Extracellular Matrix Degeneration Research Articles

Phillyrin reduces ROS production to alleviate the progression of intervertebral disc degeneration by inhibiting NF-κB pathway

BackgroundIntervertebral disc degeneration (IDD) is an increasingly important cause of low back pain (LBP) that results in substantial health and economic burdens. Inflammatory pathway activation and the production of reactive oxygen species (ROS) play vital roles in the progression of IDD. Several studies have suggested that phillyrin has a protective role and inhibits inflammation and the production of ROS. However, the role of phillyrin in IDD has not been confirmed.PurposeThe purpose of this study was to investigate the role of phillyrin in IDD and its mechanisms.Study designTo establish IDD models in vivo, ex-vivo, and in vitro to verify the function of phillyrin in IDD.MethodThe effects of phillyrin on extracellular matrix (ECM) degeneration, inflammation, and oxidation in nucleus pulposus (NP) cells were assessed using immunoblotting and immunofluorescence analysis. Additionally, the impact of phillyrin administration on acupuncture-mediated intervertebral disc degeneration (IDD) in rats was evaluated using various techniques such as MRI, HE staining, S-O staining, and immunohistochemistry (IHC).ResultPretreatment with phillyrin significantly inhibited the IL-1β-mediated reduction in the degeneration of ECM and apoptosis by alleviating activation of the NF-κB inflammatory pathway and the generation of ROS. In addition, in vivo and ex-vivo experiments verified the protective effect of phillyrin against IDD.ConclusionPhillyrin can attenuate the progression of IDD by reducing ROS production and activating inflammatory pathways.

Botulinum toxin A attenuates osteoarthritis development via inhibiting chondrocyte ferroptosis through SLC7Al1/GPX4 axis

Osteoarthritis (OA) is a prevalent joint degenerative disease, resulting in a significant societal burden. However, there is currently a lack of effective treatment option available. Previous studies have suggested that Botulinum toxin A (BONT/A), a macromolecular protein extracted from Clostridium Botulinum, may improve the pain and joint function in OA patients, but the mechanism remains elusive. This study was to investigate the impact and potential mechanism of BONT/A on OA in vivo and in vitro experiment. LPS increased the levels of ROS, Fe2+and Fe3+, as well as decreased GSH levels, the ratio of GSH / GSSH and mitochondrial membrane potential. It also enhanced the degeneration of extracellular matrix (ECM) and altered the ferroptosis-related protein expression in chondrocytes. BONT/A rescued LPS-induced decrease in collagen type II (Collagen II) expression and increase in matrix metalloproteinase 13 (MMP13), mitigated LPS-induced cytotoxicity in chondrocytes, abolished the accumulation of ROS and iron, upregulated GSH and the ratio of GSH/ GSSH, improved mitochondrial function, and promoted SLC7A11/GPX4 anti-ferroptosis system activation. Additionally, intra-articular injection of BONT/A inhibited the degradation of cartilage in OA model rats. This chondroprotective effect of BONT/A was reversed by erastin (a classical ferroptosis agonist) and enhanced by liproxstatin-1 (a classic ferroptosis inhibitor). Our research confirms that BONT/A alleviates the OA development by inhibiting the ferroptosis of chondrocytes, which revealed to be a potential therapeutic mechanism for BONT/A treating the OA.

Thioredoxin promotes the regeneration and binding of elastic fibre and basement membrane.

Thioredoxin (TRX), a ubiquitous protein with strong antioxidant activity, decreases in the skin with age. A decrease in TRX is expected to induce cellular senescence, chronic inflammation, and degeneration and loss of extracellular matrix (ECM), such as collagen and elastin within the skin. In this study, we investigated the effects of TRX addition to excised skin or skin models to understand the role of TRX on cells and ECM within the skin. To evaluate its effect on skin cells, we cultured a three-dimensional (3D) skin model in a medium containing TRX. The mRNA expression levels of proteins related to elastic and collagen fibres and the basement membrane were determined. Furthermore, 3D imaging and computational analysis were performed to evaluate the effect of TRX on the elastic fibres and extending COL VII structures in excised human skin after coculturing with TRX for 1, 4, 5 and 6 days. Thioredoxin application to a 3D skin model upregulated elastin, COLI and COLVII mRNA expression. Applying TRX to the excised skin increased the number of linear elastic fibres. This effect of TRX demonstrated a daily increment in a dose-dependent manner. Thioredoxin extended the fibrous structure of COL VII into the dermis, expanding its colocalization region with elastic fibres. These structural effects were confirmed using 3D imaging and computational methods. Thioredoxin elongates elastic fibres from the dermis to the basement membrane and extends the COL VII structure from the basement membrane to the dermis in excised human skin. These findings suggest the potential of TRX to protect the skin against age-related alterations such as wrinkles and sagging.

