Catabolic Response Research Articles

MTORC2 inhibition by JR-AB2-011 improves IL-1β-induced inflammation, catabolic response, and apoptosis in human chondrocytes through IκB-α/NF-κB p65.

Osteoarthritis (OA) is a very common chronic joint condition marked by inflammation and cartilage loss. mTOR is a well-known mediator of inflammation, cell survival, and aging; however, its role in OA has not been determined. To explore the role of mTORC2 in OA-and associated pathological changes, we examined the contribution of mTORC2-mediated Akt, rictor and IκB-α/NF-κB p65 pathway in interleukin (IL)-1β-treated human chondrocytes. We focused on the protein expression of proinflammatory cytokines and catabolic and apoptotic factors, including TNF-α, IL-6, iNOS, MMP13, Bax, and caspase3, which may occur through this signalling pathway in IL-1β-treated chondrocytes. Chondrocytes were cultured and treated with either 2 ng/mL IL‑1β alone or in combination with increasing concentrations of JR-AB2-011 (50, 100, or 250 µM), a selective mTORC2 inhibitor. The protein levels of phosphorylated (p)‑Akt, Akt, rictor, p-NF-κB p65, NF-κB p65, IκB-α, p-IκB-α, iNOS, MMP13, Bax, and caspase3 were evaluated by Western blotting. In IL-1β-stimulated chondrocytes, mTORC2 activity was increased with increased phosphorylation of Akt and expression of rictor. IL-1β increased the expression of p-IκBα, p-NF-κB p65, NF-κB p65, IL-6, TNF-α, iNOS, Bax, and caspase3 proteins and decreased the expression of IκB-α. All of these IL-1β-induced alterations were prevented by JR-AB2-011. The main novel finding in the present study is that selective mTORC2 inhibition by JR-AB2-011 prevents the inflammatory, catabolic, and apoptotic responses induced by IL-1β via modulation of IκB-α/NF-κB activity. Therefore, we demonstrated a previously unknown function of mTORC2 inhibition that seems to be a potential therapeutic target for OA.

GRAM-POSITIVE BACTERIA WITHIN THE INTERVERTEBRAL DISC AND THEIR POTENTIAL INFLUENCE

IntroductionMultiple studies have identified Cutibacterium acnes (C.acnes) and other microbes in intervertebral disc tissue using 16S DNA Sequencing and microbial cultures. However, it remains unclear whether these bacteria are native to the discs or result from perioperative contamination. Our study aimed to detect Gram-positive bacteria in non-herniated human disc samples and explore correlations with Toll-like receptors (TLR) 2, TLR4, NLRP3, and Gasdermin D.MethodsImmunohistochemical staining was conducted on 75 human IVD samples for Gram-positive bacteria, S. aureus, C.acnes, TLR2, TLR4, NLRP3, and Gasdermin D. Cell detection and classification were performed using QuPath. NP cells were treated with Lipopolysaccharide (LPS) and Peptidoglycan (PGN) in monolayer and alginate beads for up to 72 hours, followed by secretome analysis using Luminex. Statistical analysis included Kruskal-Wallis, Dunn's multiple comparison test, and Pearson correlation.ResultsImmunohistochemical staining revealed Gram-positive bacteria exclusively within cells, with C. acnes positivity ranging from 5–99% and correlating with patient age (r=0.41, p= 0.007). TLR2 positivity ranged from 5–99% and TLR4 from 3–72%, showing a strong correlation (r= 0.62, p= 1.5e-006). Females with mid-degenerative grades exhibited significantly decreased TLR2 expression compared to those without degeneration signs. Treatment with LPS and PGN increased catabolic cyto- and chemokines associated with IVD degeneration.ConclusionIn conclusion, this study confirms Gram-positive bacteria presence in non-herniated human disc samples and highlights their role in triggering a catabolic response in disc cells.No conflicts of interest Sources of fundingThis project is part of the Disc4All Training network to advance integrated computational simulations in translational medicine, applies to intervertebral disc degeneration and funded by Horizon 2020 (H2020-MSCA-ITN-ETN-2020 GA: 955735)

Peri-operative zoledronic acid attenuates peri-prosthetic osteolysis in a rat model of cemented knee replacement.

