Anti-senescence effect and molecular mechanism of the major royal jelly proteins on human embryonic lung fibroblast (HFL-I) cell line

Anti-senescence effect and molecular mechanism of the major royal jelly proteins on human embryonic lung fibroblast (HFL-I) cell line 蜂王浆主蛋白对人胚肺成纤维细胞HFL-I的抗衰老作用及其作用机制研究 1 first-author 1 1 1 1 2 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:Shenlirong@zju.edu.cn †E-mail:Shenlirong@zju.edu.cn Shenlirong@zju.edu.cn College of Biosystems Engineering and Food Science, Zhejiang University / Key Laboratory of Agro-Products Postharvest Handling of Ministry of Agriculture and Rural Affairs / Zhejiang Key Laboratory for Agri-Food Processing, Hangzhou 310058, China College of Biosystems Engineering and Food Science, Zhejiang University / Key Laboratory of Agro-Products Postharvest Handling of Ministry of Agriculture and Rural Affairs / Zhejiang Key Laboratory for Agri-Food Processing, Hangzhou 310058, China USDA ARS Nutritional Genomics Laboratory, JM-USDA Human Nutrition Research Center on Aging at Tufts University, Boston, Massachusetts 02111, the United States USDA ARS Nutritional Genomics Laboratory, JM-USDA Human Nutrition Research Center on Aging at Tufts University, Boston, Massachusetts 02111, the United States Royal jelly (RJ) from honeybee has been widely used as a health promotion supplement. The major royal jelly proteins (MRJPs) have been identified as the functional component of RJ. However, the question of whether MRJPs have anti-senescence activity for human cells remains. Human embryonic lung fibroblast (HFL-I) cells were cultured in media containing no MRJPs (A), MRJPs at 0.1 mg/ml (B), 0.2 mg/ml (C), or 0.3 mg/ml (D), or bovine serum albumin (BSA) at 0.2 mg/ml (E). The mean population doubling levels of cells in media B, C, D, and E were increased by 12.4%, 31.2%, 24.0%, and 10.4%, respectively, compared with that in medium A. The cells in medium C also exhibited the highest relative proliferation activity, the lowest senescence, and the longest telomeres. Moreover, MRJPs up-regulated the expression of superoxide dismutase-1 ( SOD1 ) and down-regulated the expression of mammalian target of rapamycin ( MTOR ), catenin beta like-1 ( CTNNB1 ), and tumor protein p53 ( TP53 ). Raman spectra analysis showed that there were two unique bands related to DNA synthesis materials, amide carbonyl group vibrations and aromatic hydrogens. These results suggest that MRJPs possess anti-senescence activity for the HFL-I cell line, and provide new knowledge illustrating the molecular mechanism of MRJPs as anti-senescence factors. 目的 2 探讨不同浓度蜂王浆主蛋白(MRJPs)对人胚肺成纤维细胞HFL-I的抗衰老作用,并对其可能的分子机理进行预测. 创新点 2 本实验采用拉曼光谱法探究添加MRJPs对HFL-I细胞内成分的影响.实验发现添加MRJPs的 HFL-I与对照组的HFL-I的拉曼光谱图中,与DNA和蛋白质合成相关的拉曼峰发生了变化.该发现在未来可作为鉴定衰老HFL-I的一种手段. 方法 2 实验以蜂王浆为原料,利用透析法得到MRJPs.通过对不同浓度MRJPs处理的HFL-I进行细胞形态学观察,测定HFL-I生命周期以及检测其β-半乳糖苷酶活性,评价了MRJPs的抗衰老效果.并且通过MTT法考察了不同浓度MRJPs对HFL-I的促增殖作用.同时,实验一方面采用实时定量聚合酶链反应(qPCR)测量了不同浓度MRJPs处理的HFL-I的细胞端粒相对长度以及 MTOR、SOD1、CTNNB1、TP53 四种衰老相关基因的相对表达量;另一方面还通过拉曼光谱实验法,进一步探索了MRJPs抗衰老的分子机理. 结论 2 本实验结果显示,添加MRJPs能降低HFL-I衰老细胞所占比例,显著延长HFL-I的生命周期,一定量的MRJPs还能够促进HFL-I的增殖.此外,MRJPs还能减缓细胞端粒长度的缩短,上调衰老相关基因SOD1的表达,下调抗衰老基因 MTOR、CTNNB1 和 TP53 的表达.通过MRJPs添加组细胞的拉曼光谱上出现的独特的碱基、酰胺羰基和芳香氢峰,推测MRJPs的抗衰老作用可能与促进DNA及蛋白质在细胞中的合成密切相关. Major royal jelly protein Human embryonic lung fibroblast (HFL-I) cell line Anti-senescence Relative proliferation activity Telomere length Molecular mechanism 蜂王浆主蛋白 人胚肺成纤维细胞 抗衰老 拉曼光谱 端粒长度 分子机理 Introduction 1 Royal jelly (RJ), secreted from hypopharyngeal and mandibular glands of nurse honeybees, contains a considerable amount of bioactive substances and has been widely used as a food (Buttstedt et al., 2014 2014 ). About 50% of the dry mass of RJ is protein (Malecová et al., 2003 2003 ), of which 80% belongs to the major royal jelly proteins (MRJPs) (Schmitzová et al., 1998 1998 ). RJ is the principal food for honeybee queens (Shen et al., 2010 2010 ). It is believed that exclusive feeding on RJ induces a honeybee larva to develop into a queen, whose lifespan can reach a maximum of eight years much longer than that of worker bees (Remolina and Hughes, 2008 2008 ). For its beneficial effect on honeybee queens, RJ has become a well-known healthcare product in many countries in Asia and Europe. Consumers anticipate that RJ could exert a similar health benefit in humans. Previous studies have shown that RJ has several kinds of bioactivity, including anti-oxidation (Jamnik et al., 2007 2007 ; Cihan et al., 2013 2013 ), neuronal function protection (Hashimoto et al., 2005 2005 ; Narita et al., 2009 2009 ; Hattori et al., 2010 2010 ; Zamani et al., 2012 2012 ), and immunoregulatory effects (Šver et al., 1996 1996 ; Oka et al., 2001 2001 ; Okamoto et al., 2003 2003 ; Majtán et al., 2006 2006 ; Mannoor et al., 2009 2009 ). Moreover, RJ enhances the reproductivity of many animals such as female sheep, rabbit (Husein and Kridli, 2002 2002 ), rat, quail, and hen (Shen et al., 2009 2009 ). Many studies have also found that RJ is effective in extending the lifespans of mice (Inoue et al., 2003 2003 ) and worms (Honda et al., 2011 2011 ). Ten proteins with a molecular weight of 49‒87 kDa have been reported in the MRJP family (Schmitzová et al., 1998 1998 ; Drapeau et al., 2006 2006 ). MRJP1, the most abundant component in the MRJP family, is the primary active substance in RJ (Kamakura, 2011 2011 ). MRJPs can stimulate DNA synthesis and increase albumin production in hepatocytes (Kamakura et al., 2001 2001 ). They also protect cells from apoptosis by the action of several important intracellular signaling factors (Kamakura, 2002 2002 ). MRJPs extracted from RJ can stimulate the proliferation of several cell lines, including hepatocytes (Kamakura et al., 2001 2001 ), human myeloid cell line U-937 (Watanabe et al., 1998 1998 ), human monocytes (Kimura et al., 2003 2003 ), and Tn-5B1-4 insect cells (Salazar-Olivo and Paz-González, 2005 2005 ). Our recent study suggests that MRJPs can increase mean lifespan and reproductivity in Drosophila (Xin et al., 2016 2016 ) and partially replace fetal bovine serum (FBS) for culturing several human cell lines and increase the cell viability (Chen et al., 2016 2016 ). The question of whether MRJPs have anti-senescence activity for human cells such as human embryonic lung fibroblast (HFL-I) cells remains. Therefore, in this study we examined the effects of MRJPs on the proliferation activity, population doubling level (PDL), senescence-associated β-galactosidase (SA-β-gal) activity, telomere length, expression of age-related genes, and Raman spectra of HFL-I cells. Materials and methods 1 Cell line and reagents 2 The HFL-I cell line was obtained from Shanghai Sixin Biotechnology Co., Ltd. (Shanghai, China). Primers were supplied by TaKaRa Co., Ltd. (Beijing, China). Mammalian DNA extraction and SA-β-gal activity kits were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Reverse transcription polymerase chain reaction (RT-PCR) and real-time quantitative PCR (qPCR) kits were supplied by TaKaRa Co., Ltd. (Beijing, China). A 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay kit was purchased from Nanjing Keygen Biotech. Co., Ltd. (Nanjing, China). FBS was purchased from HyClone Laboratories, Inc. (Logan, USA). F12K media were supplied by Gino Biotechnology Co., Ltd. (Hangzhou, China). All biochemical reagents were of the highest purity commercially available. Preparation of MRJPs 2 Fresh RJ was provided by the Hangzhou Biyuntian Heath Food Co., Ltd. (Hangzhou, China) and stored at −80 °C. For extraction, RJ was diluted in phosphate buffered saline (PBS) and extracted at 4 °C for 12 h. Then the extraction was centrifuged at 12 000 g for 30 min at 4 °C. MRJPs were obtained