2023 Volume 46 Issue 2 Pages 348-353
Royal jelly (RJ), an essential food for the queen honeybee, has a variety of biological activities. Although RJ exerts preventive effects on various lifestyle-related diseases, such as osteoporosis and obesity, no study evaluated the effect of RJ on the development of osteoarthritis (OA), the most common degenerative joint disease. Here, we showed that daily oral administration of raw RJ significantly prevented OA development in vivo following surgically-induced knee joint instability in mice. Furthermore, in vitro experiments using chondrocytes, revealed that raw RJ significantly reduced the expression of inflammatory cytokines and enzymes critical for the degradation of the extracellular matrix (ECM). Similar results were observed after treatment with 10-hydroxy-2-decenoic acid, the most abundant and unique fatty acid in raw RJ. Our results suggest that oral supplementation with RJ would benefit the maintenance of joint health and prophylaxis against OA, possibly by suppressing the activity of inflammatory cytokines and ECM-degrading enzymes.
Royal jelly (RJ), an essential product for queen reproduction and larvae development in honeybee colonies, is secreted from the hypopharyngeal and mandibular glands of worker honeybees. RJ has a variety of pharmacological and biological activities such as anti-oxidant, anti-tumor, anti-microbial, anti-inflammatory, as well as estrogen-like and insulin-like actions.1–3) Raw RJ usually contains proteins (approximately 12–15%), sugars (10–16%), lipids (3–7%), vitamins, amino acids, and minerals, though the composition could vary depending on geography and climate.4–6)
In Japan, the leading super-aged society is characterized by a disconnection between healthy longevity and life expectancy. This phenomenon is causing a decline in the QOL of elderly individuals, increasing the social-security burden.7–9) This disconnection could be caused by various lifestyle-related diseases, such as metabolic and locomotive syndromes.10) Although several independent studies indicate a protective role of RJ supplementation on postmenopausal osteoporosis, obesity, insulin resistance, and glucose intolerance, its role in the development of osteoarthritis (OA), the most common locomotive syndrome underlying chronic pain and disability in the elderly, remains unclear so far.11–14)
Currently, medications against OA focus on controlling its symptoms, such as chronic joint pain and stiffness. Therefore, the development of preventive and therapeutic approaches against cartilage degradation in joints of patients with OA has been needed for a long time.15,16) We previously revealed that β-cryptoxanthin, a natural xanthophyll carotenoid, effectively ameliorates cartilage destruction in mice with OA.17) In this study, we investigated whether oral supplementation of raw RJ has a beneficial effect in vivo using a murine model of surgically-induced knee OA. Further, we tested if RJ treatment could modify the expression of inflammatory cytokines and extracellular matrix (ECM)-degrading enzymes, which are known to contribute to cartilage degeneration in OA in vitro, using chondrocytes. Our observations indicate that daily oral RJ supplementation can ameliorate the development of OA, possibly by reducing the expression of inflammatory cytokines and ECM-degrading enzymes in chondrocytes.
The raw RJ used in this study was obtained by Katashi Nagamatsu, R&D Division, MORIKAWA KENKODO Co. Ltd., from China and deposited in Toyama Prefectural University. Murine chondrocytic ATDC5 cells were obtained from RIKEN Cell Bank. Recombinant mouse tumor necrosis factor-α (TNF-α) and 10-hydroxy-2-decenoic acid (10H2DA) were purchased from Cell Signaling Technology (MA, U.S.A.) and FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan), respectively. All other chemicals used were of the highest purity available commercially.
In Vivo ExperimentsAll animal experiments were approved by the Committee for the Ethical Use of Experimental Animals at Gifu Pharmaceutical University (#2019-138). Male C57BL/6J mice (8-week-old) underwent surgery to destabilize the knee joint as previously described.18) Model mice with OA were orally gavaged with 2.0 g/kg body weight raw RJ daily (major ingredients: protein 14%, glucose 6.5%, fructose 6.5%, polysaccharide 2.0%, and lipid 3.8%) for 8 weeks. Vehicle solution (casein 14%, glucose 6.5%, fructose 6.5%, cornstarch 2.0%, and soybean oil 3.8%) was used for control. Histological analyses were performed as previously described,17) and OA severity was quantified using the Osteoarthritis Research Society International (OARSI) histopathology grading system.19)
In Vitro StudyATDC5 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F-12 medium supplemented with 5% fetal bovine serum (FBS). To determine cell viability, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was performed, as previously described.20) For RNA analyses, reverse transcription-quantitative PCR was performed with specific primers for each gene,21) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the reference gene.
