Osteoclasts (OCs) have been the unique cell type exhibiting the bone-resorption activity in body. It is important to identify drugs to resist osteoclastogenesis to manage the bone-loss disorders. Huangqi Sanxian decoction (HQSXD) is utilized for the treatment of postmenopausal osteoporosis (PMOP) for a long history in East Asia. This work aimed to examine HQSXD's activity in OC differentiation. Based on staining with tartrate-resistant acid phosphatase (TRAP), it was found that HQSXD suppressed OC generation under the induction of RANKL produced in the bone marrow-derived monocytes/macrophages (BMMs), with no cytotoxic effect. Later analysis like molecular exploration and network pharmacology (NP) suggested the role of HQSXD in suppressing genes associated with osteoclastogenesis via PI3K/Akt-mediated mechanism dose-dependently. This work might illustrate the molecular pharmacological mechanism involved in HQSXD's effect on treating OC-associated disorders. Moreover, NP was found to modernize traditional Chinese medicine (TCM) research. Bone is constituted by minerals and proteins. In a mature skeletal system, the dynamically balanced bone formation-bone resorption can maintain persistent bone tissue metabolism, thereby maintaining bone elasticity and stiffness ,2. OsteoκB (NF-κB) ligand (RANKL) can be generated via osteoblasts (OBs) or additional bone cells, which have critical roles in OC generation [κB (RANK), and it has the major functions of inducing myeloid precursor differentiation to mature OCs [Macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-neration ,8. Of thneration . On the ture OCs . The BMMture OCs ,12. Additure OCs –15. The Radix notoginseng and Radix salviae miltiorrhizae can promote blood circulation, whereas Cistanche herba and Epimedii folium have kidney-strengthening activity. The above herbal medicines account for a superb combination that can highlight the corresponding superiority in treating OP [In the theory of traditional Chinese medicine (TCM), the kidney is suggested to regulate bone activities. A potent “kidney” is indicated to nourish the bones, on the contrary, an impotent “kidney” may aggravate bone degeneration . Blood sating OP .In this study, the findings provide a rationale for using HQSXD to treat OP in clinic.This work obtained 50 Sprague-Dawley (SD) rats , weighing approximately 100–150 g in the Animal Experiment Center of Guangdong Medical university . Afterwards, each rat was raised under 24 ± 0.5°C and 12 h/12 h light dark cycle conditions (light on: 08 : 00–20 : 00), with free access to water and food throughout the experiment. After 7 days of acclimatization, we randomized these rats in 5 groups, including blank control, raloxifene-treated positive control, and Huangqi Sanxian decoction (containing high and middle and low concentrations)-treated experimental groups. The present work gained approval from Laboratory Animal Management Committee. Each experiment was carried out following relevant regulations and rules.Radix astragali , Epimedii folium , Cistanche herba , Radix notoginseng , Radix salviae miltiorrhizae , Corydalis rhizoma , Radix angelicae Sinensis , as well as Radix clematidis  at the 15 : 10 : 10 : 5 : 10 : 10 : 8 : 10 ratio [HQSXD consisted of 8 plant extracts, namely, 10 ratio . This woμm membrane, so as to obtain HQSXD (H)-S, HQSXD (M)-S, HQSXD (L)-S, Ral -S, as well as Normal-S, separately, which were preserved under −80°C.This work randomized female rats as 5 groups, with 10 in each group. Mice were given oral administration of HQSXD solution  and Raloxifene solution (6.25 g/kg) once daily for 7 consecutive days, whereas those in normal group were given oral administration of distilled water and monitored at the same time of those in experimental groups. At 60 min post the final treatment, each rat was anesthetized to collect blood through heart puncture aseptically. After 15 min centrifugation of blood, sera were obtained from rats of the above 3 groups, followed by 30 min inactivation under the 56°C water bath as well as filtration with the 0.22 6/well) into the 96-well plate, followed by M-CSF incubation. On day 2, HQSXD at specific doses was utilized for 24-, 48-, and 72-h cell treatment. Cell viability was detected by CCK-8 assay. Afterwards, all wells were added with CCK-8 solution (10 μl), followed by another 1-h plate incubation. The infinite F200 PRO absorbance microplate reader  was employed for measuring the absorbance (OD) values at 450 nm (OD450). Finally, this work determined cell viability based on control.Primary rat osteoclast cells were obtained from bone medullary cavity of 1-Month-old neonatal Sprague-Dawley rats with 20 ng/mL M-CSF Induced. For investigating how HQSXD toxicity affected BMMs' viability, this work cultivated BMMs (1 × 106/well) into the 24-well plate, followed by cultivation with complete ɑ-MEM that contained RANKL, M-CSF, as well as HQSXD at specific doses for a 5-day period. After cell lysis, the TRAP activity assay kit was utilized for measuring TRAP activity in line with specific protocols. In brief, after removing the medium, PBS was used to rinse cells thrice, followed by 15-min lysis using a passive lysis buffer under 37°C. Thereafter, this work harvested supernatants for incubation using the para-nitrophenyl phosphate (p-NPP) for a 45-min period along with disodium tartrate. The sodium hydroxide solution was then added to terminate the reaction. The Infinite F200 PRO absorbance microplate reader  was employed to measure OD45 value for quantifying TRAP activity.This work inoculated BMMs (3 × 106/well) into the 24-well plate, followed by cultivation with complete ɑ-MEM that contained RANKL, M-CSF, as well as HQSXD at specific doses for a 5-day period with medium change at 2-day intervals. Later, cells were stained with TRAP by adopting the leukocyte acid phosphatase kit (Sigma-Aldrich) in line with specific protocols. Finally, the Olympus microscope  was utilized to visualize TRAP-positive cells with multiple nuclei (>3) and to take photographs under 40× magnification.This work inoculated BMMs  overnight under 4°C, followed by secondary antibody incubation with the Immobilon Western kit . Later, photographs were taken using the Image Quant LAS 500 imager . ImageJ  was adopted for measuring protein levels.https://tcmspw.com/tcmsp.php). In addition, osteoclastogenesis-related genes were identified from Gene Cards (https://www.genecards.org/), OMIM (https://omim.org/), and UniProt (https://www.uniprot.org/) with the keywords “osteoclast differentiation”, and “osteoclastogenesis”. The UniProt database was applied in confirming gene data like name as well as gene IDs.Chemical structure information of HQSXD was collected from the Traditional Chinese Medicine Systems database (https://string-db.org/), the protein species was set to “Homo sapiens” and “Multiple proteins” was selected, followed by the highest interaction score (0.7). This work acquired network relation information regarding HQSXD-OC target interactions with the highest confidence level (0.7). The network relationship data were optimized for PPI network graphs using Cystoscape 3.8.8 software.After importing the common targets into the STRING network platform  as well as related pathways with significant differences in the intervention of HQSXD in osteoclast were selected by KEGG pathway analysis, upon the t-test (two-sided). ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001 stood for statistical significance.Results were represented by mean ± SD from 3 separate assays. Differences were compared by one-way ANOVA or unpaired Student's According to CCK-8 assay, HQSXD produced an inhibitory effect on BMMs . Additio5After 2-day HQSXD treatment at diverse doses, the OCs-related protein levels in OCs were analyzed. As a result, HQSXD markedly suppressed the expression of related proteins  dose-dependently . This inBased on the search of TCMSP and deriving the results, 189 active ingredients were obtained, and 170 active ingredients were obtained after removing duplicate ingredients. By searching the database, there were 3707 drug targets corresponding to the active ingredients of HQSXD, and 232 remained after removing duplicates. After searching the three databases with the keyword “osteoclast differentiation”, a total of 1835 disease targets were obtained, with 1823 remaining after removing duplicates. After searching the three databases with the keyword “osteoclastogenesis”, a total of 689 disease targets were obtained, with 686 remaining after removing duplicates. Subsequently, the targets of “osteoclast differentiation” and “osteoclastogenesis” were intersected to obtain 1858 total targets of osteoclast differentiation, of which 651 were intersected by the two keywords. There were 651 intersecting targets in the two keywords.The PPI network between Huangqi Sanxian Decoction and osteoclast differentiation/production targets had 126 targets and 4662 interaction lines between the targets with an average degree of 37 .p-value for OC formation (The abovementioned hub genes were conducted KEGG pathway analysis for revealing the candidate mechanism by which HQSXD affected OC formation, and 20 pathways were enriched . After lormation .Based on KEGG analysis results, WB assay was conducted to analyze short-run protein phosphorylation mediated by RANKL, for the sake of verifying the mechanism of HQSXD in inhibiting OC generation and activity. BMMs were exposed to RANKL treatment with/without 2-day HQSXD pretreatment, and the phosphorylation of Akt was detected. Consequently, Akt phosphorylation elevated following RANKL