Exploring the pharmacological mechanism of Glycyrrhiza uralensis against KOA through integrating network pharmacology and experimental assessment.

Knee osteoarthritis (KOA), a major health and economic problem facing older adults worldwide, is a degenerative joint disease. Glycyrrhiza uralensis Fisch. (GC) plays an integral role in many classic Chinese medicine prescriptions for treating knee osteoarthritis. Still, the role of GC in treating KOA is unclear. To explore the pharmacological mechanism of GC against KOA, UPLC-Q-TOF/MS was conducted to detect the main compounds in GC. The therapeutic effect of GC on DMM-induced osteoarthritic mice was assessed by histomorphology, µCT, behavioural tests, and immunohistochemical staining. Network pharmacology and molecular docking were used to predict the potential targets of GC against KOA. The predicted results were verified by immunohistochemical staining Animal experiments showed that GC had a protective effect on DMM-induced KOA, mainly in the improvement of movement disorders, subchondral bone sclerosis and cartilage damage. A variety of flavonoids and triterpenoids were detected in GC via UPLC-Q-TOF/MS, such as Naringenin. Seven core targets (JUN, MAPK3, MAPK1, AKT1, TP53, RELA and STAT3) and three main pathways (IL-17, NF-κB and TNF signalling pathways) were discovered through network pharmacology analysis that closely related to inflammatory response. Interestingly, molecular docking results showed that the active ingredient Naringenin had a good binding effect on anti-inflammatory-related proteins. In the verification experiment, after the intervention of GC, the expression levels of pp65 and F4/80 inflammatory indicators in the knee joint of KOA model mice were significantly downregulated. GC could improve the inflammatory environment in DMM-induced osteoarthritic mice thus alleviating the physiological structure and dysfunction of the knee joint. GC might play an important role in the treatment of knee osteoarthritis.

A natural hydrogel complex improves intervertebral disc degeneration by correcting fatty acid metabolism and inhibiting nucleus pulposus cell pyroptosis.

The degeneration of intervertebral discs is strongly associated with the occurrence of pyroptosis in nucleus pulposus (NP) cells. This pyroptosis is characterized by abnormal metabolism of fatty acids in the degenerative pathological state, which is further exacerbated by the inflammatory microenvironment and degradation of the extracellular matrix. In order to address this issue, we have developed a fibrin hydrogel complex (FG@PEV). This intricate formulation amalgamates the beneficial attributes of platelet extravasation vesicles, contributing to tissue repair and regeneration. Furthermore, this complex showcases exceptional stability, gradual-release capabilities, and a high degree of biocompatibility. In order to substantiate the biological significance of FG@PEV in intervertebral disc degeneration (IVDD), we conducted a comprehensive investigation into its potential mechanism of action through the integration of RNA-seq sequencing and metabolomics analysis. Furthermore, these findings were subsequently validated through experimentation in both in vivo and in vitro models. The experimental results revealed that the FG@PEV intervention possesses the capability to reshape the inflammatory microenvironment within the disc. It also addresses the irregularities in fatty acid metabolism of nucleus pulposus cells, consequently hindering cellular pyroptosis and slowing down disc degeneration through the regulation of extracellular matrix synthesis and degradation. As a result, this injectable gel system represents a promising and innovative therapeutic approach for mitigating disc degeneration.

Advances in the study of plant-derived extracellular vesicles in the skeletal muscle system.

Plant-derived extracellular vesicles (PDEV) constitute nanoscale entities comprising lipids, proteins, nucleic acids and various components enveloped by the lipid bilayers of plant cells. These vesicles play a crucial role in facilitating substance and information transfer not only between plant cells but also across different species. Owing to its safety, stability, and the abundance of raw materials, this substance has found extensive utilization in recent years within research endeavors aimed at treating various diseases. This article provides an overview of the pathways and biological characteristics of PDEV, along with the prevalent methods employed for its isolation, purification, and storage. Furthermore, we comprehensively outline the therapeutic implications of diverse sources of PDEV in musculoskeletal system disorders. Additionally, we explore the utilization of PDEV as platforms for engineering drug carriers, aiming to delve deeper into the significance and potential contributions of PDEV in the realm of the musculoskeletal system.

MINK1 deficiency stimulates nucleus pulposus cell pyroptosis and exacerbates intervertebral disc degeneration.