Progressive osteolysis can occur at the cement-bone interface of joint replacements and the associated loss of fixation can lead to clinical loosening. We previously developed a rat hemiarthroplasty model that exhibited progressive loss of fixation with the development of cement-bone gaps under the tibial tray that mimicked patterns found in human arthroplasty retrievals. Here we explored the ability of a bisphosphonate (zoledronic acid, ZA) to attenuate cement-bone osteolysis and maintain implant stability. Sprague-Dawley rats (n = 59) received a poly(methylmethacrylate) cemented tibial component and were followed for up to 12 weeks. Treatment groups included peri-operative administration of ZA (ZA group), administration of ZA at 6 weeks postop (late ZA group), or vehicle (Veh group). There was a 60% reduction in the rate of cement-bone gap formation for the ZA group (0.15 mm3/week) compared to Veh group (0.38 mm3/week, p = 0.016). Late ZA prevented further progression of gap formation but did not reverse bone loss to the level achieved in the ZA group. Micromotion from five times body weight toggle loading was positively correlated with cement-bone gap volume (p = 0.009) and negatively correlated with the amount of cement in the metaphysis (p = 0.005). Reduced new bone formation and enduring nonviable bone in the epiphysis for the ZA group were found. This suggests that low bone turnover in the epiphysis may suppress the early catabolic response due to implantation, thereby maintaining better fixation in the epiphysis. This preclinical model presents compelling supporting data documenting improved maintenance of the cement-bone fixation with the use of peri-operative bisphosphonates.

Transcriptome analysis of avian livers reveals different molecular changes to three urban pollutants: Soot, artificial light at night and noise

Identifying key molecular pathways and genes involved in the response to urban pollutants is an important step in furthering our understanding of the impact of urbanisation on wildlife. The expansion of urban habitats and the associated human-introduced environmental changes are considered a global threat to the health and persistence of humans and wildlife. The present study experimentally investigates how short-term exposure to three urban-related pollutants -soot, artificial light at night (ALAN) and traffic noise-affects transcriptome-wide gene expression in livers from captive female zebra finches (Taeniopygia guttata). Compared to unexposed controls, 17, 52, and 28 genes were differentially expressed in soot, ALAN and noise-exposed birds, respectively. In soot-exposed birds, the enriched gene ontology (GO) terms were associated with a suppressed immune system such as interferon regulating genes (IRGs) and responses to external stimuli. For ALAN-exposed birds, enriched GO terms were instead based on downregulated genes associated with detoxification, redox, hormonal-, and metabolic processes. Noise exposure resulted in downregulation of genes associated with the GO terms: cellular responses to substances, catabolic and cytokine responses. Among the individually differentially expressed genes (DEGs), soot led to an increased expression of genes related to tumour progression. Likewise, ALAN revealed an upregulation of multiple genes linked to different cancer types. Both sensory pollutants (ALAN and noise) led to increased expression of genes linked to neuronal function. Interestingly, noise caused upregulation of genes associated with serotonin regulation and function (SLC6A4 and HTR7), which previous studies have shown to be under selection in urban birds. These outcomes indicate that short-term exposure to the three urban pollutants perturbate the liver transcriptome, but most often in different ways, which highlights future studies of multiple-stress exposure and their interactive effects, along with their long-term impacts for urban-dwelling wildlife.

Excessive load promotes temporomandibular joint chondrocyte apoptosis via Piezo1/endoplasmic reticulum stress pathway.

Excessive load on the temporomandibular joint (TMJ) is a significant factor in the development of TMJ osteoarthritis, contributing to cartilage degeneration. The specific mechanism through which excessive load induces TMJ osteoarthritis is not fully understood; however, mechanically-activated (MA) ion channels play a crucial role. Among these channels, Piezo1 has been identified as a mediator of chondrocyte catabolic responses and is markedly increased in osteoarthritis. Our observations indicate that, under excessive load conditions, endoplasmic reticulum stress in chondrocytes results in apoptosis of the TMJ chondrocytes. Importantly, using the Piezo1 inhibitor GsMTx4 demonstrates its potential to alleviate this condition. Furthermore, Piezo1 mediates endoplasmic reticulum stress in chondrocytes by inducing calcium ion influx. Our research substantiates the role of Piezo1 as a pivotal ion channel in mediating chondrocyte overload. It elucidates the link between excessive load, cell apoptosis, and calcium ion influx through Piezo1. The findings underscore Piezo1 as a key player in the pathogenesis of TMJ osteoarthritis, shedding light on potential therapeutic interventions for this condition.

