The Effect of Royal Jelly on Telomere Length and Some Biochemical Parameters in Wistar Albino Rats with Liver Damage Caused by Carbon Tetrachloride

Abstract

Royal jelly (RJ) is a natural bee product that has been used for therapeutic purposes since ancient times. The therapeutic properties of this product, which has rich biological content, are still being investigated with new approaches. In this study, the effect of RJ on telomere length, some antioxidant parameters, and lipid profile was examined. This study will contribute to the literature as it is the first to evaluate the effect of RJ on the length of telomeres in damaged liver tissues. In the study, the levels of serum triglyceride, total cholesterol (TC), high-density lipoprotein cholesterol, low-density lipoprotein cholesterol (LDL-C), aspartate transaminase (AST), alanine transaminase (ALT), telomerase, 8′-hydroxy-2′-deoxyguanosine (8-OHdG), and paraoxonase-1 (PON1) were investigated with enzyme-linked immunosorbent assay method and telomere lengths were investigated by real-time quantitative polymerase chain reaction. The increased TC, LDL-C levels, and AST and ALT activities in the serum after carbon tetrachloride (CCl₄) administration approached the control level after RJ administration. PON1 activity decreased in groups with CCl₄. PON1 activity increased after RJ administration. The level of 8-OHdG, which increased groups with CCl₄, decreased after RJ administration. According to the results of telomere length analysis in liver tissues, telomere lengths in damaged tissues were significantly shortened with CCl₄ application and increased with RJ application. Based on the findings of the study, it was concluded that RJ may have therapeutic effects on telomere lengths and some biochemistry parameters.

INTRODUCTION

Telomeres are the structures containing certain guanine-rich DNA sequences (5′-TTAGGG-3′) located at the ends of linear chromosomes.¹ These structures play a role in the protection of chromosomes. They shorten depending on the cell cycle by the size of a primer during each replication of DNA and the cell completes its life cycle after a certain loss.^1,2 Therefore, telomere length and age are associated.^3,4 The shortening of telomeres is also affected by environmental factors as well as aging.¹ Furthermore, previous studies explained that the rate of telomere shortening may differ by the type of tissue.⁵ Although there is a repair mechanism in damaged tissues, aging may cause some effects and related organ failures may occur. One of the most common organ failures is liver diseases.⁴ Although the ability of liver tissue to regenerate is strong, it decreases with age.⁶

One of the factors that slow down the aging process is telomerase. Because the elongation of telomeres is induced by the enzyme called telomerase. The RNA subunits of telomerase, which is an enzyme in ribonucleoprotein structure, act as a template for telomeric ends and ensure the protection of telomeric ends. Since DNA polymerase cannot initiate a new DNA synthesis at the 3′ end of the leading strand, telomerase overcome the end-replication problem by using their RNA sequences as templates. However, telomerase activity is limited in somatic cells and is affected by environmental factors.^1,7

Royal jelly (RJ) is secreted from the mandibular and hypopharyngeal glands of bees and is a nutrient for young larvae. This product is not only a nutrient for the queen bee but also the active agent of the mechanism required to become a queen bee.⁸ It is known that 5- to 15-day-old bee larvae turn into queen bees when fed with this food. Although worker bees have the same genetic structure, their lifespan is 6–8 weeks. Queen bees can live up to 4–5 years. RJ is the agent that enables the transformation of these two bees that have the same genome.^8,9

Although previous studies emphasized the antitumor,¹⁰ antimicrobial,¹¹ antiallergic, antioxidant,^12,13 immune-related, and life expectancy regulation¹⁰ characteristics of RJ, research on RJ is still ongoing.

This study is the first to evaluate the effect of RJ on telomere length in liver damage. Since its contribution to telomere length points to some positive factors such as antiaging and acceleration of healing, it is thought that the findings of the study will contribute to treatment approaches.

MATERIALS AND METHODS

This study was carried out with the approval of Canakkale Onsekiz Mart University Animal Experiments Local Ethics Committee (No. COMU-HADYEK-2020/04-03).

