Vitamin E Inhibits Osteoclastogenesis in Protecting Osteoporosis

*Chen Yongjie, Yu Naichun, Zhou Daguo, Li Zongguang, Gong Fengqing, Yi Weijiang, Chen Botao and Ji Guangrong*

## **Abstract**

The most common orthopedic condition affecting senior adults is osteoporosis, which is defined by a decrease in bone mass and strength as well as microstructural degradation that leads to fragility fractures. Bone remodeling is a well-planned, ongoing process that replaces deteriorated, old bone with new, healthy bone. Bone resorption and bone creation work together during the cycle of bone remodeling to preserve the bone's volume and microarchitecture. The only bone-resorbing cells in the human body, mononuclear preosteoclasts fuse to form osteoclasts, are multinucleated cells. In numerous animal models or epidemiological studies, vitamin E's anti-osteoporotic characteristics have been extensively described. This review aims to summarize recent developments in vitamin E's molecular features as a bone-protective agent. In RANKL/RANK/OPG signaling pathway, vitamin E inhibits synthesis of RANKL, stimulation of c-Fos, and increase level of OPG. Vitamin E also inhibits inflammatory cytokines, such as TNF-α, IL-1, IL-6, IL-27, and MCP-1, negative regulating the JAK–STAT, NF-κB, MAPK signaling pathways. Additionally, vitamin E decreases malondialdehyde and increases superoxide dismutase, GPx and heme oxygenase-1, in suppressing osteoclasts. In this article, we aim to give readers the most recent information on the molecular pathways that vitamin E uses to enhance bone health.

**Keywords:** vitamin E, osteoclast, osteoporosis, RANKL, inflammatory, oxidative stress

## **1. Introduction**

The most common orthopedic condition affecting elderly people is osteoporosis, which is characterized by a decline in microarchitecture and a loss of bone mass and strength. Approximately 25% of all women aged 65 or older are affected by osteoporosis, which results in the lifetime fracture risk of patients with osteoporosis being as high as 40% [1]. Osteoporosis fractures involving the hip, vertebrae, and wrist reduce life expectancy or quality of life. Some drugs classified as antiresorptive or anabolic have been used in the treatment of osteoporosis [2, 3].

Antiresorptive drugs, which include bisphosphonates, estrogens, denosumab, and selective estrogen receptor modulators (SERMs), increase bone mineral density (BMD) and reduce fragility fractures. Bisphosphonates are the most widely used antiresorptive drug, which bind avidly to bone mineral and have a continual effect in months to years. However, bisphosphonates have low bioavailability and induce gastrointestinal problems, myalgia, and flu-like symptoms. Osteoclast activity and development are significantly decreased by the monoclonal antibody denosumab, which specifically targets RANKL. Every six months, denosumab is given by subcutaneous injection. Denosumab's anti-fracture effects are comparable to those of bisphosphonates; however starting 7 months after the last injection, there is a noticeable reduction of anti-resorptive action, which can lead to clusters of rebound vertebral fractures. SERMs exhibit estrogen agonist or antagonist qualities and bind to the estrogen receptor with high affinity to prevent bone loss. However, SERMs can induce venous thromboemboembolism, stroke and hot flashes, which limit their use [4].

Anabolic agents, including teriparatide and abaloparatide, promote bone formation to reduce the risk of fractures. Teriparatide and abaloparatide are both limited to a maximum of 24 months of therapy due to elevated osteosarcoma incidence in rats treated with these medications for their entire lives. These drugs similarly induce some adverse effects, including dizziness, leg cramps, nausea, postural hypotension, and headache. Another anabolic agent is romosozumab, which blocks the actions of sclerostin to reduce bone resorption. However, the romosozumab group in the ARCH study had a higher rate of a composite endpoint that included cardiovascular death, nonfatal myocardial infarction, and nonfatal stroke. In general, different drugs have been used to treat osteoporosis (**Table 1**), but their different adverse reactions limit their long-term use against osteoporosis [2, 3, 5, 6].

Vitamin E has shown great potential in the treatment of osteoporosis. There are two subclasses of the lipid-soluble vitamin E: tocopherol (TF) and tocotrienol (T3). Each isoform can further be separated into four unique analogs, namely, alpha (α), beta (β), gamma (γ), and delta (δ). Some researchers have investigated the impact of vitamin E (TF) on osteoblast differentiation in a previous study. Early osteoblast development



#### **Table 1.**

*Principal medications for treatment of osteoporosis.*

was demonstrated to be hindered by TF, as evidenced by a decrease in alkaline phosphatase (ALP) activity and osteocalcin expression (OCN) [7]. In another study, another researcher showed that incubating human mesenchymal stem cells (MSCs) with TF boosted their proliferation. ALP and runt-related transcription factor-2 (Runx-2) expression was markedly increased in cells treated with TF. Gamma-T3 has been shown to decrease apoptosis and increase osteoblastic cell proliferation, differentiation, and mineralization. When αTF and δT3 were compared to TF and T3, αTF and δT3 had superior effects on inhibiting osteoblast differentiation. In a biomechanical strength test, bone scaffolds treated with all types of vitamin E isomers, particularly αTF and δT3, displayed enhanced elasticity [8]. According to this study, αTF and δT3 may be the isomers that affect bone most actively. Many researchers have focused on the effect of vitamin E on osteoblasts. In contrast, few *in vitro* studies have examined how vitamin E affects osteoclasts. This chapter emphasizes recent developments in our knowledge of vitamin E's molecular actions in inhibiting osteoclasts, particularly through interactions between multiple signaling pathways and signal transduction molecules.