522 Testosterone Supplementation in Patients with Burn Hypermetabolism

Abstract Introduction After major burn injuries, patients experience a hypermetabolic response that leads to hyperdynamic circulatory, physiologic, catabolic, and immune system responses. Many transdisciplinary approaches have been tested to mitigate this postburn hypermetabolic syndrome such as comprehensive burn rehabilitation, nutritional support, monitoring, and pharmacological interventions. Oxandrolone, a synthetic analog of testosterone, has been shown to have higher anabolic properties and minimal androgenic effects and studied intensely in burn care. In 2023, the FDA asked all manufacturers of oxandrolone to cease production and subsequently removed the drug from market on June 28, 2023. Our study examines the cost and operationalization of an intramuscular (IM) testosterone protocol as an alternative in male burn patients with ≥20% total body surface area (TBSA). Methods A cost analysis was conducted using the National Drug Codes (NDC) and commercially available benchmarking data. The cost of oxandrolone and testosterone were included in our analysis. Following a thorough literature review, a protocol was developed for adult male patients with >20% TBSA burn injuries. Patients undergoing gender affirming hormone therapy, untreated obstructive sleep apnea, or hormonal blockers for cancer were excluded from the protocol. Results The protocol begins with total serum testosterone levels at the time of admission and weekly thereafter for male patients with ≥20% TBSA injury. Once a testosterone level < 150 ng/dL is detected, supplementation of testosterone with 200 mg IM once weekly for two weeks is started. Weekly evaluations include total body weight, metabolic panels, and nutritional panels. Cost comparison between oxandrolone and testosterone show an equivalent unit cost of $8.62 versus $8.38, respectively. Conclusions Drug shortages and supply chain challenges are becoming more common in burn care. We hypothesize testosterone to be both a clinically effective and cost-effective alternative to oxandrolone in the setting of burn hypermetabolism in male patients with major burns. Currently, burn experts are working with endocrinologists to assess pharmacologic alternatives for female patients and have obtained IRB-approval to evaluate both safety and efficacy of testosterone supplementation in the setting of burn hypermetabolism. Applicability of Research to Practice This study evaluates the safety and efficacy of testosterone supplementation as an alternative to oxandrolone in the setting of burn hypermetabolism. This study helps provide guidance to other burn centers in filling the gap in therapy that now exists in the pharmacologic management of hypermetabolic syndrome in patients with severe burns.

Endocrine Responses to Heated Resistance Exercise in Men and Women.

Pryor, JL, Sweet, DK, Rosbrook, P, Qiao, J, Looney, DP, Mahmood, S, and Rideout, T. Endocrine responses to heated resistance exercise in men and women. J Strength Cond Res 38(7): 1248-1255, 2024-We examined the endocrine responses of 16 (female = 8) resistance trained volunteers to a single bout of whole-body high-volume load resistance exercise in hot (HOT; 40° C) and temperate (TEMP; 20° C) environmental conditions. Thermoregulatory and heart rate (HR) data were recorded, and venous blood was acquired before and after resistance exercise to assess serum anabolic and catabolic hormones. In men, testosterone increased after resistance exercise in HOT and TEMP ( p < 0.01), but postexercise testosterone was not different between condition ( p = 0.51). In women, human growth hormone was different between condition at pre-exercise ( p = 0.02) and postexercise ( p = 0.03). After controlling for pre-exercise values, the between-condition postexercise difference was abolished ( p = 0.16). There were no differences in insulin-like growth factor-1 for either sex ( p ≥ 0.06). In women, cortisol increased from pre-exercise to postexercise in HOT ( p = 0.04) but not TEMP ( p = 0.19), generating a between-condition difference at postexercise ( p < 0.01). In men, cortisol increased from pre-exercise to postexercise in HOT only ( p < 0.01). Rectal temperature increased to a greater extent in HOT compared with TEMP in both men ( p = 0.01) and women ( p = 0.02). Heart rate increased after exercise under both conditions in men and women ( p = 0.01), but only women experience greater postexercise HR in HOT vs. TEMP ( p = 0.04). The addition of heat stress to resistance exercise session did not overtly shift the endocrine response toward an anabolic or catabolic response. When acute program variables are prescribed to increase postresistance exercise anabolic hormones, adding heat stress is not synergistic but does increase physiologic strain (i.e., elevated HR and rectal temperature).

Exosomes derived from diabetic serum accelerate the progression of osteoarthritis

