YAP/TAZ Signaling Enhances Angiogenesis of Retinal Microvascular Endothelial Cells in a High-Glucose Environment.

PURPOSE: Diabetic retinopathy (DR) is a major cause of irreversible blindness in the working-age population. Neovascularization is an important hallmark of advanced DR. There is evidence that Yes-associated protein (YAP)/transcriptional co-activator with a PDZ binding domain (TAZ) plays an important role in angiogenesis and that its activity is regulated by vascular endothelial growth factor (VEGF). Therefore, the aim of this study was to investigate the effect of YAP/TAZ-VEGF crosstalk on the angiogenic capacity of human retinal microvascular endothelial cells (hRECs) in a high-glucose environment. METHODS: The expression of YAP and TAZ of hRECs under normal conditions, hypertonic conditions and high glucose were observed. YAP overexpression (OE-YAP), YAP silencing (sh-YAP), VEGF overexpression (OE-VEGF) and VEGF silencing (sh-VEGF) plasmids were constructed. Cell counting kit-8 assay was performed to detect cells proliferation ability, transwell assay to detect cells migration ability, and tube formation assay to detect tube formation ability. The protein expression of YAP, TAZ, VEGF, matrix metalloproteinase (MMP)-8, MMP-13, vessel endothelium (VE)-cadherin and alpha smooth muscle actin (α-SMA) was measured by western blot. RESULTS: The proliferation of hRECs was significantly higher in the high glucose group compared with the normal group, as well as the protein expression of YAP and TAZ (p < 0.01). YAP and VEGF promoted the proliferation, migration and tube formation of hRECs in the high glucose environment (p < 0.01), and increased the expression of TAZ, VEGF, MMP-8, MMP-13 and α-SMA while reducing the expression of VE-cadherin (p < 0.01). Knockdown of YAP effectively reversed the above promoting effects of OE-VEGF (p < 0.01) and overexpression of YAP significantly reversed the inhibition effects of sh-VEGF on above cell function (p < 0.01). CONCLUSION: In a high-glucose environment, YAP/TAZ can significantly promote the proliferation, migration and tube formation ability of hRECs, and the mechanism may be related to the regulation of VEGF expression.

[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.

MMP13 promotes the osteogenic potential of BMP9 by enhancing Wnt/ß-catenin signaling via HIF-1α upregulation in mouse embryonic fibroblasts.

Bone morphogenetic protein 9 (BMP9) has been validated as one of the most potent osteoinduction factors, but its underlying mechanism remains unclear. As a member of the matrix metalloproteinase (MMP) family, MMP13 may be involved in regulating the lineage-specific differentiation of mouse embryonic fibroblasts (MEFs). The goal of this study was to determine whether MMP13 regulates the osteoinduction potential of BMP9 in MEFs, which are multipotent progenitor cells widely used for stem cell biology research. In vitro and in vivo experiments showed that BMP9-induced osteogenic markers and/or bone were enhanced by exogenous MMP13 in MEFs, but were reduced by MMP13 knockdown or inhibition. The expression of hypoxia inducible factor 1 alpha (HIF-1α) was induced by BMP9, which was enhanced by MMP13. The protein expression of ß-catenin and phosphorylation level of glycogen synthase kinase-3 beta (GSK-3ß) were increased by BMP9 in MEFs, as was the translocation of ß-catenin from the cytoplasm to the nucleus; all these effects of BMP9 were enhanced by MMP13. Furthermore, the MMP13 effects of increasing BMP9-induced ß-catenin protein expression and GSK-3ß phosphorylation level were partially reversed by HIF-1α knockdown. These results suggest that MMP13 can enhance the osteoinduction potential of BMP9, which may be mediated, at least in part, through the HIF-1α/ß-catenin axis. Our findings demonstrate a novel role of MMP13 in the lineage decision of progenitor cells and provide a promising strategy to speed up bone regeneration.

Osteoprogenitor-GMP crosstalk underpins solid tumor-induced systemic immunosuppression and persists after tumor removal.

