatocytes and it exhibited the only anticancer effect. Conclusively, MRJP2 and its isoform X1 represent a new safe and potent

AC

CE

therapeutic approach for the most common hepatic diseases.

Acknowledgments Our sincere thanks and warm greetings are to Mr. Salem E. El-Fiky for helping,

encouraging, and providing us with the royal Jelly to complete this work. Authors Contributions M.M.A and N.H.H contributed equally in designing and conducting the experiments; analyzing and interpreting data as well as writing and revising the manuscript. 29

ACCEPTED MANUSCRIPT Conflict of interest Authors have filed an application under the Patent Cooperation Treaty (international application number: PCT/EG2017/000022, Publication Date: August 2018) concerning the results in the present paper. This research did not receive any specific grant from funding agencies in the

RI

References

PT

public, commercial, or not-for-profit sectors.

SC

[1] S. Li, H.Y. Tan, N. Wang, F. Cheung, M. Hong, Y. Feng, The potential and action mechanism of polyphenols in the treatment of liver diseases, Oxid. Med.

NU

Cell Longev. 2018 (2018) 1-3.

[2] A. Eguchi, A. Wree, A.E. Feldstein, Biomarkers of liver cell death, J. Hepatol.

MA

60 (2014) 1063–1074.

[3] R. Bataller, D.A. Brenner, Liver fibrosis, J. Clin. Investig. 115 (2005) 209–218. [4] H.B. El-Serag, K.L. Rudolph, Hepatocellular carcinoma: epidemiology and

D

molecular carcinogenesis, Gastroenterology 132 (2007) 2557-2576.

PT E

[5] A.N.K.G. Ramanathan, A.J. Nair, V.S. Sugunan, A review on royal jelly proteins and peptides, J. Funct. Foods 44 (2018) 255-264. [6] F. Fratini, G. Cilia, S. Mancinia, A. Felicioli, Royal jelly: An ancient remedy

CE

with remarkable antibacterial properties, Microbiol. Res. 192 (2016) 130-141. [7] A.G. Sabatini, Quality and standardisation of royal jelly, J. ApiProd. ApiMed. Sci. 1 (2009) 16–21.

AC

[8] M.F. Ramadan, A. Al-Ghamdi, Bioactive compounds and health-promoting properties of royal jelly: A review, J. Funct. Foods 4 (2012) 39–52.

[9] M. Khazaei, A. Ansarian, E. Ghanbari, New findings on biological actions and clinical applications of royal jelly: A Review, J. Diet Suppl. 15 (2017) 1–19. [10] J. Schmitzova, J.

Klaudiny, S. Albert, W. Schroder, W. Schreckengost,

J. Hanes, J. Judova, J. Simuth, A family of major royal jelly proteins of the honeybee Apis mellifera L, Cell Mol. Life Sci. 54 (1998) 1020–1030. [11] K. Bilikova, E. Mirgorodskaya, G. Bukovska, J. Gobom, H. Lehrach, J. Simuth, Towards functional proteomics of minority component of honeybee royal jelly:

30

ACCEPTED MANUSCRIPT The effect of post-translational modifications on the antimicrobial activity of apalbumin2, Proteomics 9 (2009) 2131–2138. [12] M.S. Hossen, T. Nahar, S.H. Gan, M.I. Khalil, Bioinformatics and Therapeutic Insights on Proteins in Royal Jelly, Curr. Proteomics. 16 (2019) 84–101. [13] M. Cemek, F. Aymelek, M.E. Buyukokuroglu, T. Karaca, A. Buyukben, F. Yilmaz, Protective potential of royal jelly against carbon tetrachloride

PT

induced-toxicity and changes in the serum sialic acid levels, Food Chem. Toxicol. 48 (2010) 2827–2832.

[14] V. Nejati, E. Zahmatkesh, M. Babaei, Protective effects of royal jelly on

RI

oxymetholone-induced liver injury in mice, Iran Biomed. J. 20 (2016) 229–234.

SC

[15] M.K. Gill, R.R. Patyar, M.R. Reshi, S. Patyar, Protective potential of royal jelly against hepatotoxicity, Int. J. Green Pharm. 11 (2017) S412- S416. K. Sasaki, A. Yukiyoshi, H. Tachibana, K. Yamada,

NU

[16] M. Nakaya, H. Onda,

Effect of royal jelly on bisphenol A-induced proliferation of human breast cancer cells, Biosci. Biotechnol. Biochem. 71 (2007) 253–255.

