Catabolic Markers Research Articles

The Role of the Bone Morphogenetic Protein Antagonist Noggin in Nucleus Pulposus Intervertebral Disc Cells.

Low back pain (LBP) is a significant global health issue, contributing to disability and socioeconomic burdens worldwide. The degeneration of the human intervertebral disc (IVD) is a critical factor in the pathogenesis of LBP. Recent studies have emphasized the significance of a specific set of genes and extracellular matrix (ECM) in IVD health. In particular, Noggin has emerged as a critical gene due to its high expression levels in healthy nucleus pulposus cells (NPCs) observed in our previous research. In this study, it was hypothesized that decreased Noggin expression in NPCs is associated with IVD degeneration and contributes to LBP development. A lentivirus-mediated RNAi was applied to knock down Noggin expression in primary NPCs from six human donors. The NPCs after transduction were evaluated through cell viability analysis, XTT assay, and cell apoptosis analyses. After two weeks, a colony formation assay was used to examine the anchor-independent growth ability of transduced cells. At the transcript level, anabolic and catabolic markers were quantified using RT-qPCR. The results demonstrated that lentivirus-mediated downregulation of Noggin significantly inhibited cell proliferation, reduced cell viability, and suppressed colony formation, while inducing apoptosis in human NPCs in vitro. Notably, it disrupted cellular anabolic processes and promoted catabolic activity in human NPCs post-transduction. Our findings indicated that the degeneration of human IVD is possibly related to decreased Noggin expression in NPCs. This research provides valuable insights into the role of Noggin in IVD homeostasis and its implications in LBP treatment.

6979 Effect of Orchiectomy on Muscle Growth and Differentiation Factors in Male Mice With Kidney Disease

Abstract Disclosure: O. Marks: None. J. Bruno: None. D. Wu: None. A. Patwardhan: None. Y. Komaru: None. G. Ling: None. E. Kefalogianni: None. R. Aurora: None. S.S. Dhindsa: None. A. Herrlich: None. Background: Sarcopenia and frailty are major clinical concerns in participants with chronic kidney disease (CKD). In males, an additional factor contributes to the development of sarcopenia. Around half of the men with CKD have subnormal serum testosterone (T) concentrations. The mechanisms that underlie the effect of low T concentrations on muscle growth in CKD are not known. In this study, we performed orchiectomy in male mice with kidney disease and evaluated the expression and protein content of the following muscle growth and differentiation factors. Methods: Forty mice were divided into 4 groups: 1) renal injury, induced by ischemia-induced reperfusion (IRI, N=11) at 2 months of age; 2) orchidectomy (ORX), performed 4 weeks after IRI (IRI + ORX, N=9); 3) sham-IRI and ORX (sham-ORX, N=8); and 4) sham-sham surgery (N=12). The mice were sacrificed when they were ∼7 months old (mean age 206±13 days). Gastrocnemius and levator ani muscles were harvested to measure expression by RT-qPCR of muscle catabolism markers (Atrogin-1 and ubiquitin ligase MuRF1), muscle growth inhibitors, myostatin and myogenic regulatory factor (Mrf4), fibroblast growth factor-2 (FGF2, a stimulator of satellite stem cells) and its receptor FGFR2, androgen receptor (AR) and IGF-1. Results: The mean BUN (blood urea nitrogen) at the time of sacrifice was higher in the IRI/ORX (32±4 mg/dl) and IRI/Sham (26±5 mg/dl) groups as compared to sham-sham (21±5 mg/dl, p<0.05 for both). There was a significant reduction (p<0.05) in the expression of myostatin (by 41%), Mrf4 (29%), atrogin-1 (51%) and MuRF1 (32%) in gastrocnemius muscle from the ORX-IRI and ORX-sham groups, while there was no difference in FGF2 and FGFR2, as compared to sham-sham group. Data from levator ani muscle (the most androgen sensitive muscle in mammals) showed increases in AR (64%) and Atrogin-1 (66%); and decreases in IGF-1 (88%) and myostatin (86%) in ORX-IRI and ORX-sham groups. Consistent with the mRNA expression, we found a drastic decline in protein content of myostatin in gastrocnemius (85%) in ORX-IRI and ORX-sham groups as compared to sham-sham (p=0.04). Conclusions: These data show that ORX in mice with renal injury is associated with changes in muscle growth and differentiation factors. Myostatin was the most affected gene, with 41-86% decline in mRNA and protein. These data are informative for attempts to develop treatments for sarcopenia in patients with kidney disease. Limitations: Our experimental model differs in some respects from hypogonadal men with CKD. ORX results in complete elimination of T, and thus much lower T concentrations than in men with kidney disease. There is a recovery of kidney function after IRI. We sacrificed the mice 5 months after renal injury was inflicted. Hence, the effect of IRI on muscle growth genes may have been different immediately following IRI. Thus, the impact of ORX was more apparent than of IRI in our study. Presentation: 6/1/2024