Remote tumors disrupt the bone marrow (BM) ecosystem (BME), eliciting the overproduction of BM-derived immunosuppressive cells. However, the underlying mechanisms remain poorly understood. Herein, we characterized breast and lung cancer-induced BME shifts pre- and post-tumor removal. Remote tumors progressively lead to osteoprogenitor (OP) expansion, hematopoietic stem cell dislocation, and CD41- granulocyte-monocyte progenitor (GMP) aggregation. The tumor-entrained BME is characterized by co-localization between CD41- GMPs and OPs. OP ablation abolishes this effect and diminishes abnormal myeloid overproduction. Mechanistically, HTRA1 carried by tumor-derived small extracellular vesicles upregulates MMP-13 in OPs, which in turn induces the alterations in the hematopoietic program. Importantly, these effects persist post-surgery and continue to impair anti-tumor immunity. Conditional knockout or inhibition of MMP-13 accelerates immune reinstatement and restores the efficacies of immunotherapies. Therefore, tumor-induced systemic effects are initiated by OP-GMP crosstalk that outlasts tumor burden, and additional treatment is required to reverse these effects for optimal therapeutic efficacy.

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.

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.

Oxidative stress as a critical factor might involve in intervertebral disc degeneration via regulating NOXs/FOXOs.

BACKGROUND: Oxidative stress is involved in many musculoskeletal diseases, such as osteoarthritis. However, the effect of oxidative stress on intervertebral disc degeneration (IDD) is still unclear. This study was aimed to provide an evidence of oxidative stress involved in IDD, and propose a new insight into pathogenesis of IDD. METHODS: Sixteen rats were randomly divided into sham and cervical muscle section (CMS) groups. The intervertebral disc degeneration scores (DDS) were assessed by histological staining at 8 weeks. Intracellular reactive oxygen species mainly comes from nicotinamide adenine dinucleotide phosphate oxidases (NOXs), while its clearance relies on antioxidant enzymes which regulated by forkhead transcription factor O (FOXOs). Thus, the oxidative stress was evaluated by the expression of NOXs and FOXOs. Meanwhile, the protein expression of Aggrecan, matrix metalloproteinase-13 (MMP-13), NOXs, FOXOs and antioxidant proteins (Manganese superoxide dismutase: MnSOD and Catalase) were tested in nucleus pulposus cells (NPCs) under tert-butyl hydroperoxide (TBHP) intervention. RESULTS: CMS induced IDD by enhancing DDS in 8 weeks, and the expression of NOX2 and NOX4 were significantly increased and the expression of FOXO3 and FOXO4 were remarkably decreased in the CMS rats. With the stimulation of TBHP, the contents of NOX2 and NOX4 in NPCs increased significantly, and the antioxidant proteins of FOXO1, FOXO3, FOXO4, MnSOD and Catalase and the matrix proteins of Aggrecan decreased remarkably, while MMP-13 significantly increased after TBHP intervention. CONCLUSIONS: The present study proposed that regulation of NOXs and FOXOs alters oxidative stress in intervertebral disc, which indicates that the intervention of oxidative stress would provide a new strategy to the treatment of IDD.

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..

Concurrent versus delayed exposure to corticosteroids in equine articular tissues cultured with local anesthetic.

OBJECTIVE: To determine the effect of concurrent versus delayed treatment with corticosteroid on equine articular tissues also treated with local anesthetic in vitro in the presence of inflammatory mediators. STUDY DESIGN: Controlled laboratory study. ANIMALS: Five geldings, one mare (aged 3-18 years). METHODS: From each horse, 24 synovial and 12 osteochondral explants were cultured in a 12-well plate (2 wells/group, 2 synovial and 1 osteochondral explant/well, total 216 explants in the study). Explants were stimulated in culture medium with 10 µg/ml recombinant equine interleukin-1ß and 10 µg/ml tumor necrosis factor-α for 48 hours, then randomly assigned to six treatments: unstimulated control, stimulated control, triamcinolone acetonide (TA, 10-6  M), mepivacaine hydrochloride (MH, 4.4 mg/ml), MH + TA (concurrent) and MH + TA (delayed). The delayed group was treated with MH and, 6 days later, treated with TA. Every 3 days for 9 days total, medium levels of lactate dehydrogenase (LDH), prostaglandin E2 (PGE2 ), matrix metalloproteinase 13 (MMP-13) and glycosaminoglycan (GAG) were quantified via ELISA. Data were analyzed with mixed-effects models with Tukey's multiple comparisons. RESULTS: Stimulation increased medium PGE2 and MMP-13 and had no effect on LDH or GAG. Treatment with MH increased LDH and decreased PGE2 and MMP-13. Treatment with TA decreased PGE2 and MMP-13. CONCLUSION: There were no differences in cytotoxicity, inflammation or matrix degradation for delayed or concurrent MH and TA treatment groups up to 9 days in culture. CLINICAL SIGNIFICANCE: The lack of an effect of concurrent versus delayed treatment might indicate that concurrent therapy is acceptable.

