Laura Gambari
Brunella Grigolo
Francesco Grassi- Laboratorio RAMSES, IRCCS Istituto Ortopedico Rizzoli, Bologna, Italy
The progressive decline of bone mass and the deterioration of bone microarchitecture are hallmarks of the bone aging. The resulting increase in bone fragility is the leading cause of bone fractures, a major cause of disability. As the frontline pharmacological treatments for osteoporosis suffer from low patients’ adherence and occasional side effects, the importance of diet regimens for the prevention of excessive bone fragility has been increasingly recognized. Indeed, certain diet components have been already associated to a reduced fracture risk. Organosulfur compounds are a broad class of molecules containing sulfur. Among them, several molecules of potential therapeutic interest are found in edible plants belonging to the Allium and Brassica botanical genera. Polysulfides derived from Alliaceae and isothiocyanates derived from Brassicaceae hold remarkable nutraceutical potential as anti-inflammatory, antioxidants, vasorelaxant and hypolipemic. Some of these effects are linked to the ability to release the gasotrasmitter hydrogen sulfide (H2S). Recent preclinical studies have investigated the effect of organosulfur compounds in bone wasting and metabolic bone diseases, revealing a strong potential to preserve skeletal health by exerting cytoprotection and stimulating the bone forming activity by osteoblasts and attenuating bone resorption by osteoclasts. This review is intended for revising evidence from preclinical and epidemiological studies on the skeletal effects of organosulfur molecules of dietary origin, with emphasis on the direct regulation of bone cells by plant-derived polysulfides, glucosinolates and isothiocyanates. Moreover, we highlight the potential molecular mechanisms underlying the biological role of these compounds and revise the importance of the so-called ‘H2S-system’ on the regulation of bone homeostasis.
Highlights
A literature search was conducted using MEDLINE database. Relevant pre-clinical and clinical studies were selected using a combination of keywords including bone, diet and/or organosulfur compounds, Allium, Brassicaceae, alliin, allicin, garlic, ajoene, diallyl trisulfide, diallyl disulfide, S-allylcysteine, diallyl sulfide, glucosinolate, thiosulfinate, sulforaphane, broccoli, methyl sulfide, isothiocyanates. Additional studies were identified by an extensive manual search of bibliographic references in original papers and reviews. Abstracts and non-English papers were not included. This study selected a total of in vitro studies (10 Alliaceae, 9 Brassicaceae); in vivo studies (17 Alliaceae, 11 Brassicaceae) and population-based studies (4 Alliaceae, 1 Brassicaceae).
Introduction
Osteoporosis (OP) is a chronic metabolic bone disease characterized by the deterioration of bone microarchitecture and a reduction in bone mass, leading to decreased bone strength and increased risk of bone fracture (1). Approximately 6 % of men and 21 % of women aged 50–84 years are diagnosed with OP and the number of fragility fractures in Europe has increased from 3.1 to nearly 4.3 million in 20 years since year 2000 (2); due to the strong correlation with the ageing of the population, the prevalence of OP is projected to further increase over the next decades (3).
At the bone tissue level, OP is characterized by increased bone porosity which results from the loss of balance between bone formation and bone resorption as aging, disuse, inflammatory diseases, hormonal imbalance or the effect of glucocorticoids impair the ability of osteoblast to keep up with the pace of bone resorption by the osteoclasts (4). Importantly, aging is associated with a decreased number of osteoprogenitor cells, inhibited proliferation, decreased mineralizing capacity, and a shift of osteogenic differentiation toward adipogenesis in senescent mesenchymal stromal cells (MSCs) (5–7).
Pharmacotherapy helps patients to prevent the occurrence or recurrence of fragility fractures and to manage symptoms. However, drugs are mostly used in patients who already show severe bone loss, and the existence of side effects, although very limited in prevalence, often leads to low patient’s adherence to anti-OP drugs (8, 9). In this context, non-pharmacological strategies aimed at preventing excessive bone loss hold relevance given that OP remains in most cases a subclinical condition until fracture occurs.
One safe way to prevent bone loss and reduce the risk of bone fracture is to positively impact bone mass through healthy lifestyles and nutrition (10, 11). In particular, the importance of defining specific diet regimens for the prevention of excessive bone fragility has been increasingly recognized (12–15). Adherence to Mediterranean diet lowered hip fracture risk (16) and certain micronutrients contained in fruit and vegetables contributed to delay bone fragility in ageing and to decrease the incidence of bone fractures (17–20). Moreover, a dietary pattern consisting of a high consumption of fruits, vegetables and seafood, has been shown to be directly associated with increased bone mineral density (BMD), independent of dietary calcium intake (21, 22).
Phytochemicals are defined as the chemical bioactive components of nutrient plants that may provide desirable health benefits beyond basic nutrition to reduce the risk of major chronic diseases. They include several classes of compounds: terpenoids, polyphenols, alkaloids, organosulfur compounds (OSCs) and phytosterols (23). Concerning OSCs, much of the research on their health benefits has been in the areas of cardiovascular diseases, cancer and neurological disorders (24–26). However, a growing body of scientific evidence supports the idea that dietary OSCs may play an important role for skeletal health by favoring bone anabolism, inhibiting bone catabolism, and preventing pathological bone loss.
This manuscript intends to provide an up-to-date review of the current evidence from preclinical (both in vitro and in vivo) and clinical studies on the skeletal effects of OSCs of dietary origin, discussing the chemical nature, the mechanism of action and the potential role of hydrogen sulfide (H2S) in their biological action. A specific focus is given to the pair glucoraphanin (GRA)-sulforaphane (SFN) as a paradigm of OSCs-H2S system in bone tissue. Finally, implications and future challenges in the field will be discussed considering the potential translation of OSCs-containing dietary components to clinical studies.
