As an endogenous gaseous signaling molecule, H2S is a hot research topic. Multiple investigations have verified its important role in the physiological and pathological processes of many tissue systems, including the visceral, cardiovascular, skeletal and nervous systems. For example, it can protect cardiomyocytes from ischemic disease . Reduction of endogenous H2S can accelerate atherosis . It can also weaken the protection against oxidative stress in the nervous system .
Estrogen promotes bone formation, which is mainly mediated by ER receptors on osteoblasts [26, 27]. It also inhibits bone absorption through the apoptosis of osteoclasts, induced by its ER receptors [28, 29]. Normally, bone metabolism depends on homeostasis and the balance of osteoblast-mediated bone formation and osteoclast-mediated bone absorption . The levels of calcium and phosphorus in the plasma are maintained in a dynamic balance with their levels in the bone, with the plasma levels reflecting the metabolism of bone tissue and the absorption–reabsorption rate of calcium and phosphorus by the small intestine and kidneys.
In this study, we investigated the effects of estrogen deficiency on the levels of H2S, calcium and phosphate in rat plasma, on the macroscopic mechanics and physics of the bones, and on related hormone secretion. We also studied how GYY4137 and alendronate sodium affected these parameters. The four groups of rats were the sham-vehicle group; ovariectomized group with vehicle treatment (OVX-vehicle); ovariectomized group alendronate sodium treatment (OVX-ALEN); and ovariectomized group with GYY4137 treatment (OVX-GYY).
The OVX-GYY group showed no significant difference compared to the sham-vehicle group in terms of macroscopic mechanics and physics. Exogenous GYY was proven to raise the level of H2S in rat plasma and improve the imbalances in calcium and phosphate caused by the lack of ovaries. It also influenced the leptin level and raised the density of the lumbar vertebrae and the maximum stress of the thighbone. The influence of estrogen deficiency on bone metabolism was partially neutralized. H&E staining and microscopic observation also proved that with the intervention of GYY, the structure and amount of trabecular bone in the OVX-GYY group was not significantly different than the results for either the sham-vehicle or OVX-ALEN groups.
Other than directly acting on bone metabolism, estrogen can increase the secretion of calcitonin, inhibit the secretion of parathyrin, promote the activation of vitamin D in the kidney, and increase calcium absorption in the small intestine through a calcium-regulating hormone.
Calcitonin is a kind of polypeptide hormone secreted by thyroid C cells. It acts on calcitonin receptors to inhibit the activation of osteoclasts, stimulate the proliferation of osteoblasts, and prevent apoptosis [31, 32]. Calcitonin can also indirectly inhibit the activation of parathyroid and vitamin D and reduce the level of calcium in the blood. Its levels have no association with age, so the basic values of calcitonin and metabolic rate show no significant change for people with osteoporosis, which concurs with the result in our study.
Parathyrin is secreted by the parathyroid in a pulsatile way, acting on the parathyrin receptors of osteoblasts to enhance bone formation. The abnormal secretion of parathyrin, such as the constant abundant secretion caused by hyperparathyroidism, can increase the activity of osteoclasts and result in bone resorption and osteoporosis. Our study demonstrated that the parathyrin level decreases over time, which might have something to do with the reduction in estrogen in the plasma.
In our OVX-ALEN group, alendronate sodium regulated calcitonin and parathyrin. However, GYY had no significant regulatory effect on these two hormones in the OVX-GYY group.
Leptin, a body-wide hormone secreted by the white adipose tissue, has a more complex regulatory relationship with bone metabolism. It is generally believed that it acts on hypothalamic long receptors (OB-Rb) to regulate bone metabolism through the neuroendocrine axis. It can also directly regulate bone metabolism through short receptors around the bone tissue. Thomas, et al.,  tested BMD and the serum leptin level of pre- and post-menopausal women and men. They found that the level of serum leptin in women was negatively associated with BMD, while there was no significant association in men. In this study, we observed that H2S might help regulate the level of leptin.