LXRα Promotes Abdominal Aortic Aneurysm Formation Through UHRF1 Epigenetic Modification of miR-26b-3p.

Abdominal aortic aneurysm (AAA) is a severe aortic disease without effective pharmacological approaches. The nuclear hormone receptor LXRα (liver X receptor α), encoded by the NR1H3 gene, serves as a critical transcriptional mediator linked to several vascular pathologies, but its role in AAA remains elusive. Through integrated analyses of human and murine AAA gene expression microarray data sets, we identified NR1H3 as a candidate gene regulating AAA formation. To investigate the role of LXRα in AAA formation, we used global Nr1h3-knockout and vascular smooth muscle cell-specific Nr1h3-knockout mice in 2 AAA mouse models induced with angiotensin II (1000 ng·kg·min; 28 days) or calcium chloride (CaCl2; 0.5 mol/L; 42 days). Upregulated LXRα was observed in the aortas of patients with AAA and in angiotensin II- or CaCl2-treated mice. Global or vascular smooth muscle cell-specific Nr1h3 knockout inhibited AAA formation in 2 mouse models. Loss of LXRα function prevented extracellular matrix degeneration, inflammation, and vascular smooth muscle cell phenotypic switching. Uhrf1, an epigenetic master regulator, was identified as a direct target gene of LXRα by integrated analysis of transcriptome sequencing and chromatin immunoprecipitation sequencing. Susceptibility to AAA development was consistently enhanced by UHRF1 (ubiquitin-like containing PHD and RING finger domains 1) in both angiotensin II- and CaCl2-induced mouse models. We then determined the CpG methylation status and promoter accessibility of UHRF1-mediated genes using CUT&Tag (cleavage under targets and tagmentation), RRBS (reduced representation bisulfite sequencing), and ATAC-seq (assay for transposase-accessible chromatin with sequencing) in vascular smooth muscle cells, which revealed that the recruitment of UHRF1 to the promoter of miR-26b led to DNA hypermethylation accompanied by relatively closed chromatin states, and caused downregulation of miR-26b expression in AAA. Regarding clinical significance, we found that underexpression of miR-26b-3p correlated with high risk in patients with AAA. Maintaining miR-26b-3p expression prevented AAA progression and alleviated the overall pathological process. Our study reveals a pivotal role of the LXRα/UHRF1/miR-26b-3p axis in AAA and provides potential biomarkers and therapeutic targets for AAA.

F-ACTIN DISTRIBUTION OF THE CHONDROCYTE CYTOSKELETON AND ITS ASSOCIATION WITH EARLY OSTEOARTHRITIC CHONDROCYTE PROPERTIES

AbstractObjectivesOsteoarthritis (OA) is a painful and debilitating disorder of diarthroidal joints. Progressive degeneration of the cartilage extracellular matrix (ECM) together with abnormal chondrocyte characteristics occur leading to a switch to a fibroblast-like phenotype and production of mechanically-weak cartilage. Early changes to chondrocytes within human cartilage have been observed including chondrocyte swelling[1] together with the development of thin cytoplasmic processes which increase in number and length with degeneration[2]. Changes to chondrocyte phenotype in degenerate cartilage are associated with F-actin redistribution and stress fibres (SF) formation, leading to morphologically-dedifferentiated (fibroblast-like) chondrocytes[3,4]. It is unclear if these processes are a consequence of ‘passive’ cell swelling into a defective ECM or an ‘active’ event driven by changes in cell metabolism resulting in alterations to cell shape. To address this, we have quantified and compared the distribution and levels of F-actin, a key cytoskeletal protein involved in the formation of cytoplasmic processes, within in situ chondrocytes in non-degenerate and mildly degenerate human cartilage.MethodsHuman femoral head cartilage was obtained from 21 patients [15 females, 6 males, average age 69.6yrs, (range 47–90yrs)] following femoral neck fracture, with Ethical Approval and patient's permission. Cartilage explants were removed from areas graded non-degenerate grade 0 (G0) or mildly degenerate grade 1 (G1) and cultured for up to 3wks in Dulbecco's Modified Eagle's Medium (DMEM) +/− 25% human serum (HS). In situ chondrocytes were stained with CMFDA (5-chloromethylfluoresceindiacetate, Cell-Tracker Green®) and phalloidin (F-actin labelling) and imaged by confocal microscopy and analysed quantitatively using ImageJ and Imaris® software.ResultsThere were significant increases in the total amount (TA) of F-actin and its distribution [intense punctuate (IP) and intense areas (IA)] between the whole chondrocyte populations of G0 and G1 cartilage (P=0.0356; 0.0112; 0.016, respectively). Where the volume of chondrocytes was divided into normal (<1000 µm³) and swollen (≥1000 µm³) cells, F-actin TA increased in swollen cells (P=0.036 within G0 and G1, and P=0.0009 between grades) compared to chondrocytes of normal volume in each grade. Moreover, IP and IA within and between G0 and G1 were higher compared to normal chondrocytes (with P<0.0001 for IP and P<0.001 for IA). In addition, tissue culture experiments demonstrated that 90% of chondrocytes with cytoplasmic processes had strong F-actin intensity (either IP or IA with P<0.0001). Furthermore, 83% of this F-actin was associated with cytoplasmic processes, with >65% situated at the base of the process (P<0.0001).ConclusionsThe increases in chondrocyte F-actin levels (TA) and its localisation (IP, IA) appear to be associated with cell swelling and development of cytoplasmic processes, which are both characteristics of early OA cartilage[1]. This suggests the formation of chondrocyte cytoplasmic processes is an ‘active’ event potentially involving changes to matrix metabolism rather than a ‘passive’ cell swelling into a defective extracellular matrix.Declaration of Interest(b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.