Increased expression of angiopoietin-like protein 4 regulates matrix metalloproteinase-13 expression in Porphyromonas gingivalis lipopolysaccharides-stimulated gingival fibroblasts and ligature-induced experimental periodontitis.

BACKGROUND: Angiopoietin-like protein 4 (ANGPTL4) is produced in chronic or acute inflammation. Although ANGPTL4 increases in the periodontal ligament fibroblasts during hypoxia, the involvement and role of ANGPTL4 in periodontitis have not been elucidated. OBJECTIVE: In this study, we investigated whether ligature-induced experimental periodontitis and/or Porphyromonas gingivalis lipopolysaccharides (Pg-LPS) would upregulate ANGPTL4 expression and whether ANGPTL4 would somehow involve in the expression of matrix metalloproteinases (MMPs) which are key molecules in the process of periodontal tissue destruction. METHODS: Experimental periodontitis was induced in 6-week-old male Sprague-Dawley rats by placing a nylon suture around the neck of the maxillary second molar. Two weeks after the induction of periodontitis, the periodontal tissue was excised and analyzed by histological/immunohistochemical staining and gene expression analyses. Human gingival fibroblasts (hGFs) were stimulated with Pg-LPS. The gene expression of ANGPTLs and receptors involved in ANGPTL4 recognition were observed. We also confirmed the changes in gene expression of MMPs upon stimulation with human ANGPTL4. Furthermore, we downregulated ANGPTL4 expression by short interfering RNA in hGFs and investigated the effect of Pg-LPS on MMP production. RESULTS: Induction of periodontitis significantly increased the expression of ANGPTL4 in the gingiva. Pg-LPS significantly increased the gene and protein expression of ANGPTL4 in hGFs but not the gene expression of other ANGPTLs or ANGPTL receptors. Recombinant human ANGPTL4 significantly increased MMP13 gene expression in hGFs. We also confirmed that MMP13 expression was increased in the gingiva during experimental periodontitis. Pg-LPS induced MMP13 gene expression in hGFs. These results suggest the pivotal role of ANGPTL4 in periodontitis. CONCLUSION: Periodontitis increases ANGPTL4 expression in the gingiva, further suggesting that increased ANGPTL4 may be a factor involved in enhancing MMP13 expression.

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.

Sappanone A Alleviated IL-1ß-Induced Inflammation in OA Chondrocytes through Modulating the NF-κB and Nrf2/HO-1 Pathways.

Objective: This study was to examine the anti-inflammatory effect of sappanone A on interleukin- (IL-) 1ß-stimulated osteoarthritis (OA) chondrocytes. Methods: Chondrocytes were pretreated with sappanone A for 2 h before subsequent IL-1ß stimulation. The mRNA expression levels of iNOs, COX-2, aggrecan, and collagen-II were measured with qRT-PCR. The levels of TNF-α, IL-6, IL-8, MMP-3, and MMP-13 were determined by ELISA. The protein levels of iNOs, COX-2, ADAMTS-4, ADAMTS-5, aggrecan, collagen-II, p-p65, p65, IκBα, Nrf2, and HO-1 were assessed by Western blot. Results: Sappanone A inhibited the IL-1ß-stimulated production of NO, PGE2, iNOS, COX-2, TNF-α, IL-6, and IL-8 in OA chondrocytes. In addition, sappanone A suppressed the expression of MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5 in IL-1ß-stimulated OA chondrocytes. The degradation of ECM components was reversed by sappanone A. Sappanone A prevented NF-κB activation while enhanced Nrf2/HO-1 activation in IL-1ß-treated chondrocytes. Conclusion: Sappanone A may be a potent therapeutic agent for OA.

Polydatin inhibits IL-1ß-mediated chondrocyte inflammation and ameliorates cartilage degradation: Involvement of the NF-κB and Wnt/ß-catenin pathways.