MA

[17] B. Filipic, L. Gradisnik, K. Rihar, E. Soos, A. Pereyra, J. Potokar, The influence of royal jelly and human interferon-alpha (HuIFN-αN3) on proliferation,

D

glutathione level and lipid peroxidation in human colorectal adenocarcinoma cells in vitro, J. Ind. Hyg. Toxicol. 66 (2015) 269-274.

PT E

[18] R.H. Whitehead, P.S. Robinson, Establishment of conditionally immortalized epithelial cell lines from the intestinal tissue of adult normal and transgenic mice, Am. J. Physiol. Gastrointest. Liver Physiol. 296 (2009) G455–G460.

CE

[19] M.M. Abu-Serie, Evaluation of the selective toxic effect of the charge switchable diethyldithiocarbamate-loaded nanoparticles between hepatic normal

AC

and cancerous cells‫‏‬, Sci. Rep.‫‏‏‬8 (2018) 1-12.

[20] S. Bogdanov, Harmonised methods of the international honey commission, Int. honey comm. (2009) 1–63.

[21] A.L. Lee, M.I. Yeh, H.M. Wen, J.C. Chern, J. Lin, W.I. Hwang, The application of capillary electrophoresis on the characterization of protein in royal jelly, J. Food Drug Anal. 7 (1999) 73–82. [22] M. M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. [23] U.K. Laemmli, F. Beguin, G. Gujer-Kellenberger, A factor preventing the major 31

ACCEPTED MANUSCRIPT head protein of bacteriophage T4 from random aggregation, J. Mol. Biol. 47 (1970) 69-85. [24] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays, J. Immunol. Methods. 65 (1983) 55–63. [25] Y. Kiso, M. Tohkin, H. Hikino, Antihepatotoxic principles of atractylodes

PT

rhizomes, J. Nat. Prod. 46 (1983) 651–654. [26] L. Marcocci, J.J. Maguire, M.T. Droylefaix, L. Packer, The nitric oxidescavenging properties of ginkgo-biloba extract Egb-761, Biochem. Biophys. Res.

RI

Commun. 201 (1994) 748–755.

SC

[27] S. Simizu, M. Imoto, N. Masuda, M. Takada, K. Umezawa, Involvement of hydrogen peroxide production in erbstatin-induced apoptosis in human small cell

NU

lung carcinoma cells, Cancer Res. 56 (1996) 4978–4982. [28] J. Yang, Y. Zhang, I-TASSER server: new development for protein structure and function predictions, Nucleic Acids Res. 43 (2015) W174–W181.

MA

[29] S.C. Lovell, I.W. Davis, W.B. Arendall, P.I. de Bakker, J.M. Word, M.G. Prisant, J.S. Richardson, D.C. Richardson, Structure validation by Calpha

D

geometry: phi,psi and Cbeta deviation, Proteins 50 (2003) 437–450. [30] C. Zhang, P.L. Freddolino, Y. Zhang, COFACTOR: Improved protein function

PT E

prediction by combining structure, sequence and protein-protein interaction information, Nucleic Acids Res. 45 (2017) W291–W299. [31] J. Yang, A. Roy, Y. Zhang, Protein-ligand binding site recognition using

CE

complementary binding-specific substructure comparison and sequence profile alignment, Bioinformatics 29 (2013) 2588–2595.

AC

[32] L.A. Salazar-Olivo, V. Paz-González, Screening of biological activities present in honeybee (Apis mellifera) royal jelly, Toxicol. in Vitro 19 (2005) 645–651.

[33] P.T. Wingfield, Protein Precipitation Using Ammonium Sulfate, Curr. Protoc. Protein Sci. 84 (2016) 1–9. [34] D. Srisuparbh, S. Klinbunga, S. Wongsiri, S. Sittipraneed, Isolation and characterization of major royal jelly cDNAs and proteins of the honey bee (Apis cerana), Biochem. Mol. Biol. J. 36 (2003) 572–259. [35] P.M. Cummins, O. Dowling, B.F. O’Connor, Ion-exchange chromatography: basic principles and application to the partial purification of soluble mammalian prolyl oligopeptidase, Methods Mol. Biol. 681 (2011) 215–228. 32

ACCEPTED MANUSCRIPT [36] F. Yu, F. Mao, L. Jianke, Royal jelly proteome comparison between A. mellifera ligustica and A. cerana cerana, J. Proteome Res. 9 (2010) 2207–2215. [37] S. Tamura, T. Kono, C. Harada, K. Yamaguchi, T. Moriyama, Estimation and characterisation of major royal jelly proteins obtained from the honeybee Apis merifera, Food Chem. 114 (2009) 1491–1497. [38] M. Rosmilah, M. Shahnaz, G. Patel, J. Lock, D. Rahman, A. Masita, A.

