Inhibits Chondrocyte Apoptosis Research Articles

Tuina Inhibits Chondrocyte Apoptosis by Regulating the FAK/PI3K/Akt Pathway in Rabbits with IVDD.

Intervertebral disc degeneration (IVDD), a key contributor to degenerative spinal diseases such as cervical spondylosis, significantly influences the quality of life of patients. Tuina, historically employed in the clinical management of cervical spondylosis, has demonstrated positive therapeutic outcomes; however, the mechanism of Tuina remains unclear. This study examined the efficacy of Tuina in correcting the imbalanced structure of the cervical spine and its impact on apoptotic chondrocytes within the cervical disc. The underlying mechanisms were explored using a rabbit model of IVDD induced by dynamic and static imbalances. The IVDD rabbit model was established by restraining the head in a downward position for 12 weeks (Model group). In the Tuina1 group, treatment was performed on the posterior cervical trapezius muscle daily for 2 weeks, whereas in the Tuina2 group, treatment was performed on both the posterior cervical trapezius and anterior sternocleidomastoid muscles daily for 2 weeks. After treatment, X-ray, micro-computed tomography (CT), histological staining, qRT-PCR, and western blotting were used to evaluate the mechanism by which Tuina inhibits chondrocyte apoptosis. The results demonstrated that Tuina treatment inhibited chondrocyte apoptosis in cervical discs by adjusting the neck structure balance, and a more significant therapeutic effect was observed in the Tuina2 group. Lateral cervical spine X-ray and CT scans in rabbits revealed notable improvements in cervical spine curvature and vertebral structure in the treatment groups compared with those in the Model group. Hematoxylin and eosin staining and TUNEL staining further confirmed the positive impact of Tuina treatment on intervertebral disc tissue morphology and chondrocyte apoptosis. Additionally, western blotting and immunohistochemical analysis showed that Tuina treatment suppressed chondrocyte apoptosis by downregulating Bax and caspase-3 while upregulating Bcl-2. Western blotting results further indicated that Tuina could activate the FAK/PI3K/Akt signaling pathway by mediating integrin-β1. Tuina treatment inhibited chondrocyte apoptosis in cervical discs by activating the FAK/PI3K/Akt signaling pathway, providing convincing evidence to support Tuina treatment as a promising method for IVDD.

CHMP5 attenuates osteoarthritis via inhibiting chondrocyte apoptosis and extracellular matrix degradation: involvement of NF-κB pathway

BackgroundOsteoarthritis (OA), the most common joint disease, is linked with chondrocyte apoptosis and extracellular matrix (ECM) degradation. Charged multivesicular body protein 5 (CHMP5), a member of the multivesicular body, has been reported to serve as an anti-apoptotic protein to participate in leukemia development. However, the effects of CHMP5 on apoptosis and ECM degradation in OA remain unclear.MethodsIn this study, quantitative proteomics was performed to analyze differential proteins between normal and OA patient articular cartilages. The OA mouse model was constructed by the destabilization of the medial meniscus (DMM). In vitro, interleukin-1 beta (IL-1β) was used to induce OA in human chondrocytes. CHMP5 overexpression and silencing vectors were created using an adenovirus system. The effects of CHMP5 on IL-1β-induced chondrocyte apoptosis were investigated by CCK-8, flow cytometry, and western blot. The effects on ECM degradation were examined by western blot and immunofluorescence. The potential mechanism was explored by western blot and Co-IP assays.ResultsDownregulated CHMP5 was identified by proteomics in OA patient cartilages, which was verified in human and mouse articular cartilages. CHMP5 overexpression repressed cell apoptosis and ECM degradation in OA chondrocytes. However, silencing CHMP5 exacerbated OA chondrocyte apoptosis and ECM degradation. Furthermore, we found that the protective effect of CHMP5 against OA was involved in nuclear factor kappa B (NF-κB) signaling pathway.ConclusionsThis study demonstrated that CHMP5 repressed IL-1β-induced chondrocyte apoptosis and ECM degradation and blocked NF-κB activation. It was shown that CHMP5 might be a novel potential therapeutic target for OA in the future.

Network pharmacology combined with experimental validation to investigate the effect of Rongjin Niantong Fang on chondrocyte apoptosis in knee osteoarthritis.