treatment. Nonetheless, HQSXD pretreatment markedly suppressed phosphorylation of those aforementioned in comparison with controls .Bone accounts for the organ undergoing constant renewal and remodeling in one's life. OCs have been identified as the necessary part for maintaining bone health, since they have characteristic bone-resorption activity . ActuallThis work evidenced that HQSXD suppressed the RANKL-mediated OC formation in vitro, and first revealed the underlying molecular mechanisms. Cell growth and viability have important effects on discovering agents against OC formation, since adverse reactions related to the frequently applied agents bisphosphonates are partially associated with their activity in OC viability ,27. As oNatural medicines can synergistically affect several targets, components as well as pathways . The comβ, and CCL2, which are all in the core PPI network (β as a downstream signaling factor of PI3K/Akt signaling pathway can regulate NFATc1 expression, and the PI3K/Akt/GSK3β/NFATc1 signaling pathway is important in osteoclast differentiation and formation [Combining the core PPI network with the results of the enrichment analysis, after reviewing the literature, the PI3K/Akt pathway was determined to be the possible molecular mechanism related to HQSXD's role in suppressing OC formation. The PI3K/Akt signaling is frequently involved in multiple signaling molecules in osteoclasts, including Akt, GSK3 network . CCL2, aormation .Therefore, we selected Akt, a core protein in the PI3K/Akt signaling pathway, for a WB assay to test the above hypothesis. As a result, HQSXD remarkably suppressed Akt's short-time phosphorylation . For bet5β are most likely the upstream and downstream signaling factors that exert their inhibitory effects. This is the first study demonstrating HQSXD's inhibition on RANKL-induced OC differentiation into BMMs as well as the related mechanisms. It might help to interpret the molecular mechanism related to HQSXD within OC-associated disorders. Moreover, NP analysis was consistent with molecular mechanism study, which indicated the possibly of NP in modernizing TCM.Therefore, we suggest that Huangqi Sanxian Tang can inhibit osteoclast differentiation by inhibiting PI3K/Akt signaling pathway, and the network pharmacology results show that CCL2 and GSK3β are most likely the upstream and downstream signaling factors that exert their inhibitory effects. Further studies are needed to investigate how HQSXD exerts its effect on osteoclast differentiation via suppressing the PI3K/Akt pathway and thus osteoclast differentiation.In this work, HQSXD shows toxicity to OBs and inhibits osteoclast differentiation. The inhibitory effect was correlated with the PI3K/Akt signaling pathway and the network pharmacology results show that CCL2 and GSK3


In addition, inhibition of STAT3/HIF-1α/VEGF and FAK/AKT signaling pathways might serve as mechanisms. Taken together, the network pharmacology analysis suggested that DSY exhibited therapeutic effects through multiple targets in the treatment of HPH. The inferences were initially confirmed by subsequent in vivo and in vitro studies. This study provides a novel perspective for studying the relevance of TCM and disease processes and illustrates the advantage of this approach and the multitargeted anti-HPH effect of DSY.Traditional Chinese medicine (TCM) plays an important role in the treatment of complex diseases, especially cardiovascular diseases. However, it is hard to identify their modes of action on account of their multiple components. The present study aims to evaluate the effects of Dan-Shen-Yin (DSY) granules on hypoxia-induced pulmonary hypertension (HPH), and then to decipher the molecular mechanisms of DSY. Systematic pharmacology was employed to identify the targets of DSY on HPH. Furthermore, core genes were identified by constructing a protein-protein interaction (PPI) network and analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes (KEGG) analysis. Related genes and pathways were verified using a hypoxia-induced mouse model and hypoxia-treated pulmonary artery cells. Based on network pharmacology, 147 potential targets of DSY on HPH were found, constructing a PPI network, and 13 hub genes were predicted. The results showed that the effect of DSY may be closely associated with AKT serine/threonine kinase 1 (AKT1), signal transducer and activator of transcription 3 (STAT3), and HIF-1 signaling pathways, as well as biological processes such as cell proliferation. Consistent with network pharmacology analysis, experiments  Pulmonary hypertension (PH) is one of the most serious chronic severe cardiopulmonary dysfunctional diseases, characterized by progressive remodeling of pulmonary arteries, resulting in elevated pulmonary vascular resistance and, eventually, in right ventricular failure and death . PH due With a long history of thousands of years, traditional Chinese medicine (TCM) plays an important role in the treatment of complex diseases worldwide. With their respective dosages according to the guidance of Chinese medicine theory and the rule of “Jun-Chen-Zuo-Shi,” also known as “King, Vassal, Assistant and Delivery servant” , the TCMWith the rapid development of bioinformatics, network pharmacology has emerged as an effective and promising approach for highly multidimensional diseases  and TCM Salvia miltiorrhiza Bunge , Santalum album L.  and Amomum villosum Lour. It is widely used in clinical practice for the treatment of cardiovascular diseases and has produced a favorable effect  is a well-known traditional Chinese formula comprising e effect . S. Miltnhancing . It is ruate HPH . Moreoveuate HPH . DSY wasuate HPH . Furtheruate HPH . Our preuate HPH . Based oin vivo and in vitro.To elucidate the effects and molecular mechanisms of DSY on HPH, we performed a network pharmacology analysis and verified by experiments. Firstly, potential targets of DSY on HPH were identified by data mining. Next, we constructed a protein-protein interaction (PPI) network of potential targets, and the core genes were extracted. Then, a significant biological functional annotation for the identified targets helped to illustrate the potential anti-HPH mechanisms of DSY. The target-function interaction network was constructed and analyzed to identify key pathways. To validate the results, we estimated the protective effects and molecule mechanisms of DSY on hemodynamics and vascular remodeling in an HPH mouse model and hypoxia-treated pulmonary artery cells. The study utilized network-based techniques to investigate the natural compounds in DSY and to illustrate its predicted modes of action, which were validated by experiments We collected targets of DSY from different databases. Firstly, the TCMSP  database  was usedHPH-related targets were collected from DisGeNET  and GeneThe intersection of obtained drug targets and HPH-related targets were considered as potential targets of DSY for the treatment of HPH. These targets were uploaded with STRING 11.0  to analyp < 0.05 were considered significantly enriched.We utilized Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis for functional annotation. To annotate the role of candidate genes and proteins associated with HPH, the potential therapeutic targets of DSY were analyzed by DAVID  as inputS. miltiorrhiza Bunge  , S. album L.   and A. villosum Lour.   were added in a mass ratio of 10:1:1. All of them were identified to comply with the relevant provisions of Chinese Pharmacopoeia (Part I) (2015 edition). Firstly, 5 times the weight of water were added to the drug and soaked for 2 h. After decocting for 1 h and filtering, dregs and filtrate were collected separately. Then, 4 times the weight of water was added to dregs got before. After decocting for 1 h and filtering, dregs and filtrate were collected again. Next, we added 3 times of water, decocted for 1 h, and filtered to collect the filtrate. Later, we distilled and concentrated all the colature under reduced pressure at 70°C and −0.05 MPa. The obtained concentrated liquid and medical auxiliary material starch were passed through a multifunctional fluidized bed and finally mixed evenly by a three-dimensional motion mixer to obtain DSY granules. Based on the total weight of the medicinal materials and the quality of the prepared granules, it is calculated that 1 g of DSY granules is equivalent to 1.26 g of crude drug  were provided by Beijing HFK Bioscience Co., Ltd. . All animal experimental procedures were approved by the Institute of Animal Care and Use Committee, Chinese Academy of Medical Sciences. The animals were allowed to acclimate for 3 days in the facility, maintained at 22°C ± 3°C and 45% ± 10% humidity, following a regular 12 h/12 h light/dark schedule. All animals had free access to water and food.Mice were randomly divided into the following four groups: group I (control), kept in normal environment for 3 weeks; group II (model), kept in normal environment for 1 week, and then exposed to 10% oxygen in a hypoxic chamber for the next 2 weeks; group III (Sildenafil), kept in normal environment for 1 week, and then exposed to 10% oxygen for next 2 weeks, with Sildenafil (30 mg/kg/d) orally for 3 weeks; group IV (DSY), kept in normal environment for 1 week, and then exposed to 10% oxygen for next 2 weeks, with DSY  orally for 3 weeks. At the end of treatment, mice were anesthetized by tribromoethanol, and then the right ventricular systolic pressure (RVSP), lung, heart, and blood samples were collected from individual mice.The 6-minute walk test (6MWT) is a commonly used test to evaluate