Inhibition of the MALT1-LPCAT3 axis protects cartilage degeneration and osteoarthritis.

The proinflammatory cytokines and arachidonic acid (AA)-derived eicosanoids play a key role in cartilage degeneration in osteoarthritis (OA). The lysophosphatidylcholine acyltransferase 3 (LPCAT3) preferentially incorporates AA into the membranes. Our recent studies showed that MALT1 [mucosa-associated lymphoid tissue lymphoma translocation protein 1]) plays a crucial role in propagating inflammatory signaling triggered by IL-1ß and other inflammatory mediators in endothelial cells. The present study shows that LPCAT3 expression was up-regulated in both human and mice articular cartilage of OA, and correlated with severity of OA. The IL-1ß-induces cell death via upregulation of LPCAT3, MMP3, ADAMTS5, and eicosanoids via MALT1. Gene silencing or pharmacological inhibition of LPCAT3 or MALT1 in chondrocytes and human cartilage explants notably suppressed the IL-1ß-induced cartilage catabolism through inhibition of expression of MMP3, ADAMTS5, and also secretion of cytokines and eicosanoids. Mechanistically, overexpression of MALT1 in chondrocytes significantly upregulated the expression of LPCAT3 along with MMP3 and ADAMTS5 via c-Myc. Inhibition of c-Myc suppressed the IL-1ß-MALT1-dependent upregulation of LPCAT3, MMP3 and ADAMTS5. Consistent with the in vitro data, pharmacological inhibition of MALT1 or gene silencing of LPCAT3 using siRNA-lipid nanoparticles suppressed the synovial articular cartilage erosion, pro-inflammatory cytokines, and eicosanoids such as PGE2, LTB4, and attenuated osteoarthritis induced by the destabilization of the medial meniscus in mice. Overall, our data reveal a previously unrecognized role of the MALT1-LPCAT3 axis in osteoarthritis. Targeting the MALT1-LPCAT3 pathway with MALT1 inhibitors or siRNA-liposomes of LPCAT3 may become an effective strategy to treat OA by suppressing eicosanoids, matrix-degrading enzymes, and proinflammatory cytokines.

[Study on the mechanism of cross-linked hyaluronic acid-dexamethasone hydrogelin post-traumatic osteoarthritis].

Objective: To explore the mechanism of cross-linked hyaluronic acid-dexamethasone hydrogel (cHA-Dex) in inhibiting chondrocyte apoptosis and alleviating early post-traumatic osteoarthritis (PTOA). Methods: To generate PTOA model, anterior cruciate ligament transection (ACLT)was performed on SD rats (n=70), and the sham surgery group (n=70) was set as control. The changes in inflammatory indicators such as interleukin-1ß (IL-1ß), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), matrix metalloproteinase-3 (MMP-3), and matrix metalloproteinase-13 (MMP-13) in the joint lavage fluid were measured at different time points (1-14 days, 5 rats at each time point) after surgery. The cHA-Dex (0.5 mg/ml) hydrogel (experimental group, n=70) and ordinary low-molecular-weight hyaluronic acid (HA) hydrogel premixed with Dex, that was, HA-Dex (0.5 mg/ml) hydrogel (control group, n=70) were injected into the joint cavity of PTOA rats, and the release amount and cumulative release amount of Dex in the joint fluid of rats at each time point(1-14 days, 5 rats at each time point) were detected to reveal the release mechanism of cHA-Dex hydrogel. The cartilage of knee joint of patients with osteoarthritis (OA) who underwent knee arthroplasty in the Second Hospital of Shanxi Medical University from January 2020 to December 2022 was taken for in vitro tissue block culture (Outbridge score=1 or 2,n=18). After the cartilage tissue block was treated with cHA-Dex hydrogel premixed with 0.1, 0.2, and 0.5 mg/ml Dex, the mRNA expression levels of IL-1ß, IL-6, TNF-α, MMP-3, and MMP-13 in the articular cartilage tissue block were detected. OA chondrocytes were isolated from cartilage samples using enzymatic hydrolysis and cultured in vitro (n=18). Chondrocytes were divided into 4 groups: saline, cHA hydrogel, Dex (0.5 mg/ml), and cHA-Dex (0.5 mg/ml) hydrogel group. The effects of different interventions on chondrocyte proliferation and apoptosis were tested. Results: The Osteoarthritis Research Society International (OARSI) score of safranine O-solid green staining in PTOA group was 3.34±0.35, and it was 1.17±0.21 in Sham group(P=0.010). The Meachim score of knee joint osteophytes in PTOA rats was significantly higher than that in the Sham group (2.66±0.41 vs 0.22±0.17, P=0.010), indicating PTOA model in rat was established successfully. The cHA-Dex hydrogel, which corresponded to the peak changes of inflammatory factors in the joints of PTOA rats in the early stage, was also released in the early stage and sustained-released in the late stage. After the OA articular cartilage tissue block was treated with cHA-Dex hydrogel premixed with 0.1, 0.2, and 0.5 mg/ml Dex, the mRNA expression levels of IL-1 ß, IL-6, TNF-α, MMP-3, and MMP-13 in the tissue block were reduced significantly (all P<0.05) and in a dose-dependent manner. Compared with Dex (0.5 mg/ml) alone group, the apoptosis rate of cHA-Dex (0.5 mg/ml) hydrogel group was significantly reduced (0.60±0.07 vs 6.63±0.98, P=0.010).Compared with the normal saline or the cHA hydrogel alone group, the cHA-Dex (0.5 mg/ml) hydrogel group had significant cell proliferation, and the difference at each time point were all significant statistically (all P<0.05). Conclusion: For the early inflammation of PTOA, cHA-Dex hydrogel can not only inhibit cartilage inflammation, but also reverse the increased apoptosis and decreased proliferation rate of chondrocytes caused by Dex, and finally alleviate the progress of PTOA by releasing Dex.