Biomimetic proteoglycans as a tool to engineer the structure and mechanics of porcine bioprosthetic heart valves.

The utility of bioprosthetic heart valves (BHVs) is limited to certain patient populations because of their poor durability compared to mechanical prosthetic valves. Histological analysis of failed porcine BHVs suggests that degeneration of the tissue extracellular matrix (ECM), specifically the loss of proteoglycans and their glycosaminoglycans (GAGs), may lead to impaired mechanical performance, resulting in nucleation and propagation of tears and ultimately failure of the prosthetic. Several strategies have been proposed to address this deterioration, including novel chemical fixatives to stabilize ECM constituents and incorporation of small molecule inhibitors of catabolic enzymes implicated in the degeneration of the BHV ECM. Here, biomimetic proteoglycans (BPGs) were introduced into porcine aortic valves ex vivo and were shown to distribute throughout the valve leaflets. Incorporation of BPGs into the heart valve leaflet increased tissue overall GAG content. The presence of BPGs also significantly increased the micromodulus of the spongiosa layer within the BHV without compromising the chemical fixation process used to sterilize and strengthen the tissue prior to implantation. These findings suggest that a targeted approach for molecularly engineering valve leaflet ECM through the use of BPGs may be a viable way to improve the mechanical behavior and potential durability of BHVs.

Studies on blocking the JAK2/STAT3 signaling pathway with elastin peptides from skipjack tuna (Katsuwonus pelamis) bulbus cordis to alleviate osteoarthritis

Osteoarthritis (OA) is a chronic disease associated with bone and muscle degeneration that causes great disturbances to daily lives. Skipjack tuna elastin peptides (STEP) is a kind of small molecule mixed peptides extracted from skipjack tuna bulbus cordis that have multiple active functions, including anti-inflammatory activity. However, it is ambiguous whether STEP reduce OA inflammation. Zebrafish served as a model in the present research for verifying the anti-inflammatory efficacy of STEP. Then, the composition of STEP was figured out by LC-MS/MS analysis, and the potential anti-inflammatory peptides were screened by molecular docking and microscale thermophoresis (MST). Finally, in vitro experiments were conducted on the potential peptides. According to the in vivo findings, STEP protected the cartilage tissues of zebrafish and regulated the expression of inflammatory factors and the JAK2/STAT3 signaling pathway. The in vitro results showed that the potential peptides (LGVGSPPGK, GFDPSR and PGAPGGGFDI) positively regulated the inflammatory responses and the JAK2/STAT3 signaling pathway and were effective in inhibiting extracellular matrix (ECM) degeneration and chondrocyte apoptosis. As a consequence, STEP possess anti-inflammatory and chondroprotective effects and could be a functional product for the alleviation of OA.

Effect of Tuina along "bladder meridian" alleviating intervertebral disc degeneration by regulating the transforming growth factor-β1/Smad signaling pathway in a rabbit model.