the activity tolerance of patients with pulmonary hypertension . Mice weMice were anesthetized with tribromoethanol. A middle incision was made at the neck to expose the right external jugular vein. A Mikro-Tip pressure catheter  was inserted into the jugular vein and then into the right ventricle (RV) for monitoring RVSP. A stable ventricular pressure wave indicates the exact position of the catheter in RV. RVSP was recorded with PowerLab  and analyzed with LabChart V8 software .Once hemodynamic data were collected, the blood was collected from the abdominal aorta. Then the mice died, and the organs were harvested. We recorded the weights of the whole heart, ventricle, right ventricle, lung, and body. The ratios of right ventricle-to-interventricular septal plus left ventricular weight (RV/LV + S), which was an indicator of right cardiac hypertrophy (RVH), were calculated.The left lung was isolated, immediately flushed with saline, and then fixed in 4% paraformaldehyde for 24 h. After being dehydrated and cleared, lungs were embedded in paraffin wax. The paraffin-embedded tissues were stained with Masson’s trichrome. The sections were observed under a light microscope for morphometric changes, and then photomicrographs were obtained.2 in humidified conditions. All experiments were carried out with cells at passages of 4–8.Human pulmonary artery endothelial cells (HPAECs)  and Human pulmonary smooth muscle cells (HPASMCs)  were purchased from ScienCell . HPAECs were cultured in Endothelial Cell Medium (ECM)  supplemented with Endothelial Cell Growth Supplement (ECGS) and 5% fetal bovine serum (FBS). HPASMCs were cultured in Dulbecco’s Modified Eagle Medium (DMEM)  supplemented with 10% FBS . The cells were cultured at 37°C with 5% CO2, 5% CO2). The others were moved into the Tri-Gas CO2 incubator  containing humidified hypoxia gas . After 48 h incubation, the cell viability was detected by the CCK8 kit. Absorbance values were read at the wave of 450 nm by a Spectra Max M5 microplate reader .The HPAECs at passage of 4–6 were seeded into normal 96 well plates at a density of 5,000 cells/well. The HPASMCs at passage of 4–6 were cultured in normal 96 well plates at a density of 4,000 cells/well. After 24 h, the cells were changed to fresh serum-free medium for 24 h. Then, HPAECs and HPASMCs were treated with or without DSY  for 2 h in the normal incubator. The cells in control groups were remained in normal conditions  as the standard. Protein samples were mixed with 5 × loading buffer and heated at 100°C for 10 min. Equal amounts of protein extracts were subjected to SDS-PAGE and electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane . After being blocked with 5% BSA for 2 h at room temperature, membranes were incubated overnight at 4°C with the following primary antibodies: β-actin , p-STAT3 , STAT3 , HIF-1α , c-Myc , VEGF , p-FAK , p-Akt , Akt  and PCNA . The membranes were then washed three times for 15 min with TBS-0.5% Tween 20 and subsequently incubated with anti-rabbit IgG  or anti-mouse IgG  secondary antibodies for 2 h at room temperature. After washing four times with TBS-0.5% Tween 20 for 20 min, immunoreactivity bands were visualized by enhanced chemiluminescence  and quantified using Image J software. Relative protein expression was normalized relative to β-actin.p-values less than 0.05. The images in this article were created using GraphPad Prism 8 .Results are expressed as the mean ± standard error of the mean (SEM). The significance of the differences between groups was determined by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test as appropriate. Differences were considered statistically significant at The whole workflow is illustrated in S. miltiorrhiza Bunge, 3 components in S. album L., and 15 components in A. villosum Lour. Based on data mining, we identified 485 pharmacological targets of DSY, 190 of which also appeared in the data set of HPH-related targets. Therefore, these 190 targets were considered to be potential targets of DSY in the treatment of HPH and were further analyzed.78 active components of DSY were obtained through screening from the TCMSP database, including 60 components in We used the STRING database to construct PPIs of these targets. As shown in For the 147 genes corresponding to potential therapeutic targets, DAVID was used to conduct functional annotation from different perspectives, respectively. There are 1,136 GO entries, of which 881 entries are related to biological processes, including negative regulation of apoptotic process, positive regulation of cell migration, positive regulation of cell proliferation, response to hypoxia and inflammatory response. 162 items are related to molecular function including enzyme binding, protein tyrosine kinase activity, protein binding, protein homodimerization activity, protein phosphatase binding and so on, and 93 cell components entries including macromolecular complex, membrane raft, plasma membrane, receptor complex, and so on . AccordiWe conducted further network analysis on several representative signal pathways and biological processes and constructed the target-function network. As shown in We monitored the body weight of mice everyday. The body weight of mice decreased after exposure to hypoxia, and then the growth slowed down. The positive drug sildenafil had no effect on body weight. DSY had an improving effect, but there was no significant difference .At the end of treatment, mice were placed on the treadmill and run until exhausted. The animal is considered exhausted if it stays on the battery lead plate for more than 10 s. Walk distance decreased in the model group (210.8 ± 50.24 vs. 331.6 ± 32.45 m). Sildenafil had no impact on walking distance, while treatment with DSY showed a tendency to extend the walking distance after exhaustion .p < 0.01). Treatment with sildenafil exhibited a decrease in RVSP, but there was no significant difference. Treatment with DSY demonstrated a prominent reduction of RVSP to 28.44 ± 0.77 mmHg from 34.64 ± 1.16 mmHg in the model group  compared with the control group  compared with the control group , which was reversed by DSY administration from 0.24 ± 0.02 to 0.17 ± 0.01  for 48 h. At the end of the experiment, the cell viability was detected by CCK8 assay. As shown in To investigate the effect of DSY on pulmonary artery cells, the cells were pretreated with DSY  for 2 h before incubating in hypoxia  protein levels and HIF-1α protein levels in lung tissues and HPAECs. As shown in AKT was shown to be an important target in network pharmacology analysis. The FAK/AKT pathway has previously been reported to promote the proliferation of HPASMCs . To deteThe past 2 decades have witnessed the coming of network science as the central paradigm behind some of the most fascinating discoveries of the 21st century . IncreasFirstly, the targets of DSY and genes related to HPH were obtained from multiple databases.78 compounds of DSY were screened out. The 190 intersection genes of DSY and HPH may be the potential therapeutic targets of DSY against HPH. GO and KEGG pathway analysis were conducted using DAVID, and networks were constructed using Cytoscape 3.7.2. When exploring the mechanism of action of DSY, we focused on analyzing the biological processes and signaling pathways associated with cardiovascular diseases, especially HPH. From the KEGG and GO biological process enrichment, the highly enriched pathways of DSY were associated with oxidative stress , cell proliferation , inflammation , and PI3K-Akt signaling pathway.The pathological mechanism of HPH is very complicated. Consistent with network analysis, previous studies have demonstrated that the development of pulmonary hypertension is highly related to oxidative stress . Our preFocal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that acts as a mediator of cell signaling downstream of growth factor and cytokine receptors . FAK hasThe current study, which investigated the impact and mechanism of DSY on HPH, achieved some findings. Firstly, DSY treatment significantly attenuated the increases in the RVSP and pulmonary artery remodeling induced by hypoxia. Next, DSY therapy downregulated HIF-1α expression and STAT3 phosphorylation in pulmonary tissues, thus implying its possible role in the HPH model, which is consistent with the network pharmacology analysis.in vivo experiment. As a well-known traditional Chinese formula, DSY is widely used in clinical practice for the treatment of coronary heart disease. Several studies have reported that it can effectively treat myocardial ischemia/reperfusion injury, ischemic myocardial injury, and angina pectoris. However, there is no related reports on DSY in the treatment of PH. Considering that excessive dose studies are meaningless, we chose the clinically equivalent dose to explore whether DSY can alleviate HPH. The clinically equivalent dose can ensure the safety of the drug and avoid the impact of adverse reactions. Fortunately, we found that it has a preventive effect on HPH. Whether DSY is dose-dependent in the treatment of HPH remains to be studied. Notably, it is unclear whether the findings in the HPH mouse model can be generalized to humans. Therefore, comprehensive in vivo and in vitro studies are required to test this hypothesis in the future.Our study has some limitations. Firstly, due to the large number of databases and the constantly updated data, the signaling networks we construct may not cover all known protein-protein interactions. Next, only one dose of DSY was investigated in the in vivo experiments. The results demonstrate that DSY can prevent the development of HPH in a hypoxia-induced mouse model and alleviate pulmonary vascular remodeling. Moreover, inhibition of the STAT3-HIF-1α signaling pathway and FAK-AKT pathway in the lung might serve as mechanisms involved in the therapeutic effects of DSY. With complex ingredients, DSY has shown a multitargeted effects on pulmonary hypertension. Multi-target therapy may be an important strategy for the treatment of pulmonary hypertension in the future.Taken together, network pharmacology provides a new approach for exploring the targets and mechanisms of TCMs, which are composed of multiple and complex ingredients. The deductions drawn from the computational analysis are, in part, validated by our subsequent


