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.

Efficient chemosensitizing and antimetastatic combinations of a naturally occurring trans-ferulic acid with different chemotherapies on an in vitro hepatocellular carcinoma model.

Trans-ferulic acid (TFA) is a polyphenolic compound present in many dietary supplements. The aim of this study was to get better chemotherapeutic outcomes through treatment protocols for human hepatocellular carcinoma (HCC). This study focused on the exploration of the in vitro influence of a combination of TFA with 5-fluorouracil (5-FU), doxorubicin (DOXO), and cisplatin (CIS) on HepG2 cell line. Treatment with 5-FU, DOXO, and CIS alone down-regulated oxidative stress and alpha-fetoprotein (AFP), and decreased cell migration through the depression of metalloproteinases (MMP-3, MMP-9, and MMP-12) expression. Co-treatment with TFA synergized the effects of these chemotherapies by decreased MMP-3, MMP-9, and MMP-12 expression, and gelatinolytic activity of both MMP-9 and MMP-2 in cancer cells. TFA significantly reduced the elevated levels of AFP and NO, and depressed cell migration ability (metastasis) in HepG2 groups. Co-treatment with TFA elevated the chemotherapeutic potency of 5-FU, DOXO, and CIS in managing HCC.

Morphofunctional analysis of fibroblast-like synoviocytes in human rheumatoid arthritis and mouse collagen-induced arthritis.

BACKGROUND: Fibroblast-like synoviocytes (FLS) play a prominent role in rheumatoid synovitis and degradation of the extracellular matrix through the production of inflammatory cytokines and metalloproteinases (MMPs). Since animal models are frequently used for elucidating the disease mechanism and therapeutic development, it is relevant to study the ultrastructural characteristics and functional responses in human and mouse FLS. The objective of the study was to analyze ultrastructural characteristics, Interleukin-6 (IL-6) and Metalloproteinase-3 (MMP-3) production and the activation of intracellular pathways in Fibroblast like synoviocytes (FLS) cultures obtained from patients with rheumatoid arthritis (RA) and from mice with collagen-induced arthritis (CIA). METHODS: FLSs were obtained from RA patients (RA-FLSs) (n = 8) and mice with CIA (CIA-FLSs) (n = 4). Morphology was assessed by transmission and scanning electron microscopy. IL-6 and MMP-3 production was measured by ELISA, and activation of intracellular signaling pathways (NF-κB and MAPK: p-ERK1/2, p-P38 and p-JNK) was measured by Western blotting in cultures of RA-FLSs and CIA-FLSs stimulated with tumor necrosis factor-alpha (TNF-α) and IL-1ß. RESULTS: RA-FLS and CIA-FLS cultures exhibited rich cytoplasm, rough endoplasmic reticula and prominent and well-developed Golgi complexes. Transmission electron microscopy demonstrated the presence of lamellar bodies, which are cytoplasmic structures related to surfactant production, in FLSs from both sources. Increased levels of pinocytosis and numbers of pinocytotic vesicles were observed in RA-FLSs (p < 0.05). Basal production of MMP-3 and IL-6 was present in RA-FLSs and CIA-FLSs. Regarding the production of MMP-3 and IL-6 and the activation of signaling pathways, the present study demonstrated a lower response to IL-1ß by CIA-FLSs than by RA-FLSs. CONCLUSION: This study provides a comprehensive understanding of the biology of RA-FLS and CIA-FLS. The differences and similarities in ultrastructural morphology and important inflammatory cytokines shown, contribute to future in vitro studies using RA-FLS and CIA-FLS, in addition, they indicate that the adoption of CIA-FLS for studies should take careful and be well designed, since they do not completely resemble human diseases.

Oroxin B alleviates osteoarthritis through anti-inflammation and inhibition of PI3K/AKT/mTOR signaling pathway and enhancement of autophagy.