Diabetes mellitus (DM) has been demonstrated to accelerate the progression of osteoarthritis (OA) by largely unknown mechanisms. Studies have shown that DM dysfunctional adipocyte-derived exosomes play a crucial role in the pathogenesis of remote organ functions. The present study aimed to clarify whether and how diabetic adipocyte-derived exosomes mediate the pathological regulation of OA. We found that intraarticular injection of DM serum exosomes in the non-diabetic mice significantly exacerbated OA injury as evidenced by a rough and fractured cartilage surface as well as increased chondrocyte apoptosis, decreased mitochondrial membrane potential (△Ψ) and increased expression of cleaved caspase-3. Mechanistic investigation identified that miR-130b-3p was significantly increased in circulating exosomes derived from DM mice and exosomes derived from HG-treated normal adipocytes, and we demonstrated that transfection of miR-130b-3p mimics significantly exacerbated the mitochondrial function of chondrocytes. Our data also indicated that miR-130b-3p impaired the △Ψ, increased cleaved caspase-3 levels, and decreased the expression of 5′-adenosine monophosphate-activated protein kinase α1 (AMPKα1), Silent mating-type information regulation 2 homolog 1 (SIRT1), and peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) in chondrocytes. Pharmacologic activation of AMPKα1 using AICAR reversed the △Ψ and catabolic responses in chondrocytes transfected with miR-130b-3p mimics. Moreover, AICAR decreased the effects of miR-130b-3p mimics on chondrocytes transfected with SIRT1-siRNA or PGC-1α-siRNA. The current study demonstrated that adipocyte-derived exosomal miR-130b-3p under DM conditions suppresses mitochondrial function in chondrocytes through targeting the AMPKα1/SIRT1/PGC1-α pathway, thus exacerbating OA injury.

Coenzyme Q10 supplementation in burn patients: a double-blind placebo-controlled randomized clinical trial

BackgroundBurn injuries are important medical problems that, aside from skin damage, cause a systemic response including inflammation, oxidative stress, endocrine disorders, immune response, and hypermetabolic and catabolic responses which affect all the organs in the body. The aim of this study was to determine the effect of coenzyme Q10 (CoQ10) supplementation on inflammation, oxidative stress, and clinical outcomes in burn patients.MethodsIn a double-blind placebo-controlled randomized clinical trial, 60 burn patients were randomly assigned to receive 100 mg CoQ10 three times a day (total 300 mg/day) or a placebo for 10 days. Inflammatory markers including erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), oxidative stress markers including total antioxidant capacity (TAC), malondialdehyde (MDA) and superoxide dismutase (SOD) activity, fasting blood glucose (FBG), blood urea nitrogen (BUN), creatinine, white blood cells (WBC), and body temperature were assessed as primary outcomes and albumin, prothrombin time (PT), partial thromboplastin time (PTT), international normalized ratio (INR), other hematological parameters, blood pressure, O2 saturation, ICU duration, and 28-mortality rate were assessed as secondary outcomes.ResultsFifty-two participants completed the trial. CRP and ESR levels were not significantly different between CoQ10 and placebo groups at the end of the study (P = 0.550 and P = 0.306, respectively). No significant differences between groups were observed for TAC (P = 0.865), MDA (P = 0.692), and SOD activity (P = 0.633) as well. Administration of CoQ10 resulted in a significant increase in albumin levels compared to placebo (P = 0.031). There was no statistically significant difference between the two groups in other measured outcomes (P > 0.05).ConclusionResults showed that in patients with burn injury, CoQ10 administration had no effect on inflammatory markers and oxidative stress, although serum albumin levels were improved after supplementation. Further studies with albumin as the primary outcome are needed to confirm this finding.

The Hepatokine RBP4 Links Metabolic Diseases to Articular Inflammation.

This study investigates the role of retinol binding protein 4 (RBP4) in an articular context. RBP4, a vitamin A transporter, is linked to various metabolic diseases. Synovial fluid RBP4 levels were assessed in crystalline arthritis (CA) patients using ELISA. RBP4's impact on articular cell types was analysed in vitro through RT-PCR and flow cytometry. Proteomic analysis was conducted on primary human osteoarthritis chondrocytes (hOACs). Synovial fluid RBP4 concentrations in CA patients correlated positively with glucose levels and negatively with synovial leukocyte count and were elevated in hypertensive patients. In vitro, these RBP4 concentrations activated neutrophils, induced the expression of inflammatory factors in hOACs as well as synoviocytes, and triggered proteomic changes consistent with inflammation. Moreover, they increased catabolism and decreased anabolism, mitochondrial dysfunction, and glycolysis promotion. Both in silico and in vitro experiments suggested that RBP4 acts through TLR4. This study identifies relevant RBP4 concentrations in CA patients' synovial fluids, linking them to hypertensive patients with a metabolic disruption. Evidence is provided that RBP4 acts as a DAMP at these concentrations, inducing robust inflammatory, catabolic, chemotactic, and metabolic responses in chondrocytes, synoviocytes, and neutrophils. These effects may explain RBP4-related metabolic diseases' contribution to joint destruction in various rheumatic conditions like CA.