Dietary sources and chemical nature of OSCs
Naturally derived OSCs are a broad class of molecules containing sulfur, predominantly found in edible plants belonging to the Allium and Brassica (also known as cruciferous vegetables) genera. These plants have been widely used throughout the centuries either as vegetables for culinary purposes as well as in folk and traditional medicine, given their renowned medicinal properties and therapeutic effects. Allium genus consists of more than 600 species which are among the oldest cultivated vegetables used as food and still represent one of the main components of the Mediterranean diet (27). Brassica genus consists of 37 species; among them, several species are known for their nutritional and therapeutic properties (28, 29). A partial list of edible plants belonging to the Allium and Brassica genera, and their main content in OSCs, is reported in Table 1.
Table 1 Most common OSCs found in edible Allium and Brassica vegetables.
In Allium, over half of the total sulfur content within the mature garlic bulb is found in the form of S-alk(en)yl cysteine sulfoxides (ASCOs) (69), non-protein sulfur amino acids which are converted to their respective thiosulfinates or propanethial-S-oxide upon tissue damage (70).
The synthesis of ASCOs in Allium species starts with the transformation of γ-glutamyl peptides (such as γ-l-glutamyl-S-methyl-L-cysteine) into sulfur-containing γ-glutamyl-S-alk(en)yl-cysteines such as γ-glutamyl-S-methyl-cysteines, γ-glutamyl-S-allyl-cysteine, γ-glutamyl-propenyl-l-cysteine sulfoxide (PeCSO). These are further deglutamylated and S-oxygenated to yield S-alk(en)yl-l-cysteine sulfoxides (71, 72). These reactions are catalyzed by γ-glutamyl transpeptidase, l-glutaminases, and oxidase in the cytoplasm of plant cells. The intact garlic bulbs contain alliin, γ-glutamyl-S-allyl-l-cysteine (GSAC), methiin, S-trans-1-propenyl-l-cysteine sulfoxide, S-2-carboxypropylglutathione, S-allylcysteine (SAC) (37).
When the bulbs are cut, crushed, chopped or chewed, the enzyme alliinase (a vacuolar lyase) is released from vacuoles and catalyzes the formation of sulfenic acids from l-cysteine sulfoxides: S-allyl-l-cysteine sulfoxide (alliin); S-methyl-l-cysteine sulfoxide (methiin); S-propyl-l-cysteine sulfoxide (propiin); S-trans-1-propenyl-l-cysteine sulfoxide (isoallin) (71, 72). Sulfenic acids spontaneously react with each other to form unstable compounds called thiosulfinates (69): eg. alliin is converted into allicin (alkenyl alkene thiosulfinate - diallyl thiosulfinate). Allicin immediately decomposes into allyl sulfide (AS), diallyl disulfide (DADS), diallyl trisulfide (DATS), diallyl tetrasulfide, dipropyl disulfide (DPDS), ajoenes, and vinyldithiins (72). The direct catabolism of γ-glutamylcysteine by γ-glutamyltranspeptidase leads to the formation of SAC and S-allylmercaptocysteine (SAMC). Allicin can react with glutathione and l-cysteine to produce S-allylmercaptoglutathione (SAMG) and SAMC, respectively (69, 72).
Among Allium, the most common ASCOs are alliin, methiin, propiin and isoalliin (70, 73, 74). However, they are differentially expressed in specific edible plants. The most abundant in garlic is alliin; in onion isoalliin, methiin, propiin are predominantly detected.
In Brassica vegetables two different kinds of OSCs are present: methiin, mainly known from Allium vegetables, and glucosinolates (S-β-thioglucoside N-hydroxhysulfates, GLS). Methiin is metabolized to (+)-S-alk(en)yl-l-cysteine sulfoxides which can degrade to volatile organosulfur compounds (VOSCs) such as S-methyl methane thiosulfinate, which is converted to dimethyl trisulfide and dimethyl disulfide.
GLS are sulfur-based compounds that consist of β-thioglycoside N-hydroxysulfates with various side chains and a sulfur-linked β-d-glycopyranose moiety. A very different profile of GLS may be found in different Brassica extracts (75). Natural isothiocyanates (ITCs) are bioactive OSCs derived from the hydrolysis of GLS by the enzyme myrosinase. In plant cells, GLS are physically separated from myrosinases and come in contact only upon tissue damage or crushing. Importantly, myrosinase is not expressed by mammalian cells; however, a small proportion is converted in the mouth by action of plant myrosinase released by chewing (76); moreover, the gut microbiota is entailed with myrosinase activity and constitutes the major site in humans where GLS are hydrolyzes to ITCs (77). While GLS are chemically stable and are characterized by a relatively long half-life, ITCs are highly reactive and short-lived in vivo (75, 78).
Effect of OSCs on bone tissue: Preclinical evidence
The effect of OSCs in bone tissue has been investigated in several preclinical models, revealing a strong potential to preserve skeletal health by stimulating the bone forming activity of osteoblasts and inhibiting the bone resorbing activity of osteoclasts, two of the key processes of bone remodeling (79).
Figures 1, 2 provide a graphical summary, respectively, of the main biological processes and molecular targets regulated by OSCs within MSCs/osteoblasts and monocytes/osteoclasts. A detailed description of these mechanisms is provided in the next paragraphs.
Figure 1 Regulation of bone remodeling processes by purified OSCs molecules. Bone remodeling is governed by the balance between bone formation by the osteoblasts (left side) and bone erosion by the osteoclasts (right side). Ancillary processes are shown. OSCs specifically regulate the following processes: promote cells proliferation and viability of mesenchymal stromal cells (1) while inhibit the proliferation and viability of monocytes (5); promote the osteogenic differentiation (2) and bone formation (3); inhibit at different stages osteoclast differentiation (6) and reduce bone erosion (7); inhibit the viability of osteocytes (4). Among the OSCs which modulate bone processes are allicin, allyl sulfide (AS), sulforaphane (SFN), glucoraphanin (GRA), diallyl sulfide (DADS). See the text for details.