TIMP-1 Protects Tight Junctions of Brain Endothelial Cells From MMP-Mediated Degradation.

OBJECTIVE: The blood-brain barrier (BBB) plays a critical role in central nervous system homeostasis, and the integrity of BBB is disrupted in many neurodegenerative diseases. Matrix metalloproteinases (MMPs) degrade the tight junctions (TJs) of endothelial cells and basement membrane components essential to BBB integrity, which leads to increased BBB permeability and allows inflammatory cells and neurotoxic substances to enter the brain. Tissue inhibitors of metalloproteinases (TIMPs), endogenous inhibitors of MMPs, regulate MMP activity, thereby maintaining BBB integrity. METHODS: The disruptive impacts of MMP-3 and MMP-9 on BBB and protective effect of TIMP-1 were investigated in a simplified in vitro model of the BBB, which was generated using rat brain microvascular endothelial cells (RBMEC). The main features of BBB formation, including permeability and the trans-endothelial electrical resistance (TEER), were monitored over time after the addition of MMP-3 and MMP-9 and their complexes with TIMP-1 inhibitor. RESULTS: Our results indicated that MMP-3 and MMP-9 caused a dose-dependent disruption of the BBB, with 1.5 µM MMPs resulting in an over threefold increase in permeability, while TIMP-1 inhibition protected the integrity of the BBB model and recovered TEER and permeability of RBMECs. The disruption and recovery of tight junction proteins of RBMECs after MMP and TIMP treatment were also detected using fluorescent microscopy. CONCLUSION: MMP-9 and MMP-3 disrupt the BBB by degrading tight junctions in endothelial cells, and TIMP-1 could inhibit the disruptive effect of MMP-3 and MMP-9 by showing potential as therapeutic protein against MMP-related diseases where BBB disruption plays a role.

Blocking TXNIP reduced IL-1ß Induced chondrocyte cell inflammation.

Osteoarthritis (OA) is the most common joint disease in the elderly and is characterized by progressive and irreversible degeneration of articular cartilage, particularly cartilage loss and callus formation. This study would like to investigate the important role and the molecular mechanism of OA progression following interleukin 1ß (IL-1ß)-induced chondrocyte injury regulated by TXNIP. In this study, high-purity mouse chondrocytes were obtained by enzymatic two-step digestion for primary culture. Toluidine blue staining and type II collagen immunofluorescence were used to identify cells through histochemical staining after slide mounting. The relative expression of TXNIP was detected by immunohistochemical staining and qRT-PCR.Aiming at the shRNA sequence of the TXNIP gene, the shRNA expression vector was constructed and packaged with lentivirus to form the lentiviral vector shTXNIP. After inhibiting the expression of TXNIP by transfecting shTXNIP into normal mouse chondrocytes, the CCK-8 kit was used for detecting its effect on cell proliferation after transfection, and the effect on chondrocyte apoptosis was detected by flow cytometry. The staining kit was used to detect the effect of TXNIP knockout on chondrocyte aging, and the differential expression of TNF, IL-6, MMP3, MMP13, ADAMTS-5 and type II collagen genes in chondrocytes was detected by RT-PCR and Western-bolt. Western blot was used to detect the expression of upstream-related protein P-ERK, downstream-related protein NLRP3 and Caspase1 after inflammatory injury of mouse articular chondrocytes. Results showed that the expression level of TXNIP in chondrocytes induced by different concentrations of il-1ß was proportional to the concentration. After silencing TXNIP by shRNA, cell proliferation increased, chondrocyte apoptosis was weakened, and chondrocyte aging was weakened. The differential expression of genes such as TNF, IL-6, MMP3, MMP13, ADAMTS-5 and type II collagen and the differential expression of protein levels were relatively decreased. In addition, the expression of the upstream-related protein P-ERK did not change much when TXNIP was silenced, and the expression levels of the downstream-related proteins NLRP3 and Caspase1 were slightly reduced. In conclusion, silencing TXNIP can inhibit il-1ß-induced chondrocyte apoptosis and aging, and has a positive effect on cell proliferation. However, this study has not clarified the molecular mechanism involved in TXNIP and the process of its signaling expression pathway.

Macrophage Migration Inhibitory Factor Promotes Expression of Matrix Metalloproteinases 1 and 3 in Spinal Cord Astrocytes following Gecko Tail Amputation.

