Icariin alleviates the apoptosis of chondrocytes in osteoarthritis through regulating SIRT-1-Nrf2-HO-1 signaling.

Icariin has shown the potential to treat osteoarthritis (OA), but the specific mechanism still needs further exploration. Therefore, this study attempted to reveal the effect and mechanism of icariin on OA based on in vitro and in vivo experiments. In vivo, a mouse model of OA was established by cutting the anterior cruciate ligament, and 10 mg/kg icariin was given to mice orally. Then, the OA injury and pathological changes of cartilage tissue in mice were identified by OA index and hematoxylin and eosin staining. In vitro, the viability of C28/I2 cells incubated with different concentrations of icariin was detected by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide assay. Subsequently, C28/I2 cells induced by IL-1ß were used as the cell model of OA, the expression of Sirtuin (SIRT)-1 in cells was knocked down, and icariin was added for intervention. Next, western blot was used to observe the expression level of sirtuin 1 (SIRT-1)-Nrf2-heme oxygenase 1 (HO-1) signaling pathway-related proteins in cells of each group. Besides, cell viability and apoptosis were detected by MTT and apoptosis assay, and DNA damage was observed by comet assay. In vivo experiments, intragastric administration of icariin could effectively reduce the OA index of mice, improve the pathological changes of cartilage tissue, and obviously activated the SIRT-1-Nrf2-HO-1 signaling pathway. In vitro experiments, icariin did not exhibit toxic effect on C28/I2 cells, but could activate the SIRT-1-Nrf2-HO-1 signaling pathway, improve the viability, reduce the level of apoptosis and relieve the DNA damage in OA cells; however, these effects were inhibited by si- SIRT-1. Icariin can improve the symptoms of OA by activating the SIRT-1-Nrf2-HO-1 signaling pathway.

Expression levels and DNA methylation profiles of the growth gene SHOX in cartilage tissues and chondrocytes.

All attempts to identify male-specific growth genes in humans have failed. This study aimed to clarify why men are taller than women. Microarray-based transcriptome analysis of the cartilage tissues of four adults and chondrocytes of 12 children showed that the median expression levels of SHOX, a growth gene in the pseudoautosomal region (PAR), were higher in male samples than in female samples. Male-dominant SHOX expression was confirmed by quantitative RT-PCR for 36 cartilage samples. Reduced representation bisulfite sequencing of four cartilage samples revealed sex-biased DNA methylation in the SHOX-flanking regions, and pyrosequencing of 22 cartilage samples confirmed male-dominant DNA methylation at the CpG sites in the SHOX upstream region and exon 6a. DNA methylation indexes of these regions were positively correlated with SHOX expression levels. These results, together with prior findings that PAR genes often exhibit male-dominant expression, imply that the relatively low SHOX expression in female cartilage tissues reflects the partial spread of X chromosome inactivation into PAR. Altogether, this study provides the first indication that sex differences in height are ascribed, at least in part, to the sex-dependent epigenetic regulation of SHOX. Our findings deserve further validation.

Intra-articular injection of miRNA-1 agomir, a novel chemically modified miRNA agonists alleviates osteoarthritis (OA) progression by downregulating Indian hedgehog in rats.

Our objective in this study is to determine whether intra-articular injection of miRNA-1 can attenuate the progression of OA in rats by down regulating Ihh. Knee chondrocytes were isolated from male Sprague-Dawley rats aged 2-3 days. Second-generation chondrocytes were transfected with miR-1 mimic and empty vector with lipo3000 for 6 h and then stimulated with 10 ng/mL IL-1ß for 24 h. OA-related and cartilage matrix genes were quantified using real-time quantitative polymerase chain reaction (RT-qPCR). Two-month-old male Sprague-Dawley rats were divided into three groups (n = 30?): sham operation group + 50 µL saline, anterior cruciate ligament transection (ACLT) group + 50 µL miR-1 agomir (concentration), and control group ACLT + 50 µL miR-1 agomir. Treatment was started one week after the operation. All animals were euthanized eight weeks after the operation. X-rays and micro-CT were used to detect imaging changes in the knee joints. FMT was used to monitor joint inflammation in vivo. Safranin O staining was used to detect morphological changes in articular cartilage. Immunohistochemistry was used to detect Col2, Col10, metalloproteinase-13 (MMP-13). RT-qPCR was used to detect gene changes includingmiR-1, Col2, Col10, MMP-13, Ihh, Smo, Gli1, Gli2, and Gli3. Overexpression of miR-1 in IL-1ß-stimulated chondrocytes reduced the levels of Ihh, MMP-13, and Col10 but increased the levels of Col2 and aggrecan. Intra-articular injection of miR-1 agomir reduced osteophyte formation, inflammation, and prevented cartilage damage. RT-qPCR results indicated that the miR-1 agomir increased articular cartilage anabolism and inhibited cartilage catabonism. miR-1 can attenuate the progression of OA by downregulating Ihh.