Data AnalysisAll results are expressed as the mean ± standard error (S.E.); the statistical significance was determined using the Student’s t-test, one-way ANOVA with Dunnett post hoc test, or two-way ANOVA with Bonferroni/Tukey–Kramer post hoc test.
Compared with vehicle, RJ administration did not significantly change the daily food intake (Fig. 1A), body weight (Fig. 1B), and weight of major organs (white adipose tissue, brown adipose tissue, liver, pancreas, heart, kidney, spleen, testis, brain, thymus, and lung; Fig. 1C). Safranin O staining showed no marked histological differences in sham-operated joints between vehicle- and RJ-treated mice (Fig. 1D, upper two panels). In the vehicle-treated mice, marked cartilage degradation was observed by Safranin O staining in joints with surgical-induced OA compared with that in sham-operated joints (Fig. 1D, left two panels). Conversely, RJ administration significantly ameliorated cartilage degradation in the joints with surgical-induced OA when compared with vehicle treatment (Fig. 1D, lower two panels). We next quantified the development of OA using the histologic scoring system recommended by OARSI. RJ-treated mice had significantly lower scores for OA damage in joints with surgical-induced OA compared with vehicle-treated mice (Fig. 1E), indicating that daily oral RJ administration prevented OA development.
(A) Daily food intake, (B) body weight, and (C) weight of major organs (vehicle, n = 12; RJ, n = 11). Representative pictures of Safranin O staining are shown in panel (D), and quantitative scores are shown in panel (E). ** p < 0.01 vs. sham-vehicle mice. ## p < 0.01 vs. OA-vehicle mice. N.S.; not significant. Bar = 500 µm (D).
Inflammatory cytokines and ECM-degrading enzymes (matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)) contribute to the cartilage degradation seen in OA.22) Further, we investigated the cellular mechanisms underlying the preventive effect of RJ on OA development observed in vivo. To this end, we tested whether RJ modifies the expression of inflammatory cytokines and ECM-degrading enzymes in chondrocytes. RJ treatment at the tested concentrations (including at 2.5 mg/mL) did not significantly affect chondrocyte viability as determined by the MTT assay (Fig. 2A). Further, we stimulated chondrocytes with TNF-α at 10 ng/mL in the presence or absence of RJ at 2.5 mg/mL to determine the expression of inflammatory cytokines and ECM-degrading enzymes. TNF-α markedly elevated the expression of inflammatory cytokines, IL-1b, and IL-6, and ECM-degrading enzymes, Adamts5, Mmp3, and Mmp13, in chondrocytes. These effects were significantly repressed by the RJ treatment (Figs. 2B–F).
(A) ATDC5 cells were cultured with RJ at a concentration range from 1 to 2.5 mg/mL. Subsequently, MTT reduction was determined (n = 3–6). ATDC5 cells were cultured with TNF-α at 10 ng/mL in either the presence or absence of RJ at 2.5 mg/mL, and subsequently, mRNA expression of (B) IL-1b, (C) IL-6, (D) Adamts5, (E) Mmp3, and (F) Mmp13 (n = 3–4), was quantified. ** p < 0.01 vs. TNF-α(−)-vehicle cells. # p < 0.05, ## p < 0.01 vs. TNF-α(+)-vehicle cells.
Raw RJ consists of water, lipids, proteins, carbohydrates, and other minor constituents. 10H2DA is the main lipid component of RJ and a member of a group of unique medium-chain fatty acids in RJ. The component possesses a variety of pharmacological and biological activities, including anti-inflammatory properties.23,24) Therefore, we evaluated whether 10H2DA could inhibit the expression of inflammatory cytokines and ECM-degrading enzymes in chondrocytes as witnessed with RJ treatment. Treatment with 10H2DA at the tested concentrations (including at 0.075 mg/mL) did not significantly alter the viability of chondrocytes (Fig. 3A). Further, we stimulated chondrocytes with TNF-α at 10 ng/mL in the absence or the presence of 10H2DA at 0.075 mg/mL, followed by determination of gene expression of the same genes as previously mentioned. 10H2DA significantly inhibited the expression of TNF-α-induced IL-6, Adamts5, Mmp3, and Mmp13 (Figs. 2B–E), reproducing the effect of raw RJ on chondrocytes.
(A) ATDC5 cells were cultured with 10H2DA at a concentration ranging from 0.025 to 0.075 mg/mL; subsequently, MTT reduction was determined (n = 6). ATDC5 cells were cultured with TNF-α at 10 ng/mL in either the presence or absence of 10H2DA at 0.075 mg/mL, and subsequently, mRNA expression of (B) IL-6, (C) Adamts5, (D) Mmp3, and (E) Mmp13 (n = 3–4) was determined. ** p < 0.01 vs. TNF-α(−)-vehicle cells. # p < 0.05, ## p < 0.01 vs. TNF-α(+)-vehicle cells.