6979 Effect of Orchiectomy on Muscle Growth and Differentiation Factors in Male Mice With Kidney Disease

Abstract Disclosure: O. Marks: None. J. Bruno: None. D. Wu: None. A. Patwardhan: None. Y. Komaru: None. G. Ling: None. E. Kefalogianni: None. R. Aurora: None. S.S. Dhindsa: None. A. Herrlich: None. Background: Sarcopenia and frailty are major clinical concerns in participants with chronic kidney disease (CKD). In males, an additional factor contributes to the development of sarcopenia. Around half of the men with CKD have subnormal serum testosterone (T) concentrations. The mechanisms that underlie the effect of low T concentrations on muscle growth in CKD are not known. In this study, we performed orchiectomy in male mice with kidney disease and evaluated the expression and protein content of the following muscle growth and differentiation factors. Methods: Forty mice were divided into 4 groups: 1) renal injury, induced by ischemia-induced reperfusion (IRI, N=11) at 2 months of age; 2) orchidectomy (ORX), performed 4 weeks after IRI (IRI + ORX, N=9); 3) sham-IRI and ORX (sham-ORX, N=8); and 4) sham-sham surgery (N=12). The mice were sacrificed when they were ∼7 months old (mean age 206±13 days). Gastrocnemius and levator ani muscles were harvested to measure expression by RT-qPCR of muscle catabolism markers (Atrogin-1 and ubiquitin ligase MuRF1), muscle growth inhibitors, myostatin and myogenic regulatory factor (Mrf4), fibroblast growth factor-2 (FGF2, a stimulator of satellite stem cells) and its receptor FGFR2, androgen receptor (AR) and IGF-1. Results: The mean BUN (blood urea nitrogen) at the time of sacrifice was higher in the IRI/ORX (32±4 mg/dl) and IRI/Sham (26±5 mg/dl) groups as compared to sham-sham (21±5 mg/dl, p<0.05 for both). There was a significant reduction (p<0.05) in the expression of myostatin (by 41%), Mrf4 (29%), atrogin-1 (51%) and MuRF1 (32%) in gastrocnemius muscle from the ORX-IRI and ORX-sham groups, while there was no difference in FGF2 and FGFR2, as compared to sham-sham group. Data from levator ani muscle (the most androgen sensitive muscle in mammals) showed increases in AR (64%) and Atrogin-1 (66%); and decreases in IGF-1 (88%) and myostatin (86%) in ORX-IRI and ORX-sham groups. Consistent with the mRNA expression, we found a drastic decline in protein content of myostatin in gastrocnemius (85%) in ORX-IRI and ORX-sham groups as compared to sham-sham (p=0.04). Conclusions: These data show that ORX in mice with renal injury is associated with changes in muscle growth and differentiation factors. Myostatin was the most affected gene, with 41-86% decline in mRNA and protein. These data are informative for attempts to develop treatments for sarcopenia in patients with kidney disease. Limitations: Our experimental model differs in some respects from hypogonadal men with CKD. ORX results in complete elimination of T, and thus much lower T concentrations than in men with kidney disease. There is a recovery of kidney function after IRI. We sacrificed the mice 5 months after renal injury was inflicted. Hence, the effect of IRI on muscle growth genes may have been different immediately following IRI. Thus, the impact of ORX was more apparent than of IRI in our study. Presentation: 6/1/2024

Targeting PAR2-mediated inflammation in osteoarthritis: a comprehensive in vitro evaluation of oleocanthal’s potential as a functional food intervention for chondrocyte protection and anti-inflammatory effects

BackgroundOsteoarthritis (OA) is a prevalent degenerative joint disease characterized by chronic inflammation and progressive cartilage degradation, ultimately leading to joint dysfunction and disability. Oleocanthal (OC), a bioactive phenolic compound derived from extra virgin olive oil, has garnered significant attention due to its potent anti-inflammatory properties, which are comparable to those of non-steroidal anti-inflammatory drugs (NSAIDs). This study pioneers the investigation into the effects of OC on the Protease-Activated Receptor-2 (PAR-2) mediated inflammatory pathway in OA, aiming to validate its efficacy as a functional food-based therapeutic intervention.MethodsTo simulate cartilage tissue in vitro, human bone marrow-derived mesenchymal stem cells (BMSCs) were differentiated into chondrocytes. An inflammatory OA-like environment was induced in these chondrocytes using lipopolysaccharide (LPS) to mimic the pathological conditions of OA. The therapeutic effects of OC were evaluated by treating these inflamed chondrocytes with various concentrations of OC. The study focused on assessing key inflammatory markers, catabolic enzymes, and mitochondrial function to elucidate the protective mechanisms of OC. Mitochondrial function, specifically mitochondrial membrane potential (ΔΨm), was assessed using Rhodamine 123 staining, a fluorescent dye that selectively accumulates in active mitochondria. The integrity of ΔΨm serves as an indicator of mitochondrial and bioenergetic function. Additionally, Western blotting was employed to analyze protein expression levels, while real-time polymerase chain reaction (RT-PCR) was used to quantify gene expression of inflammatory cytokines and catabolic enzymes. Flow cytometry was utilized to measure cell viability and apoptosis, providing a comprehensive evaluation of OC’s therapeutic effects on chondrocytes.ResultsThe results demonstrated that OC significantly downregulated PAR-2 expression in a dose-dependent manner, leading to a substantial reduction in pro-inflammatory cytokines, including TNF-α, IL-1β, and MCP-1. Furthermore, OC attenuated the expression of catabolic markers such as SOX4 and ADAMTS5, which are critically involved in cartilage matrix degradation. Importantly, OC was found to preserve mitochondrial membrane potential (ΔΨm) in chondrocytes subjected to inflammatory stress, as evidenced by Rhodamine 123 staining, indicating a protective effect on cellular bioenergetics. Additionally, OC modulated the Receptor Activator of Nuclear Factor Kappa-Β Ligand (RANKL)/Receptor Activator of Nuclear Factor Kappa-Β (RANK) pathway, suggesting a broader therapeutic action against the multifactorial pathogenesis of OA.ConclusionsThis study is the first to elucidate the modulatory effects of OC on the PAR-2 mediated inflammatory pathway in OA, revealing its potential as a multifaceted therapeutic agent that not only mitigates inflammation but also protects cartilage integrity. The preservation of mitochondrial function and modulation of the RANKL/RANK pathway further underscores OC’s comprehensive therapeutic potential in counteracting the complex pathogenesis of OA. These findings position OC as a promising candidate for integration into nutritional interventions aimed at managing OA. However, further research is warranted to fully explore OC’s therapeutic potential across different stages of OA and its long-term effects in musculoskeletal disorders.