## **2. Osteoclasts in bone homeostasis**

Bone is a dynamic tissue that changes regularly during the course of life. During the process of bone remodeling, osteoclasts remove old or damaged bone and osteoblasts replace it with fresh bone. Without appreciably altering the net bone mass and mechanical strength, the ratio of osteoblast-mediated bone formation to osteoclast-mediated bone resorption is tightly regulated in homeostatic situations [9]. However, when this equilibrium is dysregulated, aberrant bone remodeling occurs, leading to postmenopausal and secondary types of osteoporosis. Osteoclasts are the only bone-resorbing cells in the human body, which is significant since they are essential for reconstructing the skeletal system. Numerous cells originating from blood-circulating monocytes combine to become osteoclasts. They themselves come from the bone marrow. Although the majority have only 5 to 20 nuclei, osteoclasts can have up to 200 nuclei. The active area of osteoclasts is formed by numerous tiny projections (microvilli) that protrude into the surface of the bone on the side of the cell that is closest to the bone. One of the many enzymes produced by osteoclasts,

acid phosphatase dissolves both the organic collagen and the inorganic calcium and phosphorus found in bone. The osteoclast then engulfs and digests the broken-down pieces of mineralized bone inside the cytoplasmic vacuoles. Communication between osteoblasts and osteoclasts is crucial for optimizing bone remodeling during bone homeostasis. In addition to M-CSF, RANKL, and WNT5A, osteoblasts also release OPG and WNT16, which limit osteoclast activity. In contrast, S1P, CTHRC1, and C3, which encourage osteoblast differentiation, as well as SEMA4D, which inhibits osteoblast differentiation, are secreted by osteoclasts.

A hematopoietic growth factor called M-CSF enables mononuclear phagocyte lineages, which include osteoclasts, to survive, proliferate, differentiate, and move around. M-CSF binds to its specific receptor, C-FMS, on the surface of osteoclasts and monocytes/macrophages after being produced by osteoblasts and bone marrow stromal cells. Significant amounts of RANKL are expressed in lymph nodes, activated T lymphocytes, osteoblasts, and osteocytes. The main regulatory transcription factors and enzymes are activated as a result of RANKL activation of RANK, which promotes osteoclast differentiation, fusion, and proliferation. For instance, RANKL binding to RANK activates TRAF6, which has been discovered to activate the protein kinase TGF-activated kinase (TAK1). The classical IB kinase (IKK) complex is then activated by TAK1. IKK activation results in the activation of NF-κB, protein kinase Tp12, and mitogen-activated protein (MAP) kinase (MKK) kinases. MKKs trigger the activity of p38 MAP kinases and c-Jun N-terminal kinases. Extracellular signal-regulated kinase 1 (ERK1) and ERK2 are stimulated by activated Tp12, which in turn activates MEK1 and MEK2. Eventually, the MAPK and NF-κB signaling pathways are activated, increasing NFATc1, a crucial regulator of osteoclastogenesis. OPG has been shown to be a secreted glycoprotein made by numerous cell types, including osteoblasts, cells living in the liver or lungs, and B lymphocytes in the bone marrow. Due to the suppression of the development of osteoclasts, overexpression of OPG causes severe osteopetrosis. OPG is thought to act as a decoy receptor for RANKL, blocking the RANKL-RANK interaction and impairing osteoclast development and activation. Because it controls osteoblastogenesis and osteoclastogenesis through both catenin-dependent (canonical) and catenin-independent (noncanonical) pathways, the WNT pathway is essential for preserving bone homeostasis. By increasing RANK expression in osteoclasts and activating the MAPK pathway, WNT5A promotes RANKLinduced osteoclastogenesis, which is also inhibited by WNT16 both directly and indirectly. A direct method of preventing osteoclastogenesis is provided by WNT16-induced phosphorylation of JUN, which increases OPG expression in osteoblasts in addition to directly inhibiting osteoclastogenesis *via* the noncanonical JNK/MAPK pathway (**Table 2**) [10].