Toll-like receptor activation regulates the paracrine effect of adipose-derived mesenchymal stem cells on reversing osteoarthritic phenotype of chondrocytes.

BACKGROUND: The therapeutic efficacy of intra-articular mesenchymal stem cells (MSCs) injection for patients with osteoarthritis (OA) currently exhibits inconsistency, and the underlying mechanism remains elusive. It has been postulated that the immunomodulatory properties and paracrine activity of MSCs might be influenced by the inflammatory micro-environment within osteoarthritic joints, potentially contributing to this observed inconsistency. METHODS: Adipose-derived MSCs (ADSCs) were isolated from SD rats and pre-treated with Toll-like receptor 3 (TLR3) agonist Poly I:C or Toll-like receptor 4 (TLR4) agonist LPS. The pre-treated ADSCs were then co-cultured with IL-1ß-induced osteoarthritic chondrocytes using a Transwell system to analyze the paracrine effect of ADSCs on reversing the osteoarthritic phenotype of chondrocytes. RESULTS: RT-PCR and Western blot analysis revealed that Poly I:C and LPS pre-treatments up-regulated the expression of IL-10 and IL-6 in ADSCs, respectively. Furthermore, only Poly I:C-preconditioned ADSCs significantly promoted proliferation while inhibiting apoptosis in IL-1ß-treated chondrocytes. Additionally, Poly I:C-preconditioned ADSCs downregulated MMP13 expression while upregulating aggrecan and collagen II expression levels in IL-1ß-treated chondrocytes. CONCLUSIONS: TLR3 activation polarizes ADSCs into an immunomodulatory phenotype distinct from TLR4 activation, exerting differential effects on reversing the osteoarthritic phenotype of chondrocytes; thus indicating that MSCs' paracrine effect regulated by TLRs signaling impacts the efficacy of intra-articular MSCs injection.

Mesenchymal stromal cell chondrogenesis under ALK1/2/3-specific BMP inhibition: a revision of the prohypertrophic signalling network concept.

BACKGROUND: In vitro chondrogenesis of mesenchymal stromal cells (MSCs) driven by the essential chondro-inducer transforming growth factor (TGF)-ß is instable and yields undesired hypertrophic cartilage predisposed to bone formation in vivo. TGF-ß can non-canonically activate bone morphogenetic protein-associated ALK1/2/3 receptors. These have been accused of driving hypertrophic MSC misdifferentiation, but data remained conflicting. We here tested the antihypertrophic capacity of two highly specific ALK1/2/3 inhibitors - compound A (CompA) and LDN-212854 (LDN21) - in order to reveal potential prohypertrophic contributions of these BMP/non-canonical TGF-ß receptors during MSC in vitro chondrogenesis. METHODS: Standard chondrogenic pellet cultures of human bone marrow-derived MSCs were treated with TGF-ß and CompA (500 nM) or LDN21 (500 nM). Daily 6-hour pulses of parathyroid hormone-related peptide (PTHrP[1-34], 2.5 nM, from day 7) served as potent antihypertrophic control treatment. Day 28 samples were subcutaneously implanted into immunodeficient mice. RESULTS: All groups underwent strong chondrogenesis, but GAG/DNA deposition and ACAN expression were slightly but significantly reduced by ALK inhibition compared to solvent controls along with a mild decrease of the hypertrophy markers IHH-, SPP1-mRNA, and Alkaline phosphatase (ALP) activity. When corrected for the degree of chondrogenesis (COL2A1 expression), only pulsed PTHrP but not ALK1/2/3 inhibition qualified as antihypertrophic treatment. In vivo, all subcutaneous cartilaginous implants mineralized within 8 weeks, but PTHrP pretreated samples formed less bone and attracted significantly less haematopoietic marrow than ALK1/2/3 inhibitor groups. CONCLUSIONS: Overall, our data show that BMP-ALK1/2/3 inhibition cannot program mesenchymal stromal cells toward stable chondrogenesis. BMP-ALK1/2/3 signalling is no driver of hypertrophic MSC misdifferentiation and BMP receptor induction is not an adverse prohypertrophic side effect of TGF-ß that leads to endochondral MSC misdifferentiation. Instead, the prohypertrophic network comprises misregulated PTHrP/hedgehog signalling and WNT activity, and a potential contribution of TGF-ß-ALK4/5-mediated SMAD1/5/9 signalling should be further investigated to decide about its postulated prohypertrophic activity. This will help to successfully engineer cartilage replacement tissues from MSCs in vitro and translate these into clinical cartilage regenerative therapies.