Experimental design

G*Power-3.1 program was used in the experimental design. According to the principle of Cohen,¹⁴ the sample size was determined as 36 with an effect size of 0.70, an α error of 0.05, and a power of (1–β) 80%. The groups were formed to include six rats in each group. Thirty-six female Wistar albino rats weighing 200–250 g were divided into six groups, six rats in each group. Physical conditions were a temperature of 21 ± 2°C, humidity of 50 ± 5% with a cycle of 12 h light and 12 h dark. Liver damage was performed with minor modification in the Studies of Sato et al. All rats were injected intraperitoneally (IP) once a week with 10% carbon tetrachloride (CCl₄) in olive oil (Merck-289116) at a dose of 1 mL/kg body weight.¹⁵

The groups were determined as follows: control group, group A (150 mg/kg RJ), group B (350 mg/kg RJ), group CCl₄, CCl₄-A (CCl₄ with 150 mg/kg RJ), CCl₄-B (CCl₄ group with 350 mg/kg RJ). RJ (BEOO; Istanbul Technical University) was given to rats through gavage 5 days a week. Feed and water were given ad libitum.

Collection of blood and tissue samples

Rats were fasted for 12 h and anesthetized by administrating 70 mg/kg of ketamine and 10 mg/kg of xylazine (IP). After 30 min, blood was taken from their hearts through puncture and transferred into tubes without anticoagulant for serum. The tubes were centrifuged at 1400 g at 4°C for 10 min in Nüve NF 1200 centrifuge and then the serum was separated and stored at −80°C in labeled tubes.

Blood serum analysis

In the study, triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were analyzed using colorimetric kits with code numbers of Relassay RLB257, Relassay RLB248, Relassay RLB261, and Relassay RLB263, respectively. A commercial kit that utilizes the Rat Telomerase Sandwich-enzyme-linked immunosorbent assay (ELISA) principle (Elabscience E-EL-R0947). 8′-hydroxy-2′-deoxyguanosine (8-OHdG) was measured using commercial kits (Mybiosource-MBS701076) using the Competitive-ELISA principle. Bio-Tek ELx800 ELISA instrument and Bio-Tek ELx50 washer were used in the study.

Tissue homogenization

Liver tissues were homogenized using a Clinic/cell SV mini (CAT No.: 108–101) tissue extraction kit, 0.2 mm Stainless Steel Beads and, Digital Disruptor (No. 3591456) homogenizer.

Telomere length in liver

Telomere Length Quantification qPCR Assay Kit (ScienCell, ARTLQ-R8918) and Absolute Rat Telomere Length kit were used to directly measure the mean length of telomeres in the rat cell population. A single-copy reference primer, which recognizes and amplifies a 100-bp-long region on the rat chromosome 17 and serves as a reference for data normalization, was used. The genomic DNA sample with known telomere length was used as a reference to calculate the telomere length of the target samples. The calculation was as follows:

Rat diploid cells have 84 chromosome ends; therefore, the mean telomere length in each chromosome end was = (5.05 ± 0.18 Mb)/84 = 60.1 ± 2.1 kb. The mean telomere length in the target genomic DNA sample was 5.05 ± 0.18 Mb. The telomere length was calculated as 60.1 ± 2.1 kb per diploid cell or chromosome end.^2,16

Statistical analysis

Statistical analysis of the data was performed with the SPSS 23.0 package program for Microsoft. The graphs were made using the GraphPad Prism 8 program. The difference between groups was determined by a one-way analysis of variance. For significant P scores, Tukey's multiple range test was used to determine from which group the difference originated. The data were presented as mean and standard deviation (M ± SD).

RESULTS

As a result of liver damage, serum TC, TG levels, and AST and ALT activities increased significantly (P < .001, P < .01). A statistically significant decrease was observed in HDL-C level after CCl₄ application (P < .01). Although HDL-C levels approached control levels after RJ administration, this was not statistically significant. LDL-C (P < .01) and TG (P < .001) levels increased significantly with CCl₄ administration. Both LDL-C and TG levels decreased after RJ administration. TC levels increased with CCl₄ application. The highest decrease in TC with RJ application was seen in the CCl₄-B group. (Table 1).

Table 1.