Effects of aerobic exercise at different intensities on articular cartilage in mice.

Maintaining intrinsic articular cartilage homeostasis is essential for the health of cartilage. However, the impact of aerobic exercise of varying intensities on the articular cartilage homeostasis has never been studied. This study aims to elucidate the influence of different aerobic exercise intensities on the anabolic and catabolic processes within articular cartilage. Forty-eight male C57BL/6J mice, aged 7 weeks, were divided into 4 aerobic exercise groups and 1 control group. The aerobic exercise groups were subjected to both acute and chronic exercise protocols with varying intensities of 8, 12, 16, 20, and 24 mmin-1. Total RNA from the knee joint cartilage was extracted in both phases to quantify mRNA of anabolic (Sox9, Col2a1, and Acan) and catabolic (MMP-13 and ADAMTS5) markers. In the chronic exercise, articular cartilage thickness and chondrocyte density were histologically assessed. Additionally, immunohistochemical staining quantified relevant molecules involved in cartilage metabolism. Inthe acute exercise, the 8 m min-1 group exhibited reduced ADAMTS5 expression compared to the control, 16 m min-1, and 24 m min-1 groups. Chronic exercise showed enhanced articular cartilage thickness in both the 8 and 12 m min-1 groups relative to the control group. Moreover, the 8 m min-1 group demonstrated elevated aggrecan levels in comparison to both the control and 24 m min-1 groups. Additionally, the 24 m min-1 group exhibited significantly higher ADAMTS5 levels than the control group. Our findings suggest that consistent low-intensity aerobic exercise suppresses catabolic molecule expression in articular cartilage, thereby fostering anabolic activity. Conversely, continuous high-intensity aerobic exercise can potentially disrupt cartilage homeostasis by enhancing catabolic processes. This dichotomy underscores the need for balanced exercise regimens to maintain cartilage health.

TNFα-Induced Inflammation Model-Evaluation of Concentration and Passage-Dependent Effects on Bovine Chondrocytes.

Inflammation models are widely used in the in vitro investigation of new therapeutic approaches for osteoarthritis. TNFα (tumor necrosis factor alpha) plays an important role in the inflammatory process. Current inflammation models lack uniformity and make comparisons difficult. Therefore, this study aimed to systematically investigate whether the effects of TNFα are concentration-dependent and whether chondrocyte expansion has an effect on the inflammatory model. Bovine chondrocytes were enzymatically isolated, expanded to passages 1-3, and transferred into a 3D pellet culture. Chondrocyte pellets were stimulated with recombinant bovine TNFα at different concentrations for 48 h to induce inflammation. Gene expression of anabolic (collagen 2, aggrecan, cartilage oligomeric protein (COMP)), catabolic (matrix metalloproteinases (MMP3, MMP13)), dedifferentiation (collagen 1) markers, inflammation markers (interleukin-6 (IL-6), nuclear factor kappa B (NFkB), cyclooxygenase-2 (COX), prostaglandin-E-synthase-2 (PTGES2)), and the apoptosis marker caspase 3 was determined. At the protein level, concentrations of IL-6, nitric oxide (NO), and sulfated glycosaminoglycans (GAG) were evaluated. Statistical analysis was performed using the independent t-test, and significance was defined as p < 0.05. In general, TNFα caused a decrease in anabolic markers and an increase in the expression of catabolic and inflammatory markers. There was a concentration-dependent threshold of 10 ng/mL to induce significant inflammatory effects. Most of the markers analyzed showed TNFα concentration-dependent effects (COMP, PRG4, AGN, Col1, MMP3, and NFkB). There was a statistical influence of selected gene expression markers from different passages on the TNFα chondrocyte inflammation model, including Col2, MMP13, IL-6, NFkB, COX2, and PTGES2. Considering the expression of collagen 2 and MMP3, passage 3 chondrocytes showed a higher sensitivity to TNFα stimulation compared to passages 1 and 2. On the other hand, MMP13, IL-6, NFkB, and caspase 3 gene expression were lower in P3 chondrocytes compared to the other passages. On the protein level, inflammatory effects showed a similar pattern, with cytokine effects starting at 10 ng/mL and differences between the passages. TNFα had a detrimental effect on cartilage, with a clear threshold observed at 10 ng/mL. Although TNFα effects showed concentration-dependent patterns, this was not consistent for all markers. The selected passage showed a clear influence, especially on inflammation markers. Further experiments were warranted to explore the effects of TNFα concentration and passage in long-term stimulation.