from the upper layer of the extraction, which was dialyzed against 500 volumes of double-distilled water (ddH 2 O) using a 1000 Da cutoff dialysis membrane at 4 °C (Salazar-Olivo and Paz-González, 2005 2005 ). The concentration of MRJPs was measured by the Bradford method using bovine serum albumin (BSA) as a standard (Bradford, 1976 1976 ). HFL-I cultivation 2 The HFL-I cell line was cultured in F12K medium supplied with 10% (v/v) FBS at 37 °C in a humid environment with 5% (v/v) CO 2 . MRJPs and BSA solution were diluted with F12K medium by 5 mg/ml, and added to media as calculated. Five treatments were prepared as follows: (A) no MRJPs, (B) 0.1 mg/ml MRJPs, (C) 0.2 mg/ml MRJPs, (D) 0.3 mg/ml MRJPs, and (E) 0.2 mg/ml BSA. Cells were exposed to the various concentrations of MRJPs or BSA and cultured until they reached cellular senescence. Morphological observations 2 Cells were observed every day and cell images were taken using an inverted microscope. The shape, size, and refractivity of HFL-I cells were considered as indicators to evaluate the condition of the cells. Cumulative population doubling level (PDL) measurements 2 Cell passage cultivations were taken when the cell confluence level reached 80%–90%. The population doubling (PD) index was calculated according to the equation: PD=(lg H −lg O )/0.301, where H means the number of harvested cells and O means the original number of cells. An increase in PD was added to the previous PD to determine the cumulative PDL. Cells were defined as senescent when cell confluence failed to reach 90% in four weeks. The amounts of harvested and original cells were counted using a cell counter plate. Cellular proliferation activity 2 The growth-promoting activity was measured by MTT assay (Yang et al., 2013 2013 ). Logarithmic growth phase cells (2×10 3 cells/ml) suspended in 200 μl media were inoculated into 96-well plates and cultured consecutively for 4 d. The absorbance of each well at the designated time point was measured every day at 490 nm wavelength after the MTT assay. The proliferation activity was shown by the absorbance. SA-β-gal activity 2 Logarithmic growth phase cells (2×10 3 cells/ml) suspended in 200 μl media were inoculated into 96-well plates. The SA-β-gal activity kit was used after the cells were completely adherent. SA-β-gal positive (blue-stained) cells were manually counted using an inverted microscope. SA-β-gal activity was expressed as the percentage of stained cells in the total cell population. Relative telomere length 2 qPCR was used to measure the telomere length of HFL-I cells. After the indicated treatments, total DNA was extracted from cells using the mammalian DNA extraction kit. qPCR was performed using SYBR green PCR core reagents. The following primers were used: 5'-CGTTTGTTTGGGTTTGGGTT TGGGTTTGGGTTTGGGTT-3' (forward) and 5'-GC TTGCCTTACCCTTACCCTTACCCTTACCCTTAC CCT-3' (reverse); RPLP0 : 5'-CAGCAAGTGGGAA GGTGTAATCC-3' (forward) and 5'-CCCATTCTA TCATCAACGGGTACAA-3' (reverse). The PCR products were obtained at 95 °C (30 s), followed by 40 cycles of 95 °C (5 s) and 60 °C (30 s). The ΔΔcycle threshold (ΔΔ C T ) method was used to quantify the data. RPLP0 was used as a reference gene. The relative expression (RE) of telomeres was expressed as fold change and calculated according to the equation: RE=2 −ΔΔ C T . Relative expression of age-related genes 2 The expression of four age-related genes was measured using qPCR. After the indicated treatments, total RNA was extracted from cells using the TRIzol method, and reverse translated to cDNA using the RT-PCR kit. qPCR was performed using SYBR green PCR core reagents. The primers were those listed in Table 1 1 . The PCR products were obtained at 95 °C (30 s), followed by 40 cycles of 95 °C (5 s) and 60 °C (30 s). The ΔΔ C T method was used to quantify the data and β-actin ( ACTB ) was used as a normalization gene. The RE of the four genes was also calculated according to the equation: RE=2 −ΔΔ C T . Gene symbol GenBank accession No. Direction PCR primer sequence (5' to 3') Primer size (bp) 2 mTOR 2 NM_004958.3 Forward AAACTGCACGTCAGCACCATC 21 Reverse AGCCGTCTCAGCCATTCCA 19 2 SOD1 2 NM_000454.4 Forward AGTGCAGGGCATCATCAATTTC 22 Reverse CCATGCAGGCCTTCAGTCAG 20 2 CTNNB1 2 NM_001098209.1 Forward TTAGCTGGTGGGCTGCAGAA 20 Reverse GGGTCCACCACTAGCCAGTATG 22 2 TP53 2 NM_001126112.2 Forward TCGAGATGTTCCGAGAGCTGAAT 23 Reverse GTCTGAGTCAGGCCCTTCTGTCTT 24 2 ACTB 2 NM_001101.3 Forward TGGCACCCAGCACAATGAA 19 Reverse CTAAGTCATAGTCCGCCTAGAAGCA 25 Raman spectrum analysis 2 With different frequencies of incident light, Raman spectroscopy can detect and analyze scattering spectra, molecular vibration, and rotation information from live cells. This method is often used as a diagnostic tool to study cancer cell lines or a single cancer cell (Yan et al., 2005 2005 ; Li et al., 2013 2013 ). It has been used to analyze the senescence of cells in vivo (Bai et al., 2015 2015 ). Here we used Raman spectroscopy to estimate the biochemical changes in HFL-I cells in media treated with different concentrations of MRJPs. After the culture media were removed, the cells were washed twice with PBS gently. Raman spectral data were obtained using a laser Raman microscope. An excitation laser of 785 nm with power of 50 mW was focused on a single cell each time through a 10×objective lens. Raman scattering light from the sample was collected and detected by a liquid nitrogen supplied charge coupled device (CCD) with a grating (600 gr/mm) under the visible region. Each cell was exposed to the laser for 3 s, and the spectrum of this targeted cell was drawn and analyzed using Microsoft Excel. The spectral resolution error was less than 5 cm −1 , and the diameter of the laser spot at the focus point was less than 1 μm. Statistical analysis 2 The data reported represent average values from at least three independent experiments and were analyzed using Statistical Product and Service Solutions (SPSS) software. One-way analysis of variance (ANOVA) was used to evaluate differences between each group. If there was a significant difference ( P < 0.05) between groups, Duncan analysis was used to compare all the groups in pairs. The values are shown as mean±standard deviation (SD), and P < 0.05 was considered statistically significant. Results 1 Morphological phenotypes of senescent cells 2 Cellular morphological observations showed that the manifestation of HFL-I cells was influenced by MRJPs. As shown in Fig. 1a 1a , most of the cells in medium A (no MRJPs) became flat, and the cellular boundary was obscure, a cellular phenotype typical of senescent cells. Many HFL-I cells displayed the unattached state, losing their spindle shapes. However, most cells in medium C (0.2 mg/ml MRJPs; Fig. 1c 1c ) and medium D (0.3 mg/ml MRJPs; Fig. 1d 1d ) had a healthy shape and normal size. The cell boundaries were distinct. The condition of the cells in medium B (0.1 mg/ml MRJPs; Fig. 1b 1b ) and medium E (0.2 mg/ml BSA; Fig. 1e 1e ) was similar, and some of the cells in both media were in a senescent state. Lifespan-extending effect of MRJPs 2 Fig. 2 2 shows that the average PDL of cells differed significantly among the treatments ( P < 0.001). Compared with the cells in medium A, the PDL of the cells in media B, C, D, and E increased by 12.4%, 31.2%, 24.0%, and 10.4%, respectively. Compared with the cells in medium E, the PDL of the cells in media B, C, and D increased by 1.8%, 18.8%, and 12.4%, respectively. The cells in medium C had the highest PDL, followed by the cells in media D, A, and E. Thus, a concentration of 0.2 mg/ml MRJPs (medium C) was the optimal dose for lifespan-extension of HFL-I cells. The PDL represents the lifespan of