OA is the most common locomotive disorder, often requiring special assistance or nursing care in elderly individuals. Currently, no treatment or cure can reverse the cartilage destruction that characterizes the joints of patients with OA.16,25) Therefore, early preventive management using safe and inexpensive therapeutical agents to delay the progressive cartilage degradation in patients with OA is desirable. Using a murine model of OA, we recently showed that β-cryptoxanthin, a major carotenoid in vegetables and fruits, significantly prevented interleukin-1β (IL-1β)-induced increase in the expression of inflammatory cytokines and ECM-degrading enzymes in chondrocytes as well as cartilage degradation.17) The importance of the present findings is that daily oral supplementation of raw RJ significantly prevented OA development. Although RJ supplementation was shown to have preventive properties on a variety of lifestyle-related diseases, including osteoporosis and obesity, to the best of our knowledge, this study first directly demonstrates its prophylactic effect against OA development.13,14)
Furthermore, RJ treatment significantly inhibited the TNF-α-induced expression of inflammatory cytokines and enzymes critical for ECM degradation in chondrocytes. This effect was reproduced by the treatment with 10H2DA, the most abundant fatty acid in RJ, prompting us to further investigate the effects of 10H2DA on OA development in vivo in future studies. The lipid composition of RJ comprises 90–95% fatty acids, and 10H2DA (molecular formula; C10H18O3) is a unique medium-chain unsaturated fatty acid only found in RJ.26) 10H2DA reportedly exerts a variety of biological and pharmacological activities, representing one of the main bioactive components of RJ.27) Indeed, as does RJ, 10H2DA inhibits bone resorption in an ovariectomized murine model of osteoporosis,14) improving glucose intolerance and insulin resistance in obese/diabetic mice.28) Moreover, 10H2DA has not only anti-tumor, anti-angiogenic, and anti-biotic activities but anti-inflammatory activity in mammals.27) Accordingly, 10H2DA could, at least in part, be implicated in the anti-osteoarthritis property of RJ by inhibiting local inflammation in knee joints of a murine model of surgically-induced knee OA. Because raw RJ contains a variety of bioactive molecules besides 10H2DA, such as proteins, vitamins, and flavonoids, etc., further studies are warranted to determine the exact mechanism underlying its anti-osteoarthritis effect and to identify the additional components critical for this activity.1,29) Moreover, besides its direct role, it should also be noted that 10H2DA metabolites might play a pivotal role in vivo because it has been reported that the major RJ fatty acids are metabolized to dicarboxylate, absorbed into the circulation, and then excreted in the urine in humans.30)
Although we showed that RJ has anti-inflammatory properties in chondrocytes by in vitro experiments, we could not rule out the possibility that RJ indirectly ameliorates the osteoarthritic phenotypes in vivo. Indeed, the fact that inflammatory cytokines are secreted into the synovial fluid from various cell types, including synovial fibroblasts and macrophages in knee joint tissues of OA,31) hints at the idea that RJ could ameliorate the expression of inflammatory cytokines in cell types other than chondrocytes, inhibiting the expression of ECM-degrading enzymes and delaying cartilage destruction in knee joints of mice with surgically-induced knee OA. Accordingly, future studies to identify the type and localization of cytokine-producing cells in knee joint tissues of our OA mouse model using histological tools are warranted.
Although several cases of hemorrhagic colitis, asthma, and anaphylaxis by ingestion of RJ have been reported so far, in the present study, we did not observe any evident severe adverse effects following oral long-term RJ administration (2.0 g/kg, once-a-day for 8 weeks) in mice with OA.32) Collectively, we propose that long-term oral supplementation of raw RJ would be beneficial for the maintenance of joint health and serve as prophylaxis against OA through the inhibition of inflammation and ECM degradation in knee cartilage without causing severe side effects.
This work was partially supported by a Grant-in-Aid for Scientific Research (B) from the Ministry of Education, Culture, Sports, Science and Technology, Japan, to E.H.
K. Nagamatsu is an employee of MORIKAWA KENKODO Co., Ltd. K. Ikeno is an employee of AKITAYAHONTEN Co., Ltd. G. Nakamura is the employer of AKITAYAHONTEN Co., Ltd. E. Hinoi is supported by a research Grant from MORIKAWA KENKODO Co., Ltd. and AKITAYAHONTEN Co., Ltd. All other authors declare no conflict of interest.