Knee osteoarthritis (KOA) is a chronic degenerative disease that affects the quality of life of middle‑aged and elderly individuals, and is one of the major factors leading to disability. Rongjin Niantong Fang (RJNTF) can alleviate the clinical symptoms of patients with KOA, but the molecular mechanism underlying its beneficial effects on KOA remains unknown. Using pharmacological analysis and in vitro experiments, the active components of RJNTF were analyzed to explore their potential therapeutic targets and mechanisms in KOA. The potential targets and core signaling pathways by which RJNTF exerts its effects on KOA were obtained from databases such as Gene Expression Omnibus, Traditional Chinese Medicine Systems Pharmacology and Analysis Platform. Subsequently, chondrocyte apoptosis was modeled using hydrogen peroxide (H2O2). Cell Counting Kit‑8 assay involving a poly [ADP‑ribose] polymerase‑1 (PARP1) inhibitor, DAPI staining, reverse transcription‑quantitative PCR, Annexin V‑FITC/PI staining and flow cytometry, western blotting and co‑immunoprecipitation analysis were used to determine the therapeutic efficacy of RJNTF on KOA and to uncover the molecular mechanism. It was found that PARP1‑knockdown lentivirus, incubation with PARP1 inhibitor PJ34, medium and high doses of RJNTF significantly reduced H2O2‑induced chondrocyte apoptosis. Medium and high doses of RJNTF downregulated the expression of cleaved caspase‑3, cleaved PARP1 and PAR total proteins, as well as nucleus proteins of apoptosis‑inducing factor (AIF) and migration inhibitory factor (MIF), and upregulated the expression of caspase‑3, PARP1 total protein, as well as the cytoplasmic expression of AIF and MIF, suggesting that RJNTF may inhibit chondrocyte apoptosis through the PARP1/AIF signaling pathway.

Guasha improves knee osteoarthritis by inhibiting chondrocyte apoptosis and regulating expression of autophagy-related genes and proteins in rats.

To observe the effect of Guasha on inflammation factors, apoptosis and autophagy in the cartilage tissue of knee joint in rats with knee osteoarthritis (KOA), so as to explore its mechanisms underlying improvement of KOA. A total of 51 male SD rats were randomized into three groups：blank control, KOA model and Guasha (n= 17 in each group) . The rats in the blank control group received intra-articular injection of 0.9% NaCl solution in the right knee joint. The KOA model was established by intraarticular injection of glutamate sodium iodoacetic acid in the right knee joint. For rats of the Guasha group, Guasha (at a frequency of 1 time/s, and an applied pressure of 0.3-0.5 kgf) was applied to "Yanglingquan" (GB34) and "Xuehai"(SP10) areas of the right leg, once every other day, for 7 consecutive sessions. The circumference of the right knee was measured, The histopathological changes of right knee cartilage were observed after H.E. staining. The contents of inflammatory factors interleukin (IL)-1β and tumor necrosis factor (TNF)-α in the right knee articular cartilage tissue were assayed using ELISA. The expression levels of autophagy-related key molecule Beclin-1 (homologous series of yeast Atg6), light chain protease complication 3 type II/I (LC3II/LC3 I), ubiquitin binding factor 62 (P62) and cysteine aspartate protease-3 (Caspase-3) mRNAs and proteins of the right knee articular cartilage tissue were measured using real-time fluorescent quantitative PCR and Western blot, separately. The apoptosis of chondrocytes was assayed using TUNEL staining, and the immunoactivity of LC3 determined using immunofluorescence staining. After modeling, the right knee circumfe-rence of the model and Guasha groups was significantly increased compared with the blank control group (P<0.01), and after the intervention, the knee circumference of the Guasha group was markedly decreased in comparison with that of the model group (P<0.05). Results of H.E. staining showed obvious degeneration and defects in the cartilage tissue, necrosis of a large number of chondrocytes, fibrous hyperplasia, accompanied by inflammatory cell infiltration, osteoclast increase, fibroplasia and bone trabecular destruction in the model group, which was relatively milder in the Guasha group. Compared with the blank control group, the expression of Beclin-1 and LC3 mRNAs and proteins, and LC immunofluorescence intensity in the right knee articular cartilage tissue were significantly down-regulated (P<0.01, P<0.001), whereas the expression of P62 and Caspase-3 mRNAs and proteins, the apoptosis rate, contents of IL-1β and TNF-α in the right knee articular cartilage tissue considerably increased (P<0.01, P<0.001) in the model group. In contrast to the model group, the Guasha group had an apparent increase in the expression levels of Beclin-1 and LC3 mRNAs and proteins and LC immunofluorescence intensity in the right knee articular cartilage tissue (P<0.05), and a pronounced decrease in the expression of P62 and Caspase-3 mRNAs and proteins, the apoptosis rate, and contents of IL-1β and TNF-α in the right knee articular cartilage tissue (P<0.05, P<0.01). Guasha stimulation of GB34 and SP10 can improve joint cartilage damage in KOA rats, which may be associated with its functions in inhibiting the excessive release of inflammatory factors and apoptosis, possibly by down-regulating the expression of P62 and Caspase-3 mRNAs and proteins and up-regulating the expression of Beclin-1 and LC3 mRNAs and proteins, and by promoting autophagy of chondrocytes.