HFL-I. Data are expressed as mean±SD ( n =3). Different lowercase letters (a–e) above bars indicate a significant difference from each other at P < 0.05 by Duncan’s multiple range tests Proliferation effect of MRJPs 2 On the fourth day, the cells in media A, B, C, D, and E displayed differential cellular proliferation activity (absorbance): 0.39, 0.40, 0.45, 0.44, and 0.42, respectively (Fig. 3 3 ). Compared with the negative control (medium A), the relative proliferation activity (RPA) of the cells in media B, C, D, and E had increased by 1.0%, 11.9%, 11.1%, and 7.7%, respectively. In addition, the cells in medium B showed no significant difference ( P > 0.05) when compared with those in medium A after 4 d. However, the RPA of the cells in media C and D had increased significantly ( P < 0.001) compared with that of cells in medium A after 4 d. The proliferation activity represents the relative proliferation of HFL-I. Data are expressed as mean±SD ( n =8). Different lowercase letters (a–c) above bars within the same day indicate a significant difference from each other at P < 0.05 by Duncan’s multiple range test SA-β-gal activity measurement 2 The percentage of positive cells measured by SA-β-gal activity assay (Fig. 4 4 ) showed the extent of the senescence of HFL-I cells. The cells in medium C had the lowest percentage of positive cells (19.7%), followed by the cells in media D (41.0%) and B (33.7%). Cells in medium A had the highest percentage (63.7%), followed by those in medium E (43.7%). The percentage of positive cells in media B, C, D, and E had decreased by 47.1%, 69.1%, 35.6%, and 31.4% respectively, compared with that in medium A. Cells stained blue were defined as senescent. Data are expressed as mean±SD ( n =3). Different lowercase letters (a–d) above bars indicate a significant difference from each other at P < 0.05 by Duncan’s multiple range test Effect of MRJPs on telomere length 2 Fig. 5 5 shows the effects of supplemental MRJPs of different concentrations in media on the relative telomere length in HFL-I cells. The cells in medium A clearly exhibited the lowest relative length (RL). The RL of HFL-I cells in medium C was significantly different from the RLs of cells in the other media. The RLs of cells in all the media containing MRJPs were higher than those of media E and A. Compared with the RL of cells in medium A, the RLs of the cells in media B, C, D, and E increased by 6%, 50%, 10%, and 4%, respectively. Data are shown as fold-changes of the negative control group (medium A). Data are expressed as mean±SD ( n =3). Different lowercase letters (a–d) above bars indicate a significant difference from each other at P < 0.05 by Duncan’s multiple range test Effect of MRJPs on age-related gene expression 2 To understand the molecular mechanism underlying the anti-senescence effect of MRJPs on HFL-I cells, we measured the RE of mammalian target of rapamycin ( MTOR ), superoxide dismutase-1 ( SOD1 ), catenin beta like-1 ( CTNNB1 ), and tumor protein p53 ( TP53 ) in HFL-I cells (Fig. 6 6 ). Data are shown as fold-changes of the negative control group (medium A). Data are expressed as mean±SD ( n =3). Different lowercase letters (a–c) above bars within the same gene indicate a significant difference from each other at P < 0.05 by Duncan’s multiple range test Cells in medium C (0.02 mg/ml MRJPs) were used to evaluate the effect on gene expression exerted by MRJPs as they showed the longest lifespan. Cells in media A (no MRJPs) and E (0.02 mg/ml BSA) were used as negative and positive controls, respectively. Significant differences ( P < 0.05) in RE of four age-related genes were observed between cells in media C and A (negative control). Cells in medium C showed lower RE of MTOR , CTNNB1 , and TP53 than those in medium A, decreasing significantly ( P < 0.05) by 43%, 14%, and 42%, respectively. A significant increase ( P =0.0009) of about 86% in SOD1 expression was observed in cells in medium C compared with that in medium A. On the other hand, cells in medium C had a higher RE level of SOD1, and lower RE levels of MTOR and TP53 than those in medium E (positive control), while there was no significant difference in the RE of CTNNB1 ( P > 0.05). Compared with medium E, media supplemented with MRJPs showed stronger bioactivity than BSA to some extent. Raman spectrum analysis 2 To estimate the biochemical changes induced in HFL-I cells in media with different concentrations of MRJPs, we measured the mean spectra of treated cells normalized by the Raman peak of phenylalanine at 1002 cm −1 . The contributions of peaks, as identified by Li et al. ( 2013 2013 ) and Movasaghi et al. ( 2007 2007 ), are listed in Table 2 2 . The mean spectra from the different treatments were roughly similar (Fig. 7 7 ). However, bands at 1342 and 1536 cm −1 were detected only on the spectra of MRJP-treated groups and were assigned to purine (guanine and adenine) and amide carbonyl group vibrations and aromatic hydrogens, respectively. Substance changes as shown through Raman spectra. Each number in this figure represents one position of a band, as explained in Table 2 2 . a.u.: arbitrary unit The content references from Li et al. ( 2013 2013 ) and Movasaghi et al. ( 2007 2007 ) Raman shift (cm −1 ) Contribution  799 Backbone geometry and phosphate ion interactions  1002 Phenylalanine  1032 C–H/C–C stretching of phenylalanine  1155 C–C and C–N stretching of proteins  1195 Anti-symmetric phosphate vibrations  1205 Differences in collagen content  1342 Guanine and adenine  1451 CH 2 bending and CH 2 CH 3 deformation of proteins and collagens  1536 Amide carbonyl group vibrations and aromatic hydrogens  1584 C=C bending of phenylalanine  1603 C=C vibration of phenylalanine and tyrosine Discussion 1 In this study, we discovered that MRJPs may exert an anti-senescence effect on HFL-I cells. The cells in MRJP media experienced no or delayed typical cell aging, whereas cells developed senescence in MRJP-free media. The PDL assay showed that MRJPs enabled extension of the lifespan of HFL-I cells. The cells in MRJP media also had higher RPA, lower SA-β-gal activity, and longer telomere length than cells in MRJP-free media. In MRJP-supplied media, the expression of SOD1 increased and the expression of MTOR , CNNB1 , and TP53 decreased. In addition, Raman spectra analysis showed that peaks assigned to purine and amide carbonyl groups changed when MRJPs were added to the media. The HFL-I cell line has been widely used in studies investigating cell senescence, apoptosis, proliferation, growth factors, and toxicological tests. Matsuo ( 2004 2004 ) revealed that the lifespan of HFL-I cells is perturbed under oxidative stress. HFL-I is also used as a model organism in cell apoptosis tests (Xu et al., 2012 2012 ; Ren et al., 2013 2013 ; Chen et al., 2014 2014 ; Ershova et al., 2016 2016 ) and proliferation tests (Vietti et al., 2013 2013 ; Zeng et al., 2015 2015 ). We previously found that FBS culture media partially replaced by MRJPs could exert a proliferation effect on several cell lines, including the HFL-I cell line (Chen et al., 2016 2016 ). Cell senescence is followed by a series of phenomena, including morphological changes, the loss of proliferation ability, and an increase in SA-β-gal activity (Cristofalo and Pignolo, 1993 1993 ; Dimri et al., 1995 1995 ; Toussaint et al., 2000 2000 ; Liu et al., 2007 2007 ; Debacq-Chainiaux et al., 2008 2008 ; Hwang et al., 2009 2009 ). Morphological changes are widely used to identify senescent cells. One of the most apparent morphological hall-marks of senescent human diploid cells is that they usually have irregular flat and enlarged shapes (Cristofalo, 1988 1988 ; Cho et al., 2004 2004 ). Moreover, some senescent fibroblasts retract their branches resulting in a higher number of cells per unit area devoid of cell–cell contacts (Foyer et al., 2009 2009 ). In the morphological assay, senescence-related cellular morphologies partially disappeared after adding 0.2 or 0.3 mg/ml MRJPs. Thus, we discovered that MRJPs might have an anti-senescence effect on HFL-I cells. As for proliferation ability, the results of PDLs in this study imply that supplementation with MRJPs could significantly extend the mean lifespan of HFL-I cells. MTT assay after consecutive cultivation of HFL-I cells with or without different MRJPs concentrations also suggested that MRJPs might affect the proliferation of HFL-I cells. Cellular senescence occurs following cell proliferation failure or exposure to a variety of cellular stresses, e.g. expression of activated oncogenes, oxidation, or DNA damaging agents. Studies have pointed out that proliferation rate is an essential parameter to judge the senescence level of cells (Toussaint et al., 2000 2000 ) and that the SA-β-gal assay is a robust method to distinguish senescent cells from non-senescent cells in vitro (Itahana et al., 2004 2004 , 2007 2007 ; Krishnamurthy et al., 2004 2004 ; Liu et al., 2007 2007 ; Debacq-Chainiaux et al., 2009 2009 ). Our results showed that four-day incubation with addition of 0.2 or 0.3 mg/ml MRJPs significantly delayed the increase in senescent HFL-I cells, implying that MRJPs were likely to have promoted proliferation activity. From the results of SA-β-gal activity measurement, we found that the experimental MRJPs concentrations reduced the proportion of stained (senescent) cells and the best MRJP concentration was 0.2 mg/ml. Thus, MRJPs may play an important role in inhibiting the overexpression of β-galactosidase in HFL-I cells, and seem to exhibit an anti-senescence effect on HFL-I cells. Telomeres are specialized functional complexes located at the ends of eukaryotic chromosomes (Blackburn, 2001 2001 ). The shortening of telomeres could induce the DNA damage stress response and cell apoptosis. Without telomeres, terminal attrition of chromosomal DNA would lead to loss of genetic information and prevent cells from multiplying (Smogorzewska and de Lange, 2002 2002 ; D'Adda di Fagagna et al., 2003 2003 ; Takai et al., 2003 2003 ). Numerous studies have proved that telomeres shorten with the aging of cells and tissues (Allsopp et al., 1992 1992 ; Kitada et al., 1995 1995 ; Jeanclos et al., 1998 1998 ; Okuda et al., 2000 2000 ). These phenomena all support the hypothesis that telomere length limits cell mitosis (Harley, 1991 1991 ; Chan and Blackburn, 2004 2004 ; Debacq-Chainiaux et al., 2008 2008 ) and indicates the replicative potential and age of cells (Harley et al., 1990 1990 ; Toussaint et al., 2000 2000 ; von Zglinicki et al., 2005 2005 ). It has been reported that telomere length is positively associated with proliferative potential in fibroblasts (Allsopp et al., 1992 1992 ; Murillo-Ortiz et al., 2013 2013 ). The finding that cells in MRJP media have longer telomeres shows that MRJPs have the ability to inhibit the shortening of telomeres in HFL-I cells and proves the anti-senescence effect of MRJPs on HFL-I cells. Based on gene expression analysis of age-related genes and Raman spectroscopy analysis, we speculate that the regulation of age-related gene expression and the promotion of DNA and protein syntheses by MRJPs might be responsible for our observed results. MTOR , SOD1 , CTNNB1 , and TP53 are four age-related genes. MTOR plays a vital role in the mTOR/ S6K pathway. The mTOR/S6K signal pathway can be regulated by age-related factors such as stress, growth factors, nutritional conditions, and energy supplements (Sarbassov et al., 2005 2005 ; Kapahi et al., 2010 2010 ). Previous studies have indicated that the lifespan of yeasts, worms, fruit flies, and mice can be extended by inhibiting mTOR/S6K signal pathway through gene knockout, rapamycin treatment, or dietary restriction (Kapahi et al., 2010 2010 ). MRJPs may exert the anti-senescence effect through inhibiting the expression of mTOR and signal transduction in the mTOR/S6K pathway. The oxidative stress theory claims that the accumulation of oxidative damage induced by reactive oxygen species (ROS) will lead to senescence and apoptosis (Salmon et al., 2010 2010 ). However, overexpression of SOD1, an enzyme located mainly in the cytosol to remove highly toxic superoxide radicals, extends the lifespan of Drosophila , while sod1 knockdown causes a reduction of lifespan in mice and flies (Phillips et al., 1989 1989 ; Elchuri et al., 2005 2005 ; Landis and Tower, 2005 2005 ). Previous reports revealed that many age-related genes are involved in the oxidative stress response (Zahn and Kim, 2007 2007 ; Zhan et al., 2007 2007 ; Salmon et al., 2010 2010 ). Our results showed an overexpression of the SOD1 gene in the cells supplied with MRJP media. This finding supports the idea that MRJPs may protect HFL-I cells from oxidative damage through promoting the expression of the SOD1 gene and scavenging the ROS in vivo. β-Catenin encoded by the CTNNB1 gene is a signal molecule in the Wnt/β-catenin pathway. This pathway is one of the most essential signal transduction pathways in the growth process of vertebrate creatures. It has been proposed that the transduction level of the Wnt/β-catenin pathway increases with age (Brack et al., 2007 2007 ; Liu et al., 2007 2007 ; Xu et al., 2008 2008 ). Moreover, overexpression of the p53 protein is associated with overexpression of β-catenin (Damalas et al., 2001 2001 ) and the activation of the Wnt/β-catenin pathway (Mao et al., 2001 2001 ; Hiyama et al., 2010 2010 ). MRJPs might inhibit β-catenin through the down-regulation of CTNNB1 gene expression, and the Wnt/β-catenin pathway may be suppressed as a result of the lack of the signal molecule (β-catenin). p53, encoded by the TP53 gene, plays a vital role in cell cycle control, cell stress reaction, and cell gene protection (May and May, 1999 1999 ). p53 can be activated by cell stress factors such as genotoxicity, shortening of telomeres, hypoxia, and tumor gene activation (Kern et al., 1991 1991 ). Senescence is induced by the accumulation of ROS oxidative damage, which leads to cell cycle arrest caused by p21 and E2F (the target genes of p53 ) (Gewirtz et al., 2008 2008 ). Our findings suggest that MRJPs down-regulate the expression of the TP53 gene, which results in inhibition of p21 and lifespan extension of HFL-I cells. These results are consistent with the results above, showing that MRJPs induce a longer lifespan in HFL-I cells. Raman spectroscopy is a rapid and non-invasive technique, which enables us to analyze cells and tissues in vivo without additional labeling or staining (Buschman et al., 2000 2000 ; Swain et al., 2008 2008 ). Guanine and adenine are the materials of DNA replication. Amide carbonyl group and aromatic hydrogens are compounds of protein synthesis. From changes in peaks representing purine bases and amide carbonyl groups in the Raman spectroscopy assay, we inferred that MRJPs might promote the syntheses of DNA and proteins in HFL-I cells. Our findings are coincident with previous studies which suggested that MRJPs stimulate hepatocyte DNA synthesis, prolong the proliferation of hepatocytes, and increase albumin production (Kamakura et al., 2001 2001 ; Kamakura, 2002 2002 ). However, since only small peaks representing purine bases and amide carbonyl groups were observed, they might also represent products from catabolism. So in the future, more studies are needed to prove whether these changes are related to DNA and protein syntheses. In general, we successfully used Raman spectroscopy to explore differences between cells growing in media with or without MRJPs. Our results might provide a new method to characterize cellular senescence. Conclusions 1 We found that MRJPs could extend the lifespan of HFL-I cells when supplied in media. The anti-senescence effects of MRJPs were associated with up-regulation of SOD1 and down-regulations of MTOR , CTNNB1 , and TP53 , and had a stimulatory effect on DNA and protein syntheses. The anti-senescence effect of MRJPs on the HFL-I cell line may indicate a beneficial effect of MRJPs on human health. References RC Allsopp , H Vaziri , C Patterson , 等 . Telomere length predicts replicative capacity of human fibroblasts . Proc Natl Acad Sci USA , 1992 . 