Figure 2 Molecular targets of purified OSCs molecules in bone cells. Osteoblastogenesis and osteoclastogenesis are the two key processes of bone remodeling and are regulated by a tightly organized activation of specific molecular targets. This figure shows a schematic representation of a mesenchymal stromal cells/osteoblast and a monocyte/osteoclast to highlight the specific molecular targets regulated by OSCs at different stages of differentiation from precursors to fully differentiated cells. Among the OSCs which drives the modulation of specific molecular targets are allicin, allyl sulfide (AS), sulforaphane (SFN), glucoraphanin (GRA) and diallyl sulfide (DADS). The overall effects are an activation of osteogenic differentiation in mesenchymal stromal cells and both a direct and indirect inhibition of osteoclast differentiation. Follows a list of the molecular targets shown in the figure. Markers of osteoblastogenesis: osteocalcin (OCN), runt-related transcription factor 2 (RUNX-2), alkaline phosphatase (ALP), WNT1-inducible-signaling pathway protein 1 (WISP-1), bone sialoprotein (BSP), cystathionine-β-synthase (CBS), SMAD family member 1 (SMAD-1). Markers of regulators of osteoclastogenesis produced by mesenchymal stromal cells or osteoblasts: receptor activator of nuclear factor-κB ligand (RANKL), osteoprotegerin (OPG). Marker of cells survival and stress response: FAS, caspase 3/7, nuclear factor erythroid-derived 2-related factor 2 (NRF2), NAD(P)H: quinone oxidoreductase 1 (NQO1), heme oxygenase-1 (HO1), glutamate cysteine ligase catalytic subunit (GCLC), glutamate-cysteine ligase modifier subunit (GCLM), peroxiredoxin 1 (PRDX-1). Markers of osteoclasts: nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), cathepsin K (CTSK), receptor activator of NF-KB (RANK), osteoclast stimulatory transmembrane protein (OC-STAMP), dendritic cell specific transmembrane protein (DC-STAMP), osteoclasts-specific activating receptor (OSCAR), tartrate-resistant acid phosphatase (TRAP), calcitonin receptor (CTR), c-fos, tartrate-resistant acid phosphatase 5b (TRAP-5b), matrix metallopeptidase 9 (MMP-9). See the text for details.
Tables 2–5 summarize data from preclinical studies showing an effect of extracts rich in OSCs or individual OSCs molecules derived from Allium (Tables 2, 3) and Brassica vegetables (Tables 4, 5).
Table 2 Alliaceae-derived OSCs: effects on in vitro models of osteoclastogenesis and osteoblastogenesis.
Table 3 Alliaceae-derived OSCs: effects on in vivo models of bone loss.
Table 4 Brassicaceae-derived OSCs: effects on in vitro models of osteoclastogenesis and osteoblastogenesis.
Table 5 Brassicaceae-derived OSCs: effects on in vivo models of bone loss.
Importantly, while data obtained from studies on purified molecules (labeled with * in the tables) clearly attest to the effectiveness of individual OSCs, the effect of OSCs-rich extracts may result from the combined action of other phytochemicals contained in the extracts. Indeed, Allium species contains polyphenols, flavonoids, flavanols, anthocyanins, tannins, ascorbic acid, saponins and fructans (109–111); Brassica species contains ascorbic acid, phenolics, carotenoids, terpenes, phytoalexins and alkaloids (29, 112).
Regulation of osteogenesis and bone formation
Osteoblasts, the bone forming cells, regulate bone homeostasis by synthesizing a wide variety of extracellular protein of bone matrix. They differentiate from MSCs through the osteogenic differentiation process which is regulated by an orchestrated activation of several pathways. The master regulator of osteogenic differentiation is runt-related transcription factor 2 (RUNX-2), which is expressed in the early stages of differentiation and is at the intersection of several signaling pathways among which growth hormone-janus Kinase 2 (GH-JAK2), bone morphogenetic protein-SMAD (BMP-SMAD), canonical Wingless/Integrated (Wnt) and Notch signaling (113, 114). Among the genes targeted by RUNX-2 are osteocalcin (OCN), collagen I (Col I), bone sialoprotein (BSP), osteopontin (OPN), alkaline phosphatase (ALP). BSP, OPN and ALP are correlated to matrix mineralization; Coll I and OCN are among the major components of bone matrix. Wnts-β-catenin signal activates osteogenic target genes such as distal-less homeobox 5 (Dlx5) and osterix (Osx) (115) and suppresses the transcription of adipogenic transcription factors such as peroxisome proliferator-activated receptor-γ (PPAR-γ) (116). SMAD family number 1 (SMAD-1) is a critical immediate downstream mediator of BMP receptor transduction (117). Among downstream targets of canonical Wnt and BMP signaling is WNT1-inducible signaling pathway protein 1 (WISP-1), which is involved in the positive regulation of osteogenesis and negative regulation of adipogenesis (118). Interestingly, the expression of H2S generating enzymes, cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE), was found to be transcriptionally up-regulated during osteogenesis and to correlate with the biosynthesis of mineral matrix (119), thus suggesting a role for endogenous H2S in osteogenic differentiation. Osteogenic differentiation is associated to increased ALP activity and mineralization in vitro and increased BMD in vivo. Osteoblast finally differentiate toward osteocytes, multifunctional bone cells that are embedded in mineralized bone matrix. Osteocytes act as orchestrators of bone remodeling, through regulation of both osteoclast and osteoblast activity; as regulators of phosphate metabolism and calcium availability, by functioning as an endocrine cell; as mechanosensory cells (120). Key factors produced by osteocytes are sclerostin (a negative regulator of bone mass), FGF-23 (a regulator of phosphate metabolism), and the key regulator of osteoclast differentiation receptor activator of nuclear factor κβ ligand (RANKL), also produced by osteoblasts and MSCs (120, 121).
Most studies investigating OSCs extracts focused on a commonly used human osteoblastic model, the human osteosarcoma cell line (MG-63 cells). They showed increased cell proliferation and increased osteogenesis/mineralization by Allium Hookeri roots treatments (48); increased osteogenesis by Allium fistulosum (80) and Brassica Rapa L. (Jeong); while no effect on proliferation and differentiation was shown by treatment with water solution of onion crude powder (81). However, MG-63 cells are osteoblasts derived from osteosarcoma, a malignant bone tumors, thus are not fully representative of physiological osteoblasts (122). Increased cells proliferation by Allium genus was also shown by ginger and garlic extracts released by 3D-printed calcium phosphate scaffolds on human fetal osteoblast cells (82); increased osteogenesis by Allium fistulosum was also shown in the mouse C57BL/6 osteoblastic calvaria cell line (MC3T3-E1) (80). Up to date no studies on primary cultures of human MSCs have been performed with extracts derived from Alliaceae or Brassicaceae.