Osteoarthritis (OA) is a highly prevalent chronic joint disease that involves extracellular matrix (ECM) degradation and articular cartilage inflammation. Polydatin (PD) can alleviate inflammatory reactions in numerous diseases. The present study aimed to investigate the chondroprotective and anti-inflammatory effects of PD on interleukin (IL)- 1ß-treated chondrocytes in vitro and anterior cruciate ligament transection-induced rat OA models in vivo. Primary chondrocytes were isolated from SD rats and cultured. Only second-passage cells were used for subsequent experiments. Counting kit-8, quantitative real-time polymerase chain reaction, western blotting, enzyme-linked immunosorbent assay, and immunofluorescence were used to detect relevant indices. Rat OA models were established to obtain in vivo data. PD treatment decreased the production of nitric oxide (NO), prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), and IL-6 during IL-1ß-stimulated chondrocyte inflammation. Moreover, PD upregulated aggrecan and collagen II expression, whereas downregulated a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) and matrix metalloproteinase-13 (MMP-13) expression on IL-1ß-mediated chondrocytes. Additionally, PD reduced IL-1ß-stimulated NF-κB and Wnt/ß-catenin activation and nuclear translocation. The results of histological analysis and scoring revealed that OA in the rat models was effectively ameliorated by the intra-articular injection of PD. PD suppressed IL-1ß-stimulated iNOS, COX-2, NO, and PGE2 production, TNF-α, IL-6, collagen X, MMP-13, and ADAMTS-5 expression, collagen II and aggrecan degeneration by inhibiting NF-κB and Wnt/ß-catenin signaling in vitro. PD also mitigated OA progression in the rat models, thereby providing reliable data that PD could serve as a promising candidate for OA therapy.

FGF23 and SOX9 expression in haemophilic cartilage: In vitro studies of the effects of iron.

INTRODUCTION: Clarifying the links between iron and FGF23, SOX9 expression in chondrocytes would be helpful for comprehending articular cartilage degradation pathogenesis in blood-induced arthritis and exploring new protective methods. AIM: The purpose of this study was to determine iron regulation of fibroblast growth factor 23 (FGF23) and SRY-box 9 (SOX9) in human chondrocytes, an area which is unexplored in blood-induced arthritis cartilage degradation pathogenesis. METHODS: Expression of FGF23, SOX9, MMP13 and collagen Ⅱ in articular cartilage of patients with osteoarthritis (OA) or haemophilic arthritis (HA) was determined by western blot (WB). Iron-induced FGF23 and SOX9 mRNA and protein expression in primary human normal chondrocyte cells (HUM-iCell-s018) was quantified by qRT-PCR and WB, respectively. RESULTS: We found that compared with OA patients, the expression of FGF23, MMP13 in articular cartilage of patients with HA was up-regulated, while the expression of SOX9, collagen Ⅱ was down-regulated. Iron-induced FGF23 and suppressed SOX9 expression in chondrocytes in a dose-dependent manner. CONCLUSIONS: These findings demonstrated that iron was involved in hemophilic cartilage lesion directly via changing cartilage phenotype through regulation of FGF23 and SOX9 expression in chondrocytes.

[Eucommia ulmoides Oliv polysaccharide reduces the injury of IL-1ß-induced chondrocyte by inhibiting NF-κB pathway].

