Systematic Pharmacology and Experimental Validation to Reveal the Alleviation of Astragalus membranaceus Regulating Ferroptosis in Osteoarthritis.

Background: Astragalus membranaceus (AM) shows promise as a therapeutic agent for osteoarthritis (OA), a debilitating condition with high disability rates. OA exacerbation is linked to chondrocyte ferroptosis, yet the precise pharmacological mechanisms of AM remain unclear. Methods: We validated AM's protective efficacy in an anterior cruciate ligament transection (ACLT) mouse model of OA. The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) database was utilized to identify AM's active components and their targets. FerrDb (a database for regulators and markers of ferroptosis and ferroptosis-disease associations) pinpointed ferroptosis-related targets, while GeneCards, Online Mendelian Inheritance in Man (OMIM), Pharmacogenomics Knowledgebase (PharmGKB), Therapeutic Target Database (TTD), and DrugBank sourced OA-related genes. Molecular docking analysis further validated these targets. Ultimately, the validation of the results was accomplished through in vitro experiments. Results: AM exhibited anabolic effects and suppressed catabolism in OA chondrocytes. Network pharmacology identified 19 common genes, and molecular docking suggested quercetin, an AM constituent, interacts with key proteins like HO-1 and NRF2 to inhibit chondrocyte ferroptosis. In vitro experiments confirmed AM's ability to modulate the NRF2/HO-1 pathway via quercetin, mitigating chondrocyte ferroptosis. Conclusion: This study elucidates how AM regulates chondrocyte ferroptosis, impacting OA progression, providing a theoretical basis and experimental support for AM's scientific application.

Astragalus polysaccharides ameliorate osteoarthritis via inhibiting apoptosis by regulating ROS-mediated ASK1/p38 MAPK signaling pathway targeting on TXN.

This research aims to explore the potential of astragalus polysaccharides (APS) in treating osteoarthritis. The primary component of APS extracted in this study was glucose, and noticeably it had a relatively high content of glucuronic acids. In vitro, APS reduced ROS levels, protected chondrocytes from apoptosis, and promoted collagen II expression by regulating ASK1 (apoptosis-signal-regulating kinase1)/p38 cell apoptosis pathway. Further co-immunoprecipitation and immunofluorescence localization experiments demonstrated that the thioredoxin (TXN) antioxidant system was responsible for its bioactivity. Moreover, TXN silencing remarkably blocked the protective effects of APS, indicating that APS inhibited chondrocyte apoptosis by targeting TXN. In vivo, APS effectively mitigated cartilage loss and chondrocyte apoptosis and decreased expressions of p-ASK1 and p-p38. Collectively, this research first demonstrated that APS could ameliorate osteoarthritis by ASK1/p38 signaling pathway through regulating thioredoxin. In conclusion, APS holds promise as a nutraceutical supplement for osteoarthritis in future drug development.

Identification of Immune-Related Risk Genes in Osteoarthritis Based on Bioinformatics Analysis and Machine Learning.

In this research, we aimed to perform a comprehensive bioinformatic analysis of immune cell infiltration in osteoarthritic cartilage and synovium and identify potential risk genes. Datasets were downloaded from the Gene Expression Omnibus database. We integrated the datasets, removed the batch effects and analyzed immune cell infiltration along with differentially expressed genes (DEGs). Weighted gene co-expression network analysis (WGCNA) was used to identify the positively correlated gene modules. LASSO (least absolute shrinkage and selection operator)-cox regression analysis was performed to screen the characteristic genes. The intersection of the DEGs, characteristic genes and module genes was identified as the risk genes. The WGCNA analysis demonstrates that the blue module was highly correlated and statistically significant as well as enriched in immune-related signaling pathways and biological functions in the KEGG and GO enrichment. LASSO-cox regression analysis screened 11 characteristic genes from the hub genes of the blue module. After the DEG, characteristic gene and immune-related gene datasets were intersected, three genes, PTGS1, HLA-DMB and GPR137B, were identified as the risk genes in this research. In this research, we identified three risk genes related to the immune system in osteoarthritis and provide a feasible approach to drug development in the future.

METTL3 promotes SMSCs chondrogenic differentiation by targeting the MMP3, MMP13, and GATA3.