*RANX 1/2, runt-related transcription factor 1/2; CT-1, Cardiotrophin-1; OSM, oncostatin M; OSX, osterix; ATF 4, activating transcription factor 4; SATB 2, special AT-rich sequence-binding protein 2; AP-1, activator protein-1; IFN-γ, interferon-γ; TNF-α/β, tumor necrosis factor-α/β; MITF, microphthalmia transcription factor.*

#### **Table 2.**

*Key factors and signaling pathways of osteoblastogenesis or osteoclastogenesis.*

## **3. The effects of vitamin E on bone cells**

In the past, researchers have looked at the direct effects of vitamin E on bone cells. In a prior study, Soeta et al. looked at the effects of vitamin E (αTF and δT3) on osteoblast differentiation. It was demonstrated that whereas TF initially hindered osteoblast differentiation, at a later stage, osteoblast differentiation reverted to normal [7]. Ahn et al. discovered in another investigation that incubation with αTF increased the proliferation of MSCs. In cells treated with αTF, the expression of ALP and Runx-2 was dramatically upregulated. Gamma-T3 has been demonstrated to enhance osteoblastic cell proliferation, differentiation, and mineralization, as well as reduce apoptosis [11]. According to prior research, α T3's protective effects on osteoblastic cells were consistent, while TF's effects were varied. Osteoclastogenesis was inhibited by both the γT3 and αT3 isomers, with the latter showing a more significant suppression of osteoclast production and activity than the former [12].

Vitamins also affect osteoclastogenesis. Johnson showed that vitamin E administration inhibits osteoclastogenesis, potentially by preventing the development of monocytes and lymphocytes. In their study, the Ovx control group had a much higher number of osteoclasts, which were strongly inhibited by all three dosages of vitamin E, although more efficiently in the Ovx group that received 300 mg of vitamin E per kg of food. Vitamin E also reduced the increase in monocyte and lymphocyte production induced by Ovx [13]. Woon Kim discovered that tocotrienol reduced the amount of RANKL that IL-17 stimulated FLS generation. Tocotrienol reduced the activation of extracellular signal-regulated kinase, kappa B-alpha inhibitor, and mammalian target of rapamycin caused by IL-17. Osteoclasts were differentiated when monocytes were incubated with IL-17, RANKL, FLS that had been treated with IL-17, or Th17 cells, and tocotrienol inhibited this osteoclast differentiation. Tocotrienol decreased IL-17 and sRANKL synthesis as well as Th17 cell differentiation [14]. Polyethylene has been shown by Hazir et al. to increase the number of TRAP-positive cells and the expression of genes related to osteoclasts. TF therapy markedly decreased the quantity of TRAP-positive osteoclasts, bone resorption activity, antioxidant-related gene expression and mitochondrial function [15]. In Walker 256/B tumor osteolytic rats, Badraoui et al. investigated the effects of vitamin E supplementation on osteoclast resorbing activity and cytomorphometry. The results indicate that fewer osteolytic lesions were visible in W256VE. In addition, the W256VE group had less alteration of bone microarchitecture and OC activity [16]. Quercetin and vitamin E have been studied by Vakili et al. in relation to ovariectomy-induced osteoporosis.

The results showed that ovariectomy increased the total number of osteoclasts and serum osteocalcin and lowered the bone weight, bone volume, trabeculae volume, and total number of osteocytes and osteoblasts. The expression of LC3, beclin1, and caspase 3 was also upregulated in the tibia, while bcl2 expression was downregulated. Treatment with Q and vitamin E significantly improved osteoporosis by reversing these alterations [17]. In a double-blinded, randomized, placebo-controlled human trial study, vitamin E has significantly decreased the serum C-terminal telopeptide of type I collagen (CTX) [18]. These studies have indicated that vitamin E could inhibit osteoclastogenesis in the treatment of osteoporosis.

## **4. The molecular mechanism of vitamin E in osteoclastogenesis**

## **4.1 The effect of vitamin E on the M-CSF and RANKL/RANK/OPG signaling pathways**

The RANK/RANKL/OPG trimolecular complex system and M-CSF exert a considerable influence on osteoclast production and the regulation of bone resorption. The maturation of osteoclast precursor cells into mature osteoclasts in the presence of RANKL depends on the production of M-CSF by osteoblastic stromal cells. Other names include osteoclast differentiation factor (ODF), tumor necrosis factor-related activation-induced cytokine, and OPG ligand (TRANCE). When RANKL binds to its receptor, RANK, on myeloid cells, a series of intracellular signaling events are triggered, including interactions with TRAF6 adaptor molecules and the activation of NF-κB, nuclear factor of activated T cells cytoplasmic 1 (NFATc1), MAPK, and PI3K. It has been demonstrated that NF-κB regulates RANKL-induced osteoclast development, activating the Fos proto-oncogene (c-Fos) before NFATc1. The presence of NFATc1, which has been dubbed the "master regulator of osteoclast development," is critical for stem cells differentiate into osteoclasts. If RANKL and NF-κB p65 interact, it becomes transiently active in osteoclast precursors within an hour. Additionally, the c-Fos is also vital for osteoclastogenesis induced by M-CSF and RANKL. OPG is a protein that is largely secreted by osteoblast lineage cells, and it functions as a key endogenous regulator of the RANK/RANKL/OPG pathway. The same ligand, RANKL, has affinity for the receptors OPG and RANK as well. In order to stop osteoclastogenesis and the survival of existing osteoclasts, OPG acts as a decoy receptor by interacting with RANK. The ratio of RANKL/OPG may play a significant role in regulating bone resorption because OPG expression is normally downregulated and not as active as RANKL. Cortical and cancellous bone mass is considerably increased by OPG overexpression, while osteoclast levels are decreased.