Blood Serum Total Cholesterol, Triglyceride, Low-Density Lipoprotein Cholesterol, High-Density Lipoprotein Cholesterol Values, and Aspartate Transaminase and Alanine Transaminase Activities

	Control	A	B	CCl₄	CCl₄+A	CCl₄+B	P
TC (mg/dl)	72.8 ± 3.6^a	82.6 ± 4.5^a,b	88.4 ± 3.8^a,b	141.8 ± 4.7^c	91.6 ± 2.3^b,c	87.4 ± 4.7^b	<.001
TG (mg/dl)	95.2 ± 2.1^a	92.5 ± 3.1^a	91 ± 2.6^a	188.8 ± 6.7^b	110.6 ± 7^c	102.3 ± 4.5^a,c	<.001
HDL-C (mg/dl)	47.2 ± 1.7^a	46.7 ± 3.4^a	47.7 ± 1.8^a	39.7 ± 3.6^b	41.8 ± 3.6^b	42.3 ± 4.3^b	<.01
LDL-C (mg/dl)	17 ± 2.8^a	18.1 ± 1.2^a	19 ± 1.4^a	50.2 ± 2^b	42.8 ± 3.3^c	39.8 ± 3.7^c	<.01
AST (U/L)	101 ± 6.5^a	102 ± 7^a	101 ± 6.3^a	288 ± 8.4^b	187 ± 9.7^c	175 ± 6.4^c	<.001
ALT (U/L)	48.8 ± 1.7^a	51.8 ± 1.8^a	52.4 ± 1.7^a	149 ± 3.4^b	122,6 ± 3.4^c	115.65 ± 4.7^c	<.001

Different superscripts (a, b, and c) on the same row indicate that the difference between the groups is significant. Control, 150 mg/kg RJ (A), 350 mg/kg RJ (B), CCl₄, CCl₄ + 150 mg/kg RJ (CCl₄ + A), and CCl₄ + 350 mg/kg RJ (CCl₄ + B); (P < .01; P < .001; M ± SD).

ALT, alanine transaminase; AST, aspartate transaminase; CCl₄, carbon tetrachloride; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; M, mean; SD, standard deviation; TC, total cholesterol; TG, triglyceride.

As a result of liver damage, serum 8-OHdG level significantly increased in the CCl₄ group. There was a numerical decrease in the CCl₄-A group, whereas the decrease was statistically significant in the CCl₄-B group (P < .001; Fig. 1). There was no significant difference between the control group and groups A and B. As a result of liver damage, PON1 levels decreased in the CCl₄ group (P < .001). A statistically significant increase was observed in all RJ-treated groups compared with the control group (P < .001; Fig. 2).

FIG. 1.

Blood serum 8-OHdG (ng/mL). One hundred fifty milligrams per kilogram RJ (A), 350 mg/kg RJ (B), CCl₄, CCl₄ + 150 mg/kg RJ (CCl₄ + A), and CCl₄ + 350 mg/kg RJ (CCl₄ + B). Different superscripts (a and b) indicate that the difference between the groups is significant (P < .001, M ± SD). 8-OHdG, 8′ -hydroxy-2′-deoxyguanosine; CCl₄, carbon tetrachloride; M, mean; RJ, royal jelly; SD, standard deviation.

FIG. 2.

Blood serum PON1 (U/L). One hundred fifty milligrams per kilogram RJ (A), 350 mg/kg RJ (B), CCl₄, CCl₄ + 150 mg/kg RJ (CCl₄ + A), and CCl₄ + 350 mg/kg RJ (CCl₄ + B). Different superscripts (a, b, c, and d) indicate that the difference between the groups is significant (P < .001, M ± SD). PON1, paraoxonase-1.

As a result of liver tissue telomere length analysis, the groups with liver damage had lower values compared with the control group (P < .001). There was a statistically significant increase in telomere length in damaged groups for both RJ doses (P < .001; Fig. 3). When serum telomerase activities were evaluated, although an increase was observed in the CCl₄-B group, there was no statistically significant difference between the groups (Fig. 4).

FIG. 3.