[Ligusticum cycloprolactam inhibits IL-1ß-induced apoptosis and inflammation of rat chondrocytes via HMGB1/TLR4/NF-κB signaling pathway].

Chondrocytes are unique resident cells in the articular cartilage, and the pathological changes of them can lead to the occurrence of osteoarthritis(OA). Ligusticum cycloprolactam(LIGc) are derivatives of Z-ligustilide(LIG), a pharmacodynamic marker of Angelica sinensis, which has various biological functions such as anti-inflammation and inhibition of cell apoptosis. However, its protective effect on chondrocytes in the case of OA and the underlying mechanism remain unclear. This study conducted in vitro experiments to explore the molecular mechanism of LIGc in protecting chondrocytes from OA. The inflammation model of rat OA chondrocyte model was established by using interleukin-1ß(IL-1ß) to induce. LIGc alone and combined with glycyrrhizic acid(GA), a blocker of the high mobility group box-1 protein(HMGB1)/Toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB) signaling pathway, were used to intervene in the model, and the therapeutic effects were systematically evaluated. The viability of chondrocytes treated with different concentrations of LIGc was measured by the cell counting kit-8(CCK-8), and the optimal LIGc concentration was screened out. Annexin V-FITC/PI apoptosis detection kit was employed to examine the apoptosis of chondrocytes in each group. The enzyme-linked immunosorbent assay(ELISA) was employed to measure the expression of cyclooxygenase-2(COX-2), prostaglandin-2(PGE2), and tumor necrosis factor-alpha(TNF-α) in the supernatant of chondrocytes in each group. Western blot was employed to determine the protein levels of B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), caspase-3, HMGB1, TLR4, and NF-κB p65. The mRNA levels of HMGB1, TLR4, NF-κB p65, and myeloid differentiation factor 88(MyD88) in chondrocytes were determined by real-time fluorescent quantitative PCR(RT-qPCR). The safe concentration range of LIGc on chondrocytes was determined by CCK-8, and then the optimal concentration of LIGc for exerting the effect was clarified. Under the intervention of IL-1ß, the rat chondrocyte model of OA was successfully established. The modeled chondrocytes showed increased apoptosis rate, promoted expression of COX-2, PGE2, and TNF-α, up-regulated protein levels of Bax, caspase-3, HMGB1, TLR4, and NF-κB p65 and mRNA levels of HMGB1, TLR4, NF-κB p65, and MyD88, and down-regulated protein level of Bcl-2. However, LIGc reversed the IL-1ß-induced changes of the above factors. Moreover, LIGc combined with GA showed more significant reversal effect than LIGc alone. These fin-dings indicate that LIGc extracted and derived from the traditional Chinese medicine A. sinensis can inhibit the inflammatory response of chondrocytes and reduce the apoptosis of chondrocytes, and this effect may be related to the HMGB1/TLR4/NF-κB signaling pathway. The pharmacological effect of LIGc on protecting chondrocytes has potential value in delaying the progression of OA and improving the clinical symptoms of patients, and deserves further study.