Treatment with Alliaceae extracts improved bone formation in normal control rats (41, 48, 88) and mitigated the bone loss due to several pathological conditions among which osteoporosis (47, 80, 94). Similarly, extracts from Brassicaceae induced bone formation in control rats (54) and prevented bone loss in several models of osteoporosis (59, 99, 106–108). Interestingly, treatment with Lepidium sativum resulted in improved fracture healing (28, 123).
Notably, several studies focused on purified OSCs molecules, revealing a specific effect of OSCs on proliferation, osteogenic differentiation, and bone formation. Behera et al. showed increased proliferation, ALP activity and mineralization in murine MSCs derived from femur bone marrow (BMMSCs) upon allyl sulfide stimulation, with a mechanism implicating increased RUNX-2 and OCN expression (83). Thaler et al. demonstrated increased mineralization in mouse MSCs and in an ex vivo culture of calvariae explants treated with SFN (97); at the molecular level, SFN induced up-regulation of RUNX-2 in mouse MSCs (97). Gambari et al. showed increased mineralization and BSP, CBS and SMAD-1 mRNA up-regulation by GRA administration in primary human MSCs (98). Finally, with regards to osteocyte regulation, Thaler et al. showed that SFN inhibited proliferation in murine osteocyte-like cell line (MLO-Y4) (97).
Purified OSCs have also been tested in in vivo models of bone loss or osteolysis, showing beneficial effects on preserving bone mass. Oral administration of allyl sulfide in an age-associated osteoporosis mouse model resulted in increased bone density at X-ray analysis and increased serum levels of procollagen 1 intact N-terminal propeptide (P1NP; a marker of bone formation) (83). Similarly, intragastric administration of allicin increased BMD, as detected by dual energy X-ray absorptiometry, and bone strength, as measured by three-point bending assay, in a model of aging osteoporotic rats (96). Intraperitoneal administration of allicin prevented the bone loss in a mice model of lead-induced bone loss (osteoporosis induced by a toxic heavy metal), as measured by increased BMD, trabecular number (Tb.N), trabecular thickness (Tb.Th) and decreased trabecular space (Tb.Sp), quantified using micro-CT analysis (95). Finally, SFN showed to be protective against bone loss in different in vivo models. Intraperitoneal injection of SFN in lipopolysaccharide (LPS)-induced erosion of the mice calvaria bone induced increased trabecular bone volume (BV/TV), increased Tb.N and decreased Tb.Sp, as measured by micro-CT analysis (103); moreover, intraperitoneal injection of SFN in a mice model of ovariectomy-induced bone loss stimulated trabecular bone formation, increased Tb.N and decreased Tb.Sp (97); finally, the oral administration of SFN-01 (a stabilized form of SFN) in a mice model of osteoarthritis, resulted in increased trabecular bone volume and serum P1NP (105).
Regulation of osteoclastogenesis and bone resorption
Osteoclasts are bone-resorbing cells which arise from immature monocytes and mature tissue macrophages (124). Osteoclasts differentiation stems from the signaling triggered by two critical cytokines produced by MSCs, osteoblasts and osteocytes: macrophage colony-stimulating factor (M-CSF) and RANKL binding, respectively, to the receptors colony-stimulating factor-1 receptor (c-fms) and receptor activator of nuclear factor κ B (RANK) (125, 126). RANKL signaling activation induces various intracellular signal transduction cascades such as tumor necrosis factor receptor-associated factor 6 (TRAF-6), NADPH oxidase 1 (NOX-1), RAC family small GTPase 1 (RAC1), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and nuclear factor-activated T cells c1 (NFATc1), c-fos (127–129). Other receptors involved in osteoclastogenesis are calcitonin receptor (CTR), ITAM bearing Fc receptor standard g chain (FcRγ), osteoclasts-specific activating receptor (OSCAR) (126, 130); key signaling is mediated by mitogen-activated protein kinases (MAPK), and includes extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 activation. Moreover, critical to osteoclast differentiation and function are: intracellular reactive oxygen species (ROS) generation, which act as key signaling molecules (82, 88, 94); osteoclast fusion mediated among other factors, by the fusogenic molecules osteoclasts-stimulatory transmembrane protein (OC-STAMP) and dendritic cell-specific transmembrane protein (DC-STAMP) (126, 131, 132); and expression of specific enzymes such as tartrate-resistant acid phosphatase (TRAP), cathepsin K (CTSK) (126, 130), tartrate-resistant acid phosphatase 5b (TRAP5b) (83) and matrix metallopeptidase 9 (MMP-9).
Extracts from both Allium and Brassica species were shown to attenuate osteoclast differentiation in vitro in the murine macrophage cell line, RAW 264.7. In particular, extracts of onion (85), freeze dried onion juice (85), solution of onion crude powder (81) inhibited osteoclastogenesis, as measured by TRAP staining in vitro. A similar effect was achieved by an extract of Brassica oleracea but only in combination with extract from Panax ginseng (99). Using human THP1 monocytes, Bose et al. showed that ginger and garlic extracts reduce the frequency and the size of resorption pits carved by osteoclasts (82); inhibition of osteoclast number was found also by onion and commercial onion extracts in rat and rabbit osteoclasts (81). Notably, Wetli et al. demonstrated that onion extract reduced rat osteoclast differentiation and were able to isolate a specific sulfoxide component of onion powder, γ-glutamyl-trans-S-1-propenyl-l-cysteine sulfoxide (GPCS), which the authors found to be the key responsible of this biological activity (41).