Management of enteral/parenteral therapy in patients with obesity in the light of exosomes/microRNAs: a systematic review

Introduction: Alteration in the homeostasis of the intestinal microbiota is related to the prevalence of obesity, which could reach up to 57.8% of the adult world population by 2030. Likewise, the global incidence rate of type 2 diabetes, which represents 90-95 % of all diabetes cases. Extracellular vesicles (exosomes and microRNAs) have emerged as main vehicles for intercellular and intermolecular communication, including those established between the intestinal microbiota and mammals. Providing adequate nutritional therapy with essential nutrients can help mitigate the consequences of the catabolic response. Objective: It was to bring together the main considerations of enteral/parenteral nutritional therapy in patients with obesity, highlighting the management of inflammatory and metabolic processes through exosomes and microRNAs. Methods: The PRISMA Platform systematic review rules were followed. The search was carried out from August to October 2023 in the Scopus, PubMed, Science Direct, Scielo, and Google Scholar databases. The quality of the studies was based on the GRADE instrument and the risk of bias was analyzed according to the Cochrane instrument. Results and Conclusion: 118 articles were found. A total of 44 articles were evaluated in full and 39 were included in this systematic review. Considering the Cochrane tool for risk of bias, the overall assessment resulted in 12 studies with a high risk of bias and 22 studies that did not meet GRADE. Most studies showed homogeneity in their results, with X2 =52.5%&gt;50%. It was concluded that exosomes and miRNAs, through enteral/parenteral nutritional therapy, are involved in the control of weight gain, glucose homeostasis, and lipid metabolism. Some clinical studies have shown that enteral nutritional therapy is effective and safe before bariatric surgery, with ketogenic enteral nutrition leading to better clinical outcomes than hypocaloric enteral nutritional protocols in glycemic and lipid profiles to satisfy better regulation of exosomes. and microRNAs, mainly from the intestinal microbiota, for the control of inflammatory and metabolic processes.

THE ROLE OF PERICYTES IN REGULATING CARTILAGE DEGRADATION AND FIBROSIS

Pericytes are contractile, motile cells that surround the capillary. Recent studies have shown that pericytes promoted joint fibrosis and induced subchondral bone angiogenesis, indicating the role of pericytes in osteoarthritis (OA). However, whether pericytes are involved in regulating inflammatory and catabolic response, as well as fibrotic repair of cartilage is still unclear. Here we used 2D and 3D models to investigate the communication of pericytes and chondrocytes under inflammatory osteoarthritis conditions.CD34-CD146+ pericytes were isolated and sorted from human bone marrow. Human OA chondrocytes were isolated from OA joints. In 2D studies, monolayer cultured chondrocytes were treated +/- pericyte conditioned media, +/- 1ng/ml IL1β for 24h. In 3D studies, pericytes and chondrocytes were cultured within fibrin gel in 3D polyurethane scaffolds, separately or combined for 7 days, followed by treatment of +/- IL1β for another 7 days (Fig 2A). The inflammatory response, catabolic activity and expression of fibrosis markers of chondrocytes and pericytes were measured by ELISA and/or q-rtPCR.Pericytes had weak inflammatory, catabolic and fibrotic response to IL1β (data not shown). The 2D study showed that pericyte conditioned media promoted inflammation, catabolism and fibrosis markers of chondrocytes, in the absence of IL1β treatment (Figure 1). However, study in 3D showed that coculture of chondrocytes and pericytes reduced the inflammatory and catabolic response of chondrocytes to IL1β and induced fibrosis markers in chondrocytes (Figure 2).Pericytes are involved in regulating inflammatory response, catabolic response and fibrosis of chondrocytes. The opposite results from 2D and 3D experiments indicate the variety of the regulatory role of pericytes in the interaction with chondrocytes within different culture models. The underlying mechanism is under evaluation with on-going studies.AcknowledgementsThis study was funded by SINPAIN project, from European Union's Horizon Europe research and innovation programme under Grant Agreement NO. 101057778. Funded by the European Union. Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union. Neither the European Union nor the granting authority can be held responsible for them.For any figures or tables, please contact the authors directly.