Intervertebral disc (IVD) degeneration, induced by aging and irregular mechanical strain, is highly prevalent in the elderly population, serving as a leading cause of chronic low back pain and disability. Evolving evidence has revealed the involvement of nucleus pulposus (NP) pyroptosis in the pathogenesis of IVD degeneration, while the precise regulatory mechanisms of NP pyroptosis remain obscure. Misshapen/Nck-interacting kinase (NIK)-related kinase 1 (MINK1), a serine-threonine protein kinase, has the potential to modulate the activation of NLRP3 inflammasome, indicating its pivotal role in governing pyroptosis. In this study, to assess the significance of MINK1 in NP pyroptosis and IVD degeneration, NP tissues from patients with varying degrees of IVD degeneration, and IVD tissues from both aging-induced and lumbar spine instability (LSI) surgery-induced IVD degeneration mouse models, with or without MINK1 ablation, were meticulously evaluated. Our findings indicated a notable decline in MINK1 expression in NP tissues of patients with IVD degeneration and both mouse models as degeneration progresses, accompanied by heightened matrix degradation and increased NP pyroptosis. Moreover, MINK1 ablation led to substantial activation of NP pyroptosis in both mouse models, and accelerating ECM degradation and intensifying the degeneration phenotype in mechanically stress-induced mice. Mechanistically, MINK1 deficiency triggered NF-κB signaling in NP tissues. Overall, our data illustrate an inverse correlation between MINK1 expression and severity of IVD degeneration, and the absence of MINK1 stimulates NP pyroptosis, exacerbating IVD degeneration by activating NF-κB signaling, highlighting a potential innovative therapeutic target in treating IVD degeneration.

IRF1 governs the expression of SMARCC1 via the GCN5-SETD2 axis and actively engages in the advancement of osteoarthritis.

Background: Osteoarthritis (OA) is a degenerative joint disease characterized by the breakdown of joint cartilage and underlying bone. Macrophages are a type of white blood cell that plays a critical role in the immune system and can be found in various tissues, including joints. Research on the relationship between OA and macrophages is essential to understand the mechanisms underlying the development and progression of OA. Objective: This study was performed to analyze the functions of the IRF1-GCN5-SETD2-SMARCC1 axis in osteoarthritis (OA) development. Methods: A single-cell RNA sequencing (scRNA-seq) dataset, was subjected to a comprehensive analysis aiming to identify potential regulators implicated in the progression of osteoarthritis (OA). In order to investigate the role of IRF1 and SMARCC1, knockdown experiments were conducted in both OA-induced rats and interleukin (IL)-1ß-stimulated chondrocytes, followed by the assessment of OA-like symptoms, secretion of inflammatory cytokines, and polarization of macrophages. Furthermore, the study delved into the identification of aberrant epigenetic modifications and functional enzymes responsible for the regulation of SMARCC1 by IRF1. To evaluate the clinical significance of the factors under scrutiny, a cohort comprising 13 patients diagnosed with OA and 7 fracture patients without OA was included in the analysis. Results: IRF1 was found to exert regulatory control over the expression of SMARCC1, thus playing a significant role in the development of osteoarthritis (OA). The knockdown of either IRF1 or SMARCC1 disrupted the pro-inflammatory effects induced by IL-1ß in chondrocytes, leading to a mitigation of OA-like symptoms, including inflammatory infiltration, cartilage degradation, and tissue injury, in rat models. Additionally, this intervention resulted in a reduction in the predominance of M1 macrophages both in vitro and in vivo. Significant epigenetic modifications, such as abundant H3K27ac and H3K4me3 marks, were observed near the SMARCC1 promoter and 10 kb upstream region. These modifications were attributed to the recruitment of GCN5 and SETD2, which are functional enzymes responsible for these modifications. Remarkably, the overexpression of either GCN5 or SETD2 restored SMARCC1 expression in rat cartilages or chondrocytes, consequently exacerbating the OA-like symptoms. Conclusion: This research postulates that the transcriptional activity of SMARCC1 can be influenced by IRF1 through the recruitment of GCN5 and SETD2, consequently regulating the H3K27ac and H3K4me3 modifications in close proximity to the SMARCC1 promoter and 10 kb upstream region. These modifications, in turn, facilitate the M1 skewing of macrophages and contribute to the progression of osteoarthritis (OA). The Translational Potential of this Article: The study demonstrated that the regulation of SMARCC1 by IRF1 plays a crucial role in the development of OA. Knocking down either IRF1 or SMARCC1 disrupted the pro-inflammatory effects induced by IL-1ß in chondrocytes, leading to a mitigation of OA-like symptoms in rat models. These symptoms included inflammatory infiltration, cartilage degradation, and tissue injury. These findings suggest that targeting the IRF1-SMARCC1 regulatory axis, as well as the associated epigenetic modifications, could potentially be a novel approach in the development of OA therapies, offering new opportunities for disease management and improved patient outcomes.