Temporomandibular joint disc responses to installation and removal of the experimental malocclusion.

Aberrant occlusion and aging are two main risks for temporomandibular joint (TMJ) degeneration. To assess the combined impact of occlusion and age on TMJ disc. To avoid the confounding impact of gender, presently, 126 female C57BL/6J mice, 63 youngsters, 6-week old and 63 adults, 28-week old, were used. An experimental bilateral anterior crossbite (BAC) relation was created by installing metal tubes onto the mandibular incisors. Mice were sacrificed at 3, 7 and 11 weeks (n = 9). Additionally, the installed tubes were removed at 7 weeks in removal groups and the TMJs were sampled after another 4 weeks (n = 9). Disc changes were detected by histomorphology, immunohistochemistry, and western blot assays. Disc deformation was obvious in BAC groups. The typical change was hyperplasia at the posterior region of the disc where there was significant infiltration of inflammatory cells. Expressions of the inflammatory markers, including tumour necrosis factor-α and interleukin-1β, and the catabolic markers, including fibronectin (FN), FN N-terminal fragments, and vascular endothelial growth factor-A, were all increased. The changes were more obvious in adults than in youngsters. Removal of BAC attenuated inflammatory and catabolic changes in the youngsters, but the inflammatory markers recovered little in the adults. TMJ disc responds to BAC by degeneration and inflammation, and respond to BAC removal by rehabilitation. Adult discs show severer degeneration responses to BAC and a lower level of anti-inflammatory capability to BAC removal than the youngster's discs. Animals cannot be equated with humans. The human disc response to occlusion changes worth further exploration.

Palmitoylethanolamide Does Not Affect Recovery from Exercise-Induced Muscle Damage in Healthy Males.

Strenuous eccentric exercise (EE) induces microstructural muscle damage, which decreases muscle performance. Palmitoylethanolamide (PEA) exerts analgesic and anti-inflammatory effects in clinical pain conditions and preclinical models of experimentally induced-inflammation. This might hold clues for improved recovery from EE. Therefore, the current study evaluates the effect of PEA supplementation on functional and molecular responses to a single EE bout. Eleven healthy male participants were included in a double-blind crossover study in which they received PEA (350 mg Levagen+) or placebo (maltodextrin) supplements, in a randomized order. In each experimental condition participants performed an acute bout of EE (24x10 eccentric contractions of the knee extensors on an isokinetic dynamometer). At baseline, 24 (D1), 48 (D2), 72 (D3) and 120 h (D5) following EE, maximal voluntary contraction and jump height were measured. Blood samples were collected at baseline and on D1-D5, and muscle biopsies were collected at baseline and on D2. Perceived muscle soreness, sleep quality and food intake were recorded daily. Muscle strength and jump height decreased following EE (up to ~40 and ~ 17% respectively; Ptime < 0.05) in both conditions. This drop was accompanied by an increase in plasma creatine kinase and perceived muscle soreness (Ptime < 0.05). Furthermore, EE, but not PEA, increased the expression of the myogenic marker Pax7 and of the catabolic markers p-FoxO1-3a, p62 and LC3BII/I (Ptime < 0.05). PEA supplementation does not improve muscle soreness, muscle strength and jump performance following a single EE bout. Additionally, PEA supplementation had no effect on local or systemic markers of muscle damage, catabolism or regeneration.

Alpha-ketoglutarate ameliorates age-related and surgery induced temporomandibular joint osteoarthritis via regulating IKK/NF-κB signaling.

Recent studies have shed light on the important role of aging in the pathogenesis of joint degenerative diseases and the anti-aging effect of alpha-ketoglutarate (αKG). However, whether αKG has any effect on temporomandibular joint osteoarthritis (TMJOA) is unknown. Here, we demonstrate that αKG administration improves condylar cartilage health of middle-aged/aged mice, and ameliorates pathological changes in a rat model of partial discectomy (PDE) induced TMJOA. Invitro, αKG reverses IL-1β-induced/H2O2-induced decrease of chondrogenic markers (Col2, Acan and Sox9), and inhibited IL-1β-induced/ H2O2-induced elevation of cartilage catabolic markers (ADAMTS5 and MMP13) in condylar chondrocytes. In addition, αKG downregulates senescence-associated (SA) hallmarks of aged chondrocytes, including the mRNA/protein level of SA genes (p16 and p53), markers of nuclear disorders (Lamin A/C) and SA-β-gal activities. Mechanically, αKG decreases the expressions of p-IKK and p-NF-κB, protecting TMJ from inflammation and senescence-related damage by regulating the NF-κB signaling. Collectively, our findings illuminate that αKG can ameliorate age-related TMJOA and PDE-induced TMJOA, maintain the homeostasis of cartilage matrix, and exert anti-aging effects in chondrocytes, with a promising therapeutic potential in TMJOA, especially age-related TMJOA.