The aim of this study was to investigate the protective effects of Tuina (a traditional Chinese massage therapy) on intervertebral disc (IVD) degeneration and the regulatory mechanisms of the transforming growth factor-β1 (TGF-β1)/small mothers against decapentaplegic (Smad) signaling pathway. Thirty New Zealand white rabbits were randomized into five groups: the control group, model group, model + Tuina group (Tuina group), model + TGF-β1 group (TGF-β1 group), and model + TGF-β1 inhibitor SB431542 group (SB431542 group). The model was established by posterolateral annulus fibrosus puncturing (AFP). Recombinant TGF-β1 and inhibitor SB431542 was injected into the TGF-β1 group and SB431542 group with a microsyringe, respectively. The rabbits in the Tuina group received Tuina treatment along the bladder meridian for 4 weeks. Magnetic resonance imaging (MRI) was performed on rabbits before AFP and after 4 weeks of intervention. Lumbar IVDs (L2-L3 to L4-L5) were harvested after intervention. Histopathological changes in the IVDs were measured by hematoxylin and eosin (HE) staining. Type I collagen was analyzed by immunohistochemistry detection. The expression level of matrix metalloproteinase-3 (MMP3) was determined by enzyme-linked immunosorbent assay. Cell apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated nick end labeling and Western blotting. Real-time polymerase chain reaction and Western blotting were used to analyze the expression of TGF-β1 and Smad2/3/4 and a disintegrin and metalloproteinase with thrombospondin motifs 5. Posterolateral AFP induced IVD degeneration in rabbits with histopathological damage and noticeable changes in MRI images. Tuina alleviated histo-pathological changes and reversed the expression of extracellular matrix degeneration-related molecules and apoptosis-related proteins. Furthermore, AFP induced the activation of TGF-β1 and Smad2/3/4, whereas Tuina therapy markedly reduced the protein expression of Smad2/3 and the gene expression of TGF-β1 and Smad2/3/4. Additionally, the TGF-β1/Smad signaling pathway was activated in the TGF-β1 group, while the TGF-β1/Smad signaling pathway was inhibited in the SB431542 group. Posterolateral AFP induced disc degeneration as determined by MRI assessment and histological analysis. Tuina alleviated disc degeneration, possibly by inhibiting the fibrotic response mediated by the TGF-β1/Smad pathway, thus alleviating extracellular matrix degeneration and reducing cell apoptosis.

PP2 alleviates the progression of osteoarthritis by inhibiting Wnt/β-catenin and activating TGF-β/Smad signaling

ObjectiveWe aimed to explore the effect and mechanism of the Src inhibitor PP2 on osteoarthritis (OA) progression. MethodsThe protein expressions of Src, p-Src (y418) and p-FAK in normal and OA human chondrocytes were detected by immunofluorescence (IF) analysis. Chondrocytes from the femur and tibial plateau of 3-day-old mice were extracted and inoculated into 6-well plates. The chondrocytes were co-cultured with IL-1β and different doses of PP2, and then the degeneration of extracellular matrix was analyzed. A mouse OA model was induced by destabilizing medial meniscectomy of the right knee. Two weeks after the operation, different doses of PP2 were injected intraperitoneally. The drug was given three times a week for 6 weeks, and then the mice were sacrificed. Histopathological, IF and immunoblotting analyses were used to detect key OA catabolic markers and protein expression and related signaling. ResultsThe levels of Src, p-Src (y418) and p-FAK in the knee cartilage tissue of patients with OA were abnormally increased. After chondrocytes were co-treated with IL-1β and different doses of PP2, the results showed that PP2 reduced the abnormal increase of β-catenin, p-β-catenin and other proteins induced by IL-1β, and reversed the decrease of p-Smad3, aggrecan and collagen Ⅱ protein levels. Meanwhile, intraperitoneal injection of PP2 in vivo significantly reduced the degeneration of articular cartilage in the OA mouse model. ConclusionOur data indicate that targeting Src with PP2 protected against cartilage destruction in an OA mouse model by inhibiting Wnt/β-catenin and activating TGF-β/Smad signaling, suggesting that Src may be a potential therapeutic target for OA treatment.

Rat Plantar Fascia Stem/Progenitor Cells Showed Lower Expression of Ligament Markers and Higher Pro-Inflammatory Cytokines after Intensive Mechanical Loading or Interleukin-1β Treatment In Vitro.

The pathogenesis of plantar fasciitis is unclear, which hampers the development of an effective treatment. The altered fate of plantar fascia stem/progenitor cells (PFSCs) under overuse-induced inflammation might contribute to the pathogenesis. This study aimed to isolate rat PFSCs and compared their stem cell-related properties with bone marrow stromal cells (BMSCs). The effects of inflammation and intensive mechanical loading on PFSCs' functions were also examined. We showed that plantar fascia-derived cells (PFCs) expressed common MSC surface markers and embryonic stemness markers. They expressed lower Nanog but higher Oct4 and Sox2, proliferated faster and formed more colonies compared to BMSCs. Although PFCs showed higher chondrogenic differentiation potential, they showed low osteogenic and adipogenic differentiation potential upon induction compared to BMSCs. The expression of ligament markers was higher in PFCs than in BMSCs. The isolated PFCs were hence PFSCs. Both IL-1β and intensive mechanical loading suppressed the mRNA expression of ligament markers but increased the expression of inflammatory cytokines and matrix-degrading enzymes in PFSCs. In summary, rat PFSCs were successfully isolated. They had poor multi-lineage differentiation potential compared to BMSCs. Inflammation after overuse altered the fate and inflammatory status of PFSCs, which might lead to poor ligament differentiation of PFSCs and extracellular matrix degeneration. Rat PFSCs can be used as an in vitro model for studying the effects of intensive mechanical loading-induced inflammation on matrix degeneration and erroneous stem/progenitor cell differentiation in plantar fasciitis.