MiR-515-5p targeting Toll-like receptor 4 regulates myeloid differentiation primary response gene 88/nuclear factor-kappa B pathway to inhibit apoptosis and inflammatory response of osteoarthritis chondrocytes

To explore the molecular mechanism of miR-515-5p in inhibiting chondrocyte apoptosis and alleviating inflammatory response in osteoarthritis (OA). Human cartilage cell line C28/I2 was cultured in vitro and treated with 10 ng/mL interleukin 1β (IL-1β) for 24 hours to construct an in vitro OA model. C28/I2 cells were transfected with miR mimics, mimics negative control (NC), over expression (oe)-NC, and oe-Toll-like receptor 4 (TLR4), respectively, and then treated with 10 ng/mL IL-1β for 24 hours to establish OA model. Cell proliferation capacity was detected by cell counting kit 8 and 5-Ethynyl-2'-deoxyuridine, cell apoptosis and cell cycle were detected by flow cytometry, and B-cell lymphoma 2 protion (Bcl-2), Bcl-2-associated X protein (Bax), cleaved-Caspase-3, TLR4, myeloid differentiation primary response gene 88 (MyD88), p65 and phosphorylated p65 (p-p65) protein expression levels were detected by Western blot. Real-time fluorescence quantitative PCR was used to detect mRNA expression levels of miR-515-5p and TLR4, and ELISA was used to detect pro-inflammatory factor prostaglandin E2 (PGE2), tumor necrosis factor α (TNF -α), and IL-6 levels in cell supernatant. The potential binding sites between miR-515-5p and TLR4 were predicted by BiBiServ2 database, and the targeting relationship between miR-515-5p and TLR4 was verified by dual luciferase reporting assay. After the treatment of C28/I2 cells with IL-1β, the expressions of miR-515-5p and Bcl-2 protein and the proliferation ability of C28/I2 cells significantly reduced. The expression levels of Bax and cleaved-Caspase-3 protein, the levels of pro-inflammatory factors (PGE2, TNF-α, IL-6) in the supernatant of C28/I2 cells, and the apoptosis of C28/I2 cells significantly increased. In addition, the proportion of the cells at S phase and G 2 phase decreased significantly, and the proportion of cells at G 1 phase increased significantly, suggesting that the cell cycle was blocked after IL-1β treatment. After transfection with miR mimics, the expression level of miR-515-5p in the cells significantly up-regulated, partially reversing the apoptosis of OA chondrocytes induced by IL-1β, and alleviating the cycle arrest and inflammatory response of OA chondrocytes. After treating C28/I2 cells with IL-1β, the mRNA and protein levels of TLR4 significantly increased. Overexpression of miR-515-5p targeted inhibition of TLR4 expression and blocked activation of MyD88/nuclear factor κB (NF-κB) pathway. Overexpression of TLR4 could partially reverse the effect of miR mimics on IL-1β-induced apoptosis and inflammation of OA chondrocytes. miR-515-5p negatively regulates the expression of TLR4, inhibits the activation of MyD88/NF-κB pathway and apoptosis of OA chondrocytes, and effectively alleviates the inflammatory response of the cells.

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-1β (IL-1β), 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-1β, 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 β, 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.

Elucidating Cyathula Officinals' mechanism in osteoarthritis treatment: Network pharmacology and empirical evidence on anti-inflammatory actions

In this study, we explored the therapeutic potential of Cyathula Officinals (CNX) in Knee Osteoarthritis (KOA) treatment. Utilizing network pharmacology and in vitro experiments, we identified active ingredients, action targets and pathways in CNX. Our analysis, integrating databases like TCMSP, SwissTarget Prediction, Genecards, CTD, STRING, and DAVID, highlighted 396 action targets and 283 disease targets, pinpointing 64 intersection genes linked to KOA. The significant involvement of the MAPK and NF-κB pathways in CNX's anti-inflammatory action was validated through qPCR, which might underlie CNX's efficacy in inhibiting chondrocyte apoptosis and IL-6 expression. These findings suggest CNX's potential in KOA management, offering insights for its clinical application.