89 ( 21 ): 10114 - 10118 . DOI: 10.1073/pnas.89.21.10114 http://doi.org/10.1073/pnas.89.21.10114 . Telomere length predicts replicative capacity of human fibroblasts H Bai , HY Li , ZB Han , 等 . Label-free assessment of replicative senescence in mesenchymal stem cells by Raman microspectroscopy . Biomed Opt Express , 2015 . 6 ( 11 ): 4493 - 4500 . 26601012 DOI: 10.1364/BOE.6.004493 http://doi.org/10.1364/BOE.6.004493 . Label-free assessment of replicative senescence in mesenchymal stem cells by Raman microspectroscopy EH Blackburn . Switching and signaling at the telomere . Cell , 2001 . 106 ( 6 ): 661 - 673 . 11572773 DOI: 10.1016/S0092-8674(01)00492-5 http://doi.org/10.1016/S0092-8674(01)00492-5 . Switching and signaling at the telomere AS Brack , MJ Conboy , S Roy , 等 . Increased Wnt signaling during aging alters muscle stem cell fate and increases fibrosis . Science , 2007 . 317 ( 5839 ): 807 - 810 . 17690295 DOI: 10.1126/science.1144090 http://doi.org/10.1126/science.1144090 . Increased Wnt signaling during aging alters muscle stem cell fate and increases fibrosis MM Bradford . A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding . Anal Biochem , 1976 . 72 ( 1-2 ): 248 - 254 . 942051 DOI: 10.1016/0003-2697(76)90527-3 http://doi.org/10.1016/0003-2697(76)90527-3 . A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding HP Buschman , ET Marple , ML Wach , 等 . In vivo determination of the molecular composition of artery wall by intravascular Raman spectroscopy . Anal Chem , 2000 . 72 ( 16 ): 3771 - 3775 . 10959962 DOI: 10.1021/ac000298b http://doi.org/10.1021/ac000298b . In vivo determination of the molecular composition of artery wall by intravascular Raman spectroscopy A Buttstedt , RFA Moritz , S Erler . Origin and function of the major royal jelly proteins of the honeybee ( Apis mellifera ) as members of the yellow gene family . Biol Rev , 2014 . 89 ( 2 ): 255 - 269 . 23855350 DOI: 10.1111/brv.12052 http://doi.org/10.1111/brv.12052 . Origin and function of the major royal jelly proteins of the honeybee (Apis mellifera) as members of the yellow gene family SRWL Chan , EH Blackburn . Telomeres and telomerase . Philos Trans Roy Soc B Biol Sci , 2004 . 359 ( 1441 ): 109 - 122 . DOI: 10.1098/rstb.2003.1370 http://doi.org/10.1098/rstb.2003.1370 . Telomeres and telomerase D Chen , XX Xin , HC Qian , 等 . Evaluation of the major royal jelly proteins as an alternative to fetal bovine serum in culturing human cell lines . J Zhejiang Univ-Sci B (Biomed & Biotechnol) , 2016 . 17 ( 6 ): 476 - 483 . DOI: 10.1631/jzus.B1500295 http://doi.org/10.1631/jzus.B1500295 . Evaluation of the major royal jelly proteins as an alternative to fetal bovine serum in culturing human cell lines JP Chen , DG Xu , XY Yu , 等 . Discrepancy between the effects of morronside on apoptosis in human embryonic lung fibroblast cells and lung cancer A549 cells . Oncol Lett , 2014 . 7 ( 4 ): 927 - 932 . 24944645 DOI: 10.3892/ol.2014.1850 http://doi.org/10.3892/ol.2014.1850 . Discrepancy between the effects of morronside on apoptosis in human embryonic lung fibroblast cells and lung cancer A549 cells KA Cho , SJ Ryu , YS Oh , 等 . Morphological adjustment of senescent cells by modulating caveolin-1 status . J Biol Chem , 2004 . 279 ( 40 ): 42270 - 42278 . 15263006 DOI: 10.1074/jbc.M402352200 http://doi.org/10.1074/jbc.M402352200 . Morphological adjustment of senescent cells by modulating caveolin-1 status YB Cihan , A Ozturk , SS Gokalp . Protective role of royal jelly against radiation-induced oxidative stress in rats . UHOD-Uluslar Hematol , 2013 . 23 ( 2 ): 79 - 87 . DOI: 10.4999/uhod.11016 http://doi.org/10.4999/uhod.11016 . Protective role of royal jelly against radiation-induced oxidative stress in rats VJ Cristofalo . Cellular biomarkers of aging . Exp Gerontol , 1988 . 23 ( 4-5 ): 297 - 305 . 3197781 DOI: 10.1016/0531-5565(88)90032-0 http://doi.org/10.1016/0531-5565(88)90032-0 . Cellular biomarkers of aging VJ Cristofalo , RJ Pignolo . Replicative senescence of human fibroblast-like cells in culture . Physiol Rev , 1993 . 73 ( 3 ): 617 - 638 . 8332640 DOI: 10.1152/physrev.1993.73.3.617 http://doi.org/10.1152/physrev.1993.73.3.617 . Replicative senescence of human fibroblast-like cells in culture F D'Adda di Fagagna , PM Reaper , L Clay-Farrace , 等 . A DNA damage checkpoint response in telomere-initiated senescence . Nature , 2003 . 426 ( 6963 ): 194 - 198 . 14608368 DOI: 10.1038/nature02118 http://doi.org/10.1038/nature02118 . A DNA damage checkpoint response in telomere-initiated senescence A Damalas , S Kahan , M Shtutman , 等 . Deregulated β-catenin induces a p53-and ARF-dependent growth arrest and cooperates with Ras in transformation . EMBO J , 2001 . 20 ( 17 ): 4912 - 4922 . 11532955 DOI: 10.1093/emboj/20.17.4912 http://doi.org/10.1093/emboj/20.17.4912 . Deregulated β-catenin induces a p53-and ARF-dependent growth arrest and cooperates with Ras in transformation F Debacq-Chainiaux , T Pascal , E Boilan , 等 . Screening of senescence-associated genes with specific DNA array reveals the role of IGFBP-3 in premature senescence of human diploid fibroblasts . Free Radic Biol Med , 2008 . 44 ( 10 ): 1817 - 1832 . 18329388 DOI: 10.1016/j.freeradbiomed.2008.02.001 http://doi.org/10.1016/j.freeradbiomed.2008.02.001 . Screening of senescence-associated genes with specific DNA array reveals the role of IGFBP-3 in premature senescence of human diploid fibroblasts F Debacq-Chainiaux , JD Erusalimsky , J Campisi , 等 . Protocols to detect senescence-associated beta-galactosidase (SA-βgal) activity, a biomarker of senescent cells in culture and in vivo . Nat Protoc , 2009 . 4 ( 12 ): 1798 - 1806 . 20010931 DOI: 10.1038/nprot.2009.191 http://doi.org/10.1038/nprot.2009.191 . Protocols to detect senescence-associated beta-galactosidase (SA-βgal) activity, a biomarker of senescent cells in culture and in vivo GP Dimri , X Lee , G Basile , 等 . A biomarker that identifies senescent human cells in culture and in aging skin in vivo . Proc Natl Acad Sci USA , 1995 . 92 ( 20 ): 9363 - 9367 . DOI: 10.1073/pnas.92.20.9363 http://doi.org/10.1073/pnas.92.20.9363 . A biomarker that identifies senescent human cells in culture and in aging skin in vivo MD Drapeau , S Albert , R Kucharski , 等 . Evolution of the Yellow/Major Royal Jelly Protein family and the emergence of social behavior in honey bees . Genome Res , 2006 . 16 ( 11 ): 1385 - 1394 . 17065613 DOI: 10.1101/gr.5012006 http://doi.org/10.1101/gr.5012006 . Evolution of the Yellow/Major Royal Jelly Protein family and the emergence of social behavior in honey bees S Elchuri , TD Oberley , WB Qi , 等 . CuZnSOD deficiency leads to persistent and widespread oxidative damage and hepatocarcinogenesis later in life . Oncogene , 2005 . 24 ( 3 ): 367 - 380 . 15531919 DOI: 10.1038/sj.onc.1208207 http://doi.org/10.1038/sj.onc.1208207 . CuZnSOD deficiency leads to persistent and widespread oxidative damage and hepatocarcinogenesis later in life ES Ershova , VA Sergeeva , AI Chausheva , 等 . Toxic and DNA damaging efforts of a functionalized fullerene in human embryonic lung fibroblasts . Mutat Res-Genet Toxicol Environ Mutagen , 2016 . 