Osteoporosis is the most common type of systemic degenerative bone disease. It emerges at all ages but it is predominantly diagnosed in postmenopausal women and older men. Postmenopausal osteoporosis is characterized by a progressive decrease in bone mineral density (BMD), deterioration of bone microarchitecture and an imbalance of bone formation and resorption regulated by osteoblasts and osteoclasts, respectively. This is accompanied by the accumulation of bone marrow fat resulting in compromised bone strength and an increased propensity for fragility fractures (43).

Postmenopausal osteoporosis may be based on the interaction of local cellular and molecular factors, including the deficiency of estrogen and the change of immune status of postmenopausal women. For example, T lymphocyte subtypes express TNFα, which increases the apoptosis of osteoblasts and indirectly stimulates the generation of osteoblasts through RANKL produced by B cells, as well as the changes of chronic inflammation phenotypes and cytokine expression (44). Excessive accumulation of AGEs and age-related obesity lead to an increased bone turnover rate and decreased bone trabecula and cortical bone density, resulting in continuous bone destruction (31). Studies refer to ‘the three-way regulation’ model of bone metabolism, which consists of the imbalance of regulation of the coupling of bone vessels, osteoclasts and osteogenesis, which leads to a decrease in cortical thickness and an increase in cortical porosity (45,46). The histomorphology and cytokines related to bone metabolism in ovariectomized rats confirmed that the oral administration of ZnC (10–100 mg/kg/day) for 6 weeks can significantly increase zinc accumulation, bone protein synthesis and collagen contents in bone, completely block the bone loss of femoral trabecula and prevent the deterioration of cortical bone (47). Comparatively low doses of ZnC (10 and 30 mg/kg/day) possess the same ameliorating actions (48). Carnosine compounds have been proved to inhibit bone resorption in tissue culture, so reasonably, carnosine may partially inhibit osteoclast resorption. These results indicate that carnosine compounds have a direct stimulating effect on bone formation and calcification and effectively prevent BMD reduction in ovariectomized rats.

From the perspective of systemic nerve-bone axis regulation, the dysfunction of the autonomic nervous system (made up of the sympathetic and parasympathetic systems) is also frequent in elderly or postmenopausal osteoporosis patients (49). Some authors found that under the conditions of aging or menopause (estrogen deficiency), the imbalance of sympathetic and parasympathetic nerves in bones stimulates bone cells to overproduce neuropeptide Y, inhibits cAMP/PKA/CREB signaling pathway, weakens osteogenic differentiation and enhances the adipogenic differentiation of BMSC, leading to bone-lipid imbalance and bone marrow obesity (49,50). Notably, carnosine is hypothesized to inhibit sympathetic nerve activity and promote parasympathetic nerve activity (51) and its effect may be to inhibit sympathetic nerve activity through the natural killer (NK) cells activity of spleen cells or regulate the autonomic nervous system through histidine H3 receptor (52,53). In brief, carnosine may provide new research avenues for postmenopausal and age-related bone metabolism through neuro-bone axis regulation.

Carnosine compounds are important because they could exert a broad-spectrum effect on the bone remodeling of osteoblasts and osteoclasts and inhibit ovariectomy-induced osteoclastogenesis. They could be a natural and novel treatment for postmenopausal osteoporosis.

Aging is another important risk factor for primary osteoporosis. At the cellular level, an increase in the level of ROS shortens the life span of osteoblasts and inhibits their formation, reduces BMSCs differentiation and stimulates the formation of damaged DNA, proteins and lipids, leading to cell apoptosis (54). On the other hand, the levels of RANKL and sclerostin (SOST, an antagonist of Wnt intracellular signal transduction) in osteoclasts are diminished (55). RANKL can stimulate ROS production. Conversely, the formation, activation and bone-resorbing function of osteoclasts also need ROS stimulation (56). When the balance between osteoblasts and osteoclasts is disordered, osteoporosis usually results.