Liver telomere length (kb). One hundred fifty milligrams per kilogram RJ (A), 350 mg/kg RJ (B), CCl₄, CCl₄ + 150 mg/kg RJ (CCl₄ + A), and CCl₄ + 350 mg/kg RJ (CCl₄ + B). Different superscripts (a, b, c, and d) indicate that the difference between the groups is significant (P < .001, M ± SD).

FIG. 4.

Blood serum telomerase (ng/mL). One hundred fifty milligrams per kilogram RJ (A), 350 mg/kg RJ (B), CCl₄, CCl₄ + 150 mg/kg RJ (CCl₄ + A), and CCl₄ + 350 mg/kg RJ (CCl₄ + B); (P < .001, M ± SD).

DISCUSSION

Recently, the interest in natural products has been increasing. RJ is one of the most popular biological products. In previous studies, the positive effects of RJ in the treatment of many diseases such as cancer, obesity, diabetes, and Alzheimer's were mentioned. The therapeutic effect of RJ has been associated with biologically active compounds. Antioxidant and anticancer effects of 10-hydroxy-trans-2-deconoic acid in RJ have been reported.^10,17 It has also been reported to have therapeutic effects on blood biochemistry and metabolic and endocrine diseases such as obesity and diabetes.^18
–20 As in other studies performed with CCl₄ by inducing liver damage, serum HDL-C levels decreased, whereas TC, TG, and LDL-C increased.^21,22 AST and ALT activities, which are considered as biomarkers of liver damage, also increased with CCl₄ application.^23

–26 According to these data and available literature, CCl₄ caused liver damage.

Studies investigating whether RJ can be a therapeutic factor have shown that ALT, AST, TG, TC, and LDL-C parameters after RJ administration approached the values in the control group.^18,27 Studies indicated that RJ upregulates 148 genes, downregulates 119 genes related to lipid metabolism, and can regulate a total of 267 liver genes.²⁸ Therefore, RJ has an impact on the lipid profile. In this study, TC, TG, LDL-C levels, and AST and ALT activities, which increased after liver damage, approached those in the control values after RJ administration.

PON1 is an enzyme synthesized by the liver and has antioxidant activity. Since PON1 plays a role in the regulation of parenchymal cell apoptosis in the liver, it has been recognized as an important marker in liver injury. In studies conducted, decreased PON1 activity indicates liver damage,²⁹ whereas increased PON1 expression indicates a strong protective effect on liver damage.³⁰ When the effects of RJ on PON1 were examined, in a study in which liver damage was induced, it was reported that PON1 activity, which decreased with RJ application, increased significantly.³¹ Likewise, the decrease in PON1 activity in this study shows the oxidative damage caused by CCl₄. The increase in PON1 values after RJ administration is related to the fact that RJ is an active agent in antioxidant defense.

8-OHdG is a marker of oxidative stress and DNA damage.³¹ Studies emphasized that RJ makes a strong contribution to antioxidant defense. It was shown that 8-OHdG, which increases with tissue damage, decreases with the effect of RJ on antioxidant defense and raises the mean life expectancy in subjects.³² In this study, 8-OHdG levels were found to be high after CCl₄ treatment. Decreased telomere length, especially in the groups treated with CCl₄, and increased 8-OHdG level suggested that oxidative damage affects telomere structure.

Telomerase enzyme catalyzes the protection and even elongation of telomeres. Although its activity can be monitored in cancer cells, it is difficult to determine telomerase activity in some somatic cells.³³ In this study, the serum telomerase enzyme was analyzed, and no significant findings could be obtained, which might be due to the limitation of this enzyme in the study.

Telomere length is a powerful factor for strong antioxidant defense and disease prevention.³⁴ The shortening of telomeres with aging or environmental factors leads to higher susceptibility to diseases.³⁵ Although the liver is one of the organs least affected by aging, it was stated that a lifelong poor-quality diet makes the liver susceptible to damage.³⁴ It was reported that RJ administration after liver fibrosis induced by factors such as CCl₄, paracetamol, cisplatin, azathioprine, and heavy metals stimulates the antioxidant capacity of the liver and may recover biochemical abnormalities and tissue damage.^{10,12,18,33,36,37}

Although there is not adequate research to explain the relationship between telomere length and RJ, it is known that telomere shortening leads to cellular aging. Telomere shortening accelerates with aging and inhibits cell division and regeneration.³ The main starting point of this research was to research the shortening of telomeres by a factor other than aging by causing damage to the tissue and to explain the functioning mechanism of RJ, which is a factor that promotes cell regeneration,^9,10,31,38 with an uninvestigated perspective. The findings of this study showed that RJ has a positive effect on telomere length.