In vivo administration of extracts rich in OSCs decreased osteoclastogenesis and bone erosion in rodent model of osteoporosis; Huang et al. showed that ovariectomized rats fed with different concentrations of onion extracts (up to 14% wt/wt in the diet powder) were partly protected against loss of bone mass and bone material properties (94); moreover, histomorphometry revealed that treatment with onion extracts was associated with a lower number of osteoclasts in vivo (94). Similar findings were reported by Kang et al. using ovariectomized mice fed with a combination of extracts obtained from Panax ginseng and Brassica oleracea (99). Furthermore, Abdallah HM et al. reported that ovariectomized rats treated with extracts of Lepidium sativum were partly protected against osteoporosis and showed a sharply decreased RANKL/osteoprotegerin (OPG) ratio in femur bones (59).
Studies that used purified OSCs molecules further supported efficacy and specificity. Yang et al. demonstrated a dose-dependent inhibition of osteoclast differentiation and a decreased bone resorption by mature osteoclasts upon treatment with DADS (86). Monocytes proliferation and viability was inhibited by SFN (97).
Luo et al. (103) and Xue et al. (100) showed that SFN inhibits osteoclast differentiation in RAW 264.7 murine macrophagic cell line; Takagi et al. (101) and Kim et al. (102) showed similar findings in murine BM cells and so did Gambari et al. (104) in a model of osteoclast derived from human monocytes. Moreover, Chen et al. reported the inhibition of osteoclast differentiation by alliin in RAW 264.7 via scavenging of ROS signaling (87).
Mechanisms of regulation of osteoclastic differentiation by OSCs involved different molecular targets. Li et al. reported that the anti-osteoclastogenic activity of allicin in mice is associated to the activation of the SIRT1/FOXO1 pathway and ROS scavenging (95). Similarly, one key mechanism of action of SFN is the activation of the master regulator of the antioxidant defense system, nuclear factor erythroid-derived 2-related factor 2 (NRF2), and its downstream target antioxidant and detoxifying enzymes (133), which is known to actively inhibit mouse osteoclasts differentiation in vitro (104, 134). SFN modifies sulfhydryl groups in kelch-like erythroid-cell-derived protein with CNC homology (ECH)-associated protein (KEAP-1), causing KEAP-1 dislocation, NRF2 stabilization and nuclear translocation (135); moreover, SFN regulates NRF2 expression via epigenetic mechanisms (136). Coherently, SFN was shown to increase NRF2 protein accumulation in RAW 264.7 murine cell line, to increase the expression of some NRF2-mediated antioxidant genes (heme oxygenase-1, HO1; NAD(P)H: quinone oxidoreductase 1, NQO1; glutamate cysteine ligase catalytic subunit, GCLC; ligase modifier subunit, GCLM) and decrease intracellular ROS production, and the overall number of osteoclasts as shown by Xue et al. (100). Similarly, SFN was shown to inhibit the osteoclast differentiation of human monocytes while increasing NRF2 nuclear translocation and protein expression of NRF2-mediated antioxidant genes (NQO1; Peroxiredoxin 1, PRDX-1), as published by Gambari et al. (104). Finally, SFN induces Caspase 8 and 3/7, thus inducing apoptosis in a RAW 264.7 murine cell line as shown by Thaler et al. (97).
Moreover, downregulation of the key transcription factor NFATc1 is implicated in several studies showing inhibition of osteoclast development: Yang et al. reported a dose-dependent down-regulation of NFATc1 in a RAW 264.7 murine cell line after DADS treatment (86); Xue et al. (100) and Takagi et al. (101), respectively, reported similar findings in RAW 264.7 murine cell line and in murine BM cells after SFN treatment; Behera et al. in murine BM cells after allyl sulfide treatment (83). The inhibition of other key transcription factor c-Fos and Nf-kB was shown by Yang et al. in a RAW 264.7 murine cell line after DADS treatment (86).
Several other proteins implicated in the adhesion an (83, 95, 100, 137), in RAW264.7 cells and murine BM and are detailed in Tables 2, 4.
OSCs can modulate the expression of osteoclasts-specific activating receptors, necessary for the co-stimulatory signaling with immunoreceptors and prevented osteoclast fusion by inhibiting fusogenic molecules. Takagi et al. showed in RAW 264.7 murine cell line that OSCAR is inhibited by SFN (101). DC-STAMP was found inhibited in RAW 264.7 murine cell line after SFN treatment as shown by Takagi et al. (101) and by Xue et al. (100) and after DADS treatment as shown by Yang et al. (86). OC-STAMP was found inhibited in RAW 264.7 murine cell line after SFN treatment as shown by Takagi et al. (101).
Finally, OSCs compounds were shown to inhibit osteoclast differentiation via a paracrine mechanism, acting on osteoclasts-supporting cells. Thaler et al. showed that RANKL was inhibited by SFN in a murine osteocytes cell line (MLO-Y4) (97). Behera et al. showed that RANKL was inhibited while OPG was increased in supernatants of murine MSCs cells culture treated with allyl sulfide (83); and that treatment with this conditioned medium inhibited the expression of RANK and osteoclast differentiation of murine bone marrow (BM) cells (83).
Only a few in vivo studies used purified OSCs to investigate bone metabolism. In a mice model of lead-induced bone loss, intraperitoneal injection of allicin alleviates bone loss by preventing oxidative stress and osteoclastogenesis by modulating SIRT1/FOXO1 pathway (95). SFN treatment in a mouse calvaria model treated with LPS decreased the number of osteoclasts (103). Treatment of Lepidium sativum in a rat model of ovariectomy-induced osteoporosis improved mechanical properties of femurs while decreasing TRAP, serum type I collagen breakdown product (CTX-I), RANKL (59) and the number of osteoclasts (107).
H2S release from OSCs as a potential mechanism of bioactivity in bone
H2S is a pleiotropic molecule which provides numerous health benefits by improving hypertension and cardiometabolic disorders (138) (139), relieving pain (140, 141), and increasing insulin sensitivity (142); protecting against neurological diseases including Alzheimer disease (143). Moreover, H2S is critically involved in the extension of lifespan provided by caloric restriction (144, 145). Supraphysiological levels of H2S may be generated in certain pathological conditions and lead to toxicity, inducing inflammation or tissue damage (146).