Catalpol promotes articular cartilage repair by enhancing the recruitment of endogenous mesenchymal stem cells.

Articular cartilage defect is challenged by insufficient regenerative ability of cartilage. Catalpol (CA), the primary active component of Rehmanniae Radix, could exert protective effects against various diseases. However, the impact of CA on the treatment of articular cartilage injuries is still unclear. In this study, full-thickness articular cartilage defect was induced in a mouse model via surgery. The animals were intraperitoneally injected with CA for 4 or 8 weeks. According to the results of macroscopic observation, micro-computed tomography CT (µCT), histological and immunohistochemistry staining, CA treatment could promote mouse cartilage repair, resulting in cartilage regeneration, bone structure improvement and matrix anabolism. Specifically, an increase in the expression of CD90, the marker of mesenchymal stem cells (MSCs), in the cartilage was observed. In addition, we evaluated the migratory and chondrogenic effects of CA on MSCs. Different concentration of CA was added to C3H10 T1/2 cells. The results showed that CA enhanced cell migration and chondrogenesis without affecting proliferation. Collectively, our findings indicate that CA may be effective for the treatment of cartilage defects via stimulation of endogenous MSCs.

ß-catenin inhibition disrupts the homeostasis of osteogenic/adipogenic differentiation leading to the development of glucocorticoid-induced osteonecrosis of the femoral head.

Glucocorticoid-induced osteonecrosis of the femoral head (GONFH) is a common refractory joint disease characterized by bone damage and the collapse of femoral head structure. However, the exact pathological mechanisms of GONFH remain unknown. Here, we observed abnormal osteogenesis and adipogenesis associated with decreased ß-catenin in the necrotic femoral head of GONFH patients. In vivo and in vitro studies further revealed that glucocorticoid exposure disrupted osteogenic/adipogenic differentiation of bone marrow mesenchymal cells (BMSCs) by inhibiting ß-catenin signaling in glucocorticoid-induced GONFH rats. Col2+ lineage largely contributes to BMSCs and was found an osteogenic commitment in the femoral head through 9 mo of lineage trace. Specific deletion of ß-catenin gene (Ctnnb1) in Col2+ cells shifted their commitment from osteoblasts to adipocytes, leading to a full spectrum of disease phenotype of GONFH in adult mice. Overall, we uncover that ß-catenin inhibition disrupting the homeostasis of osteogenic/adipogenic differentiation contributes to the development of GONFH and identify an ideal genetic-modified mouse model of GONFH.

Liquiritin reduces chondrocyte apoptosis through P53/PUMA signaling pathway to alleviate osteoarthritis.

AIMS: The main pathological features of osteoarthritis (OA) include the degeneration of articular cartilage and a decrease in matrix synthesis. Chondrocytes, which contribute to matrix synthesis, play a crucial role in the development of OA. Liquiritin, an effective ingredient extracted from Glycyrrhiza uralensis Fisch., has been used for over 1000 years to treat OA. This study aims to investigate the impact of liquiritin on OA and its underlying mechanism. MATERIALS AND METHODS: Gait and hot plate tests assessed mouse behavior, while Micro-CT and ABH/OG staining observed joint morphological changes. The TUNEL kit detected chondrocyte apoptosis. Western blot and immunofluorescence techniques determined the expression levels of cartilage metabolism markers COL2 and MMP13, as well as apoptosis markers caspase3, bcl2, P53, and PUMA. KEGG analysis and molecular docking technology were used to verify the relationship between liquiritin and P53. KEY FINDINGS: Liquiritin alleviated pain sensitivity and improved gait impairment in OA mice. Additionally, we found that liquiritin could increase COL2 levels and decrease MMP13 levels both in vivo and in vitro. Importantly, liquiritin reduced chondrocyte apoptosis induced by OA, through decreased expression of caspase3 expression and increased expression of bcl2 expression. Molecular docking revealed a strong binding affinity between liquiritin and P53. Both in vivo and in vitro studies demonstrated that liquiritin suppressed the expression of P53 and PUMA in cartilage. SIGNIFICANCE: This indicated that liquiritin may alleviate OA progression by inhibiting the P53/PUMA signaling pathway, suggesting that liquiritin is a potential strategy for the treatment of OA.