Astragaloside IV relieves IL-1β-induced human nucleus pulposus cells degeneration through modulating PI3K/Akt signaling pathway.

Intervertebral disc degeneration (IDD) is a multifactorial disease that is associated with nucleus pulposus (NP) apoptosis and extracellular matrix (ECM) degeneration and inflammation. Astragaloside IV (AS IV) has antioxidant, free radical scavenging, anti-inflammatory and anti-apoptosis effects. This study was to investigate whether AS IV could inhibit IL-1β-mediated apoptosis of HNP cells and its possible signal transduction pathway. Human nucleus pulposus cells (HNPCs) were stimulated with AS IV or LY294002 (PI3K inhibitor), followed by exposure to IL-1β for 24 hours. CCK8, TUNEL analysis and flow cytometry, ELISA and Western blotting were used to analyze the effects of AS IV on cell proliferation, apoptosis, inflammation, ECM and PI3K/Akt pathway signaling path-related proteins in IL-1β-induced HNPCs. Compared with IL-1β-induced HNPCs, AS IV could improve the proliferation activity and the expressions of Collagen II, Aggrecan and Bcl-2 proteins, inhibit the apoptosis rate, inflammation and Bax and cleaved caspase-3 protein expression, and increase the activity of PI3K/Akt pathway. LY294002 attenuated the protective effect of AS IV against IL-1β-induced HNPCs degeneration. AS IV can inhibit IL-1β-induced HNPCs apoptosis inflammation and ECM degeneration by activating PI3K/Akt signaling pathway, which can be an effective drug to reduce disc degeneration.

Tanshinone IIA alleviates chondrocyte apoptosis and extracellular matrix degeneration by inhibiting ferroptosis.

Articular cartilage degeneration caused by chondrocyte damage is the primary pathological mechanism of osteoarthritis (OA). Oxidative stress is correlated with chondrocyte injury by potentiating ferroptosis, a newly identified form of cell death. Given the effects of Tanshinone IIA (Tan IIA) on alleviating oxidative stress, we further explored whether Tan IIA inhibited chondrocyte death and cartilage degeneration by decreasing ferroptosis. ATDC5 chondrocytes were treated with lipopolysaccharides (LPS) and Tan IIA, and cell viability was assessed using cell counting kit-8 (CCK-8) assays. Matrix metalloproteinase-13 (MMP13), a disintegrin and metalloproteinase with thrombospondin motif-5 (ADAMTS5), and type II collagen (Col II) levels were measured using quantitative real-time polymerase chain reaction (qRT‒PCR), western blotting, and immunofluorescence (IF) analysis. We demonstrated that Tan IIA treatment prominently increased ATDC5 cell viability and decreased cell apoptosis in the presence of LPS-induced stress. MMP13 and ADAMTS5 expression was increased, and Col II expression was decreased in ATDC5 cells after LPS stimulation, whereas these changes were reversed by Tan IIA. Mechanistically, Tan IIA inhibited LPS-induced ferroptosis in ATDC5 cells, as indicated by decreased levels of iron, reactive oxygen species, and malondialdehyde and increased GSH levels. Importantly, a ferroptosis agonist partially abrogated the effect of Tan IIA on alleviating chondrocyte damage and death. Taken together, these results suggest that Tan IIA ameliorates chondrocyte apoptosis and cartilage degeneration by inhibiting ferroptosis and may be a potential therapeutic agent for OA.

An in vitro and in vivo study: Valencene protects cartilage and alleviates the progression of osteoarthritis by anti-oxidative stress and anti-inflammatory effects