Protective effects of pulsed electromagnetic field therapy attenuates autophagy and apoptosis in osteoporotic osteoarthritis model rats by activating PPARγ

ABSTRACT Osteoporotic osteoarthritis (OPOA) is a specific phenotype of OA with high incidence and severe cartilage damage. This study aimed to explore the protective efficacy of PEMF on the progression of OPOA and observed the effects of PEMF on PPARγ, autophagy- and apoptosis-related proteins in OPOA rats. Rats were randomly divided into three groups: control group, OPOA group, and PEMF group (n = 6). One week after surgery, the rats in PEMF group were subjected to PEMF (3.82 mT, 8 Hz, 40 min/day and 5 day/week) for 12 weeks. Results showed that PEMF retarded cartilage degeneration and bone loss, as evidenced by pathological staining image, decreased MMP-13 expression and increased bone mineral density. PEMF inhibited the serum levels of inflammatory cytokines, and the expressions of caspase-3 and caspase-8, while upregulated the expression of PPARγ. Moreover, PEMF significantly improved the autophagy disorders, represented by decrease expressions of Beclin-1, P62, and LC3B. The research demonstrates that PEMF can effectively prevent cartilage and subchondral bone destruction in OPOA rats. The potential mechanism may be related to upregulation of PPARγ, inhibition of chondrocyte apoptosis and inflammation, and improvement of autophagy disorder. PEMF therapy thus shows promising application prospects in the treatment of postmenopausal OA.

Human Umbilical Cord Mesenchymal Stem Cells Alleviate Rat Knee Osteoarthritis via Activating Wnt/ β-catenin Signaling Pathway.

Osteoarthritis (OA) is a chronic disease characterized by joint cartilage degeneration, destruction, and osteogenic hyperplasia. Human umbilical cord mesenchymal stem cells (hUCMSCs) have attracted increasing research interest due to their high clonogenic, proliferative, and migratory potential, as well as their improved secretion of relevant chondrogenic factors. This study evaluated the therapeutic potential and underlying mechanism of hUC-MSCs in alleviating pathological symptoms of OA. For the in vivo study, OA rats were established by the Hulth method to observe the therapeutic effect of intra-articular injection of hUC-MSCs. X-ray tests, gross observations, and histological and immunohistochemical assessments were conducted in rats. Levels of interleukin-1 beta (IL-1β), IL-6, matrix metalloproteinase-13 (MMP-13), and tissue inhibitor matrix metalloproteinase-1 in rats' synovial fluid were measured using enzyme-linked immunosorbent assay kits. For the in vitro study, hUC-MSCs and chondrocytes were cultured to explore the effect and underlying mechanisms of hUC-MSCs on OA. Apoptosis, proliferation, and glycosaminoglycan (GAG) were measured in the chondrocytes. The relative expression of aggrecan, COL-2, and SOX-9 mRNA was quantified by real-time polymerase chain reaction. Expressions of Wnt/β-catenin signaling molecules were measured by Western blot. We found that intra-articular injection of hUC-MSCs reduced the combined score, increased the expression of collagen II, and decreased the expression of MMP-13, IL-1β, and IL-6 in rat knee joints. Additionally, hUC-MSCs increased the content of GAGs, inhibited chondrocyte apoptosis, and promoted chondrocyte proliferation. The expression of aggrecan, COL-2, and SOX-9 mRNA in chondrocytes was promoted by hUC-MSCs via activation of the Wnt/β-catenin signaling pathway. Overall, this study demonstrated that hUC-MSCs induce the secretion of some cytokines via the paracrine function to activate the Wnt/β-catenin signaling pathway to reduce the pathological condition of OA and maintain the proper expression of cytokines and extracellular matrix proteins.

PiRNA mmu_piR_037459 suppression alleviated the degeneration of chondrocyte and cartilage