BACKGROUND: The matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that play a variety of physiological and pathological roles in development, remodeling of tissues and diseases, mainly through degradation of various components of the extracellular matrix (ECM). Particularly, the MMPs have increasingly been found to mediate neuropathology following spinal cord injury (SCI). Proinflammatory mediators are potent activators of the MMPs. However, how the spinal cord regenerative vertebrates circumvent MMPs-mediated neuropathogenesis following SCI remains unclear. METHODS: Following the establishment of gecko tail amputation model, the correlation of MMP-1 (gMMP-1) and MMP-3 (gMMP-3) expression with that of macrophage migration inhibitory factor in gecko (gMIF) was assayed by RT-PCR, Western blot and immunohistochemistry. Transcriptome sequencing of primary astrocytes was performed to analyze the intracellular signal transduction of macrophage migration inhibitory factor (MIF). The effects of MMP-1 and MMP-3 induced by MIF on astrocyte migration were assessed by transwell migration assay. RESULTS: The expression of gMIF significantly increased at lesion site of the injured cord, in parallel with those of gMMP-1 and gMMP-3 in the gecko astrocytes (gAS). Transcriptome sequencing and in vitro cell model revealed that gMIF efficiently promoted the expression of gMMP-1 and gMMP-3 in gAS, which in turn contributed to the migration of gAS. Inhibition of gMIF activity following gecko SCI remarkably attenuated astrocytic expression of the two MMPs, and further influenced gecko tail regeneration. CONCLUSIONS: Gecko SCI following tail amputation promoted production of gMIF, which induced the expression of gMMP-1 and gMMP-3 in gAS. The gMIF-mediated gMMP-1 and gMMP-3 expression was involved in gAS migration and successful tail regeneration.

KMT5A Knockdown Suppresses Osteosarcoma Cell Proliferation and Metastasis Through Ꞵ-Catenin Signalling.

PURPOSE: Osteosarcoma (OS) is the most common malignant solid bone tumor in children and young adults. We aimed to investigate the effects and cellular mechanisms of KMT5A on OS cell activity. METHODS: The protein expression was evaluated in the clinical normal, adjacent and OS osteogenic tissues. Knockdown of KMT5A was achieved by KMT5A siRNAs in a human OS cell line, MG63, to detect cell proliferation and metastasis. RESULTS: KMT5A expression was upregulated in clinical OS tissues. Knockdown of KMT5A inhibited cell proliferation but enhanced cell death, with significantly reduced cyclinD1 and Bcl2 and increased cleaved-caspase9 levels. KMT5A knockdown also suppressed OS cell migration and invasion capacity and deceased MMP3 and vimentin expression. ß-catenin levels were upregulated in OS tissues and blocking KMT5A resulted in a significant decline in ß-catenin expression in the OS cells. Further administration of ß-catenin activator remarkably increased protein levels of KMT5A, cyclinD1, Bcl2, MMP3, and vimentin, which showed reversed effects of KMT5A knockdown on OS cell activity. CONCLUSION: KMT5A knockdown plays an inhibitory role in OS cell proliferation and metastasis through ß-catenin signalling, which provides basic evidence and suggests potential targets for OS therapeutic research.

Cyanidin-3-glucoside protects against high glucose-induced injury in human nucleus pulposus cells by regulating the Nrf2/HO-1 signaling.

Cyanidin-3-glucoside (C3G) is a well-known natural anthocyanin with antioxidant and anti-inflammatory properties. In this study, we explored the role and action mechanism of C3G in high glucose (HG)-induced damage of human nucleus pulposus cells (HNPCs). Cell viability was assessed by CCK-8 assay. TUNEL assay was performed for detecting apoptotic rate. Western blot was performed to determine the expression levels of cl-caspase-3, caspase-3, Bax, Bim, collagen II, aggrecan, MMP-3, MMP-13, and ADAMTS5. Reactive oxygen species (ROS) generation was analyzed using DCFH-DA staining. The Nrf2 was knocked down or overexpressed in HNPCs through transfection with si-Nrf2 or pcDNA3.0-Nrf2. C3G treatment (12.5, 25, and 50 µM) improved cell viability of HNPCs under HG condition. HG-induced cell apoptosis of HNPCs was attenuated by C3G with decreased apoptotic rate and relative levels of cl-caspase-3/caspase-3, Bax, and Bim. C3G treatment caused significant increase in expression levels of collagen II and aggrecan and decrease in the relative levels of MMP-3, MMP-13, and ADAMTS5. After treatment with C3G, ROS generation in HNPCs was markedly reduced. Treatment with N-acetylcysteine (NAC) reversed HG-induced cell apoptosis and extracellular matrix (ECM) degradation. C3G treatment induced the expression of Nrf2 and HO-1 in HG-induced HNPCs. Moreover, knockdown of Nrf2 reversed the inhibitory effect of C3G on ROS production. Summarily, C3G exerted a protective effect on ROS-mediated cellular damage in HNPCs under HG condition, which was attributed to the induction of the Nrf2/HO-1 signaling pathway.

Rongjin Niantong Fang ameliorates cartilage degeneration by regulating the SDF-1/CXCR4-p38MAPK signalling pathway.