Henoch–Schönlein purpura nephritis (HSPN) is considered a major cause of chronic renal failure and is the most common secondary glomerular disease in children. Huaiqihuang (HQH), a traditional Chinese herbal formula, exhibits therapeutic effects against HSPN in clinical practice. However, the potential molecular targets and mechanisms underlying HSPN treatment remain unclear.  By constructing a protein-protein interaction (PPI) network, core targets related to HQH and HSPN were identified. Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathways were analyzed to identify the main pathways related to HSPN based on the core targets. To screen the main active ingredients of HQH against HSPN, an ingredient-target-pathway network was constructed using the top 10 main pathways associated with HSPN. Then, molecular docking was performed to explore the interactions and binding patterns between molecules and proteins.  Clinical data showed that HQH combined with conventional medicine significantly reduced 24-hour urine protein excretion, urine microalbumin levels, and erythrocyte counts in the urine sediment of HSPN patients. By constructing PPI models, 15 potential core targets were identified. The top 10 main pathways showed higher enrichment ratios, including the cytokine–cytokine receptor interaction and signaling pathways related to NOD-like receptor, IL-17, etc. Through the ingredient-target-pathway network and molecular docking, we revealed that five active ingredients of HQH had good affinities with three core targets, AKT1, MMP9, and SERPINE1, which may be vital in treating HSPN.  The study preliminarily explored the active ingredients, targets, and pathways involved in HQH therapy for HSPN. The mechanism of HQH therapy may be attributed to the modulation of inflammatory response, immune response, and oxidative stress. Combined with clinical data, our results indicate that HQH is highly effective in treating HSPN. In childhood, Henoch–Schönlein purpura (HSP) is a common vasculitis disease . HSP canTripterygium glycosides [Currently, glucocorticoid steroids combined with immunosuppressive therapy are recommended to treat HSPN . Howeverycosides  and Huaiycosides , has shoTrametes robiniophila Murr (Huaier), Polygonatum sibiricum (HJ), and Lycium barbarum (GQZ). HQH has been used as an adjuvant for primary nephropathy, mycoplasma pneumonia, and bronchial asthma in children [HQH is a traditional Chinese herbal formula, which comprises children , 8. HQH children , 10. Morchildren . HoweverIt has been widely known that network pharmacology can explain the complex mechanisms and multiple effects of TCM . MoleculThis study included 30 children diagnosed with HSPN who underwent drug treatment in the Xi'an Children's Hospital from June 2021 to December 2021. All data were sourced retrospectively from the hospital information system. The control group consisted of 15 children who received conventional treatment with an oral prednisone tablet and an intravenous infusion of CYP. The remaining 15 children in the experimental group were administered HQH granules combined with the conventional treatment. Renal function indicators, including 24-hour urine protein excretion, urinary microalbumin, and erythrocyte count in urine sediment, were assessed before and after 3-month treatment. SPSS 20.0 software was used for data processing and analysis.The inclusion criteria were as follows: (1) Clinical Guidelines for Nephrology  was usedThe exclusion criteria were as follows: (1) glomerulonephritis, IgA nephropathy, and other kidney diseases; (2) allergic to the medicine during treatment; and (3) patients with other autoimmune diseases.The outcome was measured based on (1) 24-hour urine protein excretion, (2) urinary microalbumin content, and (3) erythrocyte count in urine sediment.http://tcmspw.com/tcmsp.php) conforming to oral bioavailability ≥ 30% and drug‐likeness ≥ 0.18 [https://pubchem.ncbi.nlm.nih.gov/) for subsequent prediction of potential targets and molecular docking.The Chinese State Food and Drug Administration has authorized the application of Huaier granules to treat various cancers. Evidence showed that proteoglycans were active ingredients of Huaier . The acts ≥ 0.18 . The strhttp://www.swisstargetprediction.ch) to obtain active ingredient targets. Since the targets for Huaier could not be obtained here, we searched and collected them from PubMed and China National Knowledge Infrastructure (CNKI) databases.The SDF files of active ingredients were imported into the Swiss Target Prediction database (https://www.genecards.org/), OMIM (https://www.omim.org/), and DisGeNET (http://www.disgenet.org) with the keywords “Henoch–Schönlein purpura nephritis.” The genes were limited to “Homo sapiens.” All genes were downloaded and integrated using Microsoft Excel.Disease-related genes were obtained through GeneCards (https://bioinfogp.cnb.csic.es/tools/venny_old/) were generated to determine the common targets between HQH and disease. Then, the PPI network of common targets was constructed by the STRING database (https://string-db.org/). “Homo sapiens” was chosen, and a medium confidence score > 0.4 was selected. The above results were imported into Cytoscape (v3.7.2) (https://cytoscape.org/) for visualization. These targets were defined as core targets if their degree value exceeded the average.Venn diagrams (http://metascape.org/) was used to perform GO and KEGG analysis. The core targets were imported, and “Homo sapiens” was selected. An online bioinformatics web (http://www.bioinformatics.com.cn) was used to visualize the results. The cutoff chosen for the p value was <0.01.The Metascape database (http://rcsb.org/). Additionally, the best protein crystal structure was characterized based on the images with a low resolution with observable ligands and a relatively intact structure. The ligands were prepared using the Ligand Preparation module and minimized using the CHARM force field to generate 10 conformers. Using the Receptor-Ligand Pharmacophore Generation module to define the ligand-binding site, original ligands were extracted from the target proteins. The docking results were evaluated based on -CDOCKER energy, hydrogen bond interaction, and the pattern of binding mode.Molecular docking was carried out using Discovery Studio 2021 to explore the interactions and binding patterns between molecules and proteins. The active ingredients (ligands) of HQH were stored as SDF files. The main protein targets were obtained from the Protein Data Bank (p > 0.05). However, all indices significantly decreased after 3-month treatment in both groups (p < 0.05). Moreover, the experimental group showed indices lower than the control group (p < 0.05)  years. The control group consisted of ten boys and five girls, and their ages ranged from 4.5 to 12.25 (9.03 ± 2.28) years. The degree of renal function damage is a prognostic factor for HSPN, as HSPN manifestation is primarily marked by abnormal urine, including a decrease in proteinuria and hematuria. In clinics, 24-hour urine protein excretion, urine microalbumin, and erythrocyte counts in urine sediment are used as renal function indices. Therefore, we used these three indices to evaluate the therapeutic effect of HQH in this study. Before treatment, the groups showed no significant differences in 24-hour urine protein excretion, urine microalbumin levels, and erythrocyte counts in urine sediment ( < 0.05) . TherefoThe total number of active ingredients selected from GQZ and HJ was 45 and 12, respectively . The SwiWe searched for HSPN-related genes using GeneCards, DisGeNET, and OMIM and identified 84, 29, and 63 HSPN-related genes, respectively. After removing the duplicate genes and integrating the results, 157 candidate genes were identified.