Studies on the effect of RJ on antioxidant defense, cell regeneration, and life expectancy can be associated with telomere length, which is the topic of this study because previous studies reported that there is a relationship between these factors and telomere length. Insulin-like growth factor signaling and forkhead box O transcription factor is considered as the main determinants of cellular life expectancy and it was stated that these determinants increased by the effect of RJ.^10,39,40 It was also reported that RJ acts by promoting epidermal growth factor signaling, suggesting that RJ can promote tissue regeneration.^9,38 Major-RJ-Protein1 is a major proliferation factor promoting cell proliferation. Royalactin and 10-HDA in RJ were reported to prolong lifespan in various model organisms.^10,28,38 Based on this research, it was thought that RJ might affect cell division and lifespan by positively affecting telomere length as an underlying mechanism. Undoubtedly, more research is required to definitively elucidate this mechanism.

The results obtained from the literature and the data of this study are as a shortening of telomere lengths has various effects such as aging, retarded healing process, higher susceptibility to diseases, and even shortened life span. Adverse environmental conditions, some harmful chemicals, and even stress have negative effects on telomere length. Telomerase enzyme catalyzes reactions for protecting telomeres. Unlike cancer cells, it can rarely be monitored in somatic cells. RJ has positive effects on cell proliferation and lifespan. It was determined that the doses of RJ used in the study directly or indirectly had a positive effect on the telomerase enzyme and thus contributed to the protection of telomeres. Further research is required to elucidate the mechanism of this effect.

Footnotes

AUTHOR DISCLOSURE STATEMENT

No competing financial interests exist.

FUNDING INFORMATION

This study was supported by Çanakkale Onsekiz Mart University Scientific Research department with project No. TSA-2020-3150.

References

Blackburn

. Structure and function of telomeres. Nature, 1991; 350(6319):569–573; https://doi.org/10.1038/350569a0

O'Callaghan

, Fenech

. A quantitative PCR method for measuring absolute telomere length. Biol Proced, 2011; 13(1):3; https://doi.org/10.1186/1480-9222-13-3

Bernadotte

, Mikhelson

, Spivak

. Markers of cellular senescence. Telomere shortening as a marker of cellular senescence. Aging, 2016; 8(1):3–11; https://doi.org/10.18632/aging.100871

Cherif

, Tarry

, Ozanne

, et al. Ageing and telomeres: A study into organ- and gender-specific telomere shortening. Nucleic Acids Res, 2003; 31(5):1576–1583; https://doi.org/10.1093/nar/gkg208

Tarry-Adkins

, Aiken

, Dearden

, et al. Exploring telomere dynamics in aging male rat tissues: Can tissue-specific differences contribute to age-associated pathologies. Gerontology, 2021; 67(2):233–242; https://doi.org/10.1159/000511608

, Yang

, Wang

, et al. Irisin Improves autophagy of aged hepatocytes via increasing telomerase activity in liver injury. Oxid Med Cell Longev, 2020; https://doi.org/10.1155/2020/6946037

Greider

, Blackburn

. The telomere terminal transferase of tetrahymena is a ribonucleoprotein enzyme with two kinds of primer specificity. Cell, 1987; 887–898; https://doi.org/10.1016/0092-8674(87)90576-9

Yang

, Tian

, Han

, et al. Longevity extension of worker honey bees (Apis mellifera) by royal jelly: Optimal dose and active ingredient. PeerJ. 2017; https://doi.org/10.7717/peerj.3118

Abu-Serie

, 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. Int J Biol Macromol, 2019; 782–795; https://doi.org/10.1016/j.ijbiomac.2019.01.210

10.