CONTEXT: Rongjin Niantong Fang (RJNTF) is a Traditional Chinese Medicine formulation with a good therapeutic effect on osteoarthritis (OA). However, the underlying mechanisms remain unclear. OBJECTIVE: This study investigates whether RJNTF could delay OA cartilage degeneration by regulating the SDF-1/CXCR4-p38MAPK signalling pathway. MATERIALS AND METHODS: The Sprague-Dawley (SD) rats were used to establish the OA model by a modified Hulth's method. SD rats were divided into three groups (n = 10): blank group, model group (0.9% saline, 10 mL/kg/day), and treatment group (RJNTF, 4.5 g/kg/day). After 12 weeks of treatment, each group was analysed by H&E, Safranine-O solid green, ELISA, Immunohistochemistry, and Western blot. An in vitro model was induced with 100 ng/mL SDF-1 by ELISA, the blank group, model group, RJNTF group, and inhibitor group with intervention for 12 h, each group was analysed by Immunofluorescence staining and Western blot. RESULTS: SDF-1 content in the synovium was reduced in RJNTF treatment group compared to non-treatment model group (788.10 vs. 867.32 pg/mL) and down-regulation of CXCR4, MMP-3, MMP-9, MMP-13 protein expression, along with p38 protein phosphorylated were observed in RJNTF treatment group. In vitro results showed that RJNTF (IC50 = 8.925 mg/mL) intervention could down-regulate SDF-1 induced CXCR4 and p38 protein phosphorylated and reduce the synthesis of MMP-3, MMP-9, and MMP-13 proteins of chondrocytes from SD rat cartilage tissues. DISCUSSION AND CONCLUSION: RJNTF alleviates OA cartilage damage by SDF-1/CXCR4-p38MAPK signalling pathway inhibition. Our ongoing research focuses on Whether RJNTF treats OA through alternative pathways.

High-fat diet-induced obesity regulates MMP3 to modulate depot- and sex-dependent adipose expansion in C57BL/6J mice.

Increased adipocyte size is hypothesized to signal the recruitment of adipose progenitor cells (APCs) to expand tissue storage capacity. To investigate depot and sex differences in adipose growth, male and female C57BL/6J mice (10 wk-old) were challenged with high-fat (HF) or low-fat (LF) diets (D) for 14 wk. The HFD increased gonadal (GON) depot weight by adipocyte hypertrophy and hyperplasia in females but hypertrophy alone in males. In both sexes, inguinal (ING) adipocytes were smaller than GON, and depot expansion was due to hypertrophy. Matrix metalloproteinase 3 (Mmp3), an antiadipogenic factor, and its inhibitor Timps modulate the extracellular matrix remodeling needed for depot expansion. Mmp3 mRNA was depot different (ING > GON), higher in females than males and mainly expressed in APCs. In males, HFD-induced obesity increased tissue and APC Mmp3 mRNA levels and MMP3 protein and enzymatic activity. In females however, HFD significantly decreased MMP3 protein without affecting its mRNA levels. MMP3 activity also decreased (significant in ING). Timp4 mRNA was expressed mainly in adipocytes, and HFD-induced obesity tended to increase the ratio of TIMP4 to MMP3 protein in females, whereas it decreased it in males. Overexpression of Mmp3 in 3T3-L1 preadipocytes or rhMMP3 protein added to primary human preadipocytes inhibited differentiation, whereas rhTIMP4 improved adipogenesis and attenuated the inhibitory effect of rhMMP3. These data suggest that HFD-induced obesity downregulates APC MMP3 expression to trigger adipogenesis, and adipocyte TIMP4 may modulate this process to regulate hyperplastic vs. hypertrophic adipose tissue expansion, fat distribution, and metabolic health in a sex- and depot-dependent manner.

Lack of matrix metalloproteinase 3 in mouse models of lung injury ameliorates the pulmonary inflammatory response in female but not in male mice.

BACKGROUND: The acute respiratory distress syndrome (ARDS) is a complex pulmonary disorder in which the local release of cytokines and chemokines appears central to the pathophysiology. OBJECTIVE: Based on the known role of matrix metalloproteinase-3 (MMP3) in inflammatory processes, the objective was to examine the role of MMP3 in the pathogenesis of ARDS through the modulation of pulmonary inflammation. MATERIALS AND METHODS: Female and male, wild type (MMP3+/+) and knock out (MMP3-/-) mice were exposed to two, clinically relevant models of ARDS including (i) lipopolysaccharide (LPS)-induced lung injury, and (ii) hydrochloric acid-induced lung injury. Parameters of lung injury and inflammation were assessed through measurements in lung lavage including total protein content, inflammatory cell influx, and concentrations of mediators such as TNF-α, IL-6, G-CSF, CXCL1, CXCL2, and CCL2. Lung histology and compliance were also evaluated in the LPS model of injury. RESULTS: Following intra-tracheal LPS instillation, all mice developed lung injury, as measured by an increase in lavage neutrophils, and decrease in lung compliance, with no overall effect of genotype observed. Increased concentrations of lavage inflammatory cytokines and chemokines were also observed following LPS injury, however, LPS-instilled female MMP3-/- mice had lower levels of inflammatory mediators compared to LPS-instilled female MMP3+/+ mice. This effect of the genotype was not observed in male mice. Similar findings, including the MMP3-related sex differences, were also observed after acid-induced lung injury. CONCLUSION: MMP3 contributes to the pathogenesis of ARDS, by affecting the pulmonary inflammatory response in female mice in relevant models of lung injury.