ObjectiveOsteoarthritis (OA) is a prevalent chronic degenerative joint ailment. Its primary pathological characteristics encompass degeneration of articular cartilage, inflammation of the synovium, and alterations in the subchondral bone proximate to the cartilage. Chondrocytes, as the sole cell type within articular cartilage, assume a crucial role in upholding the dynamic equilibrium between anabolic and catabolic processes within the extracellular matrix of articular cartilage. IL-1β stands as a pivotal inflammatory factor that instigates cartilage degeneration. piRNA, categorized as a subset of brief non-coding RNAs spanning nucleotide lengths of 26-31nt, assumes a significant regulatory role in cellular function. MethodsSmall RNA sequencing and quantitative PCR (qPCR) were employed to investigate the impact of the inflammatory factor IL-1β on piRNA expression within chondrocytes. The regulation of mmu_piR_037459 expression in chondrocytes was achieved using piRNA mimics and inhibitors. Additionally, collagen II expression was assessed through both qPCR and Western blot analysis. Chondrocyte apoptosis was evaluated via flow cytometry and clonogenesis assays. To assess the influence of mmu_piR_037459 on osteoarthritis, a mouse model of anterior cruciate ligament transection (ACLT) was established. Furthermore, the regulatory effect of mmu_piR_037459 on USP7 was investigated using bioinformatics and a luciferase reporter gene assay. Resultsmmu_piR_037459 inhibited the expression of collagen II in chondrocytes, inhibited the proliferation of chondrocytes, and promoted the apoptosis of chondrocytes. mmu_piR_037459 affected the function of chondrocytes by regulating the expression of USP7. Inhibition of mmu_piR_037459 expression could promote chondrocyte proliferation, inhibit chondrocyte apoptosis, and alleviate the degeneration of OA cartilage. ConclusionsThis study suggests that mmu_piR_037459 maybe a new therapeutic targets and strategies for the treatment of OA.

Stromal Cell-Derived Factor 1α Inhibits Chondrocyte Apoptosis and Promotes Autophagy Through the Akt Signaling Pathway

To investigate the effects of stromal cell-derived factor 1α (SDF-1α) on the apoptosis and autophagy of chondrocytes and the underlying mechanisms. Chondrocytes were isolated from the knee joints of neonatal mice. The chondrocytes were then stimulated with 0 (the control group), 50, 100, and 200 ng/mL of SDF-1α. CCK-8 assay was performed to determine the effects of SDF-1α stimulation for 24 h, 48 h, and 72 h on the viability of the chondrocytes. Wound healing assay was conducted to determine the effects of SDF-1α stimulation for 12 h and 24 h on chondrocyte migration. The changes in the expression of Akt signaling pathway proteins in chondrocytes were determined by Western blot assay. Chondrocytes were stimulated with 0 (the control group) and 200 ng/mL of SDF-1α. Flow cytometry was performed to determine the effect of SDF-1α on the apoptosis of chondrocytes. Transmission electron microscope was used to examine the effect of SDF-1α on chondrocyte autophagy. Immunofluorescence staining assays were performed to visualize the differences in p-Akt expression and distribution in chondrocytes treated with SDF-1α. Compared with the control group, findings for the experimental groups showed that SDF-1α at the concentrations of 50, 100, and 200 ng/mL did not decrease chondrocyte activity at any time point (P<0.01) and it consistently promoted chondrocyte migration at 24 h (P<0.05). Western blot results revealed that, in comparison to the control group, SDF-1α at concentrations of 50, 100, and 200 ng/mL significantly up-regulated the protein expression of p-Akt in chondrocytes, while no significant difference in Akt expression was observed. Flow cytometry demonstrated that SDF-1α could inhibit chondrocyte apoptosis (P<0.05) and transmission electron microscopic observation showed that SDF-1α promoted chondrocyte autophagy (P<0.05). Immunofluorescence staining showed that the expression of p-Akt in chondrocytes was concentrated in the perinuclear area of the cells and this expression was further enhanced in the perinuclear area of the chondrocytes after treatment with SDF-1α. SDF-1α inhibits chondrocyte apoptosis and promotes chondrocyte migration and autophagy through activating the Akt signaling pathway.

Comparison of the ability of exosomes and ectosomes derived from adipose-derived stromal cells to promote cartilage regeneration in a rat osteochondral defect model

BackgroundExtracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) offer promising prospects for stimulating cartilage regeneration. The different formation mechanisms suggest that exosomes and ectosomes possess different biological functions. However, little attention has been paid to the differential effects of EV subsets on cartilage regeneration.MethodsOur study compared the effects of the two EVs isolated from adipose-derived MSCs (ASCs) on chondrocytes and bone marrow–derived MSCs (BMSCs) in vitro. Additionally, we loaded the two EVs into type I collagen hydrogels to optimize their application for the treatment of osteochondral defects in vivo.ResultsIn vitro experiments demonstrate that ASC-derived exosomes (ASC-Exos) significantly promoted the proliferation and migration of both cells more effectively than ASC-derived ectosomes (ASC-Ectos). Furthermore, ASC-Exos facilitated a stronger differentiation of BMSCs into chondrogenic cells than ASC-Ectos, but both inhibited chondrocyte apoptosis to a similar extent. In the osteochondral defect model of rats, ASC-Exos promoted cartilage regeneration in situ better than ASC-Ectos. At 8 weeks, the hydrogel containing exosomes group (Gel + Exo group) had higher macroscopic and histological scores, a higher value of trabecular bone volume fraction (BV/TV), a lower value of trabecular thickness (Tb.Sp), and a better remodeling of extracellular matrix than the hydrogel containing ectosomes group (Gel + Ecto group). At 4 and 8 weeks, the expression of CD206 and Arginase-1 in the Gel + Exo group was significantly higher than that in the Gel + Ecto group.ConclusionOur findings indicate that administering ASC-Exos may be a more effective EV strategy for cartilage regeneration than the administration of ASC-Ectos.

