Study on Hops Extract for Osteoporosis

Osteoporosis (OP) is a systemic metabolic disease characterized by reduced bone mass and microscopic structural damage to bone tissue. The incidence of OP is positively correlated with age. According to an epidemiological survey conducted in 2019, the prevalence of osteoporosis in individuals aged 60 and above in China was 36%. With the aging of the population, China will face increasingly prominent osteoporosis issues [1].

Currently, the commonly used drugs for the prevention and treatment of OP are divided into two major categories: anti-resorptive agents and anabolic agents. Among the anti-resorptive agents are bisphosphonates, calcitonin, and estrogen; among the anabolic agents are parathyroid hormone analogues, strontium salts, and active vitamin D analogues. These drugs demonstrate significant clinical efficacy but are also associated with various adverse reactions [2], such as bisphosphonates causing osteonecrosis of the jaw [3] and estrogen increasing the risk of cardiovascular diseases [4]. Therefore, the development of new targeted therapies for OP holds significant clinical importance.

Flavonoids are widely present in plants in nature and have been proven to possess anti-osteoporosis activity, with minimal adverse reactions, low cost, and a broad mechanism of action, making them safe and ideal natural anti-osteoporosis agents [5]. Xanthohumol is a unique flavonoid compound found in hop extracts, exhibiting potent antioxidant stress, anti-inflammatory, anti-cancer cell proliferation, and can act on osteoblasts (OB) and osteoclasts (OC), targeting and stimulating related cytokines to promote OB proliferation and inhibit OC differentiation [6]. Relevant literature reports that patents for Xanthohumol in the prevention and treatment of OP have already been approved abroad [7]. Therefore, intensifying research on targeted therapies for OP using xanthohumol and enhancing its clinical application holds great potential. This paper summarizes the mechanisms of xanthohumol in preventing and treating OP.

1. Xanthohumol's role in preventing and treating OP at the cellular level and related signaling pathways

1.1 Xanthohumol's involvement in OB differentiation and related signaling pathways

OBs play a crucial role in skeletal development. Abnormal OB differentiation can lead to bone metabolic disorders, thereby triggering diseases such as OP [8]. Studies have shown that osteoblasts and osteoblast precursor cells exhibit competitive differentiation [9]. When osteoblast precursor cell differentiation increases, it inhibits the proliferation of OBs, thereby affecting their differentiation and maturation. Xanthohumol can promote OB proliferation and differentiation by stimulating osteoblast transcription factor 2 (runt-related transcription factor 2, Runx2), peroxisome proliferator-activated receptor γ (PPARγ) to promote OB proliferation and differentiation while inhibiting adipocyte generation [6,10].

Additionally, under oxidative stress conditions, reactive oxygen species (ROS) accumulate in the body, stimulating OB apoptosis [11]. When OB apoptosis is excessive, it leads to reduced bone formation and the onset of OP. Research has demonstrated that xanthohumol can stimulate the expression of nuclear factor erythroid-2-related factor 2 (Nrf2), enhancing the body's antioxidant stress capacity, clearing accumulated reactive oxygen species, and inhibiting OB apoptosis [12].

1.1.1 Osteogenic transcription factor Runx2

The osteogenic transcription factor Runx2 promotes cell differentiation toward osteogenic and chondrogenic directions, playing a particularly important role in OB transformation. Runx2 can promote the proliferation and division of immature OBs, converting immature bone cells into mature bone cells [13]. Under specific inductive conditions, Runx2 can also promote the transcription of specific osteogenic developmental target genes, leading to the post-transcriptional production of proteins such as osteocalcin and collagen, which play a crucial role in bone tissue formation [14]. Studies have shown that reducing Runx2 expression in mice inhibits osteoblast differentiation [15]. In summary, this indicates that Runx2 plays an important role in promoting the proliferation and differentiation of OBs.

Jeong et al. [16] investigated the effects of xanthohumol on the differentiation of mouse C2C12 cells. C2C12 cells were cultured in xanthohumol solution, and detection revealed that the activity of EPK and P38 significantly increased in a concentration-dependent manner, suggesting that xanthohumol activates Runx2 expression by upregulating the phosphorylation of EPK and P38. Xia Tian Shuang et al. [17] treated mouse osteoblasts with dexamethasone (DEX) for injury, except for the blank group. Western blot analysis showed that xanthohumol significantly promoted Runx2 expression, and bone metabolism markers such as alkaline phosphatase increased. Based on the above literature, it is suggested that xanthohumol can upregulate Runx2 expression to promote OB proliferation and differentiation, and through increased Runx2 expression, elevate osteogenic-related genes, thereby exerting a certain inhibitory effect on OP development.