β, TLR4, TNF, TGFB1, CFD, F2, IGF1, ILK, MIF, MMP9, MPO, NR3C1, PRSS1, SERPINE1, and TTR. Importantly, AKT1, MMP9, ESR1, and STAT3 were correlated with Huaier, GQZ-HJ, and HSPN.To obtain the potential key genes targeted by HQH during HSPN treatment, we considered the overlap of HQH targets and disease-related genes and identified 24 common targets correlated with HSPN , includiThe degree parameter was calculated to analyze the importance of common targets in the PPI network using Cytoscape software. A total of 24 nodes and 179 edges were visualized . Larger In total, 79 KEGG pathways were significantly enriched. These pathways were mainly related to bacterial and viral diseases and signal transduction pathways . Becausep value cutoff < 0.01, the molecular function of 15 core targets primarily focused on cytokine activity, receptor-ligand interaction, activity of growth factors, and growth factor-receptor binding. The biological processes of HSPN mainly involve the inflammatory response, regulation of cell migration, regulation of cell proliferation and apoptosis by modulating the cellular response to lipopolysaccharide, leukocyte migration, and regulation of interleukin-6 production. Cellular components were related to the side of the membrane, extracellular matrix, vesicle lumen, and platelet alpha granules. In summary, we found that the 15 core targets were enriched in signaling pathways and biological mechanisms associated with inflammation and immune response.GO analysis was performed to explore the molecular functions, biological processes, and cellular components of the core targets Figures . By settTo understand which ingredient regulates these core targets for treating HSPN through the top 10 main pathways, we constructed the ingredient-target-pathway interaction network using Cytoscape. ESR1 was excluded from the network because it was not enriched in these pathways. The remaining 14 core targets were identified by Huaier and different active ingredients from HJ and GQZ, including MOL003889, MOL009760, MOL000098, MOL001792, MOL004941, MOL009646, MOL009644, and MOL006331 . Finallyβ, TLR4, and CCL2. The three common targets, AKT1, MMP9, and STAT3, were modulated by Huaier, MOL000098, MOL006331, MOL003889, and MOL009760. In addition, MOL001792, MOL004941, MOL009646, and MOL009644 interacted with SERPINE1 and IGF1. Therefore, Huaier and active ingredients from HJ and GQZ may be utilized to treat HSPN by regulating their respective targets.Furthermore, we found that MOL003889, MOL009760, MOL006331, MOL001792, and MOL004941 were correlated to HJ, and MOL009646, MOL009644, and MOL000098 were associated with GQZ . Our resTo verify whether the eight active ingredients could interact with targets, we used molecular docking to analyze their binding mode and affinity. We set the top hit to 10 and pose cluster radius to 0.5 to ensure as diverse docking conformations as possible. A high -CDOCKER energy indicates a high affinity between the molecules and protein receptors. We selected ingredients with lower -CDOCKER energy than that of the original ligand and the target protein as potentially active molecules. Except for STAT31 and IGF1, other active ingredients showed good affinity to the targets. Five bioactive molecules were screened based on their energy, as shown in Among these, MOL000098 had the highest affinity for AKT1. MOL000098 interacted with Lys179, Asp292, Asn279, and Glu278 via hydrogen bonding and entered the active pocket of AKT1 . MOL0000HQH is effective in treating HSPN. However, its active ingredients and underlying biological mechanisms are unclear. Our study prospectively predicts the bioactive ingredients and potential targets of HQH for HSPN treatment from the perspective of network pharmacology. We found that HQH improved kidney function indices to enhance the efficacy of conventional medicine in treating children with HSPN. Second, network pharmacology demonstrated that HQH affected 15 core targets related to HSPN. Subsequently, GO and KEGG enrichment analyses showed that these core targets may participate in the immune and inflammatory responses and oxidative stress. Finally, molecular docking defined suitable active ingredients and potential target proteins. Together, our results suggest that the five ingredients from HJ and GQZ could treat HSPN by regulating AKT1, MMP9, and SERPINE1 with the exception of Huaier.HSPN is a self-limiting immune disease, which can cure spontaneously or lead to chronic kidney disease. Our results showed that HQH combined with conventional medicine significantly reduced 24-hour urine protein excretion, urine microalbumin levels, and erythrocyte counts in urine sediment. Therefore, HQH can alleviate kidney damage and enhance the effect of conventional medicines on HSPN. In addition, conventional medicines, such as CYP, increase nephrotoxicity while treating HSPN . ImportaκB signaling pathway [HQH is a Chinese herbal compound comprising Huaier, GQZ, and HJ. Huaier has demonstrated promising curative effects in clinical treatment for various cancers . The ant pathway . GQZ andConsistent with previous results, our study showed that Huaier mainly regulated immune-related targets . MOL000098 and MOL006331 could interact with AKT1 and MMP9. MOL009646 from GQZ and MOL001792 and MOL009641 from HJ could bind to SERPINE1. These components may play vital roles in HSPN treatment.β and IL-10. Patients with HSPN present high levels of IL-8, CCL2, TNF-α, IL-10, and IL-6 [β, and CCL2 [Immune abnormalities and inflammatory injuries are involved in the pathogenesis of HSPN . Childreand IL-6 , 24. HQHand IL-6 , 25. Conand CCL2 . Previouand CCL2 . UnexpecAKT1  indirectly prevents the development of glomerulosclerosis and subsequent chronic kidney disease . We obseκB pathway, which is associated with oxidative stress regulation. In the present study, the NF-κB pathway was regulated by IL1B, CXCL8, TLR4, and TNF. In addition, HIF-1 activation is closely related to inflammatory responses, while FOXO can regulate oxidative stress, autophagy, and apoptosis. Both HIF-1 and FOXO were covered by core genes from HQH. Taken together, HQH exhibits significant antioxidant effects, reduces inflammatory damage, and improves humoral immunity.Oxidative stress pathologically aggravates kidney diseases. HQH can alleviate oxidative damage . MoreoveHQH plays an important role in improving HSPN and shows promising therapeutic efficacy by modulating inflammatory response, immune response, and oxidative stress. In this study, the clinical application evidence of HQH in treating HSPN is provided. Furthermore, some shortcomings remain in the study. The prediction of the ingredients, targets, and pathways still needs further pharmacological experimental verification.


The pathogenesis of the disorders of calcium metabolism is not fully understood. This review discusses the studies in which metabolomics was applied in this area. Indeed, metabolomics could play an essential role in discovering biomarkers and elucidating pathological mechanisms. Despite the limited bibliography, the present review highlights the potential of metabolomics in identifying the biomarkers of some of the most common endocrine disorders, such as primary hyperparathyroidism (PHPT), secondary hyperparathyroidism (SHPT), calcium deficiency, osteoporosis and vitamin D supplementation. Metabolites related to above-mentioned diseorders were grouped into specific classes and mapped into metabolic pathways. Furthermore, disturbed metabolic pathways can open up new directions for the in-depth exploration of the basic mechanisms of these diseases at the molecular level. Calcium is involved in a multitude of physiological processes, including intracellular signalling, regulation of metabolism, muscle contraction and gene expression . Most caDeviations in the calcium concentration above or below the normal range are now more often diagnosed than in the past, a tendency that mainly occurs due to the increased access to laboratory tests in developed countries. Disorders associated with calcium can be classified as hypocalcemia or hypercalcemia and they usually coexist with other serum biochemical abnormalities, including the levels of phosphate, alkaline phosphatase (ALP), PTH, fibroblast growth factor 23 and vitamin D ,9.Hypercalcemia commonly occurs due to malignancy and primary hyperparathyroidism (PHPT). PHPT is a common endocrine neoplastic disorder characterized by the autonomous production of PTH with a broad spectrum of pathophysiological sequelae . It occuChanges in calcium levels can induce a wide spectrum of symptoms, including skeletal anomalies, nephrolithiasis and muscle spasms . This frMetabolomics is still a relatively new field of science that links cellular phenotypes to their genotypes and delivers biochemical information associated with the regulation of specific gene transcripts . EnvironMetabolomics has yielded valuable new insights into a number of important biological and physiological processes. In medicine, metabolic profiling can be used to propose novel diagnostic algorithms or to reveal the phenotypic differences in various disorders and then guide personalized therapy . There aThe studies reported here present the usefulness of a global human metabolic network to interpret plasma metabolites profiling in various branches of medicine . Well-cuhttp://www.metaboanalyst.ca/ accessed on 25 August 2022). Only annotated metabolites significantly discriminating studied groups were used for this analysis. The Kyoto Encyclopedia of Genes and Genomes (KEGG) based Homo sapiens library was selected for analysis with a hypergeometric test in over-representation analysis and relative-betweenness centrality in pathway typology analysis (to estimate node importance). Pathway significance was determined from pathway enrichment analysis and based on values for each compound in the dataset.For each disorder, we summarized differentiating metabolites reported in selected publications. These metabolites were classified following the Human Metabolome Data Base (HMDB) classification for polar molecules and Lipid Maps classification for lipidic molecules. This information was used to illustrate which classes of metabolites are involved in different calcium metabolic disorders the most. Moreover, HMDB identifiers for metabolites were retrieved and used for pathways analysis. Pathway analysis was performed using MetaboAnalyst 5.0 . Surprisingly plasma samples were used only in two studies. Urine samples were analyzed in four and parathyroid tissue in two studies. Moreover, only in few studies combined serum samples with urine and fecal samples were analyzed ,40,41.Further analysis revealed