Approximately 50% of ALP in the plasma comes from osteoblasts. OCN is an active polypeptide directly secreted and expressed by osteoblasts. They both reflect the activity and state of osteoblasts and represent bone formation and reconstruction . Interestingly, neither alendronate sodium nor GYY4137 exerted significant regulatory effects on ALP or OCN in the OVX groups. This demands further study.
The regulation of bone metabolism has many interactive signaling pathways at the molecular level, and these play important roles in bone formation and absorption . The bone morphogenetic protein (BMP) Smads pathway and the Wnt/β-catenin pathway mainly affect bone formation, while the pathway involving osteoprotegerin (OPG), nuclear factor κB receptor activator of the NF-kB ligand (RANKL) and nuclear factor κB receptor activator in NF-Kb (RANK) mainly affects bone absorption. Some research has shown that H2S produced by mesenchymal stem cells can regulate bone differentiation, and that the reduction of H2S can reduce the thiolation of calcium channels, influencing the internal flow of the Ca2+ pathway in the cytoderm. This reduces the differentiation of mesenchymal stem cells to osteoblasts – a process that is regulated by the Wnt/β-catenin signal pathway and mediated by PKC and ERK . H2S can also interact with Osterix by affecting the important transcription factor nuclear-binding protein (Cbfα1/Runx2) in the BMP/Smads pathway to promote osteocyte proliferation and differentiation. In addition, it can further activate the expression of specific osteoblast factors (ALP, type I collagen, and OCN) to promote bone formation . The BMP/Smads and Wnt/β-catenin signal pathways can mutually regulate each other .
In some experiments, H2S has also been observed to mediate oxidative stress injury, promote the proliferation and differentiation of osteoblasts, and alleviate inflammation through a mechanism dependent on EPK1/2 and p38 in the MAPK pathway . Osteoblasts regulate the proliferation and differentiation of osteoclasts, mainly through the OPG/RANKL/RANK signaling pathway, while H2S might regulate the activity of osteoclasts by increasing OPG expression and reducing RANKL expression in bone tissue [39,40,41]. Gambari, et al.,  also observed that H2S could directly inhibit the differentiation of osteoclasts through a mechanism dependent on NRF2. These statements are in accord with the results of this study, indicating the benefit of H2S to bone metabolism and its potential to improve the sclerotin structure.
Exogenous H2S can intervene and regulate osteoporosis in ovariectomized rats. In some cases, it could even cure osteoporosis caused by ovariectomy . The effect of H2S on osteoporosis is significantly dependency on dosage. Further studies on the effect of the H2S concentration on bone metabolism are needed. The concentration of sulfide ions in the blood of people with H2S poisoning has been reported to be 3–995 μM . The physiological concentration is quite close to the minimal concentration for H2S poisoning. At high concentrations, it can even induce lipopolysaccharide-mediated inflammation for rats and synchronously increase the H2S concentration in the plasma of patients with septic shock . The effect of exogenous H2S on important tissues and organs needs to be studied in terms of method, speed, and concentration of drug administration.
In summary, ovariectomized rats were used as subjects in this study to explore the association between the level of endogenous H2S in the plasma and osteoporosis. Through intraperitoneal injection of GYY413 as the supplement of endogenous H2S, we showed that raising and maintaining the physiological level of H2S in the plasma could regulate the metabolic balance of calcium and phosphorus. We also looked at the impact on common clinical indexes of bone metabolism: the hormones OCN, calcitonin, alkaline phosphatase, parathyrin and leptin. Our results suggest that GYY4137 is a better intervention than alendronate sodium and that it has a certain therapeutic effect on ovariectomy-induced osteoporosis.
The therapeutic effects of GYY4137 in a rat model of osteoporosis is one of our study limitations. We did not explore the therapeutic effects of GYY4137 in the clinic, so the result may differ in humans. In addition, the molecular mechanism of GYY4137 action needs to be explored. Any potential bias would also be considered in further study.