BackgroundOsteoarthritis (OA) is a heterogeneous disease involving the whole joint. The pathogenesis involves oxidative stress levels and chronic inflammation, and Valencene (VA) has excellent anti-inflammatory and antioxidant stress abilities. PurposeThe objective was to study the effects of VA therapy on combating oxidative stress and to evaluate the protective effect of chondrocytes to alleviate the progression of OA. MethodsC57BL6J mouse chondrocytes were used as the primary cells in this study. Mouse chondrocytes were stimulated with IL-1β, and VA was administered in different concentrations. Reactive oxygen species (ROS) assay kits, western blotting, cellular immunofluorescence, and scanning microscopy were used to evaluate VA's antioxidant stress mechanism, anti-inflammatory effect, and cartilage protective ability. The mouse arthritis model constructed by destabilization of medial meniscus (DMM) was observed by micro-CT scan and histology after different treatments. ResultsWe found that VA can reverse the rise of ROS under IL-1β, the degeneration of the cartilage extracellular matrix, and the production of inflammatory mediators. In terms of mechanism, VA activated NRF2/HO-1/NQO1 pathway, thus enhancing ROS clearance. The phosphorylation of IκBα is inhibited, which further reduces the downstream phosphorylation of P65 in nuclear factor-κB (NF-κB) signaling. In addition, VA inhibited mitogen-activated protein kinase (MAPK) signaling molecules P-JNK, P-ERK, and P-P38, inhibiting the production of inflammatory mediators and thus inhibiting Aggrecan and Collagen Type II （COL2）degeneration. In vivo, VA reduced DMM-induced osteophytes and spurs, suppressed subchondral bone destruction, and reduced articular cartilage erosion. ConclusionOur study demonstrated that VA is an effective candidate for OA treatment.

Donor-Matched Peripheral Blood–Derived Mesenchymal Stem Cells Combined With Platelet-Rich Plasma Synergistically Ameliorate Surgery-Induced Osteoarthritis in Rabbits: An In Vitro and In Vivo Study

Background: Osteoarthritis (OA) is a common disease that causes joint pain and disability. Stem cell therapy is emerging as a promising treatment for OA. Purpose: To evaluate the ability of peripheral blood–derived mesenchymal stem cells (PBMSCs) combined with donor-matched platelet-rich plasma (PRP) to treat OA in a rabbit model. Study Design: Controlled laboratory study. Methods: PBMSCs and donor-matched PRP were isolated and prepared from the same rabbit. PBMSCs were treated with serum-free medium, fetal bovine serum, and PRP; a series of PBMSC behaviors, including proliferation, migration, and adhesion, were compared among groups. The ability of PBMSCs or PRP alone and PBMSCs+PRP to protect chondrocytes against proinflammatory cytokine (interleukin 1β [IL-1β]) treatment was compared by analyzing reactive oxygen species (ROS)–scavenging ability and apoptosis. Real-time quantitative polymerase chain reaction and immunofluorescence were used to investigate the expression of extracellular matrix (ECM) metabolism genes and proteins, and Western blotting was used to explore the potential mechanism of the corresponding signaling pathway. In vivo, the effect of PBMSCs+PRP on cartilage and inflammation of the synovium was observed in a surgery-induced OA rabbit model via gross observation, histological and immunohistochemical staining, and enzyme-linked immunosorbent assay. Results: Proliferation, migration, and adhesion ability were enhanced in PBMSCs treated with PRP. Moreover, compared with either PBMSCs or PRP alone, PBMSCs+PRP enhanced ROS-scavenging ability and inhibited apoptosis in IL-1β–treated chondrocytes. PBMSCs+PRP also reversed the IL-1β–induced degradation of collagen type 2 and aggrecan and increased expression of matrix metalloproteinase 13, and this effect was related to increased expression of ECM synthesis and decreased expression of degradation and inflammatory genes and proteins. Mechanistically, PBMSCs+PRP reduced the phosphorylation of inhibitor of nuclear factor-κBα (IκBα), which further inhibited the phosphorylation of downstream nuclear factor–κB (NF-κB) in the NF-κB signaling pathway. In vivo, compared with PBMSCs or PRP alone, intra-articular (IA) injection of PBMSCs+PRP enhanced cartilage regeneration and attenuated synovial inflammation in OA-induced rabbits. Conclusion: These results demonstrate that PRP could enhance biological activities, including viability, migration, and adhesion, in PBMSCs. PBMSCs+PRP could rescue ECM degeneration by inhibiting inflammatory signaling in IL-1β–treated OA chondrocytes. In addition, IA injection of PBMSCs+PRP effectively attenuated OA progression in a surgery-induced OA rabbit model. Clinical Relevance: PBMSCs+PRP may provide a promising treatment for knee OA, and this study can advance the related basic research.

FBN1 knockout promotes cervical artery dissection by inducing N-glycosylation alternation of extracellular matrix proteins in rat VSMCs

FBN1 mutation promotes the degeneration of microfibril structures and extracellular matrix (ECM) integrity in the tunica media of the aorta in Marfan syndrome. However, whether FBN1 modulates cervical artery dissection (CAD) development and the potential molecular mechanisms of abnormal FBN1 in CAD remains elusive. In this study, FBN1 deficiency participated in the development of CAD and influenced the proliferation, apoptosis, and migration of vascular smooth muscle cells. FBN1 knockout induced alternations in mRNA levels of the transcriptome, protein expression of the proteome, and abundance of N-glycosylation of the N-glycoproteome. Comprehensive analysis of multiple omics showed up-regulation in mRNA levels of ECM proteins; yet, both the ECM protein levels and relative abundance of N-glycosylation were decreased. Moreover, we performed in vivo experiments to confirm the altered glycosylation of proteins in vascular smooth muscle cells. In conclusion, FBN1 deletion in vascular smooth muscle cells can result in altered N-glycosylation of ECM protein, which were critical for the stability of ECM and the process of CAD. This may open the way for a novel therapeutic strategy to treat people with CAD.