1.1.2 Lipid Transcription Factor PPARγ

Osteoblasts and adipocytes have a competitive differentiation relationship. Adipocyte differentiation is a strictly regulated transcriptional cascade process, and PPARγ is a key regulatory factor in the adipogenesis process. During adipogenic differentiation, AMP response element-binding proteins undergo protein phosphorylation, inducing the expression of CEBP-β, which in turn activates the transcription of CEBP-α and PPARγ, thereby maintaining the completion of the adipogenesis process [18]. Experimental evidence shows that when the PPARγ gene is knocked out, the adipogenesis process in embryonic stem cells is almost completely inhibited [19].

Kiyofuji et al. [20] cultured mouse 3T3-L1 cells to induce their differentiation into adipocytes, and then exposed them to different concentrations of xanthohumol. The results showed that xanthohumol expression exhibited a concentration-dependent inhibitory effect on PPARγ expression. Yang et al. [21] differentiated 3T3-L1 cells into adipocytes and intervened at different time points. Electrophoresis analysis revealed that xanthohumol significantly reduced PPARγ expression, with the most pronounced effect observed at 24 hours. The above studies indicate that xanthohumol can limit adipocyte differentiation by restricting PPARγ expression. Based on the competitive differentiation between osteoblasts and adipocytes, and since xanthohumol can promote Runx2 expression to upregulate OB differentiation, therefore, research on whether xanthohumol can promote OB differentiation by inhibiting PPARγ expression holds significant prospective significance.

1.1.3 Antioxidant stress transcription factor Nrf2

Oxidative stress refers to an imbalance between oxidative capacity and antioxidant capacity in the body, leading to excessive accumulation of ROS, causing cellular and tissue damage, and inhibiting the expression of osteogenic differentiation markers such as alkaline phosphatase, type I collagen, and Runx2, resulting in OB apoptosis and reduced bone formation, promoting the occurrence of OP [22]. Nrf2 is an important antioxidant stress transcription factor in the body. Under normal conditions, Nrf2 binds to Keap1 in the cytoplasm and is ubiquitinated and degraded; under oxidative stress stimulation, the Nrf2-Keap1 complex is dissociated and transported to the cell nucleus, where it binds to antioxidant response elements, promoting the expression of various antioxidant enzymes, such as glutathione peroxidase and glutathione S-transferase [23].

Suh et al. [24] induced ROS and mitochondrial superoxide accumulation in MC3T3-E1 osteoblasts using methylglyoxal (MG), leading to cell apoptosis. Cells were pretreated with xanthohumol and then exposed to MG. The results showed that the ROS accumulation caused by MG was significantly reduced, and the Nrf2 level increased in a concentration-dependent manner with xanthohumol concentration. Therefore, xanthohumol may promote the production of antioxidants or enhance antioxidant activity by upregulating Nrf2 expression, thereby inhibiting ROS accumulation and reducing the likelihood of oxidative stress-induced damage to OBs. Given this, xanthohumol's potent antioxidant stress capacity holds great promise for the prevention and treatment of osteoporosis.

1.2 Research on Xanthohumol's Involvement in OC Differentiation and Related Signaling Pathways

OCs are multinucleated cells derived from hematopoietic stem cells. Under the stimulation of the nuclear factor-κB receptor ligand (NF-κB ligand, RANKL) and macrophage colony-stimulating factor (M-CSF), monocytes can differentiate into mature OC. M-CSF promotes the proliferation and survival of monocytes, while RANKL induces the differentiation and maturation of OC. M-CSF). M-CSF promotes the proliferation and survival of monocytes, while RANKL induces the differentiation and maturation of OC [25]. These two signaling factors promote OC differentiation through a series of signaling pathways. Xanthohumol can inhibit the transmission of certain signaling pathways, such as NF-κB, calcium ion/calcium-dependent phospholipase/NFAT pathway (Ca2+/NFATc1), mitogen-activated protein kinase (MAPK), etc., thereby interfering with OC differentiation and maturation and inhibiting OC differentiation and maturation [6].

1.2.1 NF-κB pathway

NF-κB is an important transcription factor promoting OC differentiation. Before activation, it binds to the subunit IkB in the cytoplasm. Upon stimulation by relevant factors, IkB degrades, promoting NF-κB to enter the cell nucleus, initiating gene transcription and expression, promoting OC differentiation and maturation. RANKL binds to RANK, activating the NF-κB/IκB complex, leading to the degradation and release of IκB, and NF-κB translocates into the cell nucleus to promote OC differentiation and maturation [26]. Li et al. [27] pretreated experimental group RAW264.7 cells with Xanthohumol, followed by RANKL to induce OC differentiation. The control group was treated with RANKL alone. Detection revealed that IκB protein degradation was significantly reduced in the experimental group compared to the control group.