OBJECTIVE: To investigate the effects of Eucommia ulmoides Oliv polysaccharide on the injury of interleukin-1ß(IL-1ß)-induced chondrocyte and its possible mechanism. METHODS: ATDC5 was treated with 10 µg/ml IL-1ß to establish osteoarthritis chondrocyte inflammation model, mouse chondrocyte ATDC5 were cultured in vitro and randomly divided into blank group, model group, model+Eucommia ulmoides Oliv polysaccharide low concentration group, model+Eucommia ulmoides Oliv polysaccharide medium concentration group and model+Eucommia ulmoides Oliv polysaccharide high concentration group. The cells in the blank group were cultured with conventional medium;the cells in the model group cells were cultured with a medium containing 10 ?g/ml IL-1ß, and the cells in the model+Eucommia ulmoides Oliv polysaccharide low concentration group, model+Eucommia ulmoides Oliv polysaccharide medium concentration group and model+Eucommia ulmoides Oliv polysaccharide high concentration group were co-cultured with medium containing 100, 200, 400 µg/ml Eucommia ulmoides Oliv polysaccharide and 10 µg/ml IL-1ß. After the cells of each group were cultured for 24 h, 48 h and 72 h, CCK-8 method was used to detect cell viability. After the cells of each group were cultured for 48 h, flow cytometry and DAPI staining were used to detect cell apoptosis;ELISA method was used to detect the expression of TNF-α, NO, IFN-γ and IL-6 in cells; DCFH-DA method was used to detect the content of ROS in cells;Western blot was used to detect the protein expression of TIMP-1, MMP-13 and NF-κB signaling pathway-related P65 and p-P65;Immunofluorescence staining was used to observe the localization of NF-κB P65 cells. RESULTS: Compared with the blank group, the ATDC5 cell viability and the protein expression of TIMP-1 in the model group reduced (P<0.05), while apoptosis rate, the levels of TNF-α, NO, IFN-γ and IL-6, the content of ROS, the protein expression of MMP-13 and p-P65, and the number of P65+ in the nucleus increased(P<0.05). Compared with the model group, the ATDC5 cell viability and the protein expression of TIMP-1 in the model+Eucommia ulmoides Oliv polysaccharide low concentration group, model+Eucommia ulmoides Oliv polysaccharide medium concentration group and model+Eucommia ulmoides Oliv polysaccharide high concentration group increased (P<0.05), while apoptosis rate, the levels of TNF-α, NO, IFN-γ and IL-6, the content of ROS, the protein expression of MMP-13 and p-P65, and the number of P65+ in the nucleus reduced (P<0.05). CONCLUSION: The results showed that Eucommia ulmoides Oliv polysaccharide could promote proliferation of IL-1ß-induced chondrocyte ATDC5 and inhibit its apoptosis, inflammatory response and matrix degradation. Its mechanism may be related to the inhibition of the activation of NF-κB pathway.

YAP plays a protective role in T-2 toxin-induced inhibition of chondrocyte proliferation and matrix degradation.

Previous research has shown that T-2 toxin can damage cartilage, resulting in a disease phenotype similar to osteoarthritis. The precise molecular mechanism by which T-2 toxin causes chondrocyte injury, however, is unknown. The purpose of this study was to look into the role of YAP in T-2 toxin-induced rat chondrocyte injury. Based on research results, T-2 toxin decreased the levels of collagen II and PCNA while increasing the expression of matrix metalloproteinase MMP13. These findings supported the T-2 toxin's detrimental effect on chondrocytes. YAP's role in T-2 toxin-induced chondrocyte injury was also investigated. Total YAP and related nuclear proteins were found to decrease as the concentration of T-2 toxin increased. While PYAP expression was not significantly altered in response to T-2 toxin, the PYAP/YAP ratio decreased as the T-2 toxin concentration increased, implying that the HIPPO signaling pathway was activated. Furthermore, the YAP-specific inhibitor Verteporfin was used to investigate the role of YAP in T-2 toxin-induced chondrocyte injury. YAP inhibition increased MMP13 expression while decreasing COL2 and PCNA levels. In summary, the current study found that T-2 toxin decreased the levels of COL2 and PCNA while increasing the expression of MMP13 in chondrocytes after inhibiting YAP, providing a new insight into the mechanism of T-2 toxin-induced cartilage damage.

Ganoderic acid A improves osteoarthritis by regulating RANKL/OPG ratio.

Ganoderma mushrooms have been used to treat rheumatoid arthritis (RA) in East Asia. Whether Ganoderic acid A (GAA), the natural product extracted from Ganoderma, could be utilized to alleviate osteoarthritis (OA) is investigated in this study. Destabilization of the medial meniscus (DMM) model was constructed to reveal the in vivo effect of GAA. We found that GAA could significantly alleviate the pathology of DMM, as confirmed by the diminished maximum histologic scores. On the contrary, GAA could down-regulate the relative expression of osteoprotegerin (OPG) and up-regulate the relative expression of nuclear factor kappa-B ligand (RANKL) in DMM cartilage and human articular chondrocytes (HC-A) cells with diminished matrix metallopeptidase 13 (MMP-13) secretion in the synovial fluid. It was further demonstrated that the serum concentration of OPG was correlated with the severity of osteoarthritis. All these data reveal that GAA could improve OA by regulating the RANKL/OPG ratio to inhibit the secretion of MMP-13.