that 79% of analyses were performed using MS and only 21% employed NMR. This result is not surprising because MS dominated the field of metabolomics due to its outstanding sensitivity. MS usually is used in combination with some separation technique: LC was used in 10 publications, GC and CE in two papers. Surprisingly, there are only two studies where more than one technique was used ,41. LC iWe performed characterization and analysis of all metabolites altered in calcium metabolism disorders . InteresThe excessive secretion of PTH is a common disorder known as hyperparathyroidism. PHPT is one of the most common endocrine diseases in western countries and is caused by an abnormal proliferation of tumor parathyroid cells. Parathyroid tumors are categorized as a genetically heterogeneous group which is characterized by a significant variability in clinical features ,45. BothDue to advances in diagnostics, the clinical presentation of PHPT evolved in developed countries from a symptomatic to an incidentally discovered and asymptomatic disorder . Recent Over the last decade, studies provided numerous new insights into the differential metabolic profiles between healthy and pathological parathyroid glands. Palermo et al. in their study, attempted to use Raman spectroscopy to differentiate healthy parathyroid tissue and parathyroid adenomas (PAds). The results demonstrated the strong potential of the Raman spectroscopy method, which showed the different spectroscopic features of these two types of parathyroid tissue. This method was able to correctly classify all subjects with 100% prediction accuracy . Di MasiMetabolomics was also used in the study reporting the presence of environmental chemicals in parathyroid tumors . Lower lBattini et al. reported results from the first study checking the differences in tissue metabolomic profiles between a single PAd and multiglandular disease . MultiplAccording to widespread knowledge, there are currently no studies evaluating plasma metabolite biomarkers related to PHPT. However, their discovery may provide an important new tool for personalizing the clinical management of PHPT. Moreover, a minimally invasive method for diagnosing parathyroid carcinoma (PC) allowing proper treatment to be initiated is very much needed. PC is a rare endocrine malignancy diagnosed in the majority of cases postoperatively upon histological examination due to the lack of reliable clinical diagnostic criteria . A direcBased on these reports we selected 24 metabolites, reported as altered in PHPT. 23 of them were assigned to the HMDB identifiers and 21 were matched into the pathways. We classified them into six main categories: Carboxylic acids and derivatives, Dihydrofurans, Hydroxy acids and derivatives, Organic sulfonic acids and derivative, Organonitrogen compounds and Organooxygen compounds .Among carboxylic acids, amino acids and their derivatives were the predominant metabolites. Organo-oxygen and—nitrogen compounds there the second and third largest groups. Pathway analysis revealed several disturbed pathways, including Arginine biosynthesis, Alanine, aspartate and glutamine metabolism, aminoacyl-tRNS biosynthesis and Glyoxylate and dicarboxylate metabolism among other.2D)—the main mediator for calcium absorption in the gastrointestinal tract [One of the earliest clinical manifestations of chronic kidney disease (CKD) is SHPT. SHPT occurs due to parathyroid hyperplasia caused by stimuli such as low serum calcium levels associated with renal failure or decreased active vitamin D concentration. The reduction in CaSRs in parathyroid glands is also considered to be one of the pathogenic factors of SHPT ,6. The tal tract ,55,56. Ial tract . L-phenylalanine, D-aspartic acid and D-galactose form a group of biomarkers that might be used to distinguish patients with SHPT from healthy controls with an area under the curve (AUC) of 0.947. Following parathyroid surgery, the concentration of these metabolites restored or tended to be normal [Serum metabolites correlated with PTH in SHPT were successfully demonstrated in an untargeted metabolomic study performed by Shen involving a group of 35 patients . Allyl ie normal .Wu et al. using ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS)—based metabolic profiling, identified 32 potential biomarkers for CKD and mineral and bone disorders . The autL-phenylalanine plays the role of allosteric activator of CaSRs, inhibiting PTH secretion [L-amino acids in physiological concentrations, especially L-amino acids of the aromatic and aliphatic classes, are known as strong regulators of PTH secretion and thus whole-body calcium metabolism . L-phenyecretion . Its incecretion . The incecretion .D-aspartic acid and L-phenylalanine in abnormal bone metabolism was formerly described. The concentration of these markers may provide new information on the insensitivity of bone turnover in SHPT. Phenylketonuria is a rare inherited disorder associated with phenylalanine hydroxylase deficiency and the accumulation of phenylalanine [The involvement of lalanine . Schawahlalanine . Intereslalanine . Moreovelalanine ,62.The literature revision allowed for the collection and characterization of 76 discriminating metabolites. In this case, 74 of them were assign to the HMDB identifiers, but only 67 of the were matched into the pathways. These metabolites were assigned into 16 different classes. Once again, carboxylic acids and derivatives were the largest group followed by the Organooxygen compounds and Fatty acyls .In contrast to the PHPT, SHPT is characterized by wider group of metabolite classes. This might be related to the differences in the metabolic impact occurring in both disorders, but also to the fact the number of reported altering metabolites was three times higher for SHPT than for PHPT. Larger number of discriminating metabolites covered wider range of metabolite classes. Interestingly, despite differences in the metabolite coverage, pathway analysis revealed highly comparable results. In both cases, Arginine biosynthesis, Alanine, aspartate and glutamine metabolism, aminoacyl-tRNS biosynthesis and Glyoxylate and dicarboxylate metabolism are the most altered pathways.Hypocalcemia is considered to be the main stimulus of PTH synthesis from parathyroid glands ,63. NutrThe first study with urinary metabolic profiling for reliable calcium-deficiency biomarkers was published in 2013 . Two bioWang et al. in their study involving 200 children, investigated urinary metabolomics in patients with nutritional rickets . The autPrevious metabolomic experiments provided a set of potential diagnostic biomarkers in urine. A serum metabolomic investigation based on UPLC/Q-TOF MS/MS and multivariate statistical analyses to determine whether the biomarkers reported in urine were present in the rats’ serum with a deficiency of calcium. There were 24 calcium deficiency biomarkers performed and revealed. In previous urinary metabolomic studies, three metabolites  have also been found. The levels of phosphoric acid and indoxyl sulfate and were increased in both serum and urine, while the level of taurine was decreased in urine and at the same time it was increased in serum. The AUC combination of any two of them was higher than 0.95 . Increased levels of indoxyl sulfate lead to decreased ALP activity, the impaired mineralization of osteoblastic cells and the deregulation of the transcriptional level of collagen type 1. The combination of these changes induces the gradual deterioration of osteogenesis and the impairment of bone turnover common in patients with calcium deficiency . ClinicaAll these publications cumulatively reported 58 metabolites which level is disturbed in the calcium deficiency. We were able to assign HMDB identifiers to 48 of them and 46 were later matched into pathways. These 58 metabolites were stratified into 15 classes. Carboxylic acids and derivatives with amino acids and derivatives were the largest group. Interestingly, two next largest groups are represented by lipids and are Fatty acyls and Sterol lipids .In this case there are only few altered pathways with butanoate metabolism, TCA cycle, Purine metabolism and Alanine, aspartate and glutamate metabolism as the most impacted ones. Sterol lipids contributed to the Glyoxylate and dicarboxylate metabolism pathway.Osteoporosis is characterized by low BMD with the destruction of the bone microstructure. It can lead to increased bone fragility and low-trauma osteoporotic fractures . OsteopoAnimal models are frequently used in osteoporosis metabolomics studies. Mao et al. presented the results of a UPLC-QTOF MS analysis indicating that calcium supplementation has beneficial effect on bone loss in ovariectomized rats. The authors showed that calcium supplementation increased the levels of estradiol (E2) and led to changes in metabolites levels, increasing BMD. The administration of higher doses of calcium decreased the levels of bone-turnover markers. The glycerophospholipid metabolic pathway is closely related to calcium supplementation. The main differences in glycerophospholipids metabolism were identified in phosphatidylethanolamines, phosphatidylcholines and ceramides. Additionally, it was found that E2 may affect changes in glycerophospholipids and glycerophospholipid metabolism was considered to be the main potential target pathway of E2 . Zhao usAnimal models were also used to evaluate efficacy of traditional Chinese herb containing osthole against osteoporosis in ovariectomized rats. In this study, high throughput metabolomics method was applied to discover biomarkers