Bioreactor development for skeletal muscle hypertrophy and atrophy by manipulating uniaxial cyclic strain: proof of concept

Skeletal muscles overcome terrestrial, gravitational loading by producing tensile forces that produce movement through joint rotation. Conversely, the microgravity of spaceflight reduces tensile loads in working skeletal muscles, causing an adaptive muscle atrophy. Unfortunately, the design of stable, physiological bioreactors to model skeletal muscle tensile loading during spaceflight experiments remains challenging. Here, we tested a bioreactor that uses initiation and cessation of cyclic, tensile strain to induce hypertrophy and atrophy, respectively, in murine lineage (C2C12) skeletal muscle myotubes. Uniaxial cyclic stretch of myotubes was conducted using a StrexCell® (STB-1400) stepper motor system (0.75 Hz, 12% strain, 60 min day^-1). Myotube groups were assigned as follows: (a) quiescent over 2- or (b) 5-day (no stretch), (c) experienced 2-days (2dHY) or (d) 5-days (5dHY) of cyclic stretch, or (e) 2-days of cyclic stretch followed by a 3-day cessation of stretch (3dAT). Using ß-sarcoglycan as a sarcolemmal marker, mean myotube diameter increased significantly following 2dAT (51%) and 5dAT (94%) vs. matched controls. The hypertrophic, anabolic markers talin and Akt phosphorylation (Thr308) were elevated with 2dHY but not in 3dAT myotubes. Inflammatory, catabolic markers IL-1ß, IL6, and NF-kappaB p65 subunit were significantly higher in the 3dAT group vs. all other groups. The ratio of phosphorylated FoxO3a/total FoxO3a was significantly lower in 3dAT than in the 2dHY group, consistent with elevated catabolic signaling during unloading. In summary, we demonstrated proof-of-concept for a spaceflight research bioreactor, using uniaxial cyclic stretch to produce myotube hypertrophy with increased tensile loading, and myotube atrophy with subsequent cessation of stretch.

Protein Ingredient Quality within Infant Formulas Impacts Plasma Amino Acid Concentrations in Neonatal Minipiglets

BackgroundInfant formulas (IFs), the only adequate substitute to human milk, are complex matrices that require numerous ingredients and processing steps that may impact protein digestion and subsequent amino acid (AA) absorption. ObjectivesThe objective was to understand the impact of the protein ingredient quality within IFs on postprandial plasma AA profiles. MethodsFour isonitrogenous and isocaloric IFs were produced at a semi-industrial scale using whey proteins from different origins (cheese compared with ideal whey) and denaturation levels (IF-A, -B, -C), and caseins with different supramolecular organizations (IF-C, -D). Ten Yucatan minipiglets (12- to 27-d-old) were used as a human infant model and received each IF for 3 d according to a Williams Latin square followed by a 2-d wash-out period. Jugular plasma was regularly sampled from 10 min preprandial to 4 h postprandial on the third day to measure free AAs, urea, insulin, and glucose concentrations. Data were statistically analyzed using a mixed linear model with diet (IFs), time, and sex as fixed factors and piglet as random factor. ResultsIFs made with cheese whey (IF-A and -B) elicited significantly higher plasma total and essential AA concentrations than IFs made with ideal whey (IF-C and -D), regardless of the pre- and postprandial times. Most of the differences observed postprandially were explained by AA homeostasis modifications. IFs based on cheese whey induced an increased plasma concentration of Thr due to both a higher Thr content in these IFs and a Thr-limiting degrading capability in piglets. The use of a nonmicellar casein ingredient led to reduced plasma content of AA catabolism markers (IF-D compared with IF-C). ConclusionsOverall, our results highlight the importance of the protein ingredient quality (composition and structure) within IFs on neonatal plasma AA profiles, which may further impact infant protein metabolism.

Cytokine Receptor-like Factor 1 (CRLF1) and Its Role in Osteochondral Repair.