805 46 - 57 . 27402482 DOI: 10.1016/j.mrgentox.2016.05.004 http://doi.org/10.1016/j.mrgentox.2016.05.004 . Toxic and DNA damaging efforts of a functionalized fullerene in human embryonic lung fibroblasts CH Foyer , R Faragher , P Thornalley . Redox Metabolism and Longevity Relationships in Animals and Plants , 2009 . Taylor & Francis Group, New York, USA , . Redox Metabolism and Longevity Relationships in Animals and Plants DA Gewirtz , SE Holt , LW Elmore . Accelerated senescence: an emerging role in tumor cell response to chemotherapy and radiation . Biochem Pharmacol , 2008 . 76 ( 8 ): 947 - 957 . 18657518 DOI: 10.1016/j.bcp.2008.06.024 http://doi.org/10.1016/j.bcp.2008.06.024 . Accelerated senescence: an emerging role in tumor cell response to chemotherapy and radiation CB Harley . Telomere loss: mitotic clock or genetic time bomb? . Mutat Res , 1991 . 256 ( 2-6 ): 271 - 282 . 1722017 DOI: 10.1016/0921-8734(91)90018-7 http://doi.org/10.1016/0921-8734(91)90018-7 . Telomere loss: mitotic clock or genetic time bomb? CB Harley , AB Futcher , CW Greider . Telomeres shorten during ageing of human fibroblasts . Nature , 1990 . 345 ( 6274 ): 458 - 460 . 2342578 DOI: 10.1038/345458a0 http://doi.org/10.1038/345458a0 . Telomeres shorten during ageing of human fibroblasts M Hashimoto , M Kanda , K Ikeno , 等 . Oral administration of royal jelly facilitates mRNA expression of glial cell line-derived neurotrophic factor and neurofilament H in the hippocampus of the adult mouse brain . Biosci Biotechnol Biochem , 2005 . 69 ( 4 ): 800 - 805 . 15849420 DOI: 10.1271/bbb.69.800 http://doi.org/10.1271/bbb.69.800 . Oral administration of royal jelly facilitates mRNA expression of glial cell line-derived neurotrophic factor and neurofilament H in the hippocampus of the adult mouse brain N Hattori , S Ohta , T Sakamoto , 等 . Royal jelly facilitates restoration of the cognitive ability in trimethyltin-intoxicated mice . Evid-Based Complement Alternat Med, 2011:165968 , 2010 . DOI: 10.1093/ecam/nep029 http://doi.org/10.1093/ecam/nep029 . Royal jelly facilitates restoration of the cognitive ability in trimethyltin-intoxicated mice A Hiyama , D Sakai , MV Risbud , 等 . Enhancement of intervertebral disc cell senescence by Wnt/β-catenin signaling-induced matrix metalloproteinase expression . Arthritis Rheum , 2010 . 62 ( 10 ): 3036 - 3047 . 20533544 DOI: 10.1002/art.27599 http://doi.org/10.1002/art.27599 . Enhancement of intervertebral disc cell senescence by Wnt/β-catenin signaling-induced matrix metalloproteinase expression Y Honda , Y Fujita , H Maruyama , 等 . Lifespan-extending effects of royal jelly and its related substances on the nematode Caenorhabditis elegans . PLoS ONE , 2011 . 6 ( 8 ): e23527 21858156 DOI: 10.1371/journal.pone.0023527 http://doi.org/10.1371/journal.pone.0023527 . Lifespan-extending effects of royal jelly and its related substances on the nematode Caenorhabditis elegans MQ Husein , RT Kridli . Reproductive responses following royal jelly treatment administered orally or intramuscularly into progesterone-treated Awassi ewes . Anim Reprod Sci , 2002 . 74 ( 1-2 ): 45 - 53 . 12379374 DOI: 10.1016/S0378-4320(02)00165-3 http://doi.org/10.1016/S0378-4320(02)00165-3 . Reproductive responses following royal jelly treatment administered orally or intramuscularly into progesterone-treated Awassi ewes ES Hwang , G Yoon , HT Kang . A comparative analysis of the cell biology of senescence and aging . Cell Mol Life Sci , 2009 . 66 ( 15 ): 2503 - 2524 . 19421842 DOI: 10.1007/s00018-009-0034-2 http://doi.org/10.1007/s00018-009-0034-2 . A comparative analysis of the cell biology of senescence and aging SI Inoue , S Koya-Miyata , S Ushio , 等 . Royal jelly prolongs the life span of C3H/HeJ mice: correlation with reduced DNA damage . Exp Gerontol , 2003 . 38 ( 9 ): 965 - 969 . 12954483 DOI: 10.1016/S0531-5565(03)00165-7 http://doi.org/10.1016/S0531-5565(03)00165-7 . Royal jelly prolongs the life span of C3H/HeJ mice: correlation with reduced DNA damage K Itahana , J Campisi , GP Dimri . Mechanisms of cellular senescence in human and mouse cells . Biogerontology , 2004 . 5 ( 1 ): 1 - 10 . 15138376 DOI: 10.1023/B:BGEN.0000017682.96395.10 http://doi.org/10.1023/B:BGEN.0000017682.96395.10 . Mechanisms of cellular senescence in human and mouse cells K Itahana , J Campisi , GP Dimri . Methods to detect biomarkers of cellular senescence , 2007 . In: Tollefsbol TO (Ed.), Biological Aging: Methods and Protocols. Humana Press, Totowa , 21 - 31 . DOI: 10.1007/978-1-59745-361-5_3 http://doi.org/10.1007/978-1-59745-361-5_3 . Methods to detect biomarkers of cellular senescence P Jamnik , D Goranovič , P Raspor . Antioxidative action of royal jelly in the yeast cell . Exp Gerontol , 2007 . 42 ( 7 ): 594 - 600 . 17383134 DOI: 10.1016/j.exger.2007.02.002 http://doi.org/10.1016/j.exger.2007.02.002 . Antioxidative action of royal jelly in the yeast cell E Jeanclos , A Krolewski , J Skurnick , 等 . Shortened telomere length in white blood cells of patients with IDDM . Diabetes , 1998 . 47 ( 3 ): 482 - 486 . 9519758 DOI: 10.2337/diabetes.47.3.482 http://doi.org/10.2337/diabetes.47.3.482 . Shortened telomere length in white blood cells of patients with IDDM M Kamakura . Signal transduction mechanism leading to enhanced proliferation of primary cultured adult rat hepatocytes treated with royal jelly 57-kDa protein . J Biochem , 2002 . 132 ( 6 ): 911 - 919 . 12473193 DOI: 10.1093/oxfordjournals.jbchem.a003304 http://doi.org/10.1093/oxfordjournals.jbchem.a003304 . Signal transduction mechanism leading to enhanced proliferation of primary cultured adult rat hepatocytes treated with royal jelly 57-kDa protein M Kamakura . Royalactin induces queen differentiation in honeybees . Nature , 2011 . 473 ( 7348 ): 478 - 483 . 21516106 DOI: 10.1038/nature10093 http://doi.org/10.1038/nature10093 . Royalactin induces queen differentiation in honeybees M Kamakura , N Suenobu , M Fukushima . Fifty-seven-kDa protein in royal jelly enhances proliferation of primary cultured rat hepatocytes and increases albumin production in the absence of serum . Biochem Biophys Res Commun , 2001 . 282 ( 4 ): 865 - 874 . 11352630 DOI: 10.1006/bbrc.2001.4656 http://doi.org/10.1006/bbrc.2001.4656 . Fifty-seven-kDa protein in royal jelly enhances proliferation of primary cultured rat hepatocytes and increases albumin production in the absence of serum P Kapahi , D Chen , AN Rogers , 等 . With TOR, less is more: a key role for the conserved nutrient-sensing TOR pathway in aging . Cell Metab , 2010 . 11 ( 6 ): 453 - 465 . 20519118 DOI: 10.1016/j.cmet.2010.05.001 http://doi.org/10.1016/j.cmet.2010.05.001 . With TOR, less is more: a key role for the conserved nutrient-sensing TOR pathway in aging SE Kern , KW Kinzler , A Bruskin , 等 . Identification of p53 as a sequence-specific DNA-binding protein . Science , 1991 . 252 ( 5013 ): 1708 - 1711 . 2047879 DOI: 10.1126/science.2047879 http://doi.org/10.1126/science.2047879 . Identification of p53 as a sequence-specific DNA-binding protein M Kimura , Y Kimura , K Tsumura , 等 . 350-kDa royal jelly glycoprotein (apisin), which stimulates proliferation of human monocytes, bears the β1-3galactosylated N -glycan: analysis of the N -glycosylation site . Biosci Biotechnol Biochem , 2003 . 67 ( 9 ): 2055 - 2058 . 14520005 DOI: 10.1271/bbb.67.2055 http://doi.org/10.1271/bbb.67.2055 . 350-kDa royal jelly glycoprotein (apisin), which stimulates proliferation of human monocytes, bears the β1-3galactosylated N-glycan: analysis of the N-glycosylation site T Kitada , S Seki , N Kawakita , 等 . Telomere shortening in chronic liver diseases . Biochem Biophys Res Commun , 1995 . 