The intriguing overlap between biological effects attributed to some Allium and Brassica species and those exhibited by the gasotransmitter H2S prompted several researchers to verify the H2S releasing capacity of those molecules. Recently, the ability of releasing H2S was found as a distinctive feature of several OSCs, and a plausible mechanism for their biological effects across different organs and tissues was described. The biological relevance of H2S release by OSCs was first demonstrated by Benavides et al. in the context of a study on the vasoactivity of garlic. The authors showed that garlic polysulfides DATS and DADS, the downstream metabolites of alliin, released H2S in red blood cells; importantly, pre-treating the cells with the thiol-blocking reagent iodoacetamide inhibited the release of H2S, thereby demonstrating that the mechanism by which polysulfides release H2S is dependent on intracellular thiols, such as glutathione (GSH) (147). Chemically, this reaction involves a nucleophilic substitution from thiol at the α carbon of the H2S-donor moiety and a subsequent release of H2S (148). This mechanism is biologically relevant as the relaxation induced by both garlic extract and DADS on isolated rat aortic rings strongly correlated to the amount of H2S released. In the wake of this work, Citi et al. first revealed that a similar mechanism accounts for the ability of several Brassicaceae-derived ITCs to release pharmacologically relevant concentrations of H2S in an l-cysteine dependent manner (149): allyl isothiocyanate (AITC), 4-hydroxybenzyl isothiocyanate (HBITC), benzyl isothiocyanate (BITC), erucin (ER), SFN (149, 150). The same group reported that H2S-release is associated with the in vivo anti-hypertensive, hypoglycemic, pain-relieving, and anti-inflammatory effects of OSCs derived from the Brassicacea Eruca Sativa (138, 151–153). Interestingly, Lucarini et al. first demonstrated that GRA, a GLS, can release H2S in aqueous solution independent of myrosinase, but the chemical mechanism underlying this phenomenon is still unclear (150). Whether other Alliaceae or Brassicaceae-derived OSCs releases H2S is still unknown.
Figure 3 summarizes the known reactions leading to H2S release from polysulfides, GLS or ITCs.
Figure 3 H2S release by OSCs derived from Alliaceae and Brassicaceae. The known reactions occurring for H2S release by polysulfides and isothiocyanates are shown. Among garlic-derived polysulfides, diallyl disulfide (DADS) and diallyl trisulfide (DATS) have been shown to release H2S by reaction with glutathione (GSH) by polarographic H2S sensor (154) (147) (148). Among glucosinolates, GRA has been found to release H2S by amperometric approach (149). Similarly, several isothiocyanates showed H2S-releasing activity: allyl isothiocyanate (AITC), 4-hydroxybenzyl isothiocyanate (HBITC), benzyl isothiocyanate (BITC), erucin (ER), sulforaphane (SFN) (149) (150). While the mechanism of release is unknown for glucosinolates, the mechanism of release by isothiocyanates is dependent on L-cysteine reaction (155). Moreover, different OSCs have different kinetics of H2S release.
This mechanism holds important implications for bone. Recent findings by our group and others demonstrated that H2S plays an important role in the regulation of bone cell differentiation and function. In vitro, H2S-donors promote osteogenic differentiation and stimulate mineralization by increasing calcium intake (156) and the expression of genes directly involved in the biosynthesis of hydroxyapatite, such BSP (157). Furthermore, the expression of the enzymes CBS and CSE, which are responsible for endogenous H2S production, steadily increased during osteogenic differentiation and correlated to mineral apposition (119). Moreover, H2S-donors inhibit osteoclast maturation and resorption by activating the antioxidant response elicited by the NRF2 transcription factor (104, 158). Further attesting to the relevance of H2S in bone homeostasis, evidence from several in vivo preclinical models showed that the depletion of H2S levels is associated with loss of bone mass; similar findings were reported in ovariectomized mice (157), in H2S-deficient CBS+/− mice (156), in glucocorticoids-induced osteoporosis (159). Interestingly, when animals were treated with pharmacological H2S-donors to normalize the plasma level of H2S, bone loss was prevented or reversed (156, 157). The ability of H2S to stimulate bone formation appears to be maintained across various conditions, even unrelated to systemic or genetic disfunctions: for example, the exogenous administration of H2S by means of the pharmacological donor GYY4137 was effective to attenuate the bone loss induced by modelled microgravity (160) and to promote osteogenesis in a model of distraction osteogenesis (161).
Overall, these data demonstrate that H2S regulates osteogenesis and bone formation in both healthy and pathological conditions.
Therefore, H2S release by OSCs could account, at least in part, for their biological properties. However, up to date no clinical or preclinical in vivo studies have investigated the effect of OSCs by correlating their bioactivity to the H2S levels.
The GRA/SFN system: A case-model for OSCs bioactivity based on H2S release
GRA is a glucosinolate abundant in aerial portions, developing florets (flower buds), sprouts, seeds and mature plants of cabbage, broccoli, cauliflower, kale and Brussels sprouts (77). GRA conversion to SFN, an ITC, requires the enzyme myrosinase, an intracellular thioglucosidase, which catalyzes its hydrolysis to an unstable aglucone that spontaneously rearranges to give rise to a range of products, including SFN. SFN is the progenitor of a family of compounds widely studied in the literature mostly due to their antioxidant and anticancer properties. In mammalians, GRA conversion to SFN is primarily mediated by bacterial microflora of the gastrointestinal tract; while a small proportion is generated in the mouth by plant myrosinase when released by plants after chewing. Our current knowledge on the bioavailability and the rate of conversion of GSL into ITCs are largely based on studies on the GRA/SFN system.
Although most of GRA introduced with diet undergoes hydrolysis in the gut by microbial thioglucosidases, a fraction of GRA (around 10-15%) is absorbed directly in the stomach and in the small intestine, before the catabolic breakdown to SFN is triggered by gut microbiota (77, 162).