Background: Osteoarthritis (OA) is a common aging-related degenerative joint disease with chronic inflammation as its possible pathogenesis. Oroxin B (OB), a flavonoid isolated from traditional Chinese herbal medicine, possesses anti-inflammation properties which may be involved in regulating the pathogenesis of OA, but its mechanism has not been elucidated. Our study was the first to explore the potential chondroprotective effect and elucidate the underlying mechanism of OB in OA. Methods: In vitro, primary mice chondrocytes were stimulated with IL-1ß along with or without the administration of OB or autophagy inhibitor 3-methyladenine (3-MA). Cell viability assay was measured with a cell counting kit-8 (CCK-8). The phenotypes of anabolic-related (Aggrecan and Collagen II), catabolic-related (MMP3, MMP13, and ADAMTS5), inflammation-related (iNOS, COX-2, TNF-α, IL-6, and IL-1ß), and markers of related signaling pathways in chondrocytes with different treatment were detected through western blot, RT-qPCR, and immunofluorescent staining. In vivo, the destabilized medial meniscus (DMM) operation was performed to establish the OA mice model. After knee intra-articular injection with OB for 8 weeks, the mice's knee joints were obtained for subsequent histological staining and analysis. Results: OB reversed the expression level of anabolic-related proteins (Aggrecan and Collagen II) and catabolic-related (MMP3, MMP13, and ADAMTS5) in IL-1ß-induced chondrocytes. Mechanistically, OB suppressed the inflammatory response stimulated by IL-1ß, as the inflammation-related (iNOS, COX-2, TNF-α, IL-6, and IL-1ß) markers were downregulated after the administration of OB in IL-1ß-induced chondrocytes. Besides, the activation of PI3K/AKT/mTOR signaling pathway induced by IL-1ß could be inhibited by OB. Additionally, the autophagy process impaired by IL-1ß could be rescued by OB. What's more, the introduction of 3-MA to specifically inhibit the autophagic process impairs the protective effect of OB on cartilage. In vivo, histological staining revealed that intra-articular injection of OB attenuated the cartilage degradation, as well as reversed the expression level of anabolic and catabolic-related proteins such as Aggrecan, Collagen II, and MMP13 induced in DMM-induced OA models. Conclusions: The study verified that OB exhibited the chondroprotective effect by anti-inflammatory, inhibiting the PI3K/AKT/mTOR signaling pathway, and enhancing the autophagy process, indicating that OB might be a promising agent for the treatment of OA.

Effects of Modified Duhuo Jisheng Decoction Combined with Arthroscopic Surgery on Bone Metabolism, Oxidative Stress, and Serum TLR4 and TGF-ß1 in Patients with Knee Osteoarthritis.

Objective: To analyze the effects of modified Duhuo Jisheng Decoction combined with arthroscopic surgery on bone metabolism, oxidative stress, and serum TLR4 and TGF-ß1 in patients with knee osteoarthritis (KOA). Methods: Prospectively select 82 patients with KOA from January 2020 to January 2022 in our hospital and divide them into the control group and observation group according to the random number table method, with 41 patients in each group. The control group was treated with arthroscopic surgery alone and routine anti-infection after operation. The observation group was treated with Duhuo Jisheng Decoction on the basis of the treatment of the control group. The patients in the two groups were treated continuously for 4 weeks. The improvement of patients' symptoms was evaluated by the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC). Before treatment and 4 weeks after treatment, the scores of traditional Chinese medicine (TCM) symptoms, bone metabolism indicators (cartilage oligomeric matrix protein (COMP), collagen type II carboxy terminal peptide (ctx-II), and matrix metalloproteinase-3 (MMP-3)), oxidative stress indicators (superoxide dismutase (SOD), glutathione peroxidase (GSHPx), malondialdehyde (MDA), nitric oxide (NO)), serum Toll-like receptor 4 (TLR4), and transforming growth factor ß (TGF-ß) level were compared between the two groups. Results: After treatment, the WOMAC score of the two groups decreased (42.45 ± 10.83) in the observation group and (67.81 ± 14.63) in the control group. The WOMAC score of the observation group was lower than that of the control group (P < 0.05). After treatment, the levels of COMP, CTX-II, and MMP-3 in the two groups decreased, and the levels of COMP, CTX-II, and MMP-3 in the observation group were lower than those in the control group (P < 0.05). After treatment, the levels of SOD and GSHPx increased, while the levels of MDA and NO decreased in the two groups. The levels of SOD and GSHPx in the observation group were higher than those in the control group, while the levels of MDA and NO were lower than those in the control group (P < 0.05). After treatment, the TLR4 level in the observation group was lower than that of the control group, and the level of TGF-ß in the observation group was higher than that of the control group (P < 0.05). Conclusion: Compared with arthroscopic surgery alone, combined with modified Duhuo Jisheng Decoction can better alleviate the clinical symptoms of patients with KOA, improve their bone metabolism, oxidative stress indicators, and serum TLR4 and TGF-ß 1 level, and reduce the inflammatory injury of knee joint.