Astragalus polysaccharides ameliorate osteoarthritis via inhibiting apoptosis by regulating ROS-mediated ASK1/p38 MAPK signaling pathway targeting on TXN

This research aims to explore the potential of astragalus polysaccharides (APS) in treating osteoarthritis. The primary component of APS extracted in this study was glucose, and noticeably it had a relatively high content of glucuronic acids. In vitro, APS reduced ROS levels, protected chondrocytes from apoptosis, and promoted collagen II expression by regulating ASK1 (apoptosis-signal-regulating kinase1)/p38 cell apoptosis pathway. Further co-immunoprecipitation and immunofluorescence localization experiments demonstrated that the thioredoxin (TXN) antioxidant system was responsible for its bioactivity. Moreover, TXN silencing remarkably blocked the protective effects of APS, indicating that APS inhibited chondrocyte apoptosis by targeting TXN. In vivo, APS effectively mitigated cartilage loss and chondrocyte apoptosis and decreased expressions of p-ASK1 and p-p38. Collectively, this research first demonstrated that APS could ameliorate osteoarthritis by ASK1/p38 signaling pathway through regulating thioredoxin. In conclusion, APS holds promise as a nutraceutical supplement for osteoarthritis in future drug development.

4,8-Dicarboxyl-8,9-iridoid-1-glycoside inhibits apoptosis in human osteoarthritis chondrocytes via enhanced c-MYC-mediated cholesterol metabolism in vitro

BackgroundOsteoarthritis (OA) is a degenerative disease related to cholesterol metabolism disorders. However, current therapies for OA are insufficient and no convincing disease-modifying OA drugs exist. Therefore, we aimed to elucidate the mechanism by which borojoa iridoid glycoside (BIG) inhibits chondrocyte apoptosis in OA.MethodsBorojoa pulp was heated to 70 °C, and the main active substance in borojoa, BIG, was extracted by fractionation at an ultraviolet 254-nm absorption peak. Chondrocytes were identified by immunohistochemistry and visualized by immunofluorescence confocal microscopy. The proliferation of chondrocytes cultured with BIG was determined by MTS assay. The apoptosis of chondrocytes cultured with BIG was tested by Annexin V-FITC/PI, and the cytokine, protein, and cholesterol levels in chondrocytes were detected by ELISA, RT‒qPCR, Western blot, and biochemistry analyses. Protein‒protein interactions were verified by a coimmunoprecipitation (Co-IP) assay.ResultsBIG promoted chondrocyte proliferation and reduced apoptosis in vitro. BIG induced an alteration of the total RNA profiles in chondrocytes, and bioinformatic analysis showed that BIG inhibited chondrocyte apoptosis by promoting c-MYC expression; KEGG analysis confirmed that BIG-inhibited apoptosis was enriched in the cell cycle pathway. Flow cell cycle experiments confirmed that BIG promoted chondrocyte proliferation by significantly increasing the S phase cell number. The c-MYC inhibitor 10058-F4 stimulated the increased expression of IL-1β, IL-6, TNF-α, and AGEs and suppressed the cholesterol metabolism, which promoted chondrocyte apoptosis and autophagy. Co-IP analysis showed that BIG promoted the interaction of c-MYC and CH25H, Bcl-2, which suggests that BIG could inhibit chondrocyte apoptosis in part by enhancing c-MYC-mediated cholesterol metabolism.ConclusionsThis study confirmed that BIG promotes chondrocyte proliferation and inhibits apoptosis and autophagy, and BIG improving OA is associated with cholesterol metabolism. The results identify a potential mechanism by which BIG enhances c-MYC-mediated CH25H regulation of cholesterol metabolism in vitro and suggest that BIG might be a promising new drug against OA.

α2-Macroglobulin Promotes Chondrocyte Proliferation and Cartilage Matrix Synthesis via Inducing PCNA.