Extracellular matrix proteins regulate epithelial-mesenchymal transition in mammary epithelial cells.

Mouse mammary epithelial cells undergo transdifferentiation via epithelial-mesenchymal transition (EMT) upon treatment with matrix metalloproteinase-3 (MMP3). In rigid microenvironments, MMP3 upregulates expression of Rac1b, which translocates to the cell membrane to promote induction of reactive oxygen species and EMT. Here we examine the role of the extracellular matrix (ECM) in this process. Our data show that the basement membrane protein laminin suppresses the EMT response in MMP3-treated cells, whereas fibronectin promotes EMT. These ECM proteins regulate EMT via interactions with their specific integrin receptors. α6-integrin sequesters Rac1b from the membrane and is required for inhibition of EMT by laminin. In contrast, α5-integrin maintains Rac1b at the membrane and is required for the promotion of EMT by fibronectin. Understanding the regulatory role of the ECM will provide insight into mechanisms underlying normal and pathological development of the mammary gland.

Developing a mechanical and chemical model of degeneration in young bovine lumbar intervertebral disks and reversing loss in mechanical function.

STUDY DESIGN: A mechanical and chemical model of intervertebral disk (IVD) degeneration was developed by examining the enzymatic degradation of the nucleus pulposus (NP), and a gelatin-based restoration study was performed. OBJECTIVE: It was hypothesized that forced enzymatic degradation of the NP will mimic natural degeneration through the loss of disk height and that an injection of a gelatin solution will restore mechanical function. SUMMARY OF BACKGROUND DATA: Collagen and proteoglycans are essential for normal NP function. Their chemical destruction, combined with light mechanical loading, will mimic degeneration. Previous studies have determined that collagenase and matrix metalloproteinase-3 are directly implicated in IVD degradation; therefore, these enzymes were used in this model. MATERIALS AND METHODS: On the basis of preliminary testing, 0.5% collagenase, 1% collagenase, and 0.0025% metalloproteinase-3 in phosphate-buffered saline (PBS) were injected directly into the NP of various motion segments from a young bovine lumbar spine and subjected to light cyclic loading. To restore disk height and mechanical function, 20% gelatin in PBS at 70°C was injected into a degraded disk and subjected to the same loading conditions after an allotted hardening time. RESULTS: Mechanical testing showed statistically significant changes in disk height between control segments, 1% collagenase, and 0.5% collagenase. 0.5% collagenase had the most accurate appearance and loading pattern of degeneration upon disk transection postloading. A trend in restoration of disk function, given by the lessened loss of disk height upon loading, was observed with injection of gelatin after degradation with 0.5% collagenase. CONCLUSIONS: This study demonstrated the potential to create a degenerative model using enzymatic degradation of the NP and the possibility to restore function with an injectable therapy. Although gelatin is not a clinically viable option, it provides preliminary data for other injectable IVD therapies.

Sulforaphane activates CD8+ T cells antitumor response through IL-12RB2/MMP3/FasL-induced MDSCs apoptosis'.

BACKGROUND: Extensive attention has been given to the role of myeloid-derived suppressor cells (MDSCs) in driving tumor progression and treatment failure. Preclinical studies have identified multiple agents that eliminate MDSCs. However, none have been authorized in the cliniccal ues due to the safety reasons. In the present study, we investigated the efficacy and mechanism of sulforaphane (SFN) to eliminate MDSCs in the tumor microenvironment (TME). METHODS: We monitored SFN effect on tumor growth and the percents or apoptosis of immune cell subsets in mice models bearing LLC or B16 cells. Flow cytometry, quantitative reverse transcription-PCR, immunohistochemistry, ELISA, immunofluorescence, imaging flow cytometry and western blot were performed to validate the role of SFN on MDSCs function in vivo and in vitro. RNA sequencing was then used to interrogate the mechanisms of how SFN regulated MDSCs function. Tumor xenograft models were established to evaluate the involvement of IL-12RB2/MMP3/FasL induced MDSCs apoptosis in vivo. We verified the effect of SFN on MDSCs and CD8+ T cells in the blood samples from a phase I clinical trial (KY-2021-0350). RESULTS: In this study, we elucidated that SFN liberated CD8+ T-cell antitumor ability by reducing MDSCs abundance, leading to repressed tumor growth. SFN treatment suppressed MDSCs accumulation in the peripheral blood and tumor sites of mice, but had no effect on the bone marrow. Mechanistically, SFN activates IL-12RB2, which stimulates the MMP3/FasL signaling cascade to trigger caspase 3 cleavage and induce apoptosis in MDSCs. Clinically, SFN treatment eliminates peripheral MDSCs and increases the percentage and activation of CD8+ T cells. CONCLUSIONS: Collectively, we uncovered the role of SFN in eliminating MDSCs to emancipate CD8+ T cells through IL-12RB2/MMP3/FasL induced apoptosis, thus providing a strategy for targeting MDSCs to control tumors and improve clinical efficacy.

p38 MAPK alpha mediates cytokine-induced IL-6 and MMP-3 expression in human cardiac fibroblasts.