Objective: Synovium-derived mesenchymal stem cells (SMSCs) are multipotential non-hematopoietic progenitor cells that can differentiate into various mesenchymal lineages in adipose and bone tissue, especially in chondrogenesis. Post-transcriptional methylation modifications are relative to the various biological development procedures. N6-methyladenosine (m6A) methylation has been identified as one of the abundant widespread post-transcriptional modifications. However, the connection between the SMSCs differentiation and m6A methylation remains unknown and needs further exploration. Methods: SMSCs were derived from synovial tissues of the knee joint of male Sprague-Dawley (SD) rats. In the chondrogenesis of SMSCs, m6A regulators were detected by quantitative real-time PCR (RT-PCR) and Western blot (WB). We observed the situation that the knockdown of m6A "writer" protein methyltransferase-like (METTL)3 in the chondrogenesis of SMSCs. We also mapped the transcript-wide m6A landscape in chondrogenic differentiation of SMSCs and combined RNA-seq and MeRIP-seq in SMSCs by the interference of METTL3. Results: The expression of m6A regulators were regulated in the chondrogenesis of SMSCs, only METTL3 is the most significant factor. In addition, after the knockdown of METTL3, MeRIP-seq and RNA-seq technology were applied to analyze the transcriptome level in SMSCs. 832 DEGs displayed significant changes, consisting of 438 upregulated genes and 394 downregulated genes. DEGs were enriched in signaling pathways regulating the glycosaminoglycan biosynthesis-chondroitin sulfate/dermatan sulfate and ECM-receptor interaction via Kyoto Encyclopedia of genes and genomes (KEGG) pathway enrichment analysis. The findings of this study indicate a difference in transcripts of MMP3, MMP13, and GATA3 containing consensus m6A motifs required for methylation by METTL3. Further, the reduction of METTL3 decreased the expression of MMP3, MMP13, and GATA3. Conclusion: These findings confirm the molecular mechanisms of METTL3-mediated m6A post-transcriptional change in the modulation of SMSCs differentiating into chondrocytes, thus highlighting the potential therapeutic effect of SMSCs for cartilage regeneration.

Osteoblast cell viability over ultra-long tricalcium phosphate nanocrystal-based methacrylate chitosan composite for bone regeneration.

Bioceramic morphology plays a crucial role in bone repair and regeneration. It is extensively utilized in bone scaffold synthesis due to its better biological system activity and biocompatibility. Here, ultra-long tricalcium phosphate (UTCP) was synthesized with the assistance of the ultrasonication method. The UTCP was modified as a scaffold by the reinforcement of a methacrylate chitosan (MAC) polymer. The functionality of UTCP, UTCP/MAC, and methotrexate (MTX)-loaded composites was characterized through Fourier transform infrared spectroscopy. The crystalline natures are investigated by x-ray diffraction, and the results show the UTCP crystalline phase is not altered after the reinforcement of the MAC polymer and loading of MTX drugs. The morphological analyses were observed through electron microscopic analysis, and polymer-coated rod structures were observed. The UTCP/MAC composite mechanical stress was increased from 1813 Pa of UTCP to 4272 Pa. MTX loading and release at 79.0% within 3 h and 76.15% at 20 h, respectively, were achieved. The UTCP/MAC and UTCP/MAC/MTX's osteoblast-like (MG-63) cell viability was investigated, and the MTX-loaded UTCP/MAC composite exhibits good viability behavior up to 96.0% in 14 d. The results confirm the higher compatibility of the composite and profitable cell growth. It may be suitable for bone implantation preparation, and it helps in faster regeneration of bone tissue afterin vivoand clinical evaluation.

[Comparison of clinical efficacy of two different arthroscopic release methods for the treatment of primary frozen shoulder].

OBJECTIVE: To compare clinical effects of inside-out technique and outside-in technique for the treatment of idiopathic frozen shoulder under arthroscopy. METHODS: From April 2015 to July 2019, 65 patients with primary frozen shoulder were divided into observation group and control group according to different treatment methods. In observation group, there were 32 cases, including 14 males and 18 females, aged 48 to 64 (54.82±5.35) years old, 18 cases on the right side and 14 cases on the left side. The course of disease was 4 to 10 (7.76±1.19) months. The patients were treated with outside in technique. In control group, there were 33 cases, 16 males and 17 females, aged 45 to 62 (54.64±4.16) years old, 18 cases on the right side and 15 cases on the left side. The course of disease was 5 to 9 (7.65±1.24) months. The patients were treated with inside out technique. The operation time, hospitalization days and treatment cost were compared between the two groups. Constant-Murley function score before and after the operation andthe shoulder joint range of motion one month after operation were compared to evaluate the clinical efficacy. RESULTS: All 65 patients were followed up for 9 to 17 months with an average follow up time of (11.34±2.24) months. Compared with control group, operation time in observation group was shorter[(55.53± 10.23) min vs (85.58±13.39) min], and functional scores of Constant-Murley after surgery were significantly changed in both groups compared with that before surgery(P<0.05). There was no significant difference in functional scores of Constant-Murley between two groups (P>0.05). There was no significant differences in hospitalization time and treatment cost between two groups (P>0.05), and there was no significant difference in shoulder abduction, extension flexion and rotation activity between two groups (P>0.05), but internal rotation of observation group was improved compared with that of control group (P<0.05). CONCLUSION: The two arthroscopic release schemes have achieved satisfactory results for thetreatment of primary frozen shoulder, and the shoulder joint function and pain degree have been effectively improved. Compared with the inside-out technique, the outside in release technique is more direct, the operation is simpler and the operation time is shorter. It has certain advantages in releasing operation for primary frozen shoulder.