Ambroszkiewicz et al. observed that children with cystic fibrosis (CF), the most prevalent deadly autosomal recessive genetic illness that results in a range of longterm health issues, including bone ailments, have lower average levels of fat-soluble vitamins (A, D, and E). Children with CF have significantly lower levels of the markers for bone production (osteocalcin) and bone resorption (CTX, TRACP5b) than children without the condition. In comparison with healthy individuals, CF patients had serum levels of OPG that were much lower and RANKL that were approximately two times greater. This finding suggested a link between vitamin E and RANKL or OPG in various bone diseases [19]. In another study, Kim et al. showed that RANKL synthesis was promoted by IL-17 in fibroblast-like synoviocytes (FLS) and was inhibited by tocotrienol. Tocotrienol reduced the activation of NF-kappaB,

#### *Vitamin E Inhibits Osteoclastogenesis in Protecting Osteoporosis DOI: http://dx.doi.org/10.5772/intechopen.112760*

extracellular signal-regulated kinase, and mammalian target of rapamycin caused by IL-17. Osteoclasts were differentiated when monocytes were incubated with IL-17, RANKL, FLS that had been treated with IL-17, or Th17 cells, and tocotrienol inhibited this osteoclast differentiation [14]. Trolox is a water-soluble vitamin E analog that has been studied for its effects on osteoclastogenesis and RANKL signaling by Kim et al. By preventing RANKL production in osteoblasts, trolox effectively prevented interleukin-1-induced osteoclast development in bone marrow cell-osteoblast coculture. This decrease in RANKL was ascribed to a downregulation of cyclooxygenase-2 activity, which in turn resulted in less prostaglandin E [2] being produced. Trolox also reversibly reduced the production of osteoclasts by bone marrow macrophages stimulated by RANKL and M-CSF. Trolox targets early osteoclast precursors since its effectiveness depends on its presence early in the culture process. Trolox pretreatment had no effect on the early signaling pathways activated by RANKL, such as the MAPK, NF-κB, and Akt pathways. By inhibiting its translation, Trolox reduced the stimulation of the c-Fos protein by RANKL. Trolox's suppression of osteoclastogenesis in bone marrow macrophages was reversed by ectopic overexpression of c-Fos [20]. In cocultures of osteoblasts and bone marrow cells stimulated by either IL-1 or a combination of 1,25(OH)(2) vitamin D(3) and prostaglandin E, Ha et al. discovered that vitamin E inhibits osteoclastogenesis. This is supported by the finding that only tocotrienols prevented RANKL in osteoblasts. Additionally, c-Fos expression was suppressed by α-tocotrienols but not α-tocopherols, which may have been accomplished by preventing ERK and NF-κB activation. This may have prevented RANKL-induced osteoclast development from precursors. When c-Fos or an active version of NFATc1, a crucial downstream target of c-Fos during osteoclastogenesis, was overexpressed, this anti-osteoclastogenic impact was reversed [21]. Human osteoblastic SaOS2 cells were treated with wear particles from vitamin E-doped and regular UHMWPE, and the gene expression and protein synthesis of IL-6, RANKL, OPG, DKK-1, and Sclerostin were then evaluated. When compared to standard UHMWPE, vitamin E-blended UHMWPE lowered RANKL while increasing OPG [22]. According to research by Mohammed, annatto tocotrienol (AT) and self-emulsified annatto tocotrienol (SEAT) both increased the number of osteoblasts and the pace at which trabecular mineralization occurred. In OVX animals, AT also reduced the expression of skeletal sclerostin. Only SEAT significantly boosted the bone formation rate and decreased the RANKL/OPG ratio. SEAT increases bone growth, inhibits sclerostin expression, and lowers the RANKL/OPG ratio in rats with estrogen shortage, all of which may have skeletal anabolic effects [23].

Overall, the RANK/RANKL/OPG system is a crucial signaling channel involved in communication between bone cells, and there is substantial evidence that altering this signaling pathway has significant consequences for bone remodeling. While OPG inhibits osteoclast-induced bone resorption by negatively regulating RANKL binding to RANK and shortening the half-life of membranous RANKL, RANKL mediates osteoclastogenesis and activates mature osteoclasts. According to growing research, T3 therapy has the potential to protect bones by regulating this route. However, more research is required to support these conclusions.

#### **4.2 Vitamin E suppresses inflammation in preventing bone loss**

Inflammation, which is defined by the activation of immune cells and the consequent release of inflammatory cytokines, is the first line of defense against illnesses for both the innate and adaptive immune systems. The removal of damaged tissues