Evaluating the inhibition of IL-17A and TNFα in a cartilage explant model cultured with Th17-derived cytokines

IntroductionT-helper 17 (Th17) cells produce IL-17A playing a critical role in activating the pathogenic chain leading to joint tissue inflammation and destruction. Elevated levels of Th17 cells and IL-17A have been detected in skin lesions, blood, and synovial fluid from patients with psoriatic arthritis (PsA) and ankylosing spondylitis (AS). Moreover, IL-17A inhibitors suppress disease activity in psoriasis, PsA and AS, supporting the evidence of IL-17A contributing to the disease pathogenesis. Although, IL-17A inhibitors are widely approved, it remains unclear how the inhibitory effect of IL-17A alters the extracellular matrix (ECM) of the joint in a Th17-conditioned inflammatory milieu. Therefore, the aim of this study was to establish a cartilage model cultured with conditioned medium from Th17 cells and inhibitors to explore the effect of IL-17A inhibition on joint tissue remodeling. MethodsNaïve CD4+ T cells from healthy human buffy coat were differentiated into Th17 cells, followed by Th17 cell activation to secrete Th17-related cytokines and molecules into media. The activated Th17 cells were isolated from the conditioned media (CM) and analyzed using flow cytometry to verify Th17 cell differentiation. The CM were assessed with ELISA to quantify the concentrations of cytokines secreted into the media by the Th17 cells. Healthy bovine cartilage explants were cultured with the Th17-CM and treated with IL-17A and TNFα inhibitors for 21 days. In harvested supernatant from the cartilage cultures, MMP- and ADAMTS-mediated biomarker fragments of type II collagen, aggrecan, and fibronectin were measured by ELISA to investigate the ECM remodeling within the cartilage tissue. ResultsTh17-CM stimulated a catabolic response in the cartilage. Markers of type II collagen and aggrecan degradation were upregulated, while anabolic marker of type II collagen formation remained on similar levels as the untreated explants. The addition of IL-17A inhibitor to Th17-CM decreased the elevated type II collagen and aggrecan degradation, however, degenerative levels were still elevated compared to untreated group. The addition of TNFα inhibitor completely reduced both type II collagen and aggrecan degradation compared to untreated explants. Moreover, the TNFα inhibitor treatment did not alter the type II collagen formation compared to untreated group. ConclusionThis study suggests that inhibition of IL-17A in Th17-conditioned cartilage tissue only partially reduced the MMP-mediated type II collagen degradation and ADAMTS-mediated aggrecan degradation, while the TNFα inhibitor treatment fully reduced both MMP- and ADAMTS-mediated ECM degradation. This exploratory study where ECM biomarkers are combined with Th17-conditioned ex vivo model may hold great potential as output for describing joint disease mechanisms and predicting structural effects of treatment on joint tissue.

Role of epigenetics and the transcription factor Sp1 in the expression of the D prostanoid receptor 1 in human cartilage

D prostanoid receptor 1 (DP1), a prostaglandin D2 receptor, plays a central role in the modulation of inflammation and cartilage metabolism. We have previously shown that activation of DP1 signaling downregulated catabolic responses in cultured chondrocytes and was protective in mouse osteoarthritis (OA). However, the mechanisms underlying its transcriptional regulation in cartilage remained poorly understood. In the present study, we aimed to characterize the human DP1 promoter and the role of DNA methylation in DP1 expression in chondrocytes. In addition, we analyzed the expression level and methylation status of the DP1 gene promoter in normal and OA cartilage. Deletion and site-directed mutagenesis analyses identified a minimal promoter region (−250/−120) containing three binding sites for specificity protein 1 (Sp1). Binding of Sp1 to the DP1 promoter was confirmed using electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assays. Treatment with the Sp1 inhibitor mithramycin A reduced DP1 promoter activity and DP1 mRNA expression. Inhibition of DNA methylation by 5-Aza-2′-deoxycytidine upregulated DP1 expression, and in vitro methylation reduced the DP1 promoter activity. Neither the methylation status of the DP1 promoter nor the DP1 expression level were different between normal and OA cartilage. In conclusion, our results suggest that the transcription factor Sp1 and DNA methylation are important determinants of DP1 transcription regulation. They also suggest that the methylation status and expression level of DP1 are not altered in OA cartilage. These findings will improve our understanding of the regulatory mechanisms of DP1 transcription and may facilitate the development of intervention strategies involving DP1.

Osteoblast-lineage calcium/calmodulin-dependent kinase 2 delta and gamma regulates bone mass and quality.