The mechanism by which Naru 3 pill protects against intervertebral disc cartilage endplate degeneration based on network pharmacology and experimental verification

ContextNaru 3 pill is a traditional Mongolian medicine for the treatment of intervertebral disc degeneration (IDD), but the mechanism is not yet clear.ObjectiveThis study investigated the mechanism of Naru 3 pill in the treatment of IDD.Materials and methodsActive ingredients and related targets of Naru 3 pill, as well as IDD-related genes, were collected from public databases. The analysis was performed by protein‒protein interaction network analysis, gene ontology and Kyoto Gene and Genome Encyclopedia (KEGG) functional enrichment analysis, molecular docking and molecular dynamics simulations. Finally, the network pharmacology results were validated by in vitro experiments.ResultsNetwork analysis showed that sesamin, piperine and ellagic acid were potential key components and CASP3, BAX and BCL2 were key targets. KEGG analysis indicated the apoptotic pathway as a potential pathway. Molecular docking showed that sesamin interacted better with the targets than the other components. The results of molecular dynamics simulations indicated that the three systems BAX-sesamin, BCL2-sesamin and CASP3-sesamin were stable and reasonable during the simulation. In vitro experiments showed that sesamin had the least effect on cell growth and the most pronounced proliferation-promoting effect, and so sesamin was considered the key component. The experiments confirmed that sesamin had antiapoptotic effects and reversed the expression of CASP3, BAX and BCL2 in degeneration models, which was consistent with the network pharmacology results. Furthermore, sesamin alleviated extracellular matrix (ECM) degeneration and promoted cell proliferation in the IDD model.ConclusionThe present study suggested that Naru 3 pill might exert its therapeutic and antiapoptotic effects on IDD by delaying ECM degradation and promoting cell proliferation, which provides a new strategy for the treatment of IDD.

Intra-articular treatment of temporomandibular joint osteoarthritis by injecting actively-loaded meloxicam liposomes with dual-functions of anti-inflammation and lubrication

Temporomandibular joint (TMJ) osteoarthritis is a common osteochondral degenerative disease which can severely affect patient's mouth opening and mastication. Meloxicam (MLX), one of the most widely used non-steroidal anti-inflammatory drugs, is the main clinical therapy for the treatment of TMJ osteoarthritis. However, the clinical effect is greatly compromised because of its poor water solubility and high lipophilicity. In the present study, we developed an actively-loaded liposomal formulation, namely MLX-Ca(AC)2Lipo, using meglumine to enhance aqueous solubility and divalent metal (Ca2+) solution to improve encapsulation efficiency. By the formation of the nano-bowl shaped MLX-Ca precipitates inside the liposomes, MLX-Ca(AC)2Lipo successfully achieved an optimal encapsulation efficiency as high as 98.4% compared with previous passive loading method (60.6%). Additionally, MLX-Ca(AC)2Lipo maintained stable, and the slow drug release not only prolonged the duration of drug efficacy but also improved bioavailability. It was shown in the in vitro and in vivo tests that MLX-Ca(AC)2Lipo downregulated the synthesis of the inflammatory factors (such as prostaglandin-E2) and as a consequence reduced chondrocytes apoptosis and extracellular matrix degeneration. Furthermore, the intra-articular injection of MLX-Ca(AC)2Lipo enhanced bioinspired lubrication of TMJ, protecting the cartilage from progressive wear. In summary, MLX-Ca(AC)2Lipo with dual-functions of anti-inflammation and lubrication is a promising nanomedicine for the treatment of TMJ osteoarthritis by intra-articular injection.

Identification of Diagnostic Markers in Synovial Tissue of Osteoarthritis by Weighted Gene Coexpression Network.