Xie Juan [28] cultured RAW264.7 cells in a solution containing labeled NF-κB, followed by exposure to different concentrations of xanthohumol and RANKL solutions. The cells were lysed, and performed luciferase reporter gene analysis. The results indicated that RANKL activates NF-κB transcriptional activity, while xanthohumol exhibits concentration-dependent inhibition of RANKL-activated NF-κB transcription. Given the important role of NF-κB in OC differentiation, studying the inhibition of NF-κB differentiation is of significant importance. Based on the above studies, xanthohumol does indeed inhibit NF-κB transcription and may do so by inhibiting the degradation of IκB. However, the optimal inhibitory dose of xanthohumol for NF-κB has not yet been clearly established, so further research is needed to determine the appropriate dose of xanthohumol for inhibiting NF-κB transcription.

1.2.2 Ca2+/NFATc1 pathway

The nuclear factor of activated T-cells 1 (NFAT1) in the cytoplasm of T cells is a key transcription factor for osteoclast differentiation, primarily inducing osteoclast differentiation and maturation in the late stage. The NFAT family is primarily regulated by Ca²⁺-activated calcineurin. Upon stimulation and activation by RANKL, the serine-binding sites of NFAT are dephosphorylated, promoting NFATc1 nuclear translocation, completing gene transcription, and facilitating osteoclast differentiation and maturation. When the NFAT gene was knocked out in mice, osteoblasts could not complete OC-related differentiation under RANKL stimulation [29]. Suh et al. [30] cultured RAW264.7 cells in RANKL and then in different concentrations of xanthohumol. The results showed that at 4 μg/ml xanthohumol, NFAT1 significantly decreased, indicating that xanthohumol can inhibit OC differentiation by suppressing NFAT1 differentiation.

Other studies have shown that Ca²⁺ oscillations are key factors in maintaining NFAT transcription. When Ca²⁺ oscillations are inhibited, NFAT cannot complete transcription, and OC differentiation is also inhibited [31]. Li et al. [27] placed BMM cells in calcium flow dishes, added xanthohumol and RANKL to the experimental group to induce Ca²⁺ oscillations, and only added RANKL to the control group. The results showed that Ca²⁺ oscillations in the experimental group were significantly lower than those in the control group. The role of NFAT in OC differentiation is of great significance, and the role of Ca²⁺ oscillations in promoting NFAT transcription has been confirmed. These findings suggest that xanthohumol may inhibit OC differentiation by suppressing Ca²⁺ oscillations, indicating its great potential for the prevention and treatment of osteoporosis (OP).

1.2.3 MAPK pathway

The MAPK pathway is a crucial pathway for maintaining OC differentiation, including extracellular signal-regulated kinases (EPK), P38, and JNK. RANKL can activate the MAPK pathway, inducing EPK, NFAT1, and c-fos to stimulate OC differentiation, Among these, EPK1/2 plays a particularly crucial role in the differentiation, maturation, and apoptosis of osteoclasts [32]. M-CSF binds to c-Fms, leading to the phosphorylation of tyrosine residues at the C-terminal region of c-Fms, which then binds to MEK2 to activate the transcription of EPK1, promoting EPK1 translocation from the cytoplasm into the nucleus, initiating the phosphorylation of downstream-related factors to complete OC transcription.

When the EPK gene is knocked out in mice, OC differentiation, maturation, and transport are significantly restricted [33]. Suh et al. [30] cultured RAW264.7 cells in RANKL, followed by exposure to different concentrations of xanthohumol, and subsequently performed PCR detection. The results showed that the levels of EPK1 and c-fos were significantly inhibited. Based on the above studies, it is speculated that xanthohumol inhibits the action of RANKL on the MAPK pathway, thereby reducing the activation of downstream-related signals and limiting OC differentiation and maturation. However, due to the limited scope and depth of current research, further studies are needed to enhance the accuracy of these findings.

2 Outlook

With the accelerating aging of China's population, the prevention and treatment of osteoporosis (OP) have become a significant societal challenge. While conventional pharmaceuticals offer rapid efficacy, they are associated with numerous adverse effects and high costs, prompting growing interest in natural plant components that emphasize holistic prevention and have minimal adverse effects. Cellular experiments on xanthohumol have demonstrated its significant potential value in the prevention and treatment of OP. As a plant flavonoid component, xanthohumol possesses potent anti-inflammatory and antioxidant properties, and can regulate bone metabolism disorders through multiple pathways such as RUNX2, MAPK, and NF-κB, thereby reducing trabecular bone defects and bone mineral loss.

Additionally, it can directly act on osteoblasts (OB) and osteoclasts (OC) to influence bone matrix resorption and formation, thereby comprehensively achieving bone protective effects. While exploring and reflecting on our achievements, we must also objectively acknowledge that there are still challenges in the drug development of xanthohumol for the prevention and treatment of OP, such as insufficiently clear specific targets for xanthohumol's anti-osteoporosis activity and incomplete research on its specific molecular mechanisms. Further in-depth studies are needed. We look forward to expanding research on Xanthohumol for the prevention and treatment of OP in the future, accelerating its translation into clinical therapy, and establishing clinically effective prevention and treatment strategies based on a thorough understanding of the fundamental pathophysiological mechanisms of OP.

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