and altered pathways as potential targets. Osthole effectively altered 19 endogenous metabolites involved in several metabolic pathways, including starch and sucrose metabolism, arachidonic acid metabolism and linoleic acid metabolism .Zhang et al. aimed to find BMD-associated markers that are predictive of fracture risk . The autYou et al. in their cross-sectional study, identified a group of metabolites for characterizing low BMD in postmenopausal women . Among sAn untargeted MS-based metabolomics technique was used to analyze 69 patients’ serum samples and identify metabolic panel for patients with low bone mineral density . The resThe large population-based study conducted by Ling et al. presented evidence connecting the functional composition of the gut microbiota and metabolomics with osteoporosis. The idea of the study was derived from previous smaller studies and limited observations in humans. Fecal and serum metabolomics were applied to show that tryptophan and tyrosine metabolism and isoleucine, leucine and valine degradation were related to the identified microbiota biomarkers and to osteoporosis, respectively .In osteoporosis, the following metabolic pathways are altered: aminoacyl-tRNA biosynthesis; proline and arginine metabolism; valine, leucine and isoleucine biosynthesis; as well as biosynthesis of arginine. Interestingly, similar alterations were found in arthritis . FurtherTo conclude, despite multiple studies, it is still not possible to precisely assess the risk of potential decreases in BMD in postmenopausal women. The existing investigations yielded contradictory results. The differences between epidemiological and experimental investigations, as well as the confounding factors in sample preparation, detection and data analysis, could explain these discrepancies. Most of the published metabolomics-based studies of osteoporosis so far used untargeted metabolomic techniques and further studies of the identified metabolites are warranted to fully understand their interactions.The largest number of publications was reviewed for the osteopenia and osteoporosis, which resulted in the largest number of collected metabolites. We summarized 201 molecules, out of which 188 were assigned to the HMDB identifiers, while pathway analysis was performed for 183 compounds. All these metabolites were distributed across 21 classes. 84 molecules were assigned to the Carboxylic acids and derivatives, 21 to Fatty acyls, 16 to Organooxygen compounds and 14 to Glycerophospholipids .This large number of differentiating metabolites resulted in the big number of altered pathways. Despite relatively high number of altered lipids, majority of pathways is related to different amino acids metabolism. Similarly to the calcium deficiency, TCA cycle was also matched, however with lower significance and impact.2D3 but also other circulating vitamin D metabolites can be detected with this approach. Additional metabolites or their ratios can provide additional diagnostic information. The elevated 25-OH-D3/24,25-(OH)2D3 ratio may help to identify patients with CYP24A1 mutations. Therefore, the differential diagnosis of vitamin D-related hypercalcemia can be supported with analysis of the complete vitamin D metabolome [There is an ongoing debate on the potential benefits of vitamin D for cardiovascular disease risk, several types of cancers, all-cause mortality and other chronic illnesses. Application of LC-MS/MS in the assay of vitamin D proved that metabolites devoid of biological activity can also serve as disease indicators. Not only 25-OH-D3 or 1,25-(OH)tabolome . New memtabolome .Spermidine and spermine were found to be involved in the activation of the vitamin D receptor and the induction of spermidine N1-acetyltransferase activity was found to be triggered by 1 alpha,25(OH)2D3 ,80. ShirAnother more recent study provided new information on direct negative impact of high daily doses of vitamin D supplementation on skeletal muscles . PatientVitamin D2 and vitamin D3 are two main forms with different pharmacokinetic properties used in supplementation. Urinary metabolite profiling by liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF-MS) was used to assess the potential biological differences of vitamin D3 (15000 IUs) and D2 (20000 IUs) supplementation in prediabetic patients. Urine samples were taken before supplementation and after 12 weeks of treatment. Nevertheless, no biological differences were found between vitamin D2 and D3 supplementation at levels of circulating 25(OH)D that are equivalent. There were no differences in urinary metabolite profiles .We found 42 metabolites reported as altered in the vitamin D3 deficiency. Surprisingly, all 42 molecules were assigned to the HMDB identifiers and further matched into the pathways. These metabolites were distributed across 11 classes. Interestingly, this is the only disorder, where the most predominant group are lipids, in particular Fatty acyls .The combination of multiple omic technologies, including transcriptomics, proteomics and metabolomics, is perhaps the next major step towards a full understanding of the pathophysiology of chronic diseases. The applications of metabolomics presented in this review delineate the metabolic profile in calcium metabolism disorders. However, further studies are necessary to combine untargeted metabolomic research with targeted metabolomics to determine the significantly altered metabolites and improve the presented findings. Furthermore, efforts aimed at identifying specific metabolic pathways may serve as targets for treatment. Nevertheless, improved knowledge of the molecular alterations underlying hyperparathyroidism, the identification of PC biomarkers that could allow them to be used as discriminators of this disorder and new tools to assess the risk of fractures and decreases in BMD are undoubtedly desirable objectives. The creation of a vast biomarker library for patients with specific calcium disorders should be the purpose of future studies. To summarize, more in-depth omics studies remain to be performed for the refinement and validation of the molecular profiles described in the present review.


In recent years, clinical studies have found that there is a close relationship between osteoporosis and polycystic ovary syndrome. However, there are few literature on the pathogenesis of osteoporosis and polycystic ovary syndrome. In order to clarify their common pathogenic mechanism and provide potential targets for drugs to regulate them at the same time, bioinformatics methods are used to explore, so as to provide a new direction for the study of the relationship between diseases in the future.To screen the targets of osteoporosis and polycystic ovary syndrome by Genecards, Online Mendelian Inheritance in Man databases and Therapeutic Target Database to take the intersection of the two mappings and upload the intersection targets to the STRING database to construct protein-protein interaction network; to screen the core targets by degree value and import them to Metascape database for Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis; and finally, to construct the visualization network of core targets and pathways by Cytoscape software. Ethical approval and informed consent of patients are not required because the data used in this study is publicly available and does not involve individual patient data or privacy.The core targets of polycystic ovary syndrome and osteoporosis were insulin gene, insulin-like growth factor 1, CTNNB1, serine/threonine kinase 1, signal transducer and activator of transcription 3, LEP, etc. The biological processes involved include the regulation of protein phosphorylation, cell proliferation and differentiation, hormone endocrine, reproductive system and skeletal system. The related pathways were concentrated in Foxo signaling pathway, HTLV-I infection, PI3K-AKT signaling pathway, MAPK signaling pathway and AGE-RAGE signaling pathway in diabetic complications.There is a close relationship between osteoporosis and polycystic ovary syndrome in terms of target and molecular mechanism. This study used bioinformatics to clarify their targets and mechanisms, providing potential targets for drugs to regulate both diseases simultaneously and providing new directions to explore the relationship between the diseases. The National Institutes of Health reported that the incidence of PCOS in women of reproductive age is about 4% to 10%. Osteoporosis and polycystic ovary syndrome are two types of chronic diseases that are closely related, and a study of 11,106 women in Taiwan found an increased incidence of fractures in patients with PCOS compared to healthy women; Piovezan et al in a systematic evaluation of 31,383 women found that PCOS patients tended to have lower bone mineral density and osteocalcin; there is also literature,6 suggests an association between metabolic dysfunction and low vitamin D levels in patients with PCOS.Osteoporosis (OP) is a systemic skeletal disease characterized by a decrease in bone density and bone quality, and the associated factors are complex and diverse.Therefore, it is urgent to clarify the relationship between osteoporosis and polycystic ovary syndrome and to reduce the risk of osteoporosis in patients with PCOS. A bioinformatics approach was applied to integrate the genetic data of osteoporosis and polycystic ovary syndrome to explore the association and provide a theoretical basis for drug intervention in both diseases simultaneously.22.1https://www.genecards.org/), Online Mendelian Inheritance in Man (http://www.omim.org/), Therapeutic