Bone regulates its mass and quality in response to diverse mechanical, hormonal, and local signals. The bone anabolic or catabolic responses to these signals are often received by osteocytes, which then coordinate the activity of osteoblasts and osteoclasts on bone surfaces. We previously established that calcium/calmodulin-dependent kinase 2 (CaMKII) is required for osteocytes to respond to some bone anabolic cues in vitro. However, a role for CaMKII in bone physiology in vivo is largely undescribed. Here, we show that conditional codeletion of the most abundant isoforms of CaMKII (delta and gamma) in mature osteoblasts and osteocytes [Ocn-cre:Camk2d/Camk2g double-knockout (dCKO)] caused severe osteopenia in both cortical and trabecular compartments by 8 wk of age. In addition to having less bone mass, dCKO bones are of worse quality, with significant deficits in mechanical properties, and a propensity to fracture. This striking skeletal phenotype is multifactorial, including diminished osteoblast activity, increased osteoclast activity, and altered phosphate homeostasis both systemically and locally. These dCKO mice exhibited decreased circulating phosphate (hypophosphatemia) and increased expression of the phosphate-regulating hormone fibroblast growth factor 23. Additionally, dCKO mice expressed less bone-derived tissue nonspecific alkaline phosphatase protein than control mice. Consistent with altered phosphate homeostasis, we observed that dCKO bones were hypo-mineralized with prominent osteoid seams, analogous to the phenotypes of mice with hypophosphatemia. Altogether, these data reveal a fundamental role for osteocyte CaMKIIδ and CaMKIIγ in the maintenance of bone mass and bone quality and link osteoblast/osteocyte CaMKII to phosphate homeostasis.

Promoting the proliferation of osteoarthritis chondrocytes by resolvin D1 regulating the NLRP3/caspase-1 signaling pathway

Osteoarthritis (OA) is a degenerative joint disease commonly found in middle-aged and older people. Chondrocytes are the only cells in joint cartilage that are difficult to heal after pyroptosis, and they will aggravate the wear and tear of joint cartilage and affect the progression of OA. Pyroptosis is a novel form of programmed cell death, and the classical pyroptosis pathway is a programmed cell death pattern mediated by inflammatory cysteine protease-1. Activation of NLRP3 leads to activation and cleavage of caspase-1 precursors, which in turn leads to activation and cleavage of GSDMD proteins and the release of proinflammatory factors. Resolvin D1 (RvD1) is a specialized pro-resolving mediator (SPM) derived from omega-3 unsaturated fatty acids that reduces inflammation and catabolic responses in OA chondrocytes. However, it is unclear whether RvD1 promotes OA chondrocyte proliferation and thus joint cartilage repair. Our results show that RvD1 regulates the NLRP3/caspase-1 signaling pathway by inhibiting the expression of caspase-1, promoting the proliferation of OA chondrocytes, promoting the repair of articular cartilage in rats and delaying the progression of osteoarthritis.

Reef building corals show resilience to the hottest marine heatwave on record in the Gulf of Aqaba

Coral reefs are facing rapid deterioration, primarily due to a global rise in seawater temperature. In conjunction, the frequency and intensity of extreme high temperature events, known as marine heatwaves (MHWs), are increasing. The Gulf of Aqaba (GoA) in the northern Red Sea is home to corals known for their thermal resilience, yet concerns have been raised regarding the potential for MHWs to put this coral refuge at risk. In summer of 2021, the hottest MHW so far occurred in the GoA, with sea surface temperatures peaking at 31°C and persisting above the local summer maximum for 34 days. To assess the physiological response of the corals Stylophora pistillata and Pocillopora damicornis to this event, we analyzed the monthly content across a year of host and symbiont proteins, carbohydrates, and lipids, pre-, during, and post the MHW, as a proxy for metabolic stress. We found that the MHW was not fatal to either species and did not induce bleaching, based on algal densities and chlorophyll content. Species-specific responses were detected. In S. pistillata, host protein content decreased (33%) at the onset of the MHW (August) compared to pre-MHW levels (July). Algal symbionts of S. pistillata were unaffected by the MHW in their maximal photosynthetic efficiency (Fv/Fm) and exhibited higher carbohydrate levels (+34%) at the end of the MHW (September) compared to its onset. In contrast, no significant catabolic response was detected in P. damicornis host or symbionts, and the maximal relative electron transport rate (rETRmax) of symbionts was 37% higher during the MHW than the annual average. These results highlight the remarkable ability of common GoA corals to withstand extreme thermal anomalies, underscoring the global significance of this coral refuge.