At the molecular level, aging mainly reduces the reactivity of insulin and estrogen, which decreases bone formation and differentiation. Research shows that the level of carnosine gradually decreases with age (57). Due to carnosine's effect on glycolysis, it has been used to inhibit cell aging in cultured human fibroblasts (9). Other experiments in elderly (10-month-old) female rats found that when the bone tissues were cultured for 24 h in a medium containing ZnC (10⁻⁵ M), 4.5% glucose, 0.25% bovine serum albumin and antibiotics, the activity of ALP in osteoblasts was significantly increased and prolonged, thus lengthening the cell cycle. ZnC could also activate aminoacyl-tRNA synthetase, a key enzyme in the process of bone protein biosynthesis and translation, stimulate bone formation and mineralization and alleviate senile osteoporosis (47,58).

Mounting evidence shows that bone disuse inhibits bone formation and increases bone resorption. These, coupled with the low dynamic and insulin-mediated gravity loading effect, eventually lead to osteoporosis (59).

It has also been observed that weightlessness damages the diaphyseal part more than the metaphyseal part in the model of hindlimb suspension in rats (60). Additionally, weightlessness or long-term physical fixation does not affect the intestinal absorption of zinc, but it partially inhibits the transfer of serum zinc to bone tissue. To compensate, zinc ions overflow from the bone leading to a decrease in zinc level in the bone matrix and cell components, which inhibits bone formation. Some researchers have suggested that inflammation from bone disuse could reflect periarticular osteopenia. This uncoupled state of bone resorption and formation also leads to periarticular osteoporosis in patients with rheumatoid arthritis (61).

In vitro, ZnC (10⁻⁵ M) in a culture medium with bone tissue could stimulate the proliferation and differentiation of stem cells, the production of IGF-1, TGF-β and osteocalcin and the peptization of aminoacyl-tRNA synthetase in osteoblasts, thus increasing ALP activity and aminoacyl-tRNA content of protein in bone tissues. IGF-1 stimulated by carnosine compounds could both increase bone trabecula formation and the proliferation of osteoblasts and have an anabolic effect on bone metabolism with or without bone disuse (62).

Long-term use of glucocorticoids can reduce bone formation by directly inhibiting the proliferation and differentiation of osteoblasts (inhibiting Runx2 and Collagen I) and increasing apoptosis and bone absorption by negative feedback gonadotropin secretion, or decreasing calcium absorption and increasing calcium excretion. This eventually leads to a negative calcium balance in the body, decreased bone density and secondary development of glucocorticoid-induced osteoporosis (63). A study of hydrocortisone-induced osteopathy showed that carnosine compounds prevent the increase of PTH levels and the decrease of ALP activity and increase the DNA and zinc contents. These effects prevent disordered bone metabolism and architectural deterioration (64).

In clinical studies of peripheral osteoporosis caused by autoimmune arthritis, inflammation caused by ROS oxidative stress is the key cause of osteoporosis (65,66), whereas carnosine compounds block the decrease of ALP activity induced by IL-1β, attenuate the decrease of DNA and calcium contents, partly improve the immune response triggered by the release of matrix degradation products, inhibit inflammatory osteolysis and the loss of trabecula and collagen and alleviate the continuous destruction of bone microstructure and mechanical strength (67).

Vitamin D has extensive endocrine and autocrine functions that could promote the proliferation of osteoblasts, the differentiation and activity of osteoclasts, bone formation, absorption and coupling (68), which are essential for bone development and maintenance. Some evidence shows that 1,25 (OH)2D3 is an effective promoter of osteoblasts-osteocytes transformation including promoting osteoblasts mineralization and inducing expression of bone cell markers in pluripotent stem cells (iPSop) in vitro (69). Notably, deficiency of vitamin D or calcium caused by various factors in the body leads to osteoporosis (69). A study by Segawa et al (70) observed that an oral administration of 100 mg/kg/day of ZnC for 14 days could partially increase ALP activity and calcium contents, which directly stimulated bone formation. This was independent of the change in serum mineral homeostasis, thus it could prevent the deterioration of bone tissue caused by malnutrition.