Guo

, Wang

, Chen

, et al. Active components and biological functions of royal jelly. J Funct Foods, 2021; 82:104514; https://doi.org/10.1016/j.jff.2021.104514

11.

Fratini

, Cilia

, Mancini

, et al. Royal jelly: An ancient remedy with remarkable antibacterial properties. Microbiol Res, 2016; 192:130–141; https://dx-doi-org.web.bisu.edu.cn/10.1016/j.micres.2016.06.007

12.

Silici

, Ekmekcioglu

, Kanbur

, et al. The protective effect of royal jelly against cisplatin-induced renal oxidative stress in rats. World J Urol, 2011; 29:127–132; https://doi.org/10.1007/s00345-010-0543-5

13.

Ghanbari

, Nejati

, Khazaei

. Antioxidant and protective effects of Royal jelly on histopathological changes in testis of diabetic rats. Int J Reprod Biomed, 2016; 14: 511–518; https://doi.org/10.29252/ijrm.14.8.519

14.

Cohen

. Statistical power analysis for the behavioral sciences. A review. J Abnorm Soc Psychol, 1962; 65:145–153.

15.

Sato

, Maesawa

, Fujisawa

, et al. Prevention of critical telomere shortening by oestradiol in human normal hepatic cultured cells and carbon tetrachloride induced rat liver fibrosis. Gut, 2004; 53:1001–1009; https://doi.org/10.1136/gut.2003.027516

16.

Cawthon

. Telomere measurement by quantitative PCR. Nucleic Acids Res, 2002; 30(10):1–6. https://doi.org/10.1093/nar/30.10.e47

17.

Malkoç

, Us Altay

, Diler

, et al. The effects of royal jelly on the oxidant-antioxidant system in rats with N-methyl-N-nitrosourea-induced breast cancer. Turk J Biochem, 2018; 43(2):176–183; https://doi.org/10.1515/tjb-2017-0140

18.

Ahmed

WMS

, Khalaf

, Moselhy

, et al. Royal jelly attenuates azathioprine induced toxicity in rats. Environ Toxicol Pharmacol, 2014; 37(1):431–437; https://dx-doi-org.web.bisu.edu.cn/10.1016/j.etap.2013.12.010

19.

Pourmoradian

, Mahdavi

, Mobasseri

, et al. Effects of royal jelly supplementation on glycemic control and oxidative stress factors in type 2 diabetic female: A randomized clinical trial. Chin J Integr Med, 2014; 20; 347–352; https://doi.org/10.1007/s11655-014-1804-8

20.

Yoshida

, Hayashi

, Watadani

, et al. Royal jelly improves hyperglycemia in obese/diabetic KK-Ay mice. J Vet Med Sci, 2017; 79:299–307; https://doi.org/10.1292/jvms.16-0458

21.

Ijaz

, Iqbal

, et al. In vivo antioxidant efficacy and therapeutic potential of Artemisia brevifolia leaves extract against CCl4-induced reproductive damages in male albino rats. J King Saud Univ Sci, 2022; 34; https://doi.org/10.1016/j.jksus.2021.101816

22.

Mousa

, Mohamed

, Hagrassi

, et al. GC/MS-based metabolomics profiling approach and determination of ameliorative effect of Chiliadenus montanus extract towards CCl4 induced hepatotoxicity in Albinol rats. Egypt J Chem, 2021; 64:3499–3510; https://doi.org/10.21608/EJCHEM.2021.65354.3401

23.

Luckey

, Petersen

. Activation of Kupffer cells during the course of carbon tetrachloride-induced liver injury and fibrosis in rats. Exp Mol Pathol, 2001; 71:226–240; https://doi.org/10.1006/exmp.2001.2399

24.

Ogeturk

, Kus

, Kavakli

, et al. Effects of melatonin on carbon tetrachloride-induced changes in rat serum. J Physiol Biochem, 2004; 60:205–210; https://doi.org/10.1007/BF03167030

25.

Kumar

, Sivaraj

, Elumalai

, et al. Carbon tetrachloride-induced hepatotoxicity in rats: Protective role of aqueous leaf extracts of Coccinia grandis . Int J PharmTech Res, 2009; 1(4):1612–1615.

26.