The efficacy and safety of intravenous tranexamic acid in anterior cruciate ligament reconstruction: A randomized controlled trial protocol.

BACKGROUND: The safety and efficacy of intravenous tranexamic acid (TXA) in the anterior cruciate ligament (ACL) reconstruction remains controversial. There is an urgent need of studies that efficiently control for confounding, conduct comprehensive and consecutive observation of potential risks of the TXA administration, and investigate its clinical applicability. The purpose of this work is to assess the safety and efficacy of the intravenous TXA in decreasing perioperative blood loss in the patients undergoing ACL reconstruction. METHODS: This randomized, controlled, prospective research was carried out between January 2017 and January 2018. All the patients and their family members signed the informed consent forms, and this current work was authorized via the ethics committee of Nanjing first hospital (registration No.: NJU1003586). A total of 100 patients were divided randomly into 2 group: the control group (n = 50) and study group (n = 50). The study group receives intravenous TXA administration [1 g] before skin incision. The control group receives equivalent normal saline. Primary outcome measures including blood loss, hemoglobin decline, transfusion rate, C-reactive protein, D-dimer value, fibrinogen, prothrombin time, activated partial thromboplastin time, thrombin time, international normalized ratio and erythrocyte sedimentation rate were recorded. The measures of secondary outcomes refer to the clinical data involving the range of motion and postoperative pain score. The pain score was quantified by utilizing the 10-cm scale of visual analog. The pain strength was in the range of 0-10, where 0 is totally no pain and 10 represents the most severe pain. RESULTS: This experiment had strict inclusive criteria and exclusive criteria and a well- regulated intervention. CONCLUSION: Our results can bring a new perspective on the use of TXA after arthroscopically assisted ACL surgery. TRIAL REGISTRATION: This study protocol was registered in Research Registry (researchregistry5798).

Differential Effects of Platelets Selectively Activated by Protease-Activated Receptors on Meniscal Cells.

BACKGROUND: Meniscal injury is very common, and injured meniscal tissue has a limited healing ability because of poor vascularity. Platelets contain both pro- and anti-angiogenic factors, which can be released by platelet selective activation. HYPOTHESIS: Platelets release a high level of vascular endothelial growth factor (VEGF) when they are activated by protease-activated receptor 1 (PAR1), whereas the platelets release endostatin when they are activated by protease-activated receptor 4 (PAR4). The PAR1-treated platelets enhance the proliferation of meniscal cells in vitro and promote in vivo healing of wounded meniscal tissue. STUDY DESIGN: Controlled laboratory study. METHOD: Platelets were isolated from human blood and activated with different reagents. The released growth factors from the activated platelets were determined by immunostaining and enzyme-linked immunosorbent assay. The effects of the platelets with different treatments on meniscal cells were tested by an in vitro model of cell culture and an in vivo model of wounded meniscal healing. RESULTS: The results indicated that platelets contained both pro- and antiangiogenic factors including VEGF and endostatin. In unactivated platelets, VEGF and endostatin were contained inside of the platelets. Both VEGF and endostatin were released from the platelets when they were activated by thrombin. However, only VEGF was released from the platelets when they were activated by PAR1, and only endostatin was released from the platelets when they were activated by PAR4. The rat meniscal cells grew much faster in the medium that contained PAR1-activated platelets than in the medium that contained either PAR4-activated platelets or unactivated platelets. The wounds treated with PAR1-activated platelets healed faster than those treated with either PAR4-activated platelets or unactivated platelets. Many blood vessel-like structures were found in the wounded menisci treated with PAR1-activated platelets. CONCLUSION: The PAR1-activated platelets released high levels of VEGF, which increased the proliferation of rat meniscal cells in vitro, enhanced the vascularization of menisci in vivo, and promoted healing of wounded menisci. CLINICAL RELEVANCE: Our results suggested that selective activated platelets can be used clinically to enhance healing of wounded meniscal tissue.