Taken together, though the pathologic process of the aforementioned types of osteoporosis vary, in terms of hormone levels, cellular response and involved signaling pathways, they all exhibit impacted cellular function, abnormal bone turnover and somewhat enhanced lipogenesis and oxidative stress, which provide targets for carnosine to protect bone. In this context and based on the current evidence, carnosine complexes are more conducive to postmenopausal osteoporosis, with a potential application value in the secondary osteoporosis.

Fracture healing is a complex process based on the interaction between the inflammatory and hematopoietic stem cells and is associated with rapid osteogenic and chondrogenic differentiation of periosteal cells and continued vascular angiogenesis and ingrowth. This is subsequently followed by the remodeling of immature bone into the well-structured lamellar bone and other biochemical signal cascades (71). Animal experiments have demonstrated that macrophages are essential for both intramembranous and endochondral ossification of bone after a fracture. They contribute to the deposition of woven bone and the formation of the soft callus for endochondral ossification (72,73).

Studies have confirmed that the use of carnosine compounds could significantly promote the expression of protein components and the release of cytokines from osteoblasts in the periosteum and endosteum during bone remodeling following a fracture (7,38,74). These include bone formation stimulating factors such as PTH, IGF-1, TGF-β, OCN and 66-kDa albumin that enhance bone synthesis (75). A large amount of IGF-1 deposited in the bone matrix participates in local anabolism cooperating with PTH. The positive feedback induces bone tissues to produce more albumin, which has a synergistic effect in early and late fracture healing (76). This contributes to maintaining bone mass and bone homeostasis during bone reconstruction. The MAPKs inhibitor PD98059 or the PKC inhibitor staurosporine can completely inhibit zinc ions, thus weakening their effect on fracture healing and reducing carnosine compounds available in the body. The bioactive effect of carnosine may be partly exerted through the MAPK signaling pathway or PKC-mediated activation of albumin synthesis in bone tissue (74).

In vitro research by Hughes et al (77) supports this point of view. By using protein synthesis-promoting dietary supplements, the albumin, muscle content and BMD in fracture healing tissue and the expressions of IGF-1, IGF-2, actin, myosin and vascular endothelial growth factor (VEGF) mRNA in muscles were significantly increased. Ko et al (38) found that polaprezinc can enhance the activity of both osteoblasts and osteoclasts, thus accelerating the healing process of fractured bone.

Overall, although the specific mechanism of promoting fracture healing is unclear, there is no doubt that therapeutic or dietary supplementation of carnosine compounds benefits soft tissue repair, muscle mass recovery and bone remodeling.

OA is the most prevalent chronic joint disease, characterized by cartilage degeneration, subchondral bone osteosclerosis and synovium inflammation.

It has been proved that oral carnosine supplement could reduce the expression of IL-1α and TNF-α in serum and synovial tissues, inhibit MMPs (MMP3, MMP13) and thrombospondin type 1 motif in the extracellular matrix, the nuclear translocation of NF-κB p65 and finally reverse the inflammatory response specifically through the ROS/NF-κB signaling pathway, showing a protective effect on diabetes-induced OA (29). In a canine OA model induced by anterior cruciate ligament transection (ACLT), carnosine prevented and trapped lipid peroxide 4-HNE (18), inhibiting the production of catabolites in chondrocytes and reducing the expression of inflammatory cytokines. In addition, carnosine could markedly prevent arthritis and chondrocytes injury, through anti-inflammatory and anti-oxidation effects (78). A recent study by Lanza et al (79) further showed that a selected molecular synergy enhanced its biological activity and resistance, in which the carnosine-hyaluronic acid conjugate reduced oxidative damage to chondrocytes and cartilage degradation in OA.

A recent study from Busa et al (8) confirmed that carnosine can alleviate knee osteoarthritis pain, improve synovial protection and decrease cartilage degradation in vivo. In IL-1β stimulated fibroblast-like synoviocyte cells, carnosine reduce the expression levels of cyclooxygenase-2, MMP-3 and MMP-13, ROS level and mitochondrial membrane permeability by activating the Nrf2/heme oxygenase-1 signaling pathway.