Dnmt3b ablation affects fracture repair process by regulating apoptosis.

PURPOSE: Previous studies have shown that DNA methyltransferase 3b (Dnmt3b) is the only Dnmt responsive to fracture repair and Dnmt3b ablation in Prx1-positive stem cells and chondrocyte cells both delayed fracture repair. Our study aims to explore the influence of Dnmt3b ablation in Gli1-positive stem cells in fracture healing mice and the underlying mechanism. METHODS: We generated Gli1-CreERT2; Dnmt3bflox/flox (Dnmt3bGli1ER) mice to operated tibia fracture. Fracture callus tissues of Dnmt3bGli1ER mice and control mice were collected and analyzed by X-ray, micro-CT, biomechanical testing, histopathology and TUNEL assay. RESULTS: The cartilaginous callus significantly decrease in ablation of Dnmt3b in Gli1-positive stem cells during fracture repair. The chondrogenic and osteogenic indicators (Sox9 and Runx2) in the fracture healing tissues in Dnmt3bGli1ER mice much less than control mice. Dnmt3bGli1ER mice led to delayed bone callus remodeling and decreased biomechanical properties of the newly formed bone during fracture repair. Both the expressions of Caspase-3 and Caspase-8 were upregulated in Dnmt3bGli1ER mice as well as the expressions of BCL-2. CONCLUSIONS: Our study provides an evidence that Dnmt3b ablation Gli1-positive stem cells can affect fracture healing and lead to poor fracture healing by regulating apoptosis to decrease chondrocyte hypertrophic maturation.

α-Solanine attenuates chondrocyte pyroptosis to improve osteoarthritis via suppressing NF-κB pathway.

α-Solanine has been shown to exhibit anti-inflammatory and anti-tumour properties; however, its efficacy in treating osteoarthritis (OA) remains ambiguous. The study aimed to evaluate the therapeutic effects of α-solanine on OA development in a mouse OA model. The OA mice were subjected to varying concentrations of α-solanine, and various assessments were implemented to assess OA progression. We found that α-solanine significantly reduced osteophyte formation, subchondral sclerosis and OARSI score. And it decreased proteoglycan loss and calcification in articular cartilage. Specifically, α-solanine inhibited extracellular matrix degradation by downregulating collagen 10, matrix metalloproteinase 3 and 13, and upregulating collagen 2. Importantly, α-solanine reversed chondrocyte pyroptosis phenotype in articular cartilage of OA mice by inhibiting the elevated expressions of Caspase-1, Gsdmd and IL-1ß, while also mitigating aberrant angiogenesis and sensory innervation in subchondral bone. Mechanistically, α-solanine notably hindered the early stages of OA progression by reducing I-κB phosphorylation and nuclear translocation of p65, thereby inactivating NF-κB signalling. Our findings demonstrate the capability of α-solanine to disrupt chondrocyte pyroptosis and sensory innervation, thereby improving osteoarthritic pathological progress by inhibiting NF-κB signalling. These results suggest that α-solanine could serve as a promising therapeutic agent for OA treatment.

Protective effects of Shensuitongzhi formula on intervertebral disc degeneration via downregulation of NF-κB signaling pathway and inflammatory response.

Low back pain (LBP) is a common orthopedic disease over the world. Lumbar intervertebral disc degeneration (IDD) is regarded as an important cause of LBP. Shensuitongzhi formula (SSTZF) is a drug used in clinical treatment for orthopedic diseases. It has been found that SSTZF can have a good treatment for IDD. But the exact mechanism has not been clarified. The results showed that SSTZF protects against LSI-induced degeneration of cartilage endplates and intervertebral discs. Meanwhile, SSTZF treatment dramatically reduces the expression of inflammatory factor as well as the expression of catabolism protein and upregulates the expression of anabolism protein in LSI-induced mice. In addition, SSTZF delayed the progression of LSI-induced IDD via downregulation the level of NF-κB signaling key gene RELA and phosphorylation of key protein P65 in endplate chondrocytes. Our study has illustrated the treatment as well as the latent mechanism of SSTZF in IDD.