Scutellarein Ameliorated Chondrocyte Inflammation and Osteoarthritis in Rats.

OBJECTIVE: Osteoarthritis (OA) is a degenerative joint disorder characterized by the gradual degradation of joint cartilage and local inflammation. This study aimed to investigate the anti-OA effect of scutellarein (SCU), a single-unit flavonoid compound obtained from Scutellaria barbata D. Don, in rats. METHODS: The extracted rat chondrocytes were treated with SCU and IL-1ß. The chondrocytes were divided into control group, IL-1ß group, IL-1ß+SCU 50 µmol/L group, and IL-1ß+SCU 100 µmol/L group. Morphology of rat chondrocytes was observed by toluidine blue and safranin O staining. CCK-8 method was used to detect the cytotoxicity of SCU. ELISA, qRT-PCR, Western blotting, immunofluorescence, SAß-gal staining, flow cytometry, and bioinformatics analysis were applied to evaluate the effect of SCU on rat chondrocytes under IL-1ß intervention. Additionally, anterior cruciate ligament transection (ACL-T) was used to establish a rat OA model. Histological changes were detected by safranin O/fast green, hematoxylin-eosin (HE) staining, and immunohistochemistry. RESULTS: SCU protected cartilage and exhibited anti-inflammatory effects via multiple mechanisms. Specifically, it could enhance the synthesis of extracellular matrix in cartilage cells and inhibit its degradation. In addition, SCU partially inhibited the nuclear factor kappa-B/mitogen-activated protein kinase (NF-κB/MAPK) pathway, thereby reducing inflammatory cytokine production in the joint cartilage. Furthermore, SCU significantly reduced IL-1ß-induced apoptosis and senescence in rat chondrocytes, further highlighting its potential role in OA treatment. In vivo experiments revealed that SCU (at a dose of 50 mg/kg) administered for 2 months could significantly delay the progression of cartilage damage, which was reflected in a lower Osteoarthritis Research Society International (OARSI) score, and reduced expression of matrix metalloproteinase 13 (MMP13) in cartilage. CONCLUSION: SCU is effective in the therapeutic management of OA and could serve as a potential candidate for future clinical drug therapy for OA.

Disease-modifying potential of diphenyl diselenide in an experimental osteoarthritis model.

Oxidative stress is a key factor in the disruption of cartilage homeostasis during the development of osteoarthritis (OA). Organic selenium (Se)-containing compounds such as diselenides have excellent antioxidant activity and may prevent related diseases. We aimed to examine the benefits of the synthetic small molecule diphenyl diselenide (DPDSe) in OA models in vitro and in vivo. Our findings showed that DPDSe could maintain extracellular matrix (ECM) homeostasis and inhibit reactive oxygen species (ROS) production in IL-1ß-treated chondrocytes. In a destabilization of the medial meniscus (DMM)-induced OA mouse model, intra-articular administration of DPDSe alleviated joint degeneration, as evidenced by a decrease in the OARSI score and the restoration of collagen II (COL2) and MMP-13 expression in cartilage tissues. We confirmed that DDS activated the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in IL-1ß-treated chondrocytes, and its chondroprotective effects were significantly counteracted when Nrf2 signaling was blocked by the inhibitor ML385 or by siRNA-mediated Nrf2 knockdown. The relatively strong performance of DPDSe makes it an ideal candidate for further trials as a disease-modifying OA drug (DMOAD).

Protopine protects chondrocytes from undergoing ferroptosis by activating Nrf2 pathway.