Osteoarthritis (OA) is a serious threat to human health. However, the etiology and pathogenesis of the disease are not fully understood. Most researchers believe that the degeneration and imbalance of articular cartilage, extracellular matrix, and subchondral bone are the fundamental causes of osteoarthritis. However, recent studies have shown that synovial lesions may precede cartilage, which may be an important precipitating factor in the early stage of OA and the whole course of the disease. This study aimed to conduct an analysis based on sequence data from the Gene Expression Omnibus (GEO) database to investigate the presence of effective biomarkers in the synovial tissue of osteoarthritis for the diagnosis and control of OA progression. In this study, the differentially expressed OA-related genes (DE-OARGs) in osteoarthritis synovial tissues were extracted in the GSE55235 and GSE55457 datasets using the Weighted Gene Co-expression Network Analysis (WGCNA) and limma. Least-Absolute Shrinkage and Selection Operator (LASSO) algorithm was used to select the diagnostic genes based on the DE-OARGs by glmnet package. 7 genes were selected as diagnostic genes including SAT1, RLF, MAFF, SIK1, RORA, ZNF529, and EBF2. Subsequently, the diagnostic model was constructed and the results of the Area Under the Curve (AUC) demonstrated that the diagnostic model had high diagnostic performance for OA. Additionally, among the 22 immune cells of the Cell type Identification By Estimating Relative Subsets Of RNA Transcripts (CIBERSORT) and the 24 immune cells of the single sample Gene Set Enrichment Analysis (ssGSEA), 3 immune cells and 5 immune cells were different between the OA and normal samples, respectively. The expression trends of the 7 diagnostic genes were consistent in the GEO datasets and the results of the real-time reverse transcriptionPCR (qRT-PCR). The results of this study demonstrate that these diagnostic markers have important significance in the diagnosis and treatment of OA, and will provide further evidence for the clinical and functional studies of OA.

SPTLC2 ameliorates chondrocyte dysfunction and extracellular matrix metabolism disturbance in vitro and in vivo in osteoarthritis

Disturbances in chondrocyte extracellular matrix (ECM) metabolism in osteoarthritis (OA) are a major cause of OA and potentially lead to personal disability, placing a huge burden on society. Chondrocyte apoptosis and ECM catabolism have a major role in the OA process. Firstly, bioinformatics analysis was performed to screen differentially expressed genes (DEGs) in OA, and serine palmitoyltransferase subunit 2 (SPTLC2) was chosen, which had high-level expression in the OA cartilage tissues and OA chondrocytes. Overexpression and knockdown of SPTLC2 were achieved in OA chondrocytes and normal chondrocytes respectively to study the effect of SPTLC2 upon ECM metabolism of chondrocytes. Cell viability and apoptosis were measured using MTT and flow cytometry analyses; SPTLC2 overexpression enhanced the OA chondrocyte viability and decreased apoptotic rate. In addition, Western blot detection of ECM-related factors (Collagen I, Collage II, MMP-1, MMP-3, and MMP-13) revealed that SPTLC2 overexpression promoted the expression of collagens (Collagen I and Collage II) and suppressed matrix metalloproteinase (MMP-1, MMP-3, and MMP-13) level. In contrast, SPTLC2 knockdown in normal chondrocytes showed opposite effects on cell viability, apoptosis, and ECM degeneration. The articular cartilage of OA rats was transfected with lentivirus overexpressing SPTLC2; HE and Safranin-O fast green demonstrated that SPTLC2 overexpression could alleviate chondrocyte injuries and slow down the development of OA. In conclusion, SPTLC2 plays a role in OA and may be a potential target gene for the treatment of OA.

4-Octyl itaconate treatment inhibits mitochondrial dysfunction and ER stress via stimulating SIRT1 expression in vitro and attenuates osteoarthritis process in murine DMM model in vivo

Osteoarthritis (OA), a degenerative disease, has many pathological factors, including oxidative stress (OS), endoplasmic reticulum (ER) stress, apoptosis, and extracellular matrix (ECM) degeneration. OS regulates the pathological progression of OA via mitochondrial dysfunction, and ER stress and by modulating the derivation and elimination of reactive oxygen species (ROS). Though. 4-octyl itaconate (4-OI) is known for its effects on the treatment of various diseases, how it affects OA and its underlying mechanism still needs to be elucidated. Therefore, it is necessary to explore the effects of 4-OI both in vivo and in vitro. In our study, we used H2O2, which can accumulate ROS, OS and ER stress, to imitate the OA model in vitro. Using western blot, real-time quantitative reverse transcription polymerase chain reaction and immunofluorescence staining, we found that 125 μM 4-OI can inhibit apoptosis, ECM degeneration, mitochondrial dysfunction, ER stress and abundant accumulation of ROS in chondrocytes. We also showed that 4-OI can activate the Sirtuin1 gene (SIRT1). We used a SIRT1 inhibitor EX527 to identify the relationships among these pathological factors, and it showed that the 4-OI-mediated effect was achieved by promoting the SIRT1. In vivo, the bilateral medial meniscus was resected to establish the destabilization of the medial meniscus model wherein intraperitoneal injections of 2% and 5% 4-OI postponed the progression of OA. Collectively, this study confirmed that 4-OI can potentially impede the progression of OA by promoting the expression of SIRT1.