Aucubin Alleviates Intervertebral Disc Degeneration by Repressing NF-κB-NLRP3 Inflammasome Activation in Endplate Chondrocytes.

Background: Intervertebral disc degeneration (IDD) is a prevalent degenerative disease and often recognized as the primary cause of lower back pain (LBP). Aucubin (Au) is a natural compound with anti-inflammatory properties in various diseases. The present study aimed to confirm the therapeutic effect of Au on IDD and explore its potential mechanism in vivo and in vitro. Methods: The process of IDD was simulated using the lumbar spine instability (LSI) model. In vivo, the therapeutic effect of Au on LSI-induced mice was evaluated by micro-CT and histomorphometry. Additionally, immunohistochemistry was applied to detect the cartilage metabolism and inflammasome activation in endplate. In vitro, the cytotoxicity of Au on ATDC5 cells was detected by Cell Counting Kit-8 (CCK-8), and the biological effects of Au were evaluated by Quantitative Real-time PCR (qRT-PCR) and Western blotting. Results: Micro-CT analysis showed that Au administration significantly alleviated LSI-induced disc volume narrowing and endplate cartilage degeneration, which was further supported by Alcian Blue Hematoxylin/Orange G (ABH/OG) staining. Immunohistochemistry results verified that Au could increase the expression of Col2α1 and Aggrecan, reduce the expression of Mmp-13, and attenuate the degradation of the endplate extracellular matrix (ECM). Mechanistically, we found that Au treatment, both in vivo and in vitro, significantly inhibited NF-κB-NLRP3 inflammasome activation in chondrocytes as determined by the decreased expression of p-P65, NLRP3, and Caspase-1. Discussion: Taken together, our findings have demonstrated for the first time that Au treatment ameliorated the degeneration of cartilage endplates in IDD may by inhibiting NF-κB-NLRP3 inflammasome activation in chondrocytes and provided a potential candidate for the treatment of IDD.

Study of the curative effect of Zhang's Xibi formula and its underlying mechanism involving inhibition of inflammatory responses and delay of knee osteoarthritis.

OBJECTIVE: To verify the clinical efficacy of Zhang's Xibi formula (ZSXBF) and explain the mechanism underlying its therapeutic effect. METHODS: Preliminary elucidation of the clinical efficacy of ZSXBF in treating KOA in self-control studies, exploration of its mechanism of action with network pharmacology methods, and validation in animal experiments. RESULTS: In clinical studies, ZSXBF administration effectively improved patient quality of life and reduce pain. Network pharmacology was used to explore the possible mechanisms underlying its treatment effect, and after verification in clinical experience and animal experiments, it was found that ZSXBF regulated the expression of immune-related proteins such as IL-17, ERK1, and TP53 in mouse knee joints. CONCLUSION: ZSXBF, which is a traditional Chinese medicine compound that is used to clear heat and detoxify, can effectively improve the clinical symptoms of KOA patients, and its underlying mechanism includes the regulation of human immune-related proteins.

Inhibition of Caspase-1-mediated pyroptosis promotes osteogenic differentiation, offering a therapeutic target for osteoporosis.