Osteoarthritis is a highly prevalent joint disease; however, effective treatments are lacking. Protopine (PTP) is an isoquinoline alkaloid with potent anti-inflammatory and antioxidant properties; however, it has not been studied in osteoarthritis. This study aimed to investigate whether PTP can effectively protect chondrocytes from ferroptosis. Primary mouse chondrocytes were treated with tert-butyl hydroperoxide (TBHP) to simulate oxidative stress in an in vitro model of osteoarthritis. Two concentrations of PTP (10 and 20 µg/mL) were validated for in vitro experiments. Cellular inflammation and metabolism were detected using RT-qPCR and western blotting (WB). Ferroptosis was assessed via WB, qPCR, reactive oxygen species (ROS) levels, lipid ROS, and immunofluorescence staining. In vitro, PTP significantly ameliorated chondrocyte inflammation and cytolytic metabolism and significantly suppressed chondrocyte ferroptosis through the activation of the Nrf2 pathway. The anterior cruciate ligament transection (ACLT) mouse model was used to validate the in vivo effects of PTP. The joint cartilage was assessed using the Osteoarthritis Research Society International (OARSI) score, Safranin O staining, and immunohistochemistry. The intra-articular administration of PTP alleviated cartilage inflammation and ferroptosis, as evidenced by the expression of MMP3, MMP13, COL2A1, GPX4, and Nrf2. Overall, we find that PTP exerted anti-ferroptosis and anti-inflammatory effects on chondrocytes to protect the articular cartilage.

Compensatory growth and recovery of cartilage cytoarchitecture after transient cell death in fetal mouse limbs.

A major question in developmental and regenerative biology is how organ size and architecture are controlled by progenitor cells. While limb bones exhibit catch-up growth (recovery of a normal growth trajectory after transient developmental perturbation), it is unclear how this emerges from the behaviour of chondroprogenitors, the cells sustaining the cartilage anlagen that are progressively replaced by bone. Here we show that transient sparse cell death in the mouse fetal cartilage is repaired postnatally, via a two-step process. During injury, progression of chondroprogenitors towards more differentiated states is delayed, leading to altered cartilage cytoarchitecture and impaired bone growth. Then, once cell death is over, chondroprogenitor differentiation is accelerated and cartilage structure recovered, including partial rescue of bone growth. At the molecular level, ectopic activation of mTORC1 correlates with, and is necessary for, part of the recovery, revealing a specific candidate to be explored during normal growth and in future therapies.

Molecular mechanism of miR-203a targeting Runx2 to regulate thiram induced-chondrocyte development.

Thiram is a kind of organic compound, which is commonly used for sterilization, insecticidal and deodorization in daily life. Its toxicology has been broadly studied. Recently, more and more microRNAs have been shown to participate in the regulation of cartilage development. However, the potential mechanism by which microRNA regulates chondrocyte growth is still unclear. Our experiments have demonstrated that thiram can hamper chondrocytes development and cause a significant increase in miR-203a content in vitro and in vivo trials. miR-203a mimic significantly decrease in mRNA and protein expression of Wnt4, Runx2, COL2A1, ß-catenin and ALP, and significantly enhance the mRNA and protein levels of GSK-3ß. It has been observed that overexpression of miR-203a hindered chondrocytes development. In addition, Runx2 was confirmed to be a direct target of miR-203a by dual luciferase report gene assay. Transfection of si-Runx2 into chondrocytes reveals that significant downregulation of genes is associated with cartilage development. Overall, these results suggest that overexpression of miR-203a inhibits the expression of Runx2. These findings are conducive to elucidate the mechanism of chondrocytes dysplasia induced by thiram and provide new research ideas for the toxicology of thiram.

FTH1 protects against osteoarthritis by MAPK pathway inhibition of extracellular matrix degradation.

OBJECTIVE: Ferritin heavy chain 1 (FTH1) is an important subunit of ferro-storing proteins and is indispensable for iron metabolism. Though it has been extensively studied in numerous organs and diseases, the relationship between FTH1 and osteoarthritis (OA) is unclear. DESIGN: Primary murine chondrocytes and cartilage explants were treated with FTH1 siRNA for 72 h. Mice were injected with adenovirus expressing FTH1 after destabilized medial meniscus (DMM) surgery. These approaches were used to determine the effect of FTH1 expression on the pathophysiology of OA. RESULTS: FTH1 expression was down regulated in OA patients and mice after DMM surgery. Knock down of FTH1 induced articular cartilage damage and extracellular matrix degradation in cartilage explants. Further, over expression of FTH1 reduced the susceptibility of chondrocytes to ferroptosis and reversed decrements in SOX9 and aggrecan after DMM surgery. Moreover, FTH1 relieved OA by inhibition of the chondrocyte MAPK pathway. CONCLUSION: This study found FTH1 to play an essential role in extracellular matrix degradation, ferroptosis, and chondrocytes senescence during OA progression. Further, injection of adenovirus expressing FTH1 may be a potential strategy for OA prevention and therapy.