Yousefi-Manesh

, Shirooie

, Partoazar

, et al. Hepatoprotective effects of phosphatidylserine liposomes on carbon tetrachloride-induced hepatotoxicity in rats. J Cell Biochem, 2019; 11853–11858; https://doi.org/10.1002/jcb.28464

27.

El-Nekeety

, El-Kholy

, Abbas

, et al. Efficacy of royal jelly against the oxidative stress of fumonisin in rats. Toxicon, 2007; 50256–50269; https://doi.org/10.1016/J.TOXICON.2007.03.017

28.

Kamakura

. Royalactin induces queen differentiation in honeybees. Nature, 2011; 47:3478–3483; https://doi.org/10.1038/nature10093

29.

Yildirim

, KaradenIz

, Karakoç

, et al. Effects of royal jelly on liver paraoxonase activity in rats treated with cisplatin. Turkish J Med Sci, 2012; 42:367–375; https://doi.org/10.3906/sag-1102-1373

30.

Marsillach

, Camps

, Ferré

, et al. Paraoxonase-1 is related to inflammation, fibrosis and PPAR delta in experimental liver disease. BMC Gastroenterol, 2009; 9:1–13; https://doi.org/10.1186/1471-230X-9-3

31.

Algeda

, Ebrahim

. The efficacy of royal jelly in the restoration of liver injury in irradiated rats. Egypt J Radiat Sci Appl, 2020; https://doi.org/10.21608/ejrsa.2020.33962.1099

32.

Inoue

, Koya-Miyata

, Ushio

, et al. Royal jelly prolongs the life span of C3H/HeJ mice: Correlation with reduced DNA damage. Exp Gerontol, 2003; 38:965–969; https://doi.org/10.1016/S0531-5565(03)00165-7

33.

Brown

, Meleah Mathahs

, Broadhurst

, et al. Increased hepatic telomerase activity in a rat model of iron overload: A role for altered thiol redox state?. Free Radic Biol Med, 2007; 42:228–235; https://doi.org/10.1016/J.FREERADBIOMED.2006.10.039

34.

Varela-Lopez

, Pérez-López

, Ramirez-Tortosa

, et al. Gene pathways associated with mitochondrial function, oxidative stress and telomere length are differentially expressed in the liver of rats fed lifelong on virgin olive, sunflower or fish oils. J Nutr Biochem, 2018; 52:36–44 https://doi.org/10.1016/J.JNUTBIO.2017.09.007

35.

Epel

, Blackburn

, Lin

, et al. Accelerated telomere shortening in response to life stress. Proc Natl Acad Sci USA, 2004; 101:17312–17315; https://doi.org/10.1073/pnas.0407162101

36.

Kanbur

, Eraslan

, Beyaz

, et al. The effects of royal jelly on liver damage induced by paracetamol in mice. Exp Toxicol Pathol, 2009; 61123–61132; https://doi.org/10.1016/j.etp.2008.06.003

37.

Cemek

, Aymelek

, Büyükokuroĝlu

, et al. Protective potential of Royal jelly against carbon tetrachloride induced-toxicity and changes in the serum sialic acid levels. Food Chem Toxicol, 2010; 48:2827–2832; https://doi.org/10.1016/j.fct.2010.07.013

38.

Detienne

, De Haes

, Ernst

, et al. Royalactin extends lifespan of Caenorhabditis elegans through epidermal growth factor signaling. Exp Gerontol, 2014; 60:129–135; https://doi.org/10.1016/j.exger.2014.09.021

39.

Honda

, Fujita

, Maruyama

, et al. Lifespan-extending effects of royal jelly and its related substances on the nematode Caenorhabditis elegans . PLoS One, 2011; 6:1–10; https://doi.org/10.1371/journal.pone.0023527

40.

Wang

, Cook

, Grasso

, et al. Royal jelly-mediated prolongevity and stress resistance in caenorhabditis elegans is possibly modulated by the interplays of DAF-16, SIR-2.1, HCF-1, and 14-3-3 proteins. J Gerontol Ser A Biol Sci Med Sci, 2015; 70:827–838; https://doi.org/10.1093/gerona/glu120