and the beginning of tissue repair are both essential functions of inflammation. Continued inflammation, however, also promotes bone resorption and inhibits bone development. The main controllers of the inflammatory response seen in metabolic bone disorders, such as osteoporosis, are cytokines. Interferon, interleukin (IL), and tumor necrosis factor-alpha (TNF-α) are only a few of these cytokines. Previous research has shown that proinflammatory mediators can both positively and adversely affect osteoclast and osteoblast activities. Significant stimulators of bone resorption include TNF-α, IL-1, and IL-6. After TNF-α and IL-6 are recognized by their corresponding receptors on bone marrow stromal cells, the inhibition of MAPK, the activation of suppressor of mothers against decapentaplegic ubiquitylation regulatory factor 1 (SMURF1) and SMURF2, and the activation of signal transducers and activators of transcription (STAT) all work together to cause the downregulation of osteoblast gene products. Through the overexpression of Dickkopf-related protein 1 (DKK1) and sclerostin (SOST), proinflammatory cytokines block the transcription of osteogenic factors to block the Wnt/β-catenin pathway. Additionally, due of the link among inflammatory factors and their receptors, which promotes osteoclast development, proliferation, and activation, osteoblasts create more M-CSF and RANKL. TNF-α, IL-1, and IL-6 signal in osteoclasts and osteoclast precursors through the NF-κB, MAPK, and Janus kinase (JAK)-STAT pathways to upregulate osteoclastrelated genes and intensify osteoclastogenesis. The downregulation of c-Fos, NFATc1, and TRAF6, as well as the subsequent impairment of the activation of downstream targets, including NF-κB and JNK, has been hypothesized as the underlying mechanisms of IFN-γ in suppressing osteoclast development. Additionally, IFN-γ increases the interaction between Fas and Fas ligands to promote osteoclast precursor death and induces osteoblasts to create nitric oxide. On the other hand, IFN-γ indirectly encourages osteoclast development by promoting T-cell activation and the release of osteoclastogenic substances, including TNF-α and RANKL, by T cells. Additionally, IFN-γ stimulates the production of DC-STAMP, which is in charge of fusing mononucleated osteoclasts into mature, functioning osteoclasts. It is speculated that inflammatory cytokines play an important role in bone homeostasis.

Several different laboratory experiments have shown how vitamin E can reduce systemic inflammation. In children with biopsy-proven NAFLD, hydroxytyrosol (HXT) and vitamin E may reduce oxidative stress, insulin resistance, and steatosis, according to Mosca. Children participating in the HXT + VitE experiment had their plasma levels of IL-6, IL-1, IL-10, TNF, 4-hydroxy-2-nonenal (4-HNE), and 8-hydroxy-2'deoxyguanosine (8-OHdG) analyzed. This study showed that both the placebo (Pla) group and the HXT + VitE group exhibited changes in indicators of systemic inflammation. Levels of IL-1 and TNF were decreased in both groups; however, IL-6 and IL-10 increased significantly only in the HXT + VitE group. Systemic inflammation in children caused by NAFLD was reduced by HXT and VitE therapy [24]. Hashem pretreated rats with vitamin E (60 mg/kg) and continued giving them vitamin E until the experiment was complete to test the hypothesis that acute pancreatitis (AP) and the regulation of the TNF-AMPK axis in the presence and absence of vitamin E are related. Infiltration of inflammatory cells and severe pancreatic tissue damage, which were significantly shielded by vitamin E, served as evidence that AP had occurred. Additionally, L-arginine injections markedly lowered phospho-AMPK, IL-10 mRNA, and protein expression, which were markedly protected by vitamin E [25]. Amevor et al. exposed 400 Tianfu breeders to 0.4 g/kg quercetin (Q ) and 0.2 g/ kg vitamin E, Q (0.4 g/kg) and vitamin E (0.2 g/kg) for 14 weeks to study the effects of dietary combinations of Q and vitamin E on the intestinal structure and barrier

#### *Vitamin E Inhibits Osteoclastogenesis in Protecting Osteoporosis DOI: http://dx.doi.org/10.5772/intechopen.112760*

integrity in aged breeder chickens. The findings demonstrated that Q + vitamin E had synergistic effects on intestinal morphology by increasing villus height and crypt depth and by reducing inflammatory damage to the intestines of elderly chickens. Additionally, Q + vitamin E boosted the mRNA expression of intestinal tight junction proteins, such as occludin, ZO1, and claudin-1. Additionally, Q + vitamin E boosted the expression of anti-inflammatory genes (IL-10 and IL-4) while decreasing the mRNA expression of pro-inflammatory genes (TNF-α, IL-6, and IL-1) [26]. Cellbased, preclinical, and clinical intervention studies have investigated the mechanisms behind the impact of vitamin E on the immune system and inflammation. Vitamin E affects inflammatory mediators produced by other immune cells, which in turn affects the integrity of T-cell membranes, signal transduction, and cell division both directly and indirectly. Since it impacts the host's susceptibility to infectious diseases including respiratory infections as well as allergy and infectious diseases like asthma, vitamin E's control of immune function has therapeutic importance. In many responses to infection, inflammation, and immunological activation, IL-1 is a key player. In addition to osteoporosis and cancer-induced osteolysis, IL-1 has a role in the etiology of rheumatoid arthritis and osteolysis of orthopedic implants. It promotes osteoclast development and activity, which results in excessive bone resorption. Without the assistance of osteoblasts or stromal cells, IL-1 may directly encourage the development of osteoclasts. It works by activating NF-κB in osteoclasts and inhibiting apoptosis. It was shown that vitamin E has the capacity to prevent activated monocytes from producing IL-1. Both hemopoietic and nonhematopoietic cells produce IL-6 in response to diverse forms of stimulation. Under the control of parathyroid hormone, vitamin D3, growth factor, and other cytokines, stromal cells and osteoblasts release IL-6 during bone remodeling in nanomolar amounts. The production of IL-6 by osteoblasts in response to IL-1, TNF-α, and LPS stimulation has also been documented. Osteoclastogenesis would be encouraged, and NF-κB would be activated as a result of high cytokine levels.