Pre-clinical studies suggest that the p38 MAPK signaling pathway plays a detrimental role in cardiac remodeling, but its role in cardiac fibroblast (CF) function is not well defined. We aimed to identify the p38 MAPK subtypes expressed by human CF, study their activation in response to proinflammatory cytokines, and determine which subtypes were important for expression of specific cytokines and matrix metalloproteinases (MMPs). Quantitative real-time RT-PCR analysis of mRNA levels in human CF cultured from multiple patients revealed a consistent pattern of expression with p38α being most abundant, followed by p38γ, then p38δ and only low expression of p38ß (3% of p38α mRNA levels). Immunoblotting confirmed marked protein expression of p38α, γ and δ, with little or no expression of p38ß. Phospho-ELISA and combined immunoprecipitation/immunoblotting techniques demonstrated that the proinflammatory cytokines IL-1α and TNFα selectively activated p38α and p38γ, but not p38δ. Selective p38α siRNA gene silencing reduced IL-1α-induced IL-6 and MMP-3 mRNA expression and protein secretion, without affecting IL-1α-induced IL-1ß and MMP-9 mRNA expression. In conclusion, human CF express the α, γ and δ subtypes of p38 MAPK, and the α subtype is important for IL-1α-induced IL-6 and MMP-3 expression in this cell type.

Characterization of the Proteins Secreted by Equine Muscle-Derived Mesenchymal Stem Cells Exposed to Cartilage Explants in Osteoarthritis Model.

BACKGROUND: Osteoarthritis (OA) is a highly prevalent joint degenerative disease for which therapeutic treatments are limited or invasive. Cell therapy based on mesenchymal stem/stromal cells (MSCs) is therefore seen as a promising approach for this disease, in both human and horses. As the regenerative potential of MSCs is mainly conferred by paracrine function, the goal of this study was to characterize the secreted proteins of muscle-derived MSCs (mdMSCs) in an in vitro model of OA to evaluate the putative clinical interest of mdMSCs as cell therapy for joint diseases like osteoarthritis. METHODS: An equine osteoarthritis model composed of cartilage explants exposed to pro-inflammatory cytokines was first developed. Then, the effects of mdMSC co-culture on cartilage explant were studied by measuring the glycosaminoglycan release and the NO2- production. To identify the underlying molecular actors, stable isotope-labeling by amino acids in cell culture based secreted protein analyses were conducted, in the presence of serum. The relative abundance of highly sequenced proteins was finally confirmed by western blot. RESULTS: Co-culture with muscle-derived MSCs decreases the cytokine-induced glycosaminoglycan release by cartilage explants, suggesting a protecting effect of mdMSCs. Among the 52 equine proteins sequenced in the co-culture conditioned medium, the abundance of decorin and matrix metalloproteinase 3 was significantly modified, as confirmed by western blot analyses. CONCLUSIONS: These results suggest that muscle-derived MSCs could reduce the catabolic effect of TNFα and IL-1ß on cartilage explant by decreasing the secretion and activity of matrix metalloproteinase 3 and increasing the decorin secretion. mdMSCs capacity to reduce the catabolic consequences of cartilage exposure to pro-inflammatory cytokines. These effects can be explained by mdMSC-secreted bioactive such as TIMP-1 and decorin, known as an inhibitor of MMP3 and an anti-inflammatory protein, respectively.

Osteoblasts Regulate the Expression of ADAMTS and MMPs in Chondrocytes through ERK Signaling Pathway.