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.

Changes in salivary matrix metalloproteinase-3, -8, and -9 concentrations after 6 weeks of non-surgical periodontal therapy.

BACKGROUND: Studies using salivary inflammatory biomarkers for diagnosing and monitoring the progression of periodontal disease have garnered increased attention in recent years. The present study aimed to identify changes in clinical parameters and concentrations of salivary matrix metalloproteinases (MMPs) following 6 weeks of non-surgical periodontal therapy (NSPT). METHODS: A 6-week NSPT program was applied to 51 adults aged ≥ 20 years. The program involved scaling, root planing, and professional toothbrushing for healthy participants and those with periodontal disease. Patients with periodontal disease underwent professional toothbrushing during all three visits. Periodontal pocket depth (PD) and gingival bleeding were assessed at week 0, week 3, and week 6, and saliva samples were collected to measure the concentrations of MMP-3, -8, and -9. RESULTS: All clinical parameters were improved in the periodontal disease groups following the NSPT course. Compared with healthy participants, the patients with periodontal disease showed increased concentrations of salivary MMP-3, -8, and -9. During the 6-week program, patients with periodontal disease also showed significant reductions in PD and gingival bleeding during the third week; no significant reduction was found during the sixth week. Significant reductions in the concentrations of salivary MMP-3, -8, and -9 were also noted in the periodontal disease group at week 3. The sensitivity and specificity of MMP-3 for predicting periodontitis were 81.8% and 55.5%, respectively. CONCLUSION: The present study found that NSPT resulted in reductions of salivary MMP-3, -8, and -9, and identified the potential of MMP-3 as a biomarker in the diagnosis of periodontal disease. These findings may serve as foundational data for future studies into the development of diagnostic kits for periodontal disease.

Therapeutic potential of AAV-mediated MMP-3 secretion from corneal endothelium in treating glaucoma.

Intraocular pressure (IOP) is maintained as a result of the balance between production of aqueous humour (AH) by the ciliary processes and hydrodynamic resistance to its outflow through the conventional outflow pathway comprising the trabecular meshwork (TM) and Schlemm's canal (SC). Elevated IOP, which can be caused by increased resistance to AH outflow, is a major risk factor for open-angle glaucoma. Matrix metalloproteinases (MMPs) contribute to conventional aqueous outflow homeostasis in their capacity to remodel extracellular matrices, which has a direct impact on aqueous outflow resistance and IOP. We observed decreased MMP-3 activity in human glaucomatous AH compared to age-matched normotensive control AH. Treatment with glaucomatous AH resulted in significantly increased transendothelial resistance of SC endothelial and TM cell monolayers and reduced monolayer permeability when compared to control AH, or supplemented treatment with exogenous MMP-3.Intracameral inoculation of AAV-2/9 containing a CMV-driven MMP-3 gene (AAV-MMP-3) into wild type mice resulted in efficient transduction of corneal endothelium and an increase in aqueous concentration and activity of MMP-3. Most importantly, AAV-mediated expression of MMP-3 increased outflow facility and decreased IOP, and controlled expression using an inducible promoter activated by topical administration of doxycycline achieved the same effect. Ultrastructural analysis of MMP-3 treated matrices by transmission electron microscopy revealed remodelling and degradation of core extracellular matrix components. These results indicate that periodic induction, via use of an eye drop, of AAV-mediated secretion of MMP-3 into AH could have therapeutic potential for those cases of glaucoma that are sub-optimally responsive to conventional pressure-reducing medications.

The anti-inflammatory effects of matrix metalloproteinase-3 on irreversible pulpitis of mature erupted teeth.