LncRNA RP3-326I13.1 promotes cisplatin resistance in lung adenocarcinoma by binding to HSP90B and upregulating MMP13.

Cisplatin (DDP) resistance has become the major obstacle in the therapy of malignant tumors, including lung adenocarcinoma (LAD). Long non-coding RNAs (lncRNAs) were confirmed to be related to DDP-resistance. Studies have shown that RP3-326I13.1 (also known as PINCR) could promote the progression of colorectal cancer, and RP3-326I13.1 knockdown could induce hypersensitivity to chemotherapy drugs. While the function of RP3-326I13.1 in LAD is unclear, therefore, this study aimed to research the biological function and related molecular mechanisms of RP3-326I13.1 in DDP-resistance of LAD. QPCR analysis found that RP3-326I13.1 was highly expressed in A549/DDP cells and LAD tissues. Cytological assays found that RP3-326I13.1 pro-moted the proliferation, migration, invasion, and DDP-resistance of LAD cell lines. Moreover, knock-down of RP3-326I13.1 could induce G1 phase arrest. Nude mouse xenograft assay confirmed that RP3-326I13.1 could promote tumor growth and DDP-resistance in vivo. Mechanically, RNA pull-down and mass spectrometry analysis indicated that heat shock protein HSP 90-beta (HSP90B) could be combined with RP3-326I13.1. HSP90B knockdown inhibited the effect of RP3-326I13.1 on proliferation, invasion, and promoted LAD cell lines apoptosis. Transcriptome sequencing analysis found that MMP13 was the downstream mRNA of RP3-326I13.1. In conclusion, RP3-326I13.1 could promote DDP-resistance of LAD by binding to HSP90B and upregulating human matrix metalloproteinase-13 (MMP-13) and may serve as a therapeutic target, as well as a biomarker for predicting DDP-resistance in LAD.Abbreviations:DDP: Cisplatin; LAD: Lung adenocarcinoma; LncRNAs: Long non-coding RNAs; qPCR: real-time fluorescent quantitative PCR; HSP90B: Heat shock protein HSP 90-beta; RPMI: Roswell Park Memorial Institute; FBS: Fetal bovine serum; CT: computed tomography; MRI: magnetic resonance imaging; RECIST: Response evaluation criteria in solid tumors; NC: Negative control; OE: overexpression; shRNA: short hairpin RNA; siRNA: small interfering RNA; CCK-8: Cell Counting Kit-8; IC50: The half maximal inhibitory concentration; PBS: Phosphate buffer saline; PI: propidium iodide; SDS-PAGE: sodiumdodecylsulfate-polyacrylamide gel electrophoresis; ceRNA: Competing endogenous RNA; HE: hematoxylin-eosin; ns: no significance.

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.

miR-204-5p inhibits inflammation of synovial fibroblasts in osteoarthritis by suppressing FOXC1.

BACKGROUND: The paper is aimed at uncovering the mechanism of miR-204-5p in regulating inflammatory responses of human osteoarthritic synovial fibroblasts (SFs). METHODS: IL-1ß-induced osteoarthritic SFs were established as an osteoarthritis (OA) cell model. The osteoarthritic SFs were accordingly transfected with mimics-miR-204-5p, inhibitors-miR-204-5 or FOXC1 siRNA. MTT tested the vitality of osteoarthritic SFs by analyzing the cell optical density. The expressions of miR-204-5p, FOXC1, TNF-α, IL-6, PGE2, MMP-1, MMP-13 and COX-2 in osteoarthritic SFs were measured by qRT-PCR, Western blotting and/or ELISA. The binding of miR-204-5p to FOXC1 was verified through luciferase reporter assay. The regulatory effect of miR-204-5p on FOXC1 was also tested in normal SFs. RESULTS: miR-204-5p was under-expressed and FOXC1 was over-expressed in osteoarthritic SFs. The expressions of FOXC1, TNF-α, IL-6, PGE2, MMP-1, MMP-13 and COX-2 were up-regulated in IL-1ß-treated SFs. Up-regulation of miR-204-5p or down-regulation of FOXC1 suppressed the inflammatory responses of osteoarthritic SFs. miR-204-5p negatively regulated FOXC1 by being a sponge in osteoarthritic SFs as well as in normal SFs. CONCLUSION: miR-204-5p down-regulates FOXC1 to ameliorate inflammation of SFs in OA.