211 ( 1 ): 33 - 39 . 7779103 DOI: 10.1006/bbrc.1995.1774 http://doi.org/10.1006/bbrc.1995.1774 . Telomere shortening in chronic liver diseases J Krishnamurthy , C Torrice , MR Ramsey , 等 . Ink4a/Arf expression is a biomarker of aging . J Clin Invest , 2004 . 114 ( 9 ): 1299 - 1307 . 15520862 DOI: 10.1172/JCI22475 http://doi.org/10.1172/JCI22475 . Ink4a/Arf expression is a biomarker of aging GN Landis , J Tower . Superoxide dismutase evolution and life span regulation . Mech Ageing Dev , 2005 . 126 ( 3 ): 365 - 379 . 15664623 DOI: 10.1016/j.mad.2004.08.012 http://doi.org/10.1016/j.mad.2004.08.012 . Superoxide dismutase evolution and life span regulation ZF Li , Y Chen , YZ Li , 等 . Raman microspectroscopy as a diagnostic tool to study single living nasopharyngeal carcinoma cell lines . Biochem Cell Biol , 2013 . 91 ( 3 ): 182 - 186 . 23668791 DOI: 10.1139/bcb-2012-0024 http://doi.org/10.1139/bcb-2012-0024 . Raman microspectroscopy as a diagnostic tool to study single living nasopharyngeal carcinoma cell lines HJ Liu , MM Fergusson , RM Castilho , 等 . Augmented Wnt signaling in a mammalian model of accelerated aging . Science , 2007 . 317 ( 5839 ): 803 - 806 . 17690294 DOI: 10.1126/science.1143578 http://doi.org/10.1126/science.1143578 . Augmented Wnt signaling in a mammalian model of accelerated aging J Majtán , E Kováčová , K Bíliková , 等 . The immunostimulatory effect of the recombinant apalbumin 1–major honeybee royal jelly protein–on TNFα release . Int Immunopharmacol , 2006 . 6 ( 2 ): 269 - 278 . 16399632 DOI: 10.1016/j.intimp.2005.08.014 http://doi.org/10.1016/j.intimp.2005.08.014 . The immunostimulatory effect of the recombinant apalbumin 1–major honeybee royal jelly protein–on TNFα release B Malecová , J Ramser , JK O'Brien , 等 . Honeybee ( Apis mellifera L.) mrjp gene family: computational analysis of putative promoters and genomic structure of mrjp1 , the gene coding for the most abundant protein of larval food . Gene , 2003 . 303 165 - 175 . 12559578 DOI: 10.1016/S0378-1119(02)01174-5 http://doi.org/10.1016/S0378-1119(02)01174-5 . Honeybee (Apis mellifera L.) mrjp gene family: computational analysis of putative promoters and genomic structure of mrjp1, the gene coding for the most abundant protein of larval food MK Mannoor , I Shimabukuro , M Tsukamotoa , 等 . Honeybee royal jelly inhibits autoimmunity in SLE-prone NZB×NZW F1 mice . Lupus , 2009 . 18 ( 1 ): 44 - 52 . 19074168 DOI: 10.1177/0961203308094765 http://doi.org/10.1177/0961203308094765 . Honeybee royal jelly inhibits autoimmunity in SLE-prone NZB×NZW F1 mice CD Mao , P Hoang , PE DiCorleto . Lithium inhibits cell cycle progression and induces stabilization of p53 in bovine aortic endothelial cells . J Biol Chem , 2001 . 276 ( 28 ): 26180 - 26188 . 11337498 DOI: 10.1074/jbc.M101188200 http://doi.org/10.1074/jbc.M101188200 . Lithium inhibits cell cycle progression and induces stabilization of p53 in bovine aortic endothelial cells M Matsuo . Aging and oxidative stress resistance in human fibroblasts . J Clin Biochem Nutr , 2004 . 35 ( 2 ): 63 - 70 . DOI: 10.3164/jcbn.35.63 http://doi.org/10.3164/jcbn.35.63 . Aging and oxidative stress resistance in human fibroblasts P May , E May . Twenty years of p53 research: structural and functional aspects of the p53 protein . Oncogene , 1999 . 18 ( 53 ): 7621 - 7636 . 10618702 DOI: 10.1038/sj.onc.1203285 http://doi.org/10.1038/sj.onc.1203285 . Twenty years of p53 research: structural and functional aspects of the p53 protein Z Movasaghi , S Rehman , IU Rehman . Raman spectroscopy of biological tissues . Appl Spectrosc Rev , 2007 . 42 ( 5 ): 493 - 541 . DOI: 10.1080/05704920701551530 http://doi.org/10.1080/05704920701551530 . Raman spectroscopy of biological tissues B Murillo-Ortiz , F Albarrán-Tamayo , S López-Briones , 等 . Increased telomere length and proliferative potential in peripheral blood mononuclear cells of adults of different ages stimulated with concanavalin A . BMC Geriatr, 13:99 , 2013 . DOI: 10.1186/1471-2318-13-99 http://doi.org/10.1186/1471-2318-13-99 . Increased telomere length and proliferative potential in peripheral blood mononuclear cells of adults of different ages stimulated with concanavalin A Y Narita , S Ohta , KM Suzuki , 等 . Effects of long-term administration of royal jelly on pituitary weight and gene expression in middle-aged female rats . Biosci Biotechnol Biochem , 2009 . 73 ( 2 ): 431 - 433 . 19202272 DOI: 10.1271/bbb.80556 http://doi.org/10.1271/bbb.80556 . Effects of long-term administration of royal jelly on pituitary weight and gene expression in middle-aged female rats H Oka , Y Emori , N Kobayashi , 等 . Suppression of allergic reactions by royal jelly in association with the restoration of macrophage function and the improvement of Th1/Th2 cell responses . Int Immunopharmacol , 2001 . 1 ( 3 ): 521 - 532 . 11367535 DOI: 10.1016/S1567-5769(00)00007-2 http://doi.org/10.1016/S1567-5769(00)00007-2 . Suppression of allergic reactions by royal jelly in association with the restoration of macrophage function and the improvement of Th1/Th2 cell responses I Okamoto , Y Taniguchi , T Kunikata , 等 . Major royal jelly protein 3 modulates immune responses in vitro and in vivo . Life Sci , 2003 . 73 ( 16 ): 2029 - 2045 . 12899927 DOI: 10.1016/S0024-3205(03)00562-9 http://doi.org/10.1016/S0024-3205(03)00562-9 . Major royal jelly protein 3 modulates immune responses in vitro and in vivo K Okuda , MY Khan , J Skurnick , 等 . Telomere attrition of the human abdominal aorta: relationships with age and atherosclerosis . Atherosclerosis , 2000 . 152 ( 2 ): 391 - 398 . 10998467 DOI: 10.1016/S0021-9150(99)00482-7 http://doi.org/10.1016/S0021-9150(99)00482-7 . Telomere attrition of the human abdominal aorta: relationships with age and atherosclerosis JP Phillips , SD Campbell , D Michaud , 等 . Null mutation of copper/zinc superoxide dismutase in Drosophila confers hypersensitivity to paraquat and reduced longevity . Proc Natl Acad Sci USA , 1989 . 86 ( 8 ): 2761 - 2765 . DOI: 10.1073/pnas.86.8.2761 http://doi.org/10.1073/pnas.86.8.2761 . Null mutation of copper/zinc superoxide dismutase in Drosophila confers hypersensitivity to paraquat and reduced longevity SC Remolina , KA Hughes . Evolution and mechanisms of long life and high fertility in queen honey bees . Age , 2008 . 30 ( 2-3 ): 177 - 185 . 19424867 DOI: 10.1007/s11357-008-9061-4 http://doi.org/10.1007/s11357-008-9061-4 . Evolution and mechanisms of long life and high fertility in queen honey bees Q Ren , HS Zeng , XF Zeng . Leflunomide inhibits the apoptosis of human embryonic lung fibroblasts infected by human cytomegalovirus . Eur J Med Res , 2013 . 18 ( 1 ): 3 23369524 DOI: 10.1186/2047-783X-18-3 http://doi.org/10.1186/2047-783X-18-3 . Leflunomide inhibits the apoptosis of human embryonic lung fibroblasts infected by human cytomegalovirus LA Salazar-Olivo , V Paz-González . Screening of biological activities present in honeybee ( Apis mellifera ) royal jelly . Toxicol In Vitro , 2005 . 19 ( 5 ): 645 - 651 . 15893447 DOI: 10.1016/j.tiv.2005.03.001 http://doi.org/10.1016/j.tiv.2005.03.001 . Screening of biological activities present in honeybee (Apis mellifera) royal jelly AB Salmon , A Richardson , VI Pérez . Update on the oxidative stress theory of aging: does oxidative stress play a role in aging or healthy aging? . Free Radic Biol Med , 2010 . 48 ( 5 ): 642 - 655 . 20036736 DOI: 10.1016/j.freeradbiomed.2009.12.015 http://doi.org/10.1016/j.freeradbiomed.2009.12.015 . Update on the oxidative stress theory of aging: does oxidative stress play a role in aging or healthy aging? DD Sarbassov , SM Ali , DM Sabatini . Growing roles for the mTOR pathway . Curr Opin Cell Biol , 2005 . 