Animals with ovariectomy-induced estrogen shortage had higher levels of IL-1 and IL-6. Treatment with palm-derived T3 at 60 mg/kg was successful in preventing cytokine levels from increasing. In addition to enhancing bone microstructure in MetS mice, palm-derived T3 and annatto-derived T3 decreased inflammatory marker levels (IL-1 and IL-6). Researchers found that annatto T3 improved trabecular and cortical microstructure, raised serum procollagen I intact N-terminal propeptide while decreasing serum carboxyl-terminal telopeptide of type I collagen, and inhibited the expression of inflammation markers (MCP-1, IL-2, IL-23, IFN-γ, and TNF-α) in a distinct investigation [27–29]. Animals treated with free radicals and nicotine were also used to clarify the unique ways that vitamin E affects inflammatory indicators. Free radicals produced by ferric nitrilotriacetate increased the levels of OCN, IL-1, and IL-6 and negatively affected bone histomorphometry. By supplementation with a palm oil T3 combination at a rate of 100 mg/kg, these unfavorable alterations were reversed. Inflammatory cytokines and markers of bone resorption were both elevated after two months of nicotine administration, whereas a marker of bone production was decreased. Vitamin E at a dose of 60 mg/kg demonstrated effectiveness in enhancing all these parameters when compared to the control group. In a different investigation, it was found that 60 mg/kg of a palm T3 combination might prevent the rise in IL-1 and IL-6 caused by nicotine administration [30]. In contrast, in experimental animal models, ultrahigh molecular weight polyethylene (VE-UHMWPE) combined with vitamin E has been shown to have higher mechanical performance and less unfavorable cellular responses than ordinary polyethylene.


#### **Table 3.**

*Inflammatory factor and related signaling pathways which vitamin E regulated.*

Human macrophages stimulated by VE-UHMWPE particles showed a different transcriptional program from macrophages stimulated by UHMWPE particles. IL-27 was one of the upregulated genes, and it was discovered that in macrophages cultivated with VE-UHMWPE particles as opposed to those cultivated with UHMWPE particles, its levels were much higher. IL-27 decreased the inflammatory response induced by standard UHMWPE particles *in vitro* and prevented osteoclasts from differentiating [31].

When combined, the information from these previous articles showed that vitamin E had anti-inflammatory characteristics that can protect against osteoporosis (TNF-α, IL-1, IL-6, IL-27, MCP-1) (**Table 3**) [32]. Furthermore, other inflammatory cytokines, including IL-7, IL-8, IL-11, and IL-34, IL-3, IL-4, IL-10, IL-12, and IL-33, whether regulated by vitamin E in osteoclastogenesis need further research.

#### **4.3 Vitamin E suppresses oxidative stress in preventing bone loss**

Reactive oxygen species (ROS) and reactive nitrogen species are the two types of free radicals that are most frequently mentioned when discussing oxidative stress. An imbalance among these free radical oxidants and the cells' antioxidant defenses leads to cellular damage. Chain reactions affecting DNA, proteins, cell wall lipoproteins, and other biological components are triggered by damage from free radical molecules such as superoxide (O2−). Additionally, NF-ĸB and other pathways are activated, which upregulate the TNF-α and IL-1 downstream cytokines. Through inhibition or activation of several cellular pathways, changes in the cell and its organelles modify cellular function. These alterations result in inflammation (through IL-4, IL-6, TNF-α, and other factors), changes to crucial cellular processes, and even mitochondria-initiated cell death and apoptosis, which, in severe cases, can result in organ damage. According to the mitochondrial theory of aging, chronic cumulative oxidative stress encourages disease conditions and early aging. Oxidative stress has been linked to a wide range of chronic diseases, including osteoporosis, in a significant body of literature ever since the mitochondrial theory of aging was first proposed [33]. The mitochondria of the cell are where ROS are typically present. An oxygen molecule (O2) is combined with four electrons and four protons to create two water molecules during the electron transport process in the mitochondria of the cell. This transfer results in the formation of a superoxide ion (O2 − ) or, less frequently, a hydroxide (OH-) or peroxide ion (O2 2−), each of which is a reactive-free radical in ROS form. Normally, the antioxidant enzymes superoxide dismutase (SOD) and peroxidase convert these ROS into the less reactive molecules hydrogen peroxide (H2O2) and water (H2O). The nicotinamide adenine dinucleotide phosphate (NADP-NADPH) cycle, which neutrophils use to destroy