BACKGROUND: Pyroptosis, an emerging inflammatory form of cell death, has been previously demonstrated to stimulate a massive inflammatory response, thus hindering the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Nevertheless, the impact of pyroptosis in thwarting osteogenic differentiation and exacerbating the advancement of osteoporosis (OP) remains enigmatic. METHODS: We evaluated the expression levels of pyroptosis-associated indicators, including NOD-like receptor family pyrin domain-containing protein 3 (NLRP3), CASPASE-1, IL-1ß, and IL-18, in specimens obtained from femoral heads of OP patients, as well as in an ovariectomy-induced mouse model of OP. Subsequently, the precise roles of pyroptosis in osteogenic differentiation were investigated using bioinformatics analysis, alongside morphological and biochemical assessments. RESULTS: The pivotal pyroptotic proteins, including NLRP3, Caspase-1, IL-1ß, and IL-18, exhibited significant upregulation within the bone tissue samples of clinical OP cases, as well as in the femoral tissues of ovariectomy (OVX)-induced mouse OP model, displaying a negatively associated with compromised osteogenic capacity, as represented by lessened bone mass, suppressed expression of osteogenic proteins such as Runt-related transcription factor 2 (RUNX2), Alkaline phosphatase (ALP), Osterix (OSX), and Osteopontin (OPN), and increased lipid droplets. Moreover, bioinformatics analysis substantiated shared gene expression patterns between pyroptosis and OP pathology, encompassing NLRP3, Caspase-1, IL-1ß, IL-18, etc. Furthermore, our in vitro investigation using ST2 cells revealed that dexamethasone treatment prominently induced pyroptosis while impeding osteogenic differentiation. Notably, gene silencing of Caspase-1 effectively counteracted the inhibitory effects of dexamethasone on osteogenic differentiation, as manifested by increased ALP activity and enhanced expression of RUNX2, ALP, OSX, and OPN. CONCLUSION: Our findings unequivocally underscore that inhibition of Caspase-1-mediated pyroptosis promotes osteogenic differentiation, providing a promising therapeutic target for managing OP.

Ambient PM2.5 Exposure and Bone Homeostasis: Analysis of UK Biobank Data and Experimental Studies in Mice and in Vitro.

BACKGROUND: Previous evidence has identified exposure to fine ambient particulate matter (PM2.5) as a leading risk factor for adverse health outcomes. However, to date, only a few studies have examined the potential association between long-term exposure to PM2.5 and bone homeostasis. OBJECTIVE: We sought to examine the relationship between long-term PM2.5 exposure and bone health and explore its potential mechanism. METHODS: This research included both observational and experimental studies. First, based on human data from UK Biobank, linear regression was used to explore the associations between long-term exposure to PM2.5 (i.e., annual average PM2.5 concentration for 2010) and bone mineral density [BMD; i.e., heel BMD (n=37,440) and femur neck and lumbar spine BMD (n=29,766)], which were measured during 2014-2020. For the experimental animal study, C57BL/6 male mice were assigned to ambient PM2.5 or filtered air for 6 months via a whole-body exposure system. Micro-computed tomography analyses were applied to measure BMD and bone microstructures. Biomarkers for bone turnover and inflammation were examined with histological staining, immunohistochemistry staining, and enzyme-linked immunosorbent assay. We also performed tartrate-resistant acid phosphatase (TRAP) staining and bone resorption assay to determine the effect of PM2.5 exposure on osteoclast activity in vitro. In addition, the potential downstream regulators were assessed by real-time polymerase chain reaction and western blot. RESULTS: We observed that long-term exposure to PM2.5 was significantly associated with lower BMD at different anatomical sites, according to the analysis of UK Biobank data. In experimental study, mice exposed long-term to PM2.5 exhibited excessive osteoclastogenesis, dysregulated osteogenesis, higher tumor necrosis factor-alpha (TNF-α) expression, and shorter femur length than control mice, but they demonstrated no significant differences in femur structure or BMD. In vitro, cells stimulated with conditional medium of PM2.5-stimulated macrophages had aberrant osteoclastogenesis and differences in the protein/mRNA expression of members of the TNF-α/Traf6/c-Fos pathway, which could be partially rescued by TNF-α inhibition. DISCUSSION: Our prospective observational evidence suggested that long-term exposure to PM2.5 is associated with lower BMD and further experimental results demonstrated exposure to PM2.5 could disrupt bone homeostasis, which may be mediated by inflammation-induced osteoclastogenesis. https://doi.org/10.1289/EHP11646.

Amygdalin Delays Cartilage Endplate Degeneration and Improves Intervertebral Disc Degeneration by Inhibiting NF-κB Signaling Pathway and Inflammatory Response.