α2-Macroglobulin (A2M) can prevent cartilage degeneration by blocking many types of cartilage-degrading enzymes, but the mechanism remains to be clarified. This study aimed to test that A2M protects against cartilage degeneration by promoting chondrocyte proliferation and cartilage matrix synthesis via inducing proliferating cell nuclear antigen (PCNA). The cartilage degeneration of the anterior cruciate ligament transection (ACLT) model was evaluated by Safranin O-fast green staining, and articular cartilage degeneration was graded using the Osteoarthritis Research Society International (OARSI)-modified Mankin criteria. The chondrocyte proliferation was detected by 5-Bromodeoxyuridinc (BrdU), MTT, and Cell Counting Kit-8 (CCK8) methods. The chondrocyte apoptosis was detected by lactate dehydrogenase (LDH) assay and Annexin PI staining with the flow cytometer. The glycosaminoglycan (sGAG) and aggrecan in culture supernatant were measured by enzyme-linked immunosorbent assay (ELISA). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to analyze the type II collagen and aggrecan mRNA expression. The PCNA protein expression was analyzed by western blot and immunofluorescent staining. A2M can attenuate cartilage degeneration in ACLT rats. The OARSI scores for cartilage degeneration in the A2M group were lower than those in the phosphate-buffered saline (PBS) group. A2M can promote chondrocyte proliferation and inhibit chondrocyte apoptosis, promote the cartilage matrix synthesis in chondrocytes (type II collagen and aggrecan), and culture supernatant (sGAG and aggrecan). At the same time, it also up-regulated the PCNA protein expression in chondrocytes. A2M can promote chondrocyte proliferation and cartilage matrix synthesis via inducing PCNA expression.

Lentivirus-based shRNA of Caspase-3 gene silencing inhibits chondrocyte apoptosis and delays the progression of surgically induced osteoarthritis.

Chondrocyte apoptosis is an important pathological feature of osteoarthritis (OA). Excessive apoptosis of chondrocytes disrupts the dynamic balance of cell proliferation and apoptosis, with a marked reduction in chondrocytes and cartilage matrix disintegration, which represents the main pathology of OA. Caspases, especially Caspase-3, play a central role in cell apoptosis. In this study, a lentiviral vector was used to transduce caspase-3 short hairpin RNA (shRNA) into rat chondrocytes (RCs), and the apoptotic and phenotypic genes of RCs were analyzed using real-time PCR and western blotting in vitro. In addition, in vivo intra-articular injection of Caspase-3 shRNA lentivirus was performed in a surgically induced OA rat model. Our results showed that Caspase-3 gene silencing could down-regulate the TNF-α-mediated inflammatory gene expression of TNFR1, FADD, and IL-1β, apoptotic gene expression of APAF1, Caspase-3, and Caspase-9, thereby attenuating the apoptotic pathway in vitro. Caspase-3 gene silencing also attenuated TNF-α-mediated decreased gene expression of ACAN, Col1-a1, and Col2-a1. Furthermore, Caspase-3 gene silencing could effectively reduce the OARSI score, and gene expression of Caspase-3, Caspase-9, MMP13, and TNF-α in a surgically induced OA rat model. Caspase-3 gene silencing may serve as a novel therapeutic strategy for cartilage injury and OA.

The role of autophagy in mitigating osteoarthritis progression via regulation of chondrocyte apoptosis: A review

Osteoarthritis (OA) is the most prevalent chronic joint disease with an immense socioeconomic burden; however, no treatment has achieved complete success in effectively halting or reversing cartilage degradation, which is the central pathophysiological feature of OA. Chondrocytes loss or dysfunction is a significant contributing factor to the progressive cartilage deterioration as these sole resident cells have a crucial role to produce extracellular matrix proteins, thus maintaining cartilage structure and homeostasis. It has been previously suggested that death of chondrocytes occurring through apoptosis substantially contributes to cartilage degeneration. Although the occurrence of apoptosis in osteoarthritic cartilage and its correlation with cartilage degradation is evident, the causes of chondrocyte apoptosis leading to matrix loss are still not well-understood. Autophagy, an intracellular degradative mechanism that eliminates dysfunctional cytoplasmic components to aid cell survival in unfavourable conditions, is a potential therapeutic target to inhibit chondrocyte apoptosis and reduce OA severity. Despite accumulating evidence indicating significant cytoprotective effects of autophagy against chondrocyte apoptosis, the mechanistic link between autophagy and apoptosis in chondrocytes remains to be further explored. In this review, we summarize the relevant mechanistic events that perpetuate chondrocyte apoptosis and highlight the prominent role of autophagy in modulating these events to mitigate OA progression.