#### *Vitamin E Inhibits Osteoclastogenesis in Protecting Osteoporosis DOI: http://dx.doi.org/10.5772/intechopen.112760*

viruses and bacteria, xanthine oxidase reactions during purine catabolism, nitric oxide synthase reactions during nitric oxide synthesis, and cyclooxygenases (COX) in the pathway for prostaglandin synthesis are some additional cellular pathways that produce ROS. Despite having the potential to cause damage, ROS and RNS are essential for healthy cell function because they play a role in cellular messaging that controls processes including gene transcription and transduction, differentiation, apoptosis, and repair. To minimize the negative effects of unchecked oxidative stress, natural cellular defenses against ROS and RNS work to preserve equilibrium rather than eliminate them. Oxidative stress is accelerated by anything that speeds up mitochondrial metabolism in the cell. Examples include fractures, surgery, and trauma injuries, infections, the detoxification of drugs or alcohol, high oxygen conditions, smoke inhalation, elevated salt concentrations, and heavy metal exposure. Oxidative stress is also heightened by radiation from radiographic, solar, cosmic, or electromagnetic sources. Even consuming foods devoid of naturally occurring antioxidants, such as processed sugars and some fatty acids, increases oxidative stress. Additional factors that may contribute to an increase in oxidative stress include dehydration, stress, anxiety, and possibly inadequate sleep. OPG and RANKL expression are both susceptible to the oxidative state, which lowers OPG expression and raises RANKL expression, tipping the scales in favor of bone loss. In response to excessive oxidative stress, sclerostin and DKK-1, two WNT pathway inhibitors, involved in the osteoimmunological regulation of bone remodeling, are increased, while OPG production is decreased. Aging, which is characterized by an increase in oxidative stress, and inflammatory mediators appear to be the two situations that most strongly promote the production of WNT pathway inhibitors. Increases in pro-oxidants have been associated with decreased osteoprogenitor development into the osteoblast cell lineage. Oxidative stress inhibits rabbit bone marrow stromal cells and calvarial osteoblasts differentiate into osteoblasts, according to research by Bai et al. Differentiation markers for this include decreased levels of ALP, type-I collagen, and nuclear phosphorylation of Runx2. OPG production is lowered as a result of decreased osteoblast activity. This decrease further modifies the overall RANKL/OPG ratio, which is essential for maintaining the balance of osteoblastic and osteoclastic activity. Oxidative stress causes an increase in apoptosis in osteoblasts and osteocytes. The cytokines for osteoblastic activity are reduced with osteocyte death, which further favors osteoclastogenesis. Additionally, osteocytes that are dead or dying induce osteoclastogenesis. Antioxidants, such as GSH, N-acetylcysteine, and alpha-lipoic acid, reduce these effects.

Given the rapidly changing potential role of oxidative stress in osteoporosis, reducing oxidative stress with an antioxidative agent may be a practical way to prevent osteoporosis. Strong free radical scavenger vitamin E has been shown to have bone-protective properties by reducing oxidative stress. Osteoblasts were protected against the negative effects of H2O2 by treatment with tocotrienol (1 μM) for 24 hours in a study using an H2O2-induced osteoblast model. In ovariectomized models, tocotrienol treatments both short-term and long-term successfully increased femur bone mineral density, bone formation, and strength. In addition, osteocalcin, bone morphogenetic protein 2 (BMP-2), and RUNX-2 mRNA expression in the tibia increased after tocotrienol treatments [34]. In an *in vitro* study that looked at the effects of tocotrienol on lipid peroxidation, antioxidant enzyme activities, and apoptosis of osteoblasts exposed to H2O2, tocotrienol was able to prevent malondialdehyde (MDA) elevation, decrease osteoblast apoptosis, and increase SOD, GPx, and CAT activities. It has also been shown that other natural compounds, including tocotrienols, rice protein, melatonin, and others, lower ROS by increasing heme oxygenase-1 (HO-1)

activity, which may potentially counteract the negative effects of oxidative stress [35]. Nuclear respiratory factor 2(NRF2) significantly controls the transcription of these antioxidants. Under normal circumstances, NRF2 is recruited by Kelch-like ECH-associated protein 1 (KEAP1) into the Cul3-containing E3 ubiquitin ligase complex, where it is ubiquitylated and degraded. In contrast, oxidative stress inhibits the function of the E3 ubiquitin ligase, which causes NRF2 and KEAP1 to separate. The transcription of SOD, CAT, HO-1, GPxs is then activated once Nrf2 enters the nucleus and forms a heterodimer with tiny Maf proteins in antioxidant response elements (AREs), a conserved gene sequence in target gene promoter regions. By triggering the NRF2-KEAP1-ARE pathway, tocotrienols, rice protein, melatonin, and other naturally occurring substances that are NRF2 activators may reduce ROS. However, in Fujita's investigation, treatment with α-tocopherol had no effect on the proliferation of osteoclast precursors or the survival of mature osteoclasts. Instead, it increased the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts. Additionally, they examined whether vitamin E's antioxidant characteristics were necessary for it to trigger the fusion of osteoclasts. None of the vitamin E isoforms, with the exception of α-tocopherol, induced osteoclast fusion [36]. This result indicated that vitamin E decreases bone mass by stimulating osteoclast fusion.