Sexual dimorphism of the synovial transcriptome underpins greater PTOA disease severity in male mice following joint injury

Osteoarthritis is a disease with sex-dependent prevalence and severity in both humans and animal models. We sought to elucidate sex differences in synovitis, mechanical sensitization, structural damage, bone remodeling, and the synovial transcriptome in the anterior cruciate ligament rupture (ACLR) mouse model of post-traumatic osteoarthritis (PTOA). Male and female 12-week-old C57/BL6J mice were randomized to Sham or noninvasive ACLR with harvests at 7d or 28d post-ACLR (n=9 per sex in each group - Sham, 7d ACLR, 28d ACLR). Knee hyperalgesia, mechanical allodynia, and intra-articular MMP activity (via intravital imaging) were measured longitudinally. Trabecular and subchondral bone remodeling and osteophyte formation were assessed by µCT. Histological scoring of PTOA and synovitis and anti-MMP13 immunostaining were performed. NaV1.8-Cre;tdTomato mice were used to document localization and sprouting of nociceptors. Bulk RNA-seq of synovium in Sham, 7d, and 28d post-ACLR, and contralateral joints (n=6 per group per sex) assessed injury-induced and sex-dependent gene expression. Male mice exhibited more severe joint damage at 7d and 28d and more severe synovitis at 28d, accompanied by 19% greater MMP activity, 8% lower knee hyperalgesia threshold, and 43% lower hindpaw withdrawal threshold in injured limbs compared to female injured limbs. Females had injury-induced catabolic responses in trabecular and subchondral bone, whereas males exhibited 133% greater normalized osteophyte volume relative to females and sclerotic remodeling of trabecular and subchondral bone. NaV1.8+ nociceptor sprouting in subchondral bone and medial synovium was induced by injury and comparable between sexes. RNA-seq of synovium demonstrated similar injury-induced transcriptomic programs between the sexes at 7d, but only female mice exhibited a transcriptomic signature indicative of synovial inflammatory resolution by 28d, whereas males had persistent pro-inflammatory, pro-fibrotic, pro-neurogenic, and pro-angiogenic gene expression. Male mice exhibited more severe overall joint damage and pain behavior after ACLR, which was associated with persistent activation of synovial inflammatory, fibrotic, and neuroangiogenic processes, implicating persistent synovitis in driving sex differences in murine PTOA.

Paired growth of cultivated and halophytic wild rice under salt stress induces bacterial endophytes and gene expression responses.

Utilizing salt-affected marginal lands in coastal regions can help meet the growing demand for rice. We explored a nature-based solution involving wild halophytic rice (O. coarctata, Oc) and commercial rice BRRI Dhan 67 (O. sativa, Os) grown in close proximity to each other under salt stress. This was to investigate whether a paired planting strategy could help complement rice growth and yield under stress. We also investigated the gene expression and endophytic bacterial profiles of both Os and Oc in unpaired and paired conditions without and with salt. Paired plants exhibited lower salt damage indicators such as smaller reduction in plant height, electrolyte leakage and chlorophyll loss, as well as higher K+/Na+ ratio under saline stress. Some of the 39 endophytic bacteria in the mutualism experiment were unique to Oc and transferred to Os when paired. Differentially expressed genes in leaves of paired Os versus unpaired Os were 1097 (994 up-regulated, 101 down-regulated) without salt and 893 (763 up-regulated, 130 down-regulated) under salt stress. The presence of Oc plants under salt stress influenced major biological processes in Os, including oxidative stress; chitinase activity; phenylalanine catabolic process and response to ABA. Protein binding and serine/threonine kinase activity were primarily affected in molecular function. The downregulated WRKY transcription factor 22 in paired conditions under salt stress played a role in the MAPK signaling pathway, reducing respiratory cell death. The upregulated auxin-responsive protein IAA18 gene, involved in hormone signaling and cell enlargement, was present only in paired plants. Our findings therefore, offer insights into developing more effective cultivation strategies for sustainable rice production.

Skeletal muscle wasting after burn is regulated by a decrease in anabolic signaling in the early flow phase

Following burns a sustained catabolic stress response is activated, resulting in skeletal muscle wasting. A better understanding of the underlying mechanisms of postburn skeletal muscle wasting is essential for the development of preventive and/or therapeutic strategies. Six weeks old female rats underwent a sham, 10% or 40% total body surface area scald burn. Ten days post-injury, severely burned animals gained significantly less weight compared to sham treated and minor burned animals, reflected in a significantly lower ratio of muscle to total body weight for Soleus (SOL) and Extensor Digitorum Longus (EDL) in the severely burned group. Postburn, total fiber number was significantly lower in EDL, while in SOL the amount of type1 fibers significantly increased and type2 fibers significantly decreased. No signs of mitochondrial dysfunction (COX/SDH) or apoptosis (caspase-3) were found. In SOL and EDL, eEF2 and pAKT expression was significantly lower after severe burn. MURF1,2,3 and Atrogin-1 was significantly higher in SOL, whilst in EDL MURF1,2,3 was significantly lower postburn. In both muscles, FOXO3A was significantly lower postburn. This study identified postburn changes in muscle anthropomorphology and proteins involved in pathways regulating protein synthesis and breakdown, with more pronounced catabolic effects in SOL.