HWJMSC-EVs promote cartilage regeneration and repair via the ITGB1/TGF-ß/Smad2/3 axis mediated by microfractures.

The current first-line treatment for repairing cartilage defects in clinical practice is the creation of microfractures (MF) to stimulate the release of mesenchymal stem cells (MSCs); however, this method has many limitations. Recent studies have found that MSC-derived extracellular vesicles (MSC-EVs) play an important role in tissue regeneration. This study aimed to verify whether MSC-EVs promote cartilage damage repair mediated by MFs and to explore the repair mechanisms. In vitro experiments showed that human umbilical cord Wharton's jelly MSC-EVs (hWJMSC-EVs) promoted the vitality of chondrocytes and the proliferation and differentiation ability of bone marrow-derived MSCs. This was mainly because hWJMSC-EVs carry integrin beta-1 (ITGB1), and cartilage and bone marrow-derived MSCs overexpress ITGB1 after absorbing EVs, thereby activating the transforming growth factor-ß/Smad2/3 axis. In a rabbit knee joint model of osteochondral defect repair, the injection of different concentrations of hWJMSC-EVs into the joint cavity showed that a concentration of 50 µg/ml significantly improved the formation of transparent cartilage after MF surgery. Extraction of regenerated cartilage revealed that the changes in ITGB1, transforming growth factor-ß, and Smad2/3 were directly proportional to the repair of regenerated cartilage. In summary, this study showed that hWJMSC-EVs promoted cartilage repair after MF surgery.

MiR-98-5p plays suppressive effects on IL-1ß-induced chondrocyte injury associated with osteoarthritis by targeting CASP3.

BACKGROUND: This study aims to explore how miR-98-5p affects osteoarthritis, focusing on its role in chondrocyte inflammation, apoptosis, and extracellular matrix (ECM) degradation. METHODS: Quantitative real-time PCR was used to measure miR-98-5p and CASP3 mRNA levels in OA cartilage tissues and IL-1ß-treated CHON-001 cells. We predicted miR-98-5p and CASP3 binding sites using TargetScan and confirmed them via luciferase reporter assays. Chondrocyte viability was analyzed using CCK-8 assays, while pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α) were quantified via ELISA. Caspase-3 activity was examined to assess apoptosis, and Western blotting was conducted for protein marker quantification. RESULTS: Our results showed lower miR-98-5p levels in both OA cartilage and IL-1ß-stimulated cells. Increasing miR-98-5p resulted in reduced pro-inflammatory cytokines, decreased caspase-3 activity, and improved cell viability. Furthermore, miR-98-5p overexpression hindered IL-1ß-induced ECM degradation, evident from the decline in MMP-13 and ß-catenin levels, and an increase in COL2A1 expression. MiR-98-5p's impact on CASP3 mRNA directly influenced its expression. Mimicking miR-98-5p's effects, CASP3 knockdown also inhibited IL-1ß-induced inflammation, apoptosis, and ECM degradation. In contrast, CASP3 overexpression negated the suppressive effects of miR-98-5p. CONCLUSIONS: In conclusion, our data collectively suggest that miR-98-5p plays a protective role against IL-1ß-induced damage in chondrocytes by targeting CASP3, highlighting its potential as a therapeutic target for OA.

Circ_0044235 regulates the development of osteoarthritis by the modulation of miR-375/PIK3R3 axis.