Vitamin E has shown tremendous promise in the treatment of postmenopausal osteoporosis because it is an efficient source for halting bone loss by suppressing oxidative stress.

#### **4.4 Other mechanisms of vitamin E in preventing bone loss**

Initially, a hormone mostly made by adipose tissue, leptin, regulates satiety and energy expenditure by acting on the hypothalamus. Leptin has a variety of effects, including the promotion of hemopoietic and osteoblastic differentiation, which it uses to control bone mass. Prior studies have shown that leptin has distinct effects on bone metabolism through both central and peripheral pathways. The brain's hypothalamic leptin receptors were activated in groundbreaking *in vivo* research, which showed that doing so decreased osteoblast formation and function and boosted osteoclast activity [37]. Two downstream molecular cascades are active: (a) an increase in RANKL through the PKA signaling pathway to encourage osteoclasts' effects on bone resorption; and (b) a decrease in c-myc expression, which encourages the production of cyclin D and prevents osteoblast development. An earlier study found that compared to healthy control animals fed a standard chow diet, rats on a high-carb, high-fat diet displayed symptoms of MetS, hyperleptinemia, and hypoadiponectinemia as well as damage to the trabecular microarchitecture. The increased leptin was decreased by palm and annatto T3 treatments [28, 29]. MicroRNA (miRNA) is a small, highly conserved endogenous noncoding RNA molecule with 20–22 bases that controls the expression of proteins. The differentiation of osteoclasts and miRNA expression are closely related processes. Several miRNAs inhibit osteoclast development and function by targeting the RANK/RANKL/OPG pathway. The expression of RANKL was suppressed by miR-17/20a and miR-26a. In bone marrow-derived macrophages, other miRNAs displayed suppression on osteoclast development through the TGF-β/SMAD signaling pathway. Insufficient vitamin E led to lower levels of miR-122a and miR-125b in the rat liver. These results suggest that vitamin E may regulate miRNA expression, which may be crucial for the regulation of bone homeostasis. The precise mechanism needs to be further researched [38, 39].

*Vitamin E Inhibits Osteoclastogenesis in Protecting Osteoporosis DOI: http://dx.doi.org/10.5772/intechopen.112760*

#### **Figure 1.**

*The molecular mechanism of vitamin E on osteoblast or osteoclast. The effects of TF (red arrows) and TT (black arrows) on osteoblast and osteoclast are indicated.*

## **5. Conclusion**

Osteoporosis is the most prevalent orthopedic disease in elderly people. Vitamin E has shown great potential in treating osteoporosis. The close synchronization between bone resorption and formation is facilitated by the activation or inhibition of numerous downstream signaling pathways, which are controlled by endocrine and paracrine regulators. The molecular targets of vitamin E in regulating bone metabolism have been identified by numerous scientific studies (**Figure 1**). In order to inhibit osteoclasts, vitamin E altered the levels of inflammatory mediators, ROS, and hormones as well as the RANK/RANKL/OPG, NF-κB, MAPK, and oxidative stress signaling pathways. Vitamin E could be selected as a novel therapy against osteoporosis.

### **Acknowledgements**

Chen YJ, Yu NC, and Zhou DG gathered literatures and drafted this manuscript. Li ZG, Gong FQ, Yi WJ, and Chen BT provided valuable opinions and assistance in the process of the research. GJ supervised this study and revised this manuscript. This study was supported by the National Natural Science Foundation of China (82074233), Starting Package of Xiang'an Hospital of Xiamen University (PM201809170009), National Natural Science Foundation of Xiamen, China (3502Z20227119), and Young Investigator Research Program of Xiang'an Hospital of Xiamen University (PM202103050009).

### **Conflict of interest**

The authors declare no conflict of interest.

## **Author details**

Chen Yongjie, Yu Naichun, Zhou Daguo, Li Zongguang, Gong Fengqing, Yi Weijiang, Chen Botao and Ji Guangrong\*

Department of Orthopedics Surgery, Xiang'an Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, China

\*Address all correspondence to: jiguangrong@sina.cn

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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*Edited by Juber Akhtar, Mohammad Ahmad, Mohammad Irfan Khan and Badruddeen*

Vitamin B is a water-soluble vitamin that plays important roles in cell metabolism and synthesis of red blood cells. Many foods contain B vitamins, including meat, poultry, and fish, among others. Vitamin E is a fat-soluble vitamin with antioxidant properties that can be found in vegetable oils, cereals, meat, poultry, eggs, and fruits. Both vitamins B and E are important for human health. This book provides a comprehensive overview of vitamins B and E. It is organized into three sections on the role of vitamin B on body health and gut flora, vitamin B deficiency and its association with the disease pellagra, and the medicinal significance and complications associated with vitamin E deficiency.

Published in London, UK © 2024 IntechOpen © RusN / iStock

Vitamin B and Vitamin E - Pleiotropic and Nutritional Benefits

Vitamin B and Vitamin E

Pleiotropic and Nutritional Benefits

*Edited by Juber Akhtar, Mohammad Ahmad,* 

*Mohammad Irfan Khan and Badruddeen*