Study on the Mechanism of the Combination of Methotrexate and Leflunomide in the Treatment of Rheumatoid Arthritis Based on Network Pharmacology, Molecular Docking, and in vitro Experimental Verification.

BACKGROUND: To date, disease-modifying antirheumatic drugs (DMARDs) are widely used as the primary first-line treatment option for patients with rheumatoid arthritis (RA), and the curative effect of methotrexate (MTX) and leflunomide (LEF; MTX + LEF) is greater than that of single-agent MTX therapy, but the synergistic mechanism of MTX + LEF is unclear. METHODS: First, we explored the mechanism of action of MTX + LEF in RA through network pharmacology and molecular docking. Venn diagram analysis revealed 97 overlapping gene targets of MTX + LEF-RA and STRING, along with Cytoscape plug-in MOCDE and cytoHubba; and GO enrichment analysis revealed that the functions of 97 synergistic targets were related to 123 molecular functions (MF), 63 cell components (CC), and 1,068 biological processes (BP). The Cytoscape plug-in ClueGO demonstrated that these targets were enriched in KEGG pathways of 52 terms, whereas 9 pivotal genes were mainly involved in the signaling pathways of estrogen, Ras, Rap1, PI3K-Akt, relaxin, TNF, AMPK, FoxO, prolactin, IL-17, and adherens junction. Finally, CETSA and DARTS validated the direct binding of MTX or LEF to the selected target proteins EGFR, PPARG, MMP9, and SRC in RAW264.7 cells. RESULTS: We identified 292 MTX targets and 247 LEF targets from 7 databases. Furthermore, 2,814 potential targets of RA were identified by merging 1,925 targets from 7 databases and 999 differentially expressed genes (DEGs) between normal controls and patients with RA extracted from 5 GEO databases. Nine pivotal genes, ESR1, ALB, CASP3, EGFR, HSP90AA1, SRC, MMP9, PPARG, and IGF1, were identified. Molecular docking verified that both MTX and LEF strongly bind to most of the 9 pivotal proteins except ESR1 and IGF1. CONCLUSION: These results contribute to our understanding of the enhancement mechanism of MTX combined with LEF and provide a targeted basis for the clinical treatment of RA.

The effects of different doses of teriparatide on bisphosphonate-related osteonecrosis of the jaw in mice.

OBJECTIVES: This study aimed to investigate the therapeutic effect of different doses of teriparatide (TPTD) on bisphosphonate-related osteonecrosis of the jaw (BRONJ). MATERIALS AND METHODS: To establish the BRONJ model, 20 mice were randomly divided into two groups: a group that received tail vein administration of zoledronic acid with dexamethasone (ZA-125 µg/kg, DEX 5 mg/kg) and a group that received saline weekly. The mice subsequently underwent bilateral maxillary first molar extraction. After 8 weeks of modelling administration, the maxilla samples were examined by micro-computed tomography and histological staining (haematoxylin and eosin, Masson's trichrome and tartrate-resistant acid phosphatase) and the cytokine level was measured (enzyme-linked immunosorbent assay and Western blot). To determine the role of TPTD in BRONJ, the same protocol as previously described was applied in 100 mice (80 received ZA + DEX, and 20 received saline). After 8 weeks of modelling administration, 80 ZA + DEX mice were randomly divided into four groups: three groups with subcutaneous administration of TPTD (i.e. T1-3, T2-10 and T3-30 µg kg-1  day-1 ) and one group with saline daily for the next 8 weeks. The other 20 saline mice continued to receive saline daily. RESULTS: In Part 1, the level of receptor activator of nuclear factor-kappa Β ligand and the numbers of osteoclasts differed between the model and control groups. In Part 2, we found that TPTD had a positive effect on BRONJ in a mouse model based on clinical and histomorphological observations. Among the three treatment groups, the T1 and T2 groups significantly differed from the model group, whereas the T3 group showed no statistical differences. CONCLUSION: Subcutaneous administration of TPTD has a beneficial effect on BRONJ in mice. Nevertheless, further studies are needed to determine whether the therapeutic effect on BRONJ is dose-dependent.