PT

Noormalin, Characterization of major allergens of royal jelly Apis mellifera, Trop. Biomed. 25 (2008) 243–251.

[39] B. Kuster, M. Mann, 18O-labeling of N-glycosylation sites to improve the

RI

identification of gel-separated glycoproteins using peptide mass mapping and

SC

database searching, Anal. Chem. 71 (1999) 1431–140.

[40] K. Weber, M. Osborn, The Reliability of Molecular Weight Determinations

NU

Sulfate-Polyacrylamide Gel Electrophoresis, J. Biol. Chem. 244 (1969) 4406– 4412.

[41] L. Zhang, B. Han, R. Li, X. Lu, A. Nie, L. Guo, Y. Fang, M. Feng, J. Li,

MA

Comprehensive identification of novel proteins and N-glycosylation sites in royal jelly, BMC. Genomics. 15 (2014) 1-14.

D

[42] L.W.D. Weber, M. Boll, A. Stampfl, Hepatotoxicity and mechanism of action of haloalkanes: Carbon tetrachloride as a toxicological model, Crit. Rev.

PT E

Toxicol. 33 (2003) 105–136. [43] M.J. Morgan, Z. Liu, Crosstalk of reactive oxygen species and NF-κB signaling, Cell Res. 21 (2011) 103–115.

CE

[44] W.K. Saeed, D.W. Jun, Necroptosis: An emerging type of cell death in liver diseases, World J. Gastroenterol. 20 (2014) 12526–12532.

AC

[45] H.A. Khan, M.Z. Ahmad, J.A. Khan, M.I. Arshad, Crosstalk of liver immune cells and cell death mechanisms in different murine models of liver injury and its clinical relevance, Hepatobiliary Pancreat. Dis. Int. 16 (2017) 245–256.

[46] K. Pradeep, C.V.R. Mohan, K. Gobianand, S. Karthikeyan, Effect of Cassia fistula Linn. leaf extract on diethylnitrosamine induced hepatic injury in rats, Chem. Biol. Interact. 167 (2007) 12–8. [47] N.Z. Shaban, M.A.L. El-Kersh, F.H. El-Rashidy, N.H. Habashy, Protective role of Punica granatum (pomegranate) peel and seed oil extracts on diethylnitrosamine and phenobarbital-induced hepatic injury in male rats, Food Chem. 141 (2013) 1587–1596. 33

ACCEPTED MANUSCRIPT [48] A.A. El-Nekeety, W. El-Kholy, N.F. Abbas, A. Ebaid, H.A. Amra, M.A. Abdel-Wahhab, Efficacy of royal jelly against the oxidative stress of fumonisin in rats, Toxicon 50 (2007) 256-269. [49] M. Kanbur, G. Eraslan, L. Beyaz, S. Silici, B.C. Liman, S. Altinordulu, A. Atasever. The effects of royal jelly on liver damage induced by paracetamol in mice, Exp. Toxicol. Pathol. 61 (2009) 123-132.

PT

[50] H. Guo, Y. Kouzuma, M. Yonekura, Structures and properties of antioxidative peptides derived from royal jelly protein, Food Chem. 113 (2009) 238–245. [51] C. Zhang, J. Liu, X. Jiang, N. Haydar, C. Zhang, H. Shan, J. Zhu, Modulation

RI

of integrin activation and signaling by α1/α1′-helix unbending at the junction, J.

[52] K. Kotoh, M.

SC

Cell Sci. 126 (2013) 5735–5747.

Nakamuta, M. Kohjima, M. Fukushima, S. Morizono,

NU

N. Kobayashi, M. Enjoji, H. Nawata, Arg-Gly-Asp (RGD) peptide ameliorates carbon tetrachloride-induced liver fibrosis via inhibition of collagen production and acceleration of collagenase activity, Int. J. Mol. Med. 14 (2004) 1049–1053.

MA

[53] N. Dixit, S. Baboota, K. Kohli, S. Ahmad, J. Ali, Silymarin: A review of pharmacological aspects and bioavailability enhancement approaches, Indian J.

D

Pharmacol. 39 (2007) 172-179.