OBJECTIVE: Degradative enzymes such as matrix metalloproteinase (MMP) and disintegrin metalloproteinase with platelet thrombin-sensitive protein-like motifs (ADAMTS) play a key role in the development of osteoarthritis (OA). We aimed to investigate the effects of OA subchondral osteoblasts on the expression of ADAMTS4, ADAMTS5, MMP-3, MMP-9, and MMP-13 in chondrocytes and the regulation of mitogen-activated protein kinase (MAPK) signaling pathway. METHODS: A rat knee OA model was constructed by cutting the anterior cruciate ligament of the knee joints, and normal rat articular cartilage chondrocytes (N-ACC), OA rat articular cartilage chondrocytes (O-ACC), normal subchondral bone osteoblasts (N-SBO), and OA subchondral bone osteoblasts (O-SBO) were isolated and extracted. The expressions of O-ACC and O-SBO COL1 and COL2 were detected respectively. Chondrocytes were identified by immunofluorescence of COL2 and toluidine blue staining, and osteoblasts were identified by COL1 immunofluorescence, alkaline phosphatase (ALP), and Alizarin Red staining. Gene expression of COL1, COL2, and aggrecan in normal chondrocytes and OA chondrocytes, and gene expression of osteoblast ALP and osteocalcin (OCN) were detected by RT-PCR to identify the two chondrocytes and the two osteoblast phenotypes. The constructing N-ACC group, O-ACC group, N-ACC + N-SBO group, N-ACC + O-SBO group, O-ACC + N-SBO group, O-ACC + O-SBO group, I + N-ACC + O-SBO group, and I + O-ACC + O-SBO group cell cultures, and the expression of ERK, ADAMTS4, ADAMTS5, MMP-3, MMP-9, and MMP-13 genes in chondrocytes cultured for 0, 24, 48, and 72 h were detected by RT-PCR. The protein expressions of pERK, ADAMTS4, ADAMTS5, MMP-3, MMP-9, and MMP-13 were detected by Western blot. RESULTS: · The X-ray showed that the knee joint space of the affected limb became narrow.. · The results of RT-PCR of COL2 and aggrecan gene in OA and normal chondrocytes suggest that the relative expression of COL2 in OA articular chondrocytes (0.24 ± 0.07) is significantly lower than that in normal cartilage (0.61 ± 0.07) (p < 0.05). The relative expression of AGG (0.37 ± 0.16) in OA chondrocytes was significantly lower than that of normal chondrocytes AGG (1.30 ± 0.25) (p < 0.05). The expression of COL1 was very low, and was not statistically significant.. · The results of RT-PCR of the osteoblast ALP and OCN gene indicated that gene expression of ALP (12.30 ± 1.17) and OCN (20.47 ± 4.19)was upregulated when compared with the relative expression of ALP (4.66 ± 0.71) (p < 0.05) and OCN (12.17 ± 2.76) (p < 0.05) in normal osteoblasts, indicating that osteoblasts of OA have greater osteogenic potential than normal osteoblasts.. · The expressions of ADAMTS4, ADAMTS5, MMP-3, MMP-9, and MMP-13 genes and proteins in OA chondrocytes or normal chondrocytes were basically unchanged when they were cocultured with normal osteoblasts. Indirect coculture of OA osteoblasts and chondrocytes could promote the expression of ADAMTS4, ADAMTS5, MMP-3, MMP-9, and MMP-13 genes and proteins in chondrocytes. Overexpression of ADAMTS and MMP in coculture systems can be reversed by MAPK-ERK inhibitors.. CONCLUSIONS: · OA subchondral bone osteoblasts can promote the overexpression of ADAMTS and MMPs in chondrocytes.. · The ERK signaling pathway may be involved in the regulation of the effect of subchondral bone osteoblasts on chondrocytes..

IRF1 promotes the chondrogenesis of human adipose-derived stem cells through regulating HILPDA.

BACKGROUND: Osteoarthritis is a main cause of deformity in aging people. The chondrogenesis of human adipose-derived stem cells (hADSCs) has a positive effect on the cure of osteoarthritis. However, the regulatory mechanism of hADSC chondrogenesis still needs further exploration. This research investigates the role of interferon regulatory factor 1 (IRF1) in the chondrogenesis of hADSCs. METHODS: hADSCs were purchased and cultured. The interaction between IRF1 and hypoxia inducible lipid droplet associated (HILPDA) was predicted by bioinformatics analysis, and verified through dual-luciferase reporter and chromatin immunoprecipitation assays. The expressions of IRF1 and HILPDA in osteoarthritis cartilage samples were measured through qRT-PCR. After hADSCs were transfected or further induced for chondrogenesis, the chondrogenesis was visualized by Alcian blue staining, and the expressions of IRF1, HILPDA and chondrogenesis-related factors (SOX9, Aggrecan, COL2A1, MMP13, MMP3) were determined through qRT-PCR or Western blot. RESULTS: HILPDA bound to IRF1 in hADSCs. IRF1 and HILPDA levels were up-regulated during the chondrogenesis of hADSCs. Overexpressions of IRF1 and HILPDA promoted the chondrogenesis of hADSCs with the up-regulation of SOX9, Aggrecan and COL2A1 and the down-regulation of MMP13 and MMP3; however, IRF1 silencing generated the opposite effects. Besides, HILPDA overexpression reversed the effects of IRF1 silencing on inhibiting chondrogenesis of hADSCs and regulating the expressions of chondrogenesis-related factors. CONCLUSION: IRF1 promotes the chondrogenesis of hADSCs through up-regulating HILPDA level, providing novel biomarkers for treating osteoarthritis.

Tenacissoside G alleviated osteoarthritis through the NF-κB pathway both in vitro and in vivo.

