With the increasing incidence of osteoporosis year by year, osteoporosis has become a worldwide health problem and social public problem[17]. Exploring the mechanism of GSK and other TCM can provide greater advantages and theoretical supplement for TCM treatment and prevention of osteoporosis. Clearly, proteins and small molecule metabolites are closely associated with the occurrence of diseases, thus the combination of proteomics and metabonomics are matched our attention. It has been reported that the specific mechanism of GSK in the prevention and treatment of OP has not yet been explained, and its molecular mechanism has not been clarified.
Based on the combined analysis of proteomics and metabonomics, the results showed that the expression trend of differential proteins Cant1, Gstz1, Aldh3b1, Bid, and Slc1a3 in the common metabolic pathway of differential proteins and metabolites between GSK/OP and OP/SHAM was different, that is, they were corrected in GSK group. Moreover, Cant1 was involved in purine metabolism and pyrimidine metabolism, and Aldh3b1 was involved in tyrosine metabolism, histidine metabolism, beta-Alanine metabolism and phenylalanine metabolism.After GSK treatment, Slc1a3 was surely correlated with L-histidine (P=0.045), Aldh3b1 was significantly correlated with 1-stearyl-sn-glycerol 3-phosphocholine (P=0.045). Combined with proteomic analysis, Gstz1 and Aldh3b1 were located in the functional nodes of protein-protein interaction network. In addition, the expression trend of 12 metabolites (Tyramine、Thymidine、Deoxycytidine、Cytosine、L-Aspartate、Ergothioneine、L-Histidine、L-Anserine、P-antothenate、Arachidonic Acid (peroxide free)、Benzoic acid、DL-Phenylalanine) was differential in the common enrichment metabolic pathway between GSK /OVX and OVX/SHAM. The above metabolic pathways are primarily involved in nucleotide metabolism, amino acid metabolism, immune system, cell process, and other signaling pathways, indicating that GSK may treat osteoporosis in rats through these.
Uric acid is the final product of purine metabolism in the human body, which is mainly decomposed by enzymes from nucleic acids and other purine compounds metabolized by cells and purines in food. Uric acid is a reducing substance in the human body, which participates in the redox reactions and scavenging oxygen free radicals, thereby inhibiting oxidative stress[18, 19]. A number of studies have reported that serum uric acid (UA) is involved in the pathogenesis of OP by affecting oxidative stress and inflammatory cascades[20]. Higher serum UA levels appear to be protective for bone loss in peri- and postmenopausal women[21]. Cant1, a calcium-activated nucleotide, is essential for glycosaminoglycan synthesis in cartilage[22]. In this study, the key differential protein Cant1 was involved in purine metabolism and was down-regulated after GSK treatment.
Amino acid metabolism is an important part of life activities. As the basic unit of macromolecular protein, it is essential for the normal metabolism of the human body[23]. Previous studies[24, 25] showed that Amino acid metabolism is important to osteoporosis. In the correlation analysis, the significant differential proteins and metabolites have a strong correlation with Histidine metabolism and β-alanine metabolism. Histidine is an essential amino acid in the human body during childhood. In adulthood, the human body can synthesize it by itself and histidine becomes a nonessential amino acid. Histamine is formed under the action of histidine decarboxylase and is a factor that regulates inflammation and allergic reactions[26]. An increase in the amount of bone cortex and bone minerals was found in mice deficient in histamine synthesis, revealing that the lack of histamine may increase the bone density and bone formation of histamine deficient mice by stimulating the synthesis of calcitriol[27].
The results showed that after treatment with GSK, Aldh3b1 in the Histidine metabolism pathway was downregulated, L-Aspartate was downregulated, and Ergothioneine, L-Histidine, and L-Anserine were upregulated. β-Alanine plays a key role in bone metabolism, mainly by improving the production of insulin and insulin-like growth factor-1, as well as the synthesis of collagen and muscle proteins. Studies[28] have shown that β-Alanine may be a potential biomarker of osteoporosis, and β-Alanine levels increase significantly in postmenopausal osteoporosis patients with low bone density. Tyrosine is an important amino acid in the body and the main raw material for the synthesis of thyroxine[29]. In previous studies, it has been found that GSK may participate in bone metabolism by regulating tyrosine metabolism and differential proteins. In addition, Aldh3b1 is also involved in the metabolism of Tyrosine metabolism, Histidine metabolism, beta-Alanine metabolism, and Phenylalanine metabolism. We speculate that Aldh3b1 may be closely related to the metabolism of amino acids. Aldh3b1 is a member of the ALDH family. Its function is related to the scavenging of reactive oxygen species[30]. In postmenopausal women, the level of estrogen and antioxidants decreased with age, and the ROS accumulated in the body could not be cleared in time, which induced the occurrence of oxidative stress and led to the damage of osteoblasts and osteocytes[31, 32]. ALDH can reduce oxidative stress through a variety of aldehyde metabolism[33].
In this study, the expression trend of arachidonic acid (AA) in the platelet activation pathway and inflammatory mediator regulation of TRP channels changed after GSK treatment. AA and its products play an important role in regulating inflammatory response[34], vascular elasticity, platelet activation, or bone remodeling. AA belongs to polyunsaturated fatty acids (PUFAs), one of the indispensable free fatty acids in the human body. According to the position of the first double bond position[35], PUFAs can be divided into n-3pufas and n-6pufas, and n-6pufas mainly include linoleic acid and AA. Recent research highlights the potential role of PUFA in the inflammatory regulation of bone remodeling via several cellular pathways[36–38]. The combined use of n-3 PUFA and E2 exerted synergistic bone-protective efficacy through upregulation of RUNX2, an essential transcription factor for bone formation, as well as the suppression of bone-resorbing cytokine IL-1β[39].