Microfiber-Reinforced Composite Hydrogels Loaded with Rat Adipose-Derived Stem Cells and BMP-2 for the Treatment of Medication-Related Osteonecrosis of the Jaw in a Rat Model.

Severe adverse reactions of bisphosphonates and anti-resorptive or anti-angiogenic medications, termed medication-related osteonecrosis of the jaw (MRONJ), have been reported. MRONJ are difficult to completely cure and could cause great pain to patients. Recent studies have shown that mesenchymal stem cell (MSC) therapies are effective for treating MRONJ, but the method of intravenous injection is unstable and increases the risk of producing tumors. In the present study, low-acyl gellan gum (LAGG) hydrogels were modified with hemicellulose polysaccharide microfibers (PMs) to improve the performance of supporting three-dimensional (3D) cell growth. LAGG-PM composite hydrogels were found to be nontoxic to rat adipose-derived stem cells (rADSCs) in vitro. The hydrogels also promoted the secretion of angiogenic factors, induced osteoclastogenesis by conditioned medium, and supported osteogenic marker expression after the addition of human bone morphogenetic protein-2 (BMP-2). Due to its injectability, the LAGG-PM composite hydrogel incorporated with rADSCs and BMP-2 could be applied into the MRONJ lesion site, which promoted mucosal recovery, bone tissue reconstruction, and osteoclastogenesis. This study confirms the potential applications of LAGG-PM composite hydrogels as 3D cell culture platforms and delivery vehicles for the treatment of MRONJ in a rat model.

Skeletal Site-specific Effects of Zoledronate on in vivo Bone Remodeling and in vitro BMSCs Osteogenic Activity.

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) has been associated with long-term oral or intravenous administration of nitrogen-containing bisphosphonates (BPs). However, the pathogenesis of BRONJ remains unknown, and definitively effective treatment has not yet been established. Bisphosphonate-related osteonecrosis (BRON) tends to occur in maxillofacial bones. Why this occurs is still unclear. Here we show that zoledronate (Zol) treatment suppresses alveolar bone remodeling after tooth typical clinical and radiographic hallmarks of the human BRONJ, whereas enhances peripheral bone quantity in bone remodeling following injury in the same individuals, shown as increased cortical bone thickness, increased trabecular bone formation and accelerated bone defect repair. We find that the RANKL/OPG ratio and Wnt-3a expression are suppressed at the extracted alveolar sites in Zol-treated rats compared with those at the injured sites of peripheral bones. We also show that Zol-treated bone marrow stromal cell (BMSCs) derived from jaw and peripheral bones exhibit differences in cell proliferation, alkaline phosphatase (ALP) activity, expression of osteogenic and chondrogenic related marker genes, and in vivo bone formation capacity. Hopefully, this study will help us better understand the pathogenesis of BRONJ, and deepen the theoretical research.

[Degradation of the mixed pharmaceuticals with low concentration in aqueous by solar/ferrioxalate system and process evaluation through toxicity bioassay].

Under the sunlight irradiation, the four common trace pollutants such as metronidazole, diclofenac, sulfamethoxazole and ibuprofen were degradated and mineralized by the hydroxyl radical (*OH) generated from decomposition of H2O2 catalyzed by ferrioxalate (FeOx), and the toxicity of the water solution containing degradated products and intermediates were evaluated. The factors affecting the removal of the TOC, such as the initial concentration of H2O2, FeOx, and the pH, were investigated through an indicator of total organic carbon. The disappearing rate of pollutants in aqueous solution was explained according to the chemical structure of the pharmaceuticals; the biotoxicity of the pharmaceuticals and the intermediates were evaluated by EC50 value of pharmaceutical solution to the Chlorella. The appropriate operating conditions were achieved at pH 3 with initial concentrations of 300 mg x L(-1) H2O2 and 75 mg x L(-1) FeOx at the conditions such as the initial concentration of four drugs were 20 mg x L(-1), respectively. The order of the degradation rate for the pharmaceuticals is metronidazole > ibuprofen > sulfamethoxazole > diclofenac. During the reaction, the biological toxicity increases with time and then decreases rapidly, along with appearance and disappearance of intermediates. Finally, a model on reaction mechanism was proposed, where Solar/FeOx/H2O2 system was used for the degradation of the pharmaceuticals with low concentration in aqueous solution.