Thus, considering the aforementioned protective effects of carnosine in against cartilage degeneration, abnormal subchondral bone remodeling and synovitis, it may become a potential choice for OA treatment, particularly when chemically conjugated with some selected agent with enhanced antioxidative and anti-inflammation effects.

Bone is the main target organ for tumor metastasis and the presence of bone metastases is generally a sign of poor prognosis for advanced malignant tumors. Preclinical and clinical studies show that carnosine has active antitumor and immunomodulatory effects (22) that may directly or indirectly affect the proliferation and metastasis of bone tumor cells, in which carnosine inhibits the glycolytic pathway and/or possibly participates in the regulation of biological activity mediated by mTOR, hypoxia-inducible factor α, STAT3 and MAPK (80). Primary bone tumors are highly invasive and strongly dependent on ATP produced by glycolysis. They can penetrate and destroy the bone cortex and expand into adjacent soft tissues, finally, affecting the whole bone (81). The viability of tumor cells is reduced in the presence of carnosine. Carnosine inhibits tumor growth, is anti-antigenic, improves genomic stability, reduces telomere impairment and protects cells from DNA damage induced by genotoxic substances (82).

Additionally, carnosine selectively inhibits the growth of transformed cells and ATP-dependent glycolysis activity, thus inhibiting the growth of tumor cells. Relevant studies have confirmed that carnosine activates the STAT3 signaling pathway, inhibits neuronal cell apoptosis after acute cerebral ischemia, increases the level of p27 (a cell cycle regulator) and shortens the growth cycle and proliferation of tumor cells (83,84). However, the overexpression of STAT3 activated by carnosine is related to the poor prognosis of osteosarcoma. A study by Gao et al (85) indicated that carnosine could inhibit the proliferation and invasion of osteosarcoma cells, promote cell apoptosis and suppress the growth and activity of tumors by mediating the Wnt3a/β-catenin signaling pathway. Hwang et al (86) demonstrated that carnosine could be anti-antigenic by inhibiting the ERK/AKT/eNOS signaling pathway and MMP-2 mediated by VEGF, thus inhibiting the viability and activity of bladder cancer cells.

Further studies show that the implantation of bone metastases from malignant tumors may induce the imbalance of bone homeostasis and the change of the mechanical stress of osteoblasts, leading to osteoblastic and osteolytic lesions, thus accelerating the progression of tumor. Hsieh et al (87) found that carnosine could inhibit the migration and invasion of human colorectal HCT-116 cells by inhibiting pro-inflammatory cytokines, oxidative stress, NF-κB and MAPK pathway activities and regulating MMPs and epithelial-mesenchymal transition related molecules such as E-cadherin, Twist-1 and MMP-9. Another important study found that the immunomodulatory action of carnosine may significantly inhibit the sympathetic nerve activity of the spleen, thereby inhibiting cancer cell proliferation by increasing NK cell activity (52).

Last but certainly not least, from the perspective of antitumor therapy options, the current evidence shows that carnosine mitigates cyclophosphamide (CTX) induced G₂/M arrest in bone marrow cells to inhibit its apoptosis and genotoxicity, downregulates the expressions of cytochrome c, Bcl-2-associated X protein (BAX) and p21, inhibits DNA damage and ROS production, protects bone marrow cells from CTX-induced oxidative stress and partly restores CTX-induced suppression of hematopoietic function (10). Additionally, the combination of carnosine and 5-fluorouracil decreases hypoxia-inducible factor 1 and multi-drug resistant protein MDR1-pg expression, overcomes the acquired resistance of some chemotherapy drugs and continually improves the anti-tumor effect of standard chemotherapy (88). Adding carnosine to adjuvant chemotherapy and radiotherapy could increase its efficiency and alleviate radiation-induced skin damage and decreased immunity. In addition, it does not have any toxic effects and could therefore act as a ‘security guard’ for normal tissues.

Carnosine could minimize the adverse effects of antitumor therapy in both experimental and clinical research. Exploring further the molecular mechanisms of carnosine in antitumor growth and bone metastasis may provide a new preventive agent for bone tumor therapy.