BACKGROUND: Circular RNAs (circRNAs) play an important role in osteoarthritis (OA). However, the role of circRNA in OA is still unclear. Here, we explored the role and mechanism of circ_0044235 in OA. METHODS: CHON-001 cells were treated with IL-1ß to establish OA model in vitro. The levels of circ_0044235, miR-375 and phosphoinositide 3-kinase (PI3K) regulatory subunit 3 (PIK3R3) were detected by quantitative real-time PCR. Cell count kit-8 assay and flow cytometry assay were used to detect cell viability and apoptosis. The concentrations of inflammation factors were determined by enzyme-linked immunosorbent assay. Western blot was used to detect protein levels. The interaction between miR-375 and circ_0044235 or PIK3R3 was analyzed by dual-luciferase reporter assay and RNA immunoprecipitation assay. RESULTS: Circ_0044235 was significantly decreased in OA cartilage tissue and IL-1ß-treated CHON-001 cells. Overexpression of circ_0044235 promoted IL-1ß-stimulated CHON-001 cell viability and inhibited apoptosis, inflammation, and extracellular matrix (ECM) degradation. In mechanism analysis, circ_0044235 could act as a sponge for miR-375 and positively regulate PIK3R3 expression. In addition, miR-375 ameliorated the effect of circ_0044235 overexpression on IL-1ß-mediated CHON-001 cells injury. In addition, miR-375 inhibition mitigated IL-1ß-induced CHON-001 cell injury, while PIK3R3 silencing restored the effect. CONCLUSION: Circ_0044235 knockdown alleviated IL-1ß-induced chondrocytes injury by regulating miR-375/PIK3R3 axis, confirming that circ_0044235 might be a potential target for OA treatment.

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 middle­aged 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 Kit­8 assay involving a poly [ADP­ribose] polymerase­1 (PARP1) inhibitor, DAPI staining, reverse transcription­quantitative PCR, Annexin V­FITC/PI staining and flow cytometry, western blotting and co­immunoprecipitation analysis were used to determine the therapeutic efficacy of RJNTF on KOA and to uncover the molecular mechanism. It was found that PARP1­knockdown lentivirus, incubation with PARP1 inhibitor PJ34, medium and high doses of RJNTF significantly reduced H2O2­induced chondrocyte apoptosis. Medium and high doses of RJNTF downregulated the expression of cleaved caspase­3, cleaved PARP1 and PAR total proteins, as well as nucleus proteins of apoptosis­inducing factor (AIF) and migration inhibitory factor (MIF), and upregulated the expression of caspase­3, 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.

Mechanosensitive protein polycystin-1 promotes periosteal stem/progenitor cells osteochondral differentiation in fracture healing.

Background: Mechanical forces are indispensable for bone healing, disruption of which is recognized as a contributing cause to nonunion or delayed union. However, the underlying mechanism of mechanical regulation of fracture healing is elusive. Methods: We used the lineage-tracing mouse model, conditional knockout depletion mouse model, hindlimb unloading model and single-cell RNA sequencing to analyze the crucial roles of mechanosensitive protein polycystin-1 (PC1, Pkd1) promotes periosteal stem/progenitor cells (PSPCs) osteochondral differentiation in fracture healing. Results: Our results showed that cathepsin (Ctsk)-positive PSPCs are fracture-responsive and mechanosensitive and can differentiate into osteoblasts and chondrocytes during fracture repair. We found that polycystin-1 declines markedly in PSPCs with mechanical unloading while increasing in response to mechanical stimulus. Mice with conditional depletion of Pkd1 in Ctsk+ PSPCs show impaired osteochondrogenesis, reduced cortical bone formation, delayed fracture healing, and diminished responsiveness to mechanical unloading. Mechanistically, PC1 facilitates nuclear translocation of transcriptional coactivator TAZ via PC1 C-terminal tail cleavage, enhancing osteochondral differentiation potential of PSPCs. Pharmacological intervention of the PC1-TAZ axis and promotion of TAZ nuclear translocation using Zinc01442821 enhances fracture healing and alleviates delayed union or nonunion induced by mechanical unloading. Conclusion: Our study reveals that Ctsk+ PSPCs within the callus can sense mechanical forces through the PC1-TAZ axis, targeting which represents great therapeutic potential for delayed fracture union or nonunion.

Cartilage stem/progenitor cells-derived exosomes facilitate knee cartilage repair in a subacute osteoarthritis rat model.