17 ( 6 ): 596 - 603 . 16226444 DOI: 10.1016/j.ceb.2005.09.009 http://doi.org/10.1016/j.ceb.2005.09.009 . Growing roles for the mTOR pathway J Schmitzová , J Klaudiny , Š Albert , 等 . A family of major royal jelly proteins of the honeybee Apis mellifera L . Cell Mol Life Sci , 1998 . 54 ( 9 ): 1020 - 1030 . 9791542 DOI: 10.1007/s000180050229 http://doi.org/10.1007/s000180050229 . A family of major royal jelly proteins of the honeybee Apis mellifera L LR Shen , LW Zhang , MH Ding , 等 . Research progress on nutritional and health-care functions and Molecular mechanism of royal Jelly . J Agric Sci Technol , 2009 . 11 ( 4 ): 41 - 47 . (in Chinese) DOI: 10.3969/j.issn.1008-0864.2009.04.007 http://doi.org/10.3969/j.issn.1008-0864.2009.04.007 . Research progress on nutritional and health-care functions and Molecular mechanism of royal Jelly LR Shen , WG Zhang , F Jin , 等 . Expression of recombinant AccMRJP1 protein from royal jelly of Chinese Honeybee in Pichia pastoris and its proliferation activity in an insect cell line . J Agric Food Chem , 2010 . 58 ( 16 ): 9190 - 9197 . 23654243 DOI: 10.1021/jf1007133 http://doi.org/10.1021/jf1007133 . Expression of recombinant AccMRJP1 protein from royal jelly of Chinese Honeybee in Pichia pastoris and its proliferation activity in an insect cell line A Smogorzewska , T de Lange . Different telomere damage signaling pathways in human and mouse cells . EMBO J , 2002 . 21 ( 16 ): 4338 - 4348 . 12169636 DOI: 10.1093/emboj/cdf433 http://doi.org/10.1093/emboj/cdf433 . Different telomere damage signaling pathways in human and mouse cells L Šver , N Oršolić , Z Tadić , 等 . A royal jelly as a new potential immunomodulator in rats and mice . Comp Immunol Microbiol Infect Dis , 1996 . 19 ( 1 ): 31 - 38 . 8654043 DOI: 10.1016/0147-9571(95)00020-8 http://doi.org/10.1016/0147-9571(95)00020-8 . A royal jelly as a new potential immunomodulator in rats and mice RJ Swain , G Jell , MM Stevens . Non-invasive analysis of cell cycle dynamics in single living cells with Raman micro-spectroscopy . J Cell Biochem , 2008 . 104 ( 4 ): 1427 - 1438 . 18348254 DOI: 10.1002/jcb.21720 http://doi.org/10.1002/jcb.21720 . Non-invasive analysis of cell cycle dynamics in single living cells with Raman micro-spectroscopy H Takai , A Smogorzewska , T de Lange . DNA damage foci at dysfunctional telomeres . Curr Biol , 2003 . 13 ( 17 ): 1549 - 1556 . 12956959 DOI: 10.1016/S0960-9822(03)00542-6 http://doi.org/10.1016/S0960-9822(03)00542-6 . DNA damage foci at dysfunctional telomeres O Toussaint , EE Medrano , T von Zglinicki . Cellular and molecular mechanisms of stress-induced premature senescence (SIPS) of human diploid fibroblasts and melanocytes . Exp Gerontol , 2000 . 35 ( 8 ): 927 - 945 . 11121681 DOI: 10.1016/S0531-5565(00)00180-7 http://doi.org/10.1016/S0531-5565(00)00180-7 . Cellular and molecular mechanisms of stress-induced premature senescence (SIPS) of human diploid fibroblasts and melanocytes G Vietti , S Ibouraadaten , M Palmai-Pallag , 等 . Towards predicting the lung fibrogenic activity of nomaterials: experimental validation of an in vitro fibroblast proliferation assay . Part Fibre Toxicol, 10:52 , 2013 . DOI: 10.1186/1743-8977-10-52 http://doi.org/10.1186/1743-8977-10-52 . Towards predicting the lung fibrogenic activity of nomaterials: experimental validation of an in vitro fibroblast proliferation assay T von Zglinicki , G Saretzki , J Ladhoff , 等 . Human cell senescence as a DNA damage response . Mech Ageing Dev , 2005 . 126 ( 1 ): 111 - 117 . 15610769 DOI: 10.1016/j.mad.2004.09.034 http://doi.org/10.1016/j.mad.2004.09.034 . Human cell senescence as a DNA damage response K Watanabe , H Shinmoto , M Kobori , 等 . Stimulation of cell growth in the U-937 human myeloid cell line by honey royal jelly protein . Cytotechnology , 1998 . 26 ( 1 ): 23 - 27 . 22359003 DOI: 10.1023/A:1007928408128 http://doi.org/10.1023/A:1007928408128 . Stimulation of cell growth in the U-937 human myeloid cell line by honey royal jelly protein XX Xin , Y Chen , D Chen , 等 . Supplementation with major royal-jelly proteins increases lifespan, feeding, and fecundity in Drosophila . J Agric Food Chem , 2016 . 64 ( 29 ): 5803 - 5812 . 27388939 DOI: 10.1021/acs.jafc.6b00514 http://doi.org/10.1021/acs.jafc.6b00514 . Supplementation with major royal-jelly proteins increases lifespan, feeding, and fecundity in Drosophila M Xu , Q Yu , R Subrahmanyam , 等 . β-Catenin expression results in p53-independent DNA damage and oncogene-induced senescence in prelymphomagenic thymocytes in vivo . Mol Cell Biol , 2008 . 28 ( 5 ): 1713 - 1723 . 18160717 DOI: 10.1128/MCB.01360-07 http://doi.org/10.1128/MCB.01360-07 . β-Catenin expression results in p53-independent DNA damage and oncogene-induced senescence in prelymphomagenic thymocytes in vivo ZL Xu , LS Chou , J Sun . Effects of SiO 2 nanoparticles on HFL-I activating ROS-mediated apoptosis via p53 pathway . J Appl Toxicol , 2012 . 32 ( 5 ): 358 - 364 . 21766316 DOI: 10.1002/jat.1710 http://doi.org/10.1002/jat.1710 . Effects of SiO2 nanoparticles on HFL-I activating ROS-mediated apoptosis via p53 pathway XL Yan , RX Dong , L Zhang , 等 . Raman spectra of single cell from gastrointestinal cancer patients . World J Gastroenterol , 2005 . 11 ( 21 ): 3290 - 3292 . 15929185 DOI: 10.3748/wjg.v11.i21.3290 http://doi.org/10.3748/wjg.v11.i21.3290 . Raman spectra of single cell from gastrointestinal cancer patients FF Yang , W Huang , YF Li , 等 . Anti-tumor effects in mice induced by survivin-targeted siRNA delivered through polysaccharide nanoparticles . Biomaterials , 2013 . 34 ( 22 ): 5689 - 5699 . 23632321 DOI: 10.1016/j.biomaterials.2013.03.047 http://doi.org/10.1016/j.biomaterials.2013.03.047 . Anti-tumor effects in mice induced by survivin-targeted siRNA delivered through polysaccharide nanoparticles JM Zahn , SK Kim . Systems biology of aging in four species . Curr Opin Biotechnol , 2007 . 18 ( 4 ): 355 - 359 . 17681777 DOI: 10.1016/j.copbio.2007.07.004 http://doi.org/10.1016/j.copbio.2007.07.004 . Systems biology of aging in four species Z Zamani , P Reisi , H Alaei , 等 . Effect of royal jelly on spatial learning and memory in rat model of streptozotocin-induced sporadic Alzheimer's disease . Adv Biomed Res, 1:26 , 2012 . DOI: 10.4103/2277-9175.98150 http://doi.org/10.4103/2277-9175.98150 . Effect of royal jelly on spatial learning and memory in rat model of streptozotocin-induced sporadic Alzheimer's disease AH Zeng , YY Ou , MM Guo , 等 . Human embryonic lung fibroblasts treated with artesunate exhibit reduced rates of proliferation and human cytomegalovirus infection in vitro . J Thorac Dis , 2015 . 7 ( 7 ): 1151 - 1157 . 26380730 DOI: 10.3978/j.issn.2072-1439.2015.07.05 http://doi.org/10.3978/j.issn.2072-1439.2015.07.05 . Human embryonic lung fibroblasts treated with artesunate exhibit reduced rates of proliferation and human cytomegalovirus infection in vitro M Zhan , H Yamaza , Y Sun , 等 . Temporal and spatial transcriptional profiles of aging in Drosophila melanogaster . Genome Res , 2007 . 17 ( 8 ): 1236 - 1243 . 17623811 DOI: 10.1101/gr.6216607 http://doi.org/10.1101/gr.6216607 . Temporal and spatial transcriptional profiles of aging in Drosophila melanogaster Compliance with ethics guidelines Chen-min JIANG, Xin LIU, Chun-xue LI, Hao-cheng QIAN, Di CHEN, Chao-qiang LAI, and Li-rong SHEN declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1800257 Project supported by the Science and Technology Project of Zhejiang Province (No. 2017C32033) and the National Natural Science Foundation of China (No. 3127848) 2018-04-29 2018-09-09 2022-01-05 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2018 2018 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 12 19