Matrix metalloproteinases (MMPs) are involved in extracellular matrix degradation and the modulation of cell behavior. These proteinases have also been implicated in tissue repair and regeneration. Our previous studies have demonstrated that MMP-3 elicits stimulatory effects on the proliferation and the migration of endothelial cells as well as anti-apoptotic effects on these cells in vitro. In addition, we found that MMP-3 enhanced the regeneration of lost pulp tissue in a rat incisor pulp injury model. However, continuously erupting rodent incisors exhibit significantly different pulp organization compared with mature erupted teeth. Therefore, we have further extended these studies using a canine irreversible pulpitis model to investigate the effects of MMP-3. In this study, the crowns of the canine mature premolars were removed and the pulp tissues were amputated. The amputated pulp tissues remained exposed for 24 or 72 hours to induce mild or severe irreversible pulpitis, respectively, followed by sealing of the cavities. In both models, the whole pulp tissues became necrotic by day 14. In this mild pulpitis model, the regeneration of pulp tissue with vasculature and nerves was observed until 14 days after sealing with MMP-3, followed by extracellular matrix formation in the regenerated pulp tissues until day 28. The treatment with MMP-3 resulted in a decrease in the number of macrophage and antigen-presenting cells and a significant inhibition of IL-6 expression on day 3. The inhibition of MMP-3 activity abolished these anti-inflammatory effects. Immunofluorescence staining demonstrated that MMP-3 was involved in the modification of serum-derived hyaluronan-associated proteins and hyaluronan (SHAP-HA) complexes possibly through the degradation of versican. These results demonstrate that MMP-3 can act as an anti-inflammatory agent and suggest that MMP-3 might represent a useful therapy for the treatment of mild irreversible pulpitis.

Chemonucleolysis with human stromelysin-1.

STUDY DESIGN: Immunohistologic analysis was performed on surgically removed samples of herniated nucleus pulposus to examine the expression of stromelysin-1. We performed in vitro and in vivo experiments to determine whether recombinant human (rh) stromelysin-1 is capable of degrading nucleus pulposus. OBJECTIVE: To analyze the production of stromelysin-1 in various types of herniated nucleus pulposus, and to examine the effects of this recombinant protein on nucleus pulposus tissues. SUMMARY OF BACKGROUND DATA: The authors previously demonstrated a progressive decrease in herniated nucleus pulposus size in some of the transligamentous and sequestration types of herniated nucleus pulposus using magnetic resonance imaging. An increased production of stromelysin-1, a cartilage proteoglycan degrading enzyme, in herniated nucleus pulposus was reported recently. The authors speculated that if stromelysin-1 is involved in the degradation of herniated nucleus pulposus, stromelysin-1 itself may be used as a chemonucleolytic agent. METHODS: Immunohistologic analysis using streptoavidin-biotin method was performed on 20 herniated nucleus pulposus samples to investigate the expression of stromelysin-1. Five herniated nucleus pulposus samples were incubated in a tissue culture medium in the presence or absence of rh stromelysin-1. After 24 hours of incubation, their weight changes were measured, and the loss of proteoglycan was assessed by Safranin O staining. Rat nucleus pulposus tissues were obtained from coccygeal intervertebral discs, and autologous subcutaneous transplantation was performed. Rh stromelysin-1 was injected into the grafted materials, and the reduction in size was followed by two-dimensional measurements from the skin surface, using engineer's calipers. RESULTS: Immunohistologic analysis demonstrated the production of stromelysin-1 in the granulation tissues of herniated nucleus pulposus. When stromelysin-1 was injected into the murine nucleus pulposus tissues, they reduced in size more rapidly than the control group. In addition, human herniated nucleus pulposus materials obtained at surgery showed significant weight loss when treated with stromelysin-1 in an organ culture system. Safranin O staining revealed extensive depletion of proteoglycan in these herniated nucleus pulposus samples. CONCLUSIONS: Stromelysin-1 is a possible key enzyme in herniated nucleus pulposus resorption, and stromelysin-1 may be a good candidate for use in chemonucleolysis. Administration of human stromelysin-1 may physiologically facilitate the resorption process of herniated nucleus pulposus, increase the healing rate and decrease complications after chemonucleolysis.