Umbilical cord mesenchymal stem cells relieve osteoarthritis in rats through immunoregulation and inhibition of chondrocyte apoptosis

This study aims to investigate the effectiveness of umbilical cord mesenchymal stem cells (UCMSCs) in treating osteoarthritis (OA). Sprague–Dawley rats were used in in vivo experiments and divided into four groups: normal, OA model, saline, and UCMSC-treated groups (n = 6). An OA model was established by injecting iodoacetic acid into the joint cavity. The results indicate that UCMSC transplantation significantly reduced joint surface and articular cartilage damage, and the levels of IL-1β, TNF-α, and MMP13 in the joint fluid were significantly reduced after UCMSC treatment. In vitro experiments showed that co-culturing UCMSCs and chondrocytes promoted the expression of aggrecan, COL2, SOX9, and BCL-2; downregulated the expression of BAX and BAD in chondrocytes; and promoted the expression of IL-10 and TGF-β1 in UCMSCs. Additionally, the supernatant of UCMSCs inhibited the expression of IL-1β and TNF-α in the articular cavity and promoted the expression of COL2 and aggrecan in vivo. These effects were impaired when IL-10 and TGF-β1 were removed. Collectively, UCMSC transplantation appears to improve joint pathology, reduce inflammatory factors, and decrease chondrocyte apoptosis, likely through the involvement of IL-10 and TGF-β1, thus providing a potential therapeutic option for patients with OA.

ALKBH5-mediated m6A demethylation of HS3ST3B1-IT1 prevents osteoarthritis progression

HS3ST3B1-IT1 was identified as a downregulated long noncoding RNA in osteoarthritic cartilage. However, its roles and mechanisms in the pathogenesis of osteoarthritis (OA) are unclear. In this study, we demonstrated that the expressions of HS3ST3B1-IT1 and its maternal gene HS3ST3B1 were downregulated and positively correlated in osteoarthritic cartilage. Overexpression of HS3ST3B1-IT1 significantly increased chondrocyte viability, inhibited chondrocyte apoptosis, and upregulated extracellular matrix (ECM) proteins, whereas HS3ST3B1-IT1 knockdown had the opposite effects. In addition, HS3ST3B1-IT1 significantly ameliorated monosodium-iodoacetate-induced OA invivo. Mechanistically, HS3ST3B1-IT1 upregulated HS3ST3B1 expression by blocking its ubiquitination-mediated degradation. Knockdown of HS3ST3B1 reversed the effects of HS3ST3B1-IT1 on chondrocyte viability, apoptosis, and ECM metabolism. AlkB homolog 5 (ALKBH5)-mediated N6-methyladenosine (m6A) demethylation stabilized HS3ST3B1-IT1 RNA. Together, our data revealed that ALKBH5-mediated upregulation of HS3ST3B1-IT1 suppressed OA progression by elevating HS3ST3B1 expression, suggesting that HS3ST3B1-IT1/HS3ST3B1 may serve as potential therapeutic targets for OA treatment.

Tranexamic Acid Causes Chondral Injury Through Chondrocytes Apoptosis Induced by Activating Endoplasmic Reticulum Stress

To investigate whether tranexamic acid (TXA) is cytotoxic in chondrocyte and cartilage tissues, as well as explore the mechanisms behind the possible toxicity in detail. We detected the cell viability of chondrocytes invitro and the change of morphology and specific invivo contents of cartilage after TXA treatment. Furthermore, we detected apoptosis in cartilage. We used apoptosis-specific staining, reactive oxygen species detection, mitochondrial membrane potential detection, flow cytometry, and western blot for apoptosis detection. Finally, we detected the activation of endoplasmic reticulum stress (ERS) in TXA-treated chondrocytes to clarify the mechanism behind chondrocyte apoptosis. TXA presented an increasing toxic effect with increasing concentrations, especially in the 100 mg/mL group. In addition, we found that 50 mg/mL and 100 mg/mL TXA significantly increased apoptosis in cartilage and subchondral bone. TXA could induce chondrocyte apoptosis in cell and protein levels with reactive oxygen species generation and mitochondrial membrane depolarization. An apoptosis inhibitor could inhibit the induced apoptosis. Next, TXA induced calcium overload in chondrocytes and increased ERS-specific protein expression, whereas ERS inhibitor blocked ERS activation and further inhibited chondrocyte apoptosis. We concluded that TXA had a toxic effect on chondrocytes by inducing apoptosis through ERS activation, especially in 50 mg/mL and 100 mg/mL groups. We recommend TXA concentrations of less than 50 mg/mL in joint surgeries. It is still unclear whether TXA has a toxic effect on cartilage when topically used in joint surgeries. The concentration also varies. This study provides additional evidence that TXA at high concentrations will cause cartilage damage, which will help to provide a new understanding of the clinical administration of TXA.