Cartilage defects in the knee are often associated with the progression of degenerative osteoarthritis (OA), and cartilage repair is a useful strategy for managing this disease. However, cartilage repair is challenging because of the unique environment within the tissue. Recently, stem cell-based therapies have shed new light on this issue. In this study, we prepared exosomes (EXOs) from cartilage stem/progenitor cells (CSPCs) and found that treatment with EXOs increased the viability, migration, and proliferation of cultured primary chondrocytes. In a subacute OA rat model, the application of EXOs facilitated cartilage regeneration as evidenced by histological staining. Exosomal protein analysis together with bioinformatics suggested that cyclin-dependent kinase 9 (CDK9) is a key factor for chondrocyte growth and migration. Functional studies confirmed this prediction, that is, inhibiting CDK9 reduced the beneficial effects induced by EXOs in primary chondrocytes; while overexpression of CDK9 recapitulated the EXOs-induced phenotypes. RNA-Seq data showed that a set of genes involved in cell growth and migration were up-regulated by EXOs in chondrocytes. These changes could be partially reproduced by CDK9 overexpression. Overall, our data suggest that EXOs derived from primary CSPCs hold great therapeutic potential for treating cartilage defect-associated disorders such as degenerative OA, and that CDK9 is a key factor in this process.

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Runt-related transcription factor (RUNX1) is a transcription factor closely involved in hematopoiesis. RUNX1 gene mutation plays an essential pathogenic role in the initiation and development of hematological tumors, especially in acute myeloid leukemia. Recent studies have shown that RUNX1 is also involved in the regulation of bone development and the pathological progression of bone-related diseases. RUNX1 promotes the differentiation of mesenchymal stem cells into chondrocytes and osteoblasts and modulates the maturation and extracellular matrix formation of chondrocytes. The expression of RUNX1 in mesenchymal stem cells, chondrocytes, and osteoblasts is of great significance for maintaining normal bone development and the mass and quality of bones. RUNX1 also inhibits the differentiation and bone resorptive activities of osteoclasts, which may be influenced by sexual dimorphism. In addition, RUNX1 deficiency contributes to the pathogenesis of osteoarthritis, delayed fracture healing, and osteoporosis, which was revealed by the RUNX1 conditional knockout modeling in mice. However, the roles of RUNX1 in regulating the hypertrophic differentiation of chondrocytes, the sexual dimorphism of activities of osteoclasts, as well as bone loss in diabetes mellitus, senescence, infection, chronic inflammation, etc, are still not fully understood. This review provides a systematic summary of the research progress concerning RUNX1 in the field of bone biology, offering new ideas for using RUNX1 as a potential target for bone related diseases, especially osteoarthritis, delayed fracture healing, and osteoporosis.

Overexpression of RAD54L attenuates osteoarthritis by suppressing the HIF-1α/VEGF signaling pathway: Bioinformatics analysis and experimental validation.

Osteoarthritis (OA) is a widespread chronic, progressive, degenerative joint disease that causes pain and disability. Current treatments for OA have limited effectiveness and new biomarkers need to be identified. Bioinformatics analysis was conducted to explore differentially expressed genes and DNA repair/recombination protein 54 L (RAD54L) was selected. We firstly overexpressed RAD54L in interleukin-1ß (IL-1ß)-induced human articular chondrocytes or in OA rats to investigate its effect on OA. Chondrocyte viability and apoptotic rate were measured by Cell Counting Kit-8 and flow cytometry, respectively. Then we evaluated OA severity in vivo by Hematoxylin-eosin staining and Osteoarthritis Research Society International standards. The expression of inflammatory mediators was tested by enzyme-linked immunosorbent assay. Finally, western blot was performed to determine the relative expression level of hypoxia-inducible factors 1α (HIF-1α) and vascular endothelial growth factor (VEGF). Overexpression of RAD54L promoted cell viability and attenuated apoptosis in IL-1ß-induced human chondrocytes. A lower Osteoarthritis Research Society International score and a remarkable alleviation of chondrocyte disordering and infiltration of inflammatory cells were found in cartilage tissues of OA rats after overexpressing RAD54L. The inflammatory response induced by OA was decreased by RAD54L overexpression in vitro and in vivo. In addition, RAD54L overexpression decreased the relative expression level of HIF-1α and VEGF. Overexpression of RAD54L could attenuate OA by suppressing the HIF-1α/VEGF signaling pathway, indicating that