Background: Intervertebral disc degeneration (IDD) is a major cause of lower back pain (LBP), in which inflammatory is frequently involved. Amygdalin (AMD) is a naturally occurring compound that exerts anti-fibrotic, anti-inflammatory, analgesic, and immunomodulatory effects in various diseases. The purpose of this study was to investigate the therapeutic effects and molecular mechanisms of AMD on Lumbar spine instability (LSI)-induced IDD in mice. Methods: In this study, we first explored the effects of AMD in vivo, and then further explored the mechanism of its effects both in vivo and in vitro. Ten-week-old male C57BL/6J mice were administrated with AMD. At 10 weeks after LSI, spinal were collected for tissue analyses, including histology, micro-CT, and immunohistochemistry for Col2, Mmp-13, TNF-α, and p-P65. Additionally, we also evaluated the mRNA and protein expression level of p-P65 and p-IKBα after being treated with AMD in vitro. Results: Histological staining, micro-CT and immunohistochemical analysis showed that AMD treatment significantly inhibited the expression of TNF-α and Mmp-13, increased the expression of Col2 as well as attenuated the calcification of cartilage endplates, eventually to delayed the progression of IDD. Meanwhile, in vivo and in vitro fluorescence imaging revealed that AMD markedly inhibited the AMD significantly inhibited the LSI-induced increase in TNF-α expression and P65and IKBα phosphorylation. Discussion: Our findings suggest that AMD partly inhibits the activation of NF-κB signaling pathway to reduce the release of inflammatory mediators and delay the degeneration of cartilage endplate in IDD model mice. Therefore, AMD may be a potential candidate for the treatment of IDD.

AR/PCC herb pair inhibits osteoblast pyroptosis to alleviate diabetes-related osteoporosis by activating Nrf2/Keap1 pathway.

Osteoporosis is a prevalent complication of diabetes, characterized by systemic metabolic impairment of bone mass and microarchitecture, particularly in the spine. Anemarrhenae Rhizoma/Phellodendri Chinensis Cortex (AR/PCC) herb pair has been extensively employed in Traditional Chinese Medicine to manage diabetes; however, its potential to ameliorate diabetic osteoporosis (DOP) has remained obscure. Herein, we explored the protective efficacy of AR/PCC herb pair against DOP using a streptozotocin (STZ)-induced rat diabetic model. Our data showed that AR/PCC could effectively reduce the elevated fasting blood glucose and reverse the osteoporotic phenotype of diabetic rats, resulting in significant improvements in vertebral trabecular area percentage, trabecular thickness and trabecular number, while reducing trabecular separation. Specifically, AR/PCC herb pair improved impaired osteogenesis, nerve ingrowth and angiogenesis. More importantly, it could mitigate the aberrant activation of osteoblast pyroptosis in the vertebral bodies of diabetic rats by reducing increased expressions of Nlrp3, Asc, Caspase1, Gsdmd and IL-1ß. Mechanistically, AR/PCC activated antioxidant pathway through the upregulation of the antioxidant response protein Nrf2, while concurrently decreasing its negative feedback regulator Keap1. Collectively, our in vivo findings demonstrate that AR/PCC can inhibit osteoblast pyroptosis and alleviate STZ-induced rat DOP, suggesting its potential as a therapeutic agent for mitigating DOP.

Disruption of Col9a2 expression leads to defects in osteochondral homeostasis and osteoarthritis-like phenotype in mice.

Background/Objective: As one of the branched chains of Type IX collagen (Col9), Collagen IX alpha2 (Col9a2) has been reported to be associated with several orthopedic conditions. However, the relationship between Col9a2 and knee osteoarthritis (KOA) remains to be elucidated. Methods: To probe the relationship between Col9a2 and KOA, we performed a systematic analysis of Col9a2-deficient (Col9a2-/-) mice using whole-mount skeletal staining, Micro-CT (µCT), biomechanics, histomorphometry, immunohistochemistry (IHC), immunofluorescence (IF) and Elisa. Results: We found that the subchondral bone (SCB) in the knee joint of Col9a2-/- mice became sparse and deformed in the early stage, with altered bone morphometric parameters, reduced load-bearing capacity, dysfunctional bone homeostasis (decreased osteogenesis capacity and elevated bone resorption capacity), diminished cartilage proteoglycans and disrupted cartilage extracellular matrix (ECM) anabolism and catabolism compared with the Col9a2+/+ mice. In the late stage, the cartilage degeneration in Col9a2-/- mice were particularly pronounced compared to Col9a2+/+ mice, as evidenced by severe cartilage destruction and a marked reduction in cartilage thickness and area. Conclusion: Overall, Col9a2 is essential for maintaining osteochondral homeostasis in the knee joint of mice, and the absence of this gene is accompanied by distinct sclerosis of the SCB and a reduction in load-bearing capacity; in the late stage, in the lack of SCB stress inhibition, excessive load is consistently exerted on the cartilage, ultimately leading to osteoarthritic-like articular cartilage damage.