Gastric acidity appears to attenuate GSL bioavailability (163). However, GRA is not destroyed by digestive enzymes during passage through the digestive tract and is able to reach the rat cecum intact, when is hydrolyzed to SFN which is able to cross the cecal enterocyte for systemic absorption and enterohepatic circulation (164, 165). Conversion of GRA to bioactive SFN by the rat cecal microbiota requires four or more days after broccoli consumption and is reversible (166); however, recent randomized clinical trials have ascertained that upon ingestion of GRA-enriched soups, increased SFN levels were detectable as early as 30’ in plasma and 1h in the urine of patients (162). Attesting the tissue systemic absorption of SFN and ITCs in general, they have been detected in both plasma and synovial fluid of osteoarthritis patients undergoing consumption of GLS-rich diets for 2 weeks (167). On the other hand, the direct delivery of SFN from foods is possible and was demonstrated in recent clinical studies (168, 169) where SFN was shown to be readily bioavailable (170); however, SFN is unstable, requires storage at freezing temperature, and SFN-enriched extracts are difficult to prepare and very expensive (163).
Although most of the research on the biological effects of SFN is focused on cancer because of its effect on cell cycle and apoptosis (171–173), it also regulates bone cells: in vitro, SFN inhibits monocyte cell proliferation and osteoclast differentiation in multiple ways, detailed above (100–104), while increases mineralization in mouse MSCs and in an ex vivo culture of calvariae explants (97). Notably, in one in vivo study the administration of SFN for 5 weeks to normal and ovariectomized mice lead to an approximate 20% increase in bone mass (97), shifting the balance of bone homeostasis and favoring bone acquisition and/or mitigation of bone resorption.
Of note, our group recently demonstrated that GRA obtained from Tuscan black kale promotes osteogenesis in human MSCs, independent of SFN, and this effect is associated to the release of H2S and an increased H2S uptake inside the cells (98). This is relatively unexpected as GLS have been considered for many years a relatively inert precursor of reactive derivatives ITCs. Although the chemistry underlying this phenomenon is still unclear and will require further investigation, this finding suggests that GLS may exert inherent biological activity based on their capacity to release H2S.
As the hydrolytic product of GRA, SFN, had been already shown to inhibit the activity of osteoclast in bone, it can be suggested that the ‘GRA-SFN system’ exerts a beneficial effect on bone both at level of GLS and of its cognate ITC. The routes of absorption of GRA and SFN as well as the proposed mode of action on bone cells is summarized in Figure 4.
Figure 4 A general model describing the routes of absorption of GRA and SFN and a proposed mechanism of action on bone cells based on H2S-release. Briefly, upon chewing of plants belonging to Brassica genus, myrosinase (MYR, green) is released and can convert glucoraphanin (GRA) to sulforaphane (SFN) (1). GRA can be adsorbed in the stomach or in the small intestine (2). Microbacterial thioglucosidases (MYR, red) converts GRA to SFN which is further adsorbed in large quantities (3). SFN and GRA are released by circulation in bone tissue where can release H2S and exert anabolic and anticatabolic properties on bone cells (4). The mechanism by which H2S can be directly released from GRA has not been clarified yet.
Clinical studies
OSCs and chronic diseases
Despite this review focuses primarily on the skeletal effects of OSCs, much of the clinical research on the health benefits of OSCs is aimed at metabolic or cardiovascular disease and cancer.
Vegetables or extracts rich in OSCs improved dyslipidemia, insulin resistance, hypertension and cardiovascular risk linked to atherosclerotic plaques in human studies.
Among interventional, randomized clinical trials, Jeon et al. evidenced that ethanol extracts from Brassica rapa, administrated as a part of the diet of overweight human for 10 weeks, induce a significant increase in the HLDL-cholesterol concentration and a significant reduction in the total cholesterol/HDL-cholesterol ratio, free fatty acid, and adipsin levels (174). A randomized double-blind trial, performed by Bahadoran et. al., investigated the effects of broccoli sprouts powder containing high concentration of SFN for four weeks in type 2 diabetic patients and showed that broccoli sprouts improve insulin resistance by decreasing serum insulin concentration and ‘homeostatic model assessment for insulin resistance’ (HOMA-IR) score (175).
In a prospective cohort study on Australian women aged 70 years and older, without clinical atherosclerotic vascular disease (ASVD) or diabetes mellitus at baseline, Blekkenhorst et al. investigated the occurrence of ASVD‐related deaths during 15 years of follow‐up and correlated it with several dietary intake, through a multivariable‐adjusted model. Among the nutrients tested, intakes of cruciferous and Allium vegetables were inversely associated with ASVD mortality supporting the evidence that the effect of increased intake of cruciferous and Allium vegetables lowered cardiovascular disease risk (176).
In cancer, treatment with OSCs-rich food showed promising results as chemopreventive.
A placebo double-blind randomized controlled trial on men scheduled for prostate biopsy and treated with broccoli sprout extract (BSE) supplementation (providing SFN and myrosinase) for 4.4 wk, performed by Zhang et. al., showed that BSE supplementation correlated with changes in gene expression but not with other prostate cancer immunohistochemistry biomarkers (173). In a double-blind placebo randomized clinical trial in patients with colorectal adenomas-precancerous lesions of the large bowel treated with aged garlic extract (AGE), Tanaka et al. demonstrated that AGE significantly reduced the size and number of colon adenomas in patients after 12 month (25). Several epidemiological studies showed that SFN consumption has been reported to be associated with a lower risk of cancer development (breast, lung, stomach, esophagus, mammary glands, gastric, colorectal, prostate, skin, head and neck, and liver) (172). In a large cohort study Millen et al. correlated the presence of adenoma with food intake of several fruit and vegetables, as assessed by a food-frequency questionnaire, and showed that onions and garlic were significantly related to lower risk of adenoma (177). Notably, a randomized double-blinded intervention study, performed by Traka et. al., showed that consuming GRA-rich broccoli for 12 months reduced the risk of prostate cancer progression (178). In particular, patients administrated with a weekly portion of soup made from a standard broccoli or 2 experimental broccoli genotypes with enhanced concentrations of GRA, showed dose-dependent attenuated activation of gene expression associated to oncogenic pathways in transperineal biopsies; and an inverse association between consumption of cruciferous vegetables and cancer progression was observed (178).
Overall, these studies highlighted the significant role of diet administration of OSCs in several chronic diseases and substantiate the relevance of creating specific dietary regimen for their prevention.