[54] C. Carvalho, R.X. Santos, S. Cardoso, S. Correia, P.J. Oliveira, M.S. Santos,

PT E

P.I. Moreira, Doxorubicin: The Good, the Bad and the Ugly Effect, Curr. Med. Chem. 16 (2009) 3267–3285. [55] C.M. Pfeffer, A.T.K. Singh, Apoptosis: A Target for Anticancer Therapy, Int.

CE

J. Mol. Sci.19 (2018) 1-10. [56] R.B. Badisa, S.F. Darling-Reed, P. Joseph, J.S. Cooperwood, L.M. Latinwo,

AC

C.B. Goodman, Selective cytotoxic activities of two novel synthetic drugs on human breast carcinoma MCF-7 cells, Anticancer Res. 29 (2009) 2993–2996.

[57] M.M. Abu-Serie, E.M. El-Fakharany, Efficiency of novel nanocombinations of bovine milk proteins (lactoperoxidase and lactoferrin) for combating different human cancer cell lines, Sci. Rep. 7 (2017) 16769. [58] M.M. Abu-Serie, F.H. El-Rashidy, In vitro collapsing colon cancer cells by selectivity of disulfiram-loaded charge switchable nanoparticles against cancer stem cells, Recent Pat. Anticancer Drug Discov. 12 (2017) 260-271. [59] S. Mahoney, F. Arfuso, M. Millward, A. Dharmarajan, The effects of phenoxodiol on the cell cycle of prostate cancer cell lines, Cancer Cell Int. 14 34

ACCEPTED MANUSCRIPT (2014) 1-12. [60] S. Simizu, M. Takada, K. Umezawa, M. Imoto, Requirement of caspase-3(like) protease-mediated hydrogen peroxide production for apoptosis induced by various anticancer drugs, J. Biol. Chem. 273 (1998) 26900–26907. [61] E. Czeczuga-Semeniuk, S. Wołczynski, M. Dabrowska, J. Dzieciol, T.Anchim, The effect of doxorubicin and retinoids on proliferation, necrosis and apoptosis

PT

in MCF-7 breast cancer cells, Folia. Histochem. Cytobiol. 42 (2004) 221–227. [62] M. Valko, D. Leibfritz, J. Moncol, M.T. Cronin, M. Mazur, J. Telser, Free radicals and antioxidants in normal physiological functions and human disease,

RI

Int. J. Biochem. Cell Biol. 39 (2007) 44–84.

SC

[63] S. Luanpitpong, P. Chanvorachote, U. Nimmannit, S.S. Leonard, C. Stehlik, L. Wang, Y. Rojanasakul, Mitochondrial superoxide mediates doxorubicin-induced

NU

keratinocyte apoptosis through oxidative modification of ERK and Bcl-2 ubiquitination, Biochem. Pharmacol. 83 (2012) 1643–1654. [64] K. Ramasamy, R. Agarwal, Multitargeted therapy of cancer by silymarin,

AC

CE

PT E

D

MA

Cancer Lett. 269 (2008) 352–362.

35

ACCEPTED MANUSCRIPT Figure legends Fig. 1. Identification of royal jelly protein fractions. (A, B) MALDI-TOF MS spectrum of MRJP2 and MRJP2 isoform X1, respectively "i" Amino acid sequence with the matched peptides of the protein, "ii" Mass spectra of the protein tryptic peptides. (C) SDS-PAGE (12%), Lane 1, molecular mass standards; Lane 2, 3, 4, 5, crude protein

PT

fraction, PF50, MRJP2 isoform X1 and MRJP2, respectively.

RI

Fig. 2. MRJP2 and its isoform X1 3D prediction structures and their Ramachandran

SC

plots. (A, E) Backbone structure of MRJP2 and its isoform X1, respectively (B, F) Ramachandran plots of MRJP2 and its isoform X1, respectively (C, G) Surface view of

NU

the 3D structure colored by the hydrophilic (blue), hydrophobic (brown), and neutral

MA

(white) residues (D, H) Surface view of the 3D structure colored by the charge potential (red negative, blue positive, white neutral).

D

Fig. 3. Flow cytometric analysis and morphological investigation of the untreated and

PT E

treated necrotic hepatocytes before and after exposure to CCl4 (72 h) alone or CCl4 with different RJ isolated fractions in comparison with the standard drugs (SM and DOX).