Target Database (http://db.idrblab.net/ttd/) database screening of disease related targets. To improve the accuracy, the top 300 target genes with high Relevance Score were selected from the Genecards database, and the results were combined and de-weighted, and then the Uniprot database was used to screen the clearly identified target genes as the final targets for polycystic ovary syndrome and osteoporosis.The “osteoporosis” “ polycystic ovary syndrome” as keywords retrieval Genecards (2.2http://www.biocloud.net/) platform to obtain the intersecting genes, and the intersecting genes were uploaded to the STRING database (https://string-db.org/cgi/input.pl) to construct a PPI network. Download the PPI network in tsv format and import it into Cytoscape 3.8.0 software to better visualize the protein-protein interaction relationships. Using Network analyzer plug-in, adjust the size and color of the targets according to the node connectivity (degree), and adjust the thickness and color of the connecting lines according to the combined score, and finally select the targets with the degree value ≥18 to make the core target map of osteoporosis – polycystic ovary syndrome.The target genes of osteoporosis and polycystic ovary syndrome were imported into the BMK Cloud (2.3http://metascape.org/), and set P < .01 and enrichment factor > 1.5 for GO and KEGG analysis. The first 40 GO, KEGG pathways of the two diseases were selected and intersected by logP ascending order, and imported into Cytoscape 3.8.0 software to construct the interaction pathway map of osteoporosis – polycystic ovary syndrome.The target genes of osteoporosis and polycystic ovary syndrome were imported into Metascape database , 19 (KEGG) interaction pathways were screened Fig. , and imp44.1 Clinical studies,14 PCOS patients were found to have different degrees of bone mineral density loss, which is closely related to leptin resistance and hyperinsulinemia. Therefore, in this study, bioinformatics was used to find the intersection and common pathways in the vast gene network of the two diseases to provide potential targets for drug therapy. A total of 70 intersecting targets were found between polycystic ovary syndrome and osteoporosis, accounting for about 10.38%, with core targets such as insulin gene (INS), insulin-like growth factor 1 (IGF1), CTNNB1, Serine/threonine kinase 1 (AKT1), signal transducer and activator of transcription 3 (STAT3), LEP, etc.; The biological processes involved include the regulation of protein phosphorylation, cell proliferation and differentiation, hormone endocrine, reproductive system and skeletal system; KEGG pathway analysis showed that the Foxo signaling pathway, HTLV-I infection, PI3K-AKT signaling pathway, MAPK signaling pathway and AGE-RAGE signaling pathway in diabetic complications and many other signaling pathways.Recent studies have found that the two types of diseases, polycystic ovary syndrome and osteoporosis, are interrelated and affect each other. The INS is also considered as one of the candidate genes for PCOS. Studies,17 have confirmed that decreased insulin receptor activity and PI-3K activity lead to the decrease of glucose uptake rate. Therefore, PCOS patients are prone to metabolic abnormalities such as obesity and decreased glucose tolerance. Meanwhile, the high glucose environment alters osteoblast activity and induces osteoblast apoptosis, leading to reduced bone mineral density and increased fracture risk.,19 IGF1, an osteogenic differentiation factor, upregulates the expression of protein kinase and alkaline phosphatase mesenchymal stem cells and participates in cartilage anabolism and repair. Animal experiments,22 found that different concentrations of IGF1 play a positive role in regulating the expression of bone genes in cartilage layer and subchondral bone, and the bone thickness of IGF1 knockout mice is significantly lower than that of normal mice, which confirmed the importance of IGF1 in bone growth; meanwhile, the occurrence of hyperandrogenism in PCOS is closely related to hyperinsulinemia, 44–77% of PCOS patients can be combined with hyperinsulinemia, and the higher insulin stimulates IGF1, promotes the release of LH, and enhances the activity of 17α-hydroxylase in follicular membrane cells, which increases the synthesis and secretion of androgens, thus causing the development of hyperandrogenemia; AKT1 is an isomer of Akt, mediates many downstream pathways regulated by PI3K, regulates the growth of follicles, the proliferation of granulosa luteal cells and the dynamic balance between bone formation and bone resorption. Some studies speculate that the high expression of AKT1 is one of the reasons affecting the quality of follicles and the function of granulosa luteal cells in patients with PCOS. There are also literature,27 reported that activated AKT1 can control the proliferation and differentiation of osteoblasts and osteoclasts through the RANK/RANKL signaling pathway and PI3K/AKT signaling pathway to affect bone metabolism, and Mukherjee et al also found that osteoblast formation was significantly inhibited by culturing AKT1 knockout bone marrow stromal cells or mesenchymal stem cells. The STAT3 dimer formed by the dimerization of STAT3 can activate STAT, promote the transcription of STAT3 target genes, form JAK2/STAT3 signal pathway mediated by IL-6, inhibit the expression of TNF, VEGF and other inflammatory factors, and improve leptin resistance in PCOS patients. Meanwhile, STAT3 dimer can also promote the proliferation and differentiation of Th17 cells, promote the secretion of IL-17 and TNF-α. It affects the formation of osteoclasts and bone resorption process, and inhibits the apoptosis of osteoblasts by promoting the expression of apoptosis inhibitory protein Bcl-2.As an endocrine disease with complex etiology, polycystic ovary syndrome is closely related to obesity and insulin resistance, and the INS is also considered to be one of the candidate genes for the development of PCOS. It can prevent the effect of high glucose environment on osteoblast apoptosis, and regulate the free oxygen concentration in osteoblasts to promote osteoblast differentiation. It can also reduce the damage to cartilage caused by inflammation. He Tingting et al found that the level of anti-apoptotic gene Foxo1 in PCOS patients was lower than that in normal women, suggesting that the low expression of Foxo may be one of the reasons for ovarian granulosa cell autophagy and follicular development disorder in PCOS patients. As an important pathway to regulate cell proliferation, differentiation, metabolism and other basic functions, PI3K/AKT pathway can regulate the function of ovarian granulosa cell, affect hormone levels in vivo, and also affect bone metabolism by controlling the proliferation and differentiation of osteoblasts and osteoclasts, and is a key pathway to affect the functional coordination between osteoblasts and osteoclasts. Mei Shao et al found that PI3K/AKT signaling pathway could inhibit apoptosis and autophagy of ovarian granulosa cells in PCOS patients, promote cell differentiation and proliferation, and improve polycystic ovary syndrome. Animal experiments demonstrated that activation of PI3K/AKT signaling pathway can bidirectionally regulate osteoblast differentiation and osteoclast apoptosis. Dina et al and Tao Ling et al found that the activation of PTHrP gene in HTLV-I infected patients can affect bone mineral deposition, promote osteolysis and cause osteoporosis. MAPK signaling pathway is an important pathway that regulates the proliferation and differentiation of osteoclasts and osteoblasts, and can also promote the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), accelerate calcium deposition, and prevent the occurrence of osteoporosis. The high insulin status of PCOS patients leads to abnormal activation of MAPK signaling pathway, which affects the growth and differentiation of ovarian granulosa cells. Xu Jinbang et al found that the artificial cycle of acupuncture and drugs can regulate the mitotic activity of ovarian granulosa cells through MAPK signaling pathway, and improve the reproductive endocrine disorders of PCOS patients.Foxo signaling pathway, as one of the pathways mediating the inflammatory response, can improve insulin resistance, regulate glucose metabolism in PCOS patients.5In this study, we searched for common targets and pathways of action between polycystic ovary syndrome and osteoporosis through bioinformatics, and expressed the interrelationship between them more clearly through visualization network, which also provides potential targets for drugs to regulate both diseases simultaneously. It is found that insulin resistance, aggravation of glucose metabolism disorder and imbalance of hormone level in polycystic ovary syndrome will affect the proliferation and differentiation of osteoblasts and osteoclasts. Foxo signal pathway, PI3K/AKT signal pathway and JAK2/STAT3 signal pathway all play a direct or indirect role. Of course, with the continuous development of bioinformatics and disease databases, the reliability and accuracy of data will continue to improve, and further studies are needed to make the conclusions more reliable.Conceptualization: Chun-xiao Dang, Jin-xing Liu.Data curation: Chun-xiao Dang, Ding Wang, Xiao Yu.Formal analysis: Chun-xiao Dang, Ding Wang.Funding acquisition: Peng-fei Liu, Jin-xing Liu, Xiao Yu.Investigation: Chun-xiao Dang, Peng-fei Liu.Methodology: Chun-xiao Dang, Yu Xiao.Writing – original draft: Chun-xiao Dang, Ding Wang.Writing – review & editing: Peng-fei Liu, Jin-xing Liu.