OSCs in the prevention of bone loss and skeletal frailty
A few clinical trials or population-based studies have revealed positive relationships between the consumption of vegetables, bone density, muscle strength and fractures in women/men, as summarized in Table 6.
Table 6 Clinical studies on musculoskeletal effects of OSCs-rich food and extracts.
Matheson et al. used a food frequency questionnaire added to the Nutritional Health and Nutrition Examination Survey (2003–2004) to examine the correlation between habitual consumption of onion over the past 12 months to BMD (N unweighted =507; N weighed =35.7 million). They found that higher consumption of onion increased the BMD by 5% (179). Law et al. administrated onion juice to healthy men and women and post-menopausal women for 8 weeks and investigated the association with bone BMD; the results found that the BMD of 3 postmenopausal women was mildly improved at the end of the treatment (85).
In an intriguing study, Blekkenhorst et al. used a food frequency questionnaire to examine the associations of vegetable and fruit intakes, separately, and specific types of vegetables and fruits with fracture-related hospitalizations in a prospective cohort of elderly women (mean age ≥ 70; n=1468); the authors found that the consumption of vegetable, but not fruit, is associated to a lower incidence of fracture; of note, the habitual consumption of cruciferous vegetables and Allium vegetables was significantly inversely associated with all fractures (18); importantly, these results were adjusted for energy intake and physical activity.
In musculoskeletal ageing, sarcopenia and declining physical activity are often associated with osteoporosis as the clinical hallmarks of frailty (180).
Interestingly, a prospective cohort study performed on elderly women (mean age ≥ 70; n=1429) investigated the correlation between vegetable consumption and incident falls-related hospitalization over a time-period of 14 years. The authors found that hospitalizations were lower in participants consuming more vegetables, but the consumption of cruciferous vegetables was most strongly associated with lower falls-related hospitalization (181) and was associated with increased muscle strength.
Finally, cross-sectional study, by Gu et. al., demonstrated a positive correlation between raw garlic consumption, assessed using a food frequency questionnaire, and handgrip strength in both males and females (182). The results were adjusted for age, body mass index, smoking status, alcohol-consumption status, education levels, employment status, household income, family history of diseases (cardiovascular disease, hypertension, hyperlipidemia, and diabetes), metabolic syndromes, physical activity, total energy intake, dietary pattern, onion intake. Although this study did not directly assess indexes of bone quantity, it supports an overall protective effect of OSCs-rich vegetables on the musculoskeletal system (181).
Perspectives and challenges
The present literature revision stems from the increasing appreciation of the link between dietary habits, and particularly the use of phytochemicals, and bone health. We show that a growing body of evidence supports a beneficial effect of dietary OSCs on skeletal health. Of note, although a few population-based studies offer interesting clues on the clinical relevance of OSCs-rich vegetables for the prevention of bone fragility (18, 85, 179, 183), no clinical studies have been performed yet to specifically address the potential protective role of OSCs against osteoporosis or bone fractures; this goal would require a study design including a controlled intake of OSCs-rich nutrients for long time-periods and/or the evaluation of purified OSCs molecules.
The ability of OSCs to work a as dietary source of the bioactive molecule H2S provide interesting future perspectives. OSCs-rich vegetables appear as the ideal candidate for clinical investigations on whether nutrients rich in sulfur can affect the pool of circulating reactive sulfur species (RSS), which include H2S; this may have a broad implication for the prevention of those pathologies, sometimes referred to as ‘H2S-poor diseases’, where the onset of the disease was associated to a lower systemic concentration of RSS compared to healthy controls. Increasing systemic RSS levels may also have important implication for bone-wasting diseases such as osteoporosis: indeed, animal studies have established that the bone loss associated to estrogen deficiency or to corticosteroid therapy is associated to a low systemic level of H2S (157, 159). However, these preclinical data still await confirmation in observational clinical studies in humans. To obtain reliable data on this topic, it will be critical to include in the study design a robust analytical methodology to quantitatively measure the different sulfur species in human serum or plasma since they may hold different importance in different pathologies (184, 185) and the high reactivity of these gaseous molecules implies a complex chemistry (186).
Further investigations may be addressed to the evaluation of the effect of these compounds on the gut-bone axis. OSCs show a considerable ability to modulate the gut microbiome and its secondary metabolites (187–190) and to mitigate the gut-based inflammatory response; given the paramount importance of metabolites and cytokines originated from the gut on the regulation on bone metabolism (191), it is conceivable that dietary OSCs may modulate the bone-bioactive components of the microbiota.
In the end, it is apparent that members of the OSCs family of phytochemicals affect bone homeostasis in several ways and may provide new insights into the potential bone health benefits of plant-derived food and leading to a more effective prevention of osteoporosis by non-pharmacological tools. This review may be useful to fuel clinical trials that may use a robust set of outcome measurements, aiming at assessing both bone quantity and bone quality before and after specific nutrition protocols; correlation between nutrients intake, H2S blood levels and bone status would help to define preventive/clinical dietary protocols for patients with an increased risk of bone fragility.
Author contributions
FG and LG contributed to the conception and design of the review. FG ad LG wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
Glossary
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Keywords: organosulfur compounds (OSCs), osteoporosis, hydrogen sulfide (H2S), Brassicaceae, Allium, glucosinolates, isothiocyanates, polysulfides
Citation: Gambari L, Grigolo B and Grassi F (2022) Dietary organosulfur compounds: Emerging players in the regulation of bone homeostasis by plant-derived molecules. Front. Endocrinol. 13:937956. doi: 10.3389/fendo.2022.937956
Received: 06 May 2022; Accepted: 23 August 2022;
Published: 15 September 2022.
Edited by:
Simona Bertoli, University of Milan, ItalyReviewed by:
Franziska S. Hanschen, Leibniz Institute of Vegetable and Ornamental Crops, GermanyHanyao Huang, Sichuan University, China
Copyright © 2022 Gambari, Grigolo and Grassi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Laura Gambari, laura.gambari@ior.it; Francesco Grassi, francesco.grassi@ior.it