Since cytokine receptor-like factor 1 (CRLF1) has been implicated in tissue regeneration, we hypothesized that CRLF1 released by mesenchymal stem cells can promote the repair of osteochondral defects. The degree of a femoral osteochondral defect repair in rabbits after intra-articular injections of bone marrow-derived mesenchymal stem cells (BMSCs) that were transduced with empty adeno-associated virus (AAV) or AAV containing CRLF1 was determined by morphological, histological, and micro computer tomography (CT) analyses. The effects of CRLF1 on chondrogenic differentiation of BMSCs or catabolic events of interleukin-1beta-treated chondrocyte cell line TC28a2 were determined by alcian blue staining, gene expression levels of cartilage and catabolic marker genes using real-time PCR analysis, and immunoblot analysis of Smad2/3 and STAT3 signaling. Intra-articular injections of BMSCs overexpressing CRLF1 markedly improved repair of a rabbit femoral osteochondral defect. Overexpression of CRLF1 in BMSCs resulted in the release of a homodimeric CRLF1 complex that stimulated chondrogenic differentiation of BMSCs via enhancing Smad2/3 signaling, whereas the suppression of CRLF1 expression inhibited chondrogenic differentiation. In addition, CRLF1 inhibited catabolic events in TC28a2 cells cultured in an inflammatory environment, while a heterodimeric complex of CRLF1 and cardiotrophin-like Cytokine (CLC) stimulated catabolic events via STAT3 activation. A homodimeric CRLF1 complex released by BMSCs enhanced the repair of osteochondral defects via the inhibition of catabolic events in chondrocytes and the stimulation of chondrogenic differentiation of precursor cells.

Endocannabinoid remodeling in murine cachexic muscle associates with catabolic and metabolic regulation

Muscle degeneration is a common feature in cancer cachexia that cannot be reversed. Recent advances show that the endocannabinoid system, and more particularly cannabinoid receptor 1 (CB1), regulates muscle processes, including metabolism, anabolism and regenerative capacity. However, it is unclear whether muscle endocannabinoids, their receptors and enzymes are responsive to cachexia and exercise. Therefore, this study investigated whether cachexia and exercise affected muscle endocannabinoid signaling, and whether CB1 expression correlated with markers of muscle anabolism, catabolism and metabolism. Male BALB/c mice were injected with PBS (CON) or C26 colon carcinoma cells (C26) and had access to wheel running (VWR) or remained sedentary (n = 5–6/group). Mice were sacrificed 18 days upon PBS/tumor cell injection. Cachexic mice exhibited a lower muscle CB1 expression (−43 %; p < 0.001) and lower levels of the endocannabinoid anandamide (AEA; −22 %; p = 0.044), as well as a lower expression of the AEA-synthesizing enzyme NAPE-PLD (−37 %; p < 0.001), whereas the expression of the AEA degrading enzyme FAAH was higher (+160 %; p < 0.001). The 2-AG-degrading enzyme MAGL, was lower in cachexic muscle (−34 %; p = 0.007), but 2-AG and its synthetizing enzyme DAGLβ were not different between CON and C26. VWR increased muscle CB1 (+25 %; p = 0.005) and increased MAGL expression (+30 %; p = 0.035). CB1 expression correlated with muscle mass, markers of metabolism (e.g. p-AMPK, PGC1α) and of catabolism (e.g. p-FOXO, LC3b, Atg5). Our findings depict an emerging role of the endocannabinoid system in muscle physiology. Future studies should elaborate how this translates into potential therapies to combat cancer cachexia, and other degenerative conditions.

Lei's formula attenuates osteoarthritis mediated by suppression of chondrocyte senescence via the mTOR axis: in vitro and in vivo experiments.

Lei's formula (LSF), a traditional Chinese herbal remedy, is recognized for its remarkable clinical effectiveness in treating osteoarthritis (OA). Despite its therapeutic potential, the exact molecular mechanisms underlying LSF's action in OA have remained enigmatic. Existing research has shed light on the role of the mTOR signaling pathway in promoting chondrocyte senescence, a central factor in OA-related cartilage degeneration. Consequently, targeting mTOR to mitigate chondrocyte senescence presents a promising avenue for OA treatment. The primary objective of this study is to establish LSF's chondroprotective potential and confirm its anti-osteoarthritic efficacy through mTOR inhibition. In vivo assessments using an OA mouse model reveal substantial articular cartilage degeneration. However, LSF serves as an effective guardian of articular cartilage, evidenced by reduced subchondral osteosclerosis, increased cartilage thickness, improved surface smoothness, decreased OARSI scores, elevated expression of cartilage anabolic markers (Col2 and Aggrecan), reduced expression of catabolic markers (Adamts5 and MMP13), increased expression of the chondrocyte hypertrophy marker (Col10), and decreased expression of chondrocyte senescence markers (P16 and P21). In vitro findings demonstrate that LSF shields chondrocytes from H2O2-induced apoptosis, inhibits senescence, enhances chondrocyte differentiation, promotes the synthesis of type II collagen and proteoglycans, and reduces cartilage degradation. Mechanistically, LSF suppresses chondrocyte senescence through the mTOR axis, orchestrating the equilibrium between chondrocyte anabolism and catabolism, ultimately leading to reduced apoptosis and decelerated OA cartilage degradation. LSF holds significant promise as a therapeutic approach for OA treatment, offering new insights into potential treatments for this prevalent age-related condition.

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.

A MODEL TO EXPLORE INTERVERTEBRAL DISC CELL ACTIVITY IN ADVERSE BIOCHEMICAL ENVIRONMENTS

Intervertebral disc (IVD) degeneration (IDD) involves imbalance between the anabolic and the catabolic processes that regulate the extracellular matrix of its tissues. These processes are complex, and improved integration of knowledge is needed. Accordingly, we present a nucleus pulposus cell (NPC) regulatory network model (RNM) that integrates critical biochemical interactions in IVD regulation and can replicate experimental results. The RNM was built from a curated corpus of 130 specialized journal articles. Proteins were represented as nodes that interact through activation and inhibition edges. Semi-quantitative steady states (SS) of node activations were calculated. Then, a full factorial sensitivity analysis (SA) identified which out of the RNM 15 cytokines, and 4 growth factors affected most the structural proteins and degrading enzymes. The RNM was further evaluated against metabolic events measured in non-healthy human NP explant cultures, after 2 days of 1ng/ml IL-1B catabolic induction. The RNM represented successfully an anabolic basal SS, as expected in normal IVD. IL-1B was able to increase catabolic markers and angiogenic factors and decrease matrix proteins. Such activity was confirmed by the explant culture measurements. The SA identified TGF-β and IL1RA as the two most powerful rescue mediators. Accordingly, TGFβ signaling-based IDD treatments have been proposed and IL-1RA gene therapy diminished the expression of proteases. It resulted challenging to simulate rescue strategies by IL-10, but interestingly, IL-1B could not induce IL-10 expression in the explant cultures. Our RNM was confronted to independent in vitro measurements and stands for a unique model, to integrate soluble protein signaling and explore IDD.Acknowledgements: European Commission (Disc4All-ITN-ETN-955735)

MESENCHYMAL STEM CELLS MAINTAIN ARTICULAR STABILIZATION AND PROMOTE ENDOGENOUS CARTILAGE REPAIR AFTER HIGH TIBIAL OSTEOTOMY: A SECOND-LOOK ARTHROSCOPY STUDY

Varus malalignment increases the susceptibility of cartilage to mechanical overloading, which stimulates catabolic metabolism to break down the extracellular matrix and lead to osteoarthritis (OA). The altered mechanical axis from the hip, knee to ankle leads to knee joint pain and ensuing cartilage wear and deterioration, which impact millions of the aged population. Stabilization of the remaining damaged cartilage, and prevention of further deterioration, could provide immense clinical utility and prolong joint function. Our previous work showed that high tibial osteotomy (HTO) could shift the mechanical stress from an imbalanced status to a neutral alignment. However, the underlying mechanisms of endogenous cartilage stabilization after HTO remain unclear. We hypothesize that cartilage-resident mesenchymal stem cells (MSCs) dampen damaged cartilage injury and promote endogenous repair in a varus malaligned knee. The goal of this study is to further examine whether HTO-mediated off-loading would affect human cartilage-resident MSCs' anabolic and catabolic metabolism. This study was approved by IACUC at Xi'an Jiaotong University. Patients with medial compartment OA (52.75±6.85 yrs, left knee 18, right knee 20) underwent open-wedge HTO by the same surgeons at one single academic sports medicine center. Clinical data was documented by the Epic HIS between the dates of April 2019 and April 2022 and radiographic images were collected with a minimum of 12 months of follow-up. Medial compartment OA with/without medial meniscus injury patients with unilateral Kellgren /Lawrence grade 3–4 was confirmed by X-ray. All incisions of the lower extremity healed well after the HTO operation without incision infection. Joint space width (JSW) was measured by uploading to ImageJ software. The Knee injury and Osteoarthritis Outcome Score (KOOS) toolkit was applied to assess the pain level. Outerbridge scores were obtained from a second-look arthroscopic examination. RNA was extracted to quantify catabolic targets and pro-inflammatory genes (QiaGen). Student's t test for two group comparisons and ANOVA analysis for differences between more than 2 groups were utilized. To understand the role of mechanical loading-induced cartilage repair, we measured the serial changes of joint space width (JSW) after HTO for assessing the state of the cartilage stabilization. Our data showed that HTO increased the JSW, decreased the VAS score and improved the KOOS score significantly. We further scored cartilage lesion severity using the Outerbridge classification under a second-look arthroscopic examination while removing the HTO plate. It showed the cartilage lesion area decreased significantly, the full thickness of cartilage increased and mechanical strength was better compared to the pre-HTO baseline. HTO dampened medial tibiofemoral cartilage degeneration and accelerate cartilage repair from Outerbridge grade 2 to 3 to Outerbridge 0 to 1 compared to untreated varus OA. It suggested that physical loading was involved in HTO-induced cartilage regeneration. Given that HTO surgery increases joint space width and creates a physical loading environment, we hypothesize that HTO could increase cartilage composition and collagen accumulation. Consistent with our observation, a group of cartilage-resident MSCs was identified. Our data further showed decreased expression of RUNX2, COL10 and increased SOX9 in MSCs at the RNA level, indicating that catabolic activities were halted during mechanical off-loading. To understand the role of cartilage-resident MSCs in cartilage repair in a biophysical environment, we investigated the differentiation potential of MSCs under 3-dimensional mechanical loading conditions. The physical loading inhibited catabolic markers (IL-1 and IL-6) and increased anabolic markers (SOX9, COL2).Knee-preserved HTO intervention alleviates varus malalignment-related knee joint pain, improves daily and recreation function, and repairs degenerated cartilage of medial compartment OA. The off-loading effect of HTO may allow the mechanoregulation of cartilage repair through the differentiation of endogenous cartilage-derived MSCs.