BACKGROUND: Osteoarthritis (OA) is a degenerative joint disease characterized by the destruction of articular cartilage. Tenacissoside G is a flavonoid isolated from the dry roots of Marsdenia tenacissima (Roxb) and has been shown to have anti-inflammatory effects. However, there is no report on the protective effects of Tenacissoside G on OA. OBJECTIVES: To identify the effects and mechanism of Tenacissoside G on OA. METHODS: In vitro, primary mouse chondrocytes were induced with IL-1ß to establish OA model. mRNA expression of MMP-13, MMP-3, TNF-α, IL-6 and iNOS, was detected by PCR. Protein expression of Collagen-II, MMP-13, p65, p-p65, and IκBα was detected by Western blot. Collagen-II in chondrocytes was also detected by immunofluorescence. In vivo, we established DMM OA mice model. The preventive effect of Tenacissoside G on OA was observed by micro-CT and histological analysis. RESULTS: In vitro, Tenacissoside G significantly inhibited the expression of iNOS, TNF-α, IL-6, MMP-3, MMP-13 and the degradation of collagen-II, Tenacissoside G also significantly suppressed NF-κB activation in chondrocytes by IL-1ß-stimulated. In vivo, we demonstrated Tenacissoside G can decrease articular cartilage damage and reduce OARSI score. CONCLUSION: These results suggest that Tenacissoside G may serve as a potential drug for the prevention and treatment of OA.

[Research of epigallocatechin gallate in delaying chondrocyte senescence].

Objective: To investigate the effect of epigallocatechin gallate (EGCG) on chondrocyte senescence and its mechanism. Methods: The chondrocytes were isolated from the articular cartilage of 4-week-old Sprague Dawley rats, and cultured with type Ⅱcollagenase and passaged. The cells were identified by toluidine blue staining, alcian blue staining, and immunocytochemical staining for type Ⅱ collagen. The second passage (P2) cells were divided into blank control group, 10 ng/mL IL-1ß group, and 6.25, 12.5, 25.0, 50.0, 100.0, and 200.0 µmol/L EGCG+10 ng/mL IL-1ß group. The chondrocyte activity was measured with cell counting kit 8 after 24 hours of corresponding culture, and the optimal drug concentration of EGCG was selected for the subsequent experiment. The P2 chondrocytes were further divided into blank control group (group A), 10 ng/mL IL-1ß group (group B), EGCG+10 ng/mL IL-1ß group (group C), and EGCG+10 ng/mL IL-1ß+5 mmol/L 3-methyladenine (3-MA) group (group D). After cultured, the degree of cell senescence was detected by ß-galactosidase staining, the autophagy by monodansylcadaverine method, and the expression levels of chondrocyte-related genes [type Ⅱ collagen, matrix metalloproteinase 3 (MMP-3), MMP-13] by real-time fluorescent quantitative PCR, the expression levels of chondrocyte-related proteins (Beclin-1, LC3, MMP-3, MMP-13, type Ⅱ collagen, P16, mTOR, AKT) by Western blot. Results: The cultured cells were identified as chondrocytes. Compared with the blank control group, the cell activity of 10 ng/mL IL-1ß group significantly decreased ( P<0.05). Compared with the 10 ng/mL IL-1ß group, the cell activity of EGCG+10 ng/mL IL-1ß groups increased, and the 50.0, 100.0, and 200.0 µmol/L EGCG significantly promoted the activity of chondrocytes ( P<0.05). The 100.0 µmol/L EGCG was selected for subsequent experiments. Compared with group A, the cells in group B showed senescence changes. Compared with group B, the senescence rate of chondrocytes in group C decreased, autophagy increased, the relative expression of type Ⅱ collagen mRNA increased, and relative expressions of MMP-3 and MMP-13 mRNAs decreased; the relative expressions of Beclin-1, LC3, and type Ⅱ collagen proteins increased, but the relative expressions of P16, MMP-3, MMP-13, mTOR, and AKT proteins decreased; the above differences were significant ( P<0.05). Compared with group C, when 3-MA was added in group D, the senescence rate of chondrocytes increased, autophagy decreased, and the relative expressions of the target proteins and mRNAs showed an opposite trend ( P<0.05). Conclusion: EGCG regulates the autophagy of chondrocytes through the PI3K/AKT/mTOR signaling pathway and exerts anti-senescence effects.

Chondrotoxic effects of intra-articular injection of local anaesthetics in the rabbit temporomandibular joint.

The aim of this study was to investigate the chondrotoxic effects of a single-dose intra-articular injection of articaine, lidocaine, and bupivacaine on the rabbit temporomandibular joint (TMJ). Twenty-four rabbits were divided into four groups: control (group 1), articaine (group 2), lidocaine (group 3), and bupivacaine (group 4). Synovial fluid samples and venous blood were taken to evaluate matrix metalloproteinase 3 (MMP-3) levels. One millilitre of local anaesthetic solution was injected in the study groups and saline solution in the control group. The rabbits were euthanized after 4 weeks and the mandibular condyles and articular discs were evaluated. On histological examination, the study group samples had irregular joint surfaces, decreased collagen, and a thinner cartilage layer. Apoptotic cells were evaluated with the TUNEL method. TUNEL-positive apoptotic cell counts were higher in all study groups compared to the control group, and the difference was significant (P < 0.001). The mean preoperative serum MMP-3 level for all groups was 5.71 ± 3.33 ng/mL, while the mean postoperative level was 22.61 ± 6.36 ng/mL; this difference was significant (P < 0.001). A single-dose intra-articular injection of local anaesthetic had apoptotic effects on chondrocytes, leading to degenerative changes in the TMJ articular structures. Articaine was found to have less harmful effects than lidocaine and bupivacaine. Intra-articular injection of local anaesthetics should be limited in the TMJ because of the potential toxic effects.