CE

(A) Annexin V/PI flow charts for normal and necrotic hepatocytes before and after treatment with RJ-PFs and the standard drugs (B) Quantification of the % necrotic cells

AC

in normal and necrotic hepatocytes before and after treatment with RJ-PFs and the standard drugs (C) Morphological alterations in the hepatocytes before and after exposure to CCl4 (72 h) alone or CCl4 with RJ-PFs in comparison with the standard drugs. Values are presented as mean ± SE and different letters specify the significance at P < 0.05. Fig. 4. Morphological and biochemical investigation of the anti-hepatotoxicity potential of the effective RJ protein fractions in comparison with the standard drugs (SM and

36

ACCEPTED MANUSCRIPT DOX). (A) AO/EB nuclear double staining of the hepatocytes for detection of necrosis (B) Albumin level and transaminases activities (C) MLKL, TNF-α and NO levels (D) Flow cytometric histograms of the fluorescence for ROS detection (E) Quantitative results of the flow cytometric detection of ROS. Values are presented as mean ± SE and different letters specify the significance at P < 0.05. VC, viable cells; EN, Early necrotic

PT

cells; LN, Late necrotic cells.

RI

Fig. 5. Detection of the apoptotic HepG2 cells. (A) Flow cytometric charts using

SC

annexin V/PI double staining for the control and treated cancer cells with different RJ fractions in comparison with the standard drugs (SM and DOX). (B) Quantification of

NU

the % apoptotic cells in the control and treated cancer cells (C) Morphology of HepG2

MA

cells with and without the different established treatments. Values are presented as mean ± SE and different letters specify the significance at P < 0.05.

D

Fig. 6. Investigation of the anticancer role of the effective RJ protein fractions in

PT E

comparison with the standard drugs (SM and DOX). (A) AO/EB double staining of the apoptotic cells (B) Fold change in Bcl-2 and p53 expression levels (C) Percentage of

CE

caspase 3/7 activation (D) Ki-67 immunostaining of cancer cells (E) Percentage of Ki67 immunostained cells. VC, viable cells; EA, Early apoptotic cells; LA, Late apoptotic

AC

cells.

37

ACCEPTED MANUSCRIPT Table 1 The percentage yield of royal jelly (RJ) isolated fractions, their safe doses (EC 100) in normal hepatocytes, EC50 values against CCl4-induced hepatotoxicity (CIH), and IC50 values against HepG2 cell line in comparison with SM and DOX drugs. HepG2 Selectivity index (SI) -

EC100 (µg/mL)

CIH EC50 (mg/mL)

-

727.26±41.96a

-

IC50 (mg/mL) -

9.50±0.01

1300.13±16.84b

-

-

-

Lipids

0.17±0.00

326.07±11.0

-

-

-

Soluble proteins (Crude protein fraction)

14.30±0.01 1333.95±29.95b

-

-

0.21±0.00ab 0.02±0.00a

1.33±0.04‫‏‬a 0.91± 0.02‫‏‬a

-

0.28±0.00a Nil

SC

RI

125.62±4.91d 20.69±0.40e

1287.36±1.71b 1298.85±9.87b 1321.33±15.37b 1333.64±13.42b 1307.03±10.56b 1330.59±10.41b 1332.35±11.10b SE (n=3). Different

6.16±0.07b 2.67±0.28c 2.34±0.27‫‏‬b 5.32±0.08c 0.55±0.00b 12.05±0.22‫‏‬c 6.69±0.12d 1.92±0.13d 2.78±0.14‫‏‬b letters are significantly different in the same

NU

0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 0.34±0.05 0.48±0.00 6.46±0.04 2.86±0.00 1.15±0.03 1.43±0.02 1.28±0.01 as mean ±

c

MA

-

AC

CE

SM DOX RJ protein fractions [% of ammonium sulfate] 5 [0-5] 10 [5-10] 15 [10-15] 20 [15-20] 25 [20-25] 30 [25-30] 40 [30-40] 50 [40-50] 60 [50-60] MRJP2 MRJP2 X1 Results are presented column at P < 0.05.

D

Carbohydrates

PT E

RJ

38

PT

Yield (g%)

RJ fractions

ACCEPTED MANUSCRIPT Highlights 

A new simple method for purification of the most active royal jelly (RJ) proteins. These proteins identified as MRJP2 and its isoform X1.



These proteins exhibited a high anti-hepatic necrosis effect (in vitro study).



This anti-necrotic potential mediated by suppression TNF-α, MLKL, and ROS

PT



CE

PT E

D

MA

NU

SC

Moreover, they showed in vitro potent apoptosis-dependent anticancer activity.

AC



RI

levels.

39

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6