Cyaonoside A-loaded composite hydrogel microspheres to treat osteoarthritis by relieving chondrocyte inflammation.

Cyaonoside A (CyA), derived from the natural Chinese medicine, Cyathula officinalis Kuan, which was for a long time used to treat knee injuries and relieve joint pain in traditional Chinese medicine, showed an unclear mechanism for protecting cartilage. In addition, CyA was poorly hydrosoluble and incapable of being injected directly into the joint cavity, which limited its clinical application. This study reveals that CyA resisted IL-1β-mediated chondrogenic inflammation and apoptosis. Next, transcriptome sequencing is used to explore the potential mechanisms underlying CyA regulation of MSC chondrogenic differentiation. Based on these findings, CyA-loaded composite hydrogel microspheres (HLC) were developed and they possessed satisfactory loading efficiency, a suitable degradation rate and good biocompatibility. HLC increased chondrogenic anabolic gene (Acan, COL2A, and SOX9) expression, while downregulating the expression of the catabolic marker MMP13 in vitro. In the osteoarthritis mouse model, HLC demonstrated promising therapeutic capabilities by protecting the integrity of articular cartilage. In conclusion, this study provides insights into the regulatory mechanisms of CyA for chondrocytes and proposes a composite hydrogel microsphere-based advanced therapeutic strategy for osteoarthritis.

The silencing of NREP aggravates OA cartilage damage through the TGF-β1/Smad2/3 pathway in chondrocytes

BackgroundOsteoarthritis (OA) is a common chronic degenerative joint disease. Due to the limited understanding of its complex pathological mechanism, there is currently no effective treatment that can alleviate or even reverse cartilage damage associated with OA. With improvement in public databases, researchers have successfully identified the key factors involved in the occurrence and development of OA through bioinformatics analysis. The aim of this study was to screen for the differentially expressed genes (DEGs) between the normal and OA cartilage through bioinformatics, and validate the function of the TGF-β1/Smad2/3 pathway-related neuron regeneration related protein (NREP) in the articular cartilage. MethodsThe DEGs between the cartilage tissues of OA patients and healthy controls were screened by bioinformatics, and functionally annotated by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. The expression levels of the DEG in human and murine OA cartilage was verified by reverse transcription-quantitative PCR (RT-qPCR), Western blotting and immunohistochemistry (IHC). RT-qPCR, Western-blotting, Cell Counting Kit-8(CCK8) and EdU assays were used to evaluate the effects of knocking down NREP in normal chondrocytes, and the molecular mechanisms were investigated by RT-qPCR, Western blotting and IHC. ResultsIn this study, we identified NREP as a DEG in OA through bioinformatics analysis, and found that NREP was downregulated in the damaged articular cartilage of OA patients and mouse model with surgically-induced OA. In addition, knockdown of NREP in normal chondrocytes reduced their proliferative capacity, which is the pathological basis of OA. At the molecular level, knock-down of NREP inactivated the TGF-β1/Smad2/3 pathway, resulting in the downregulation of the anabolic markers Col2a1 and Sox9, and an increase in the expression of the catabolic markers MMP3 and MMP13. ConclusionNREP plays a key role in the progression of OA by regulating the TGF-β1/Smad2/3 pathway in chondrocytes, and warrants further study as a potential therapeutic target.

Fatty acid binding protein 4 (FABP4) induces chondrocyte degeneration via activation of the NF-κb signaling pathway.

The pathogenesis of osteoarthritis (OA) is still unclear. Fatty acid binding protein 4 (FABP4), a novel adipokine, has been found to play a role in OA. This study aimed to explore the role of NF-κB in FABP4-induced OA. In the invivo study, four pairs of 12-week-old male FABP4 knockout (KO) and wild-type (WT) mice were included. The activation of NF-κB was assessed. In parallel, 24 6-week-old male C57/Bl6 mice were fed a high-fat diet (HFD) and randomly allocated to four groups: daily oral gavage with (1) PBS solution; (2) QNZ (NF-κB-specific inhibitor, 1 mg/kg/d); (3) BMS309403 (FABP4-specific inhibitor, 30 mg/kg/d); and (4) BMS309403 (30 mg/kg/d) + QNZ (1 mg/kg/d). The diet and treatment were sustained for 4 months. The knee joints were obtained to assess cartilage degradation, NF-κB activation, and subchondral bone sclerosis. In the invitro study, a mouse chondrogenic cell line (ATDC5) was cultured. FABP4 was supplemented to stimulate chondrocytes, and the activation of NF-κB was investigated. In parallel, QNZ and NF-κB-specific siRNA were used to inhibit NF-κB. Invivo, the FABP4 WT mice had more significant NF-κB activation than the KO mice. Dual inhibition of FABP4 and NF-κB alleviated knee OA in mice. FABP4 has no significant effect on the activation of the JNK signaling pathway. Invitro, FABP4 directly activated NF-κB in chondrocytes. The use of QNZ and NF-κB-siRNA significantly alleviated the expression of catabolic markers of chondrocytes induced by FABP4. FABP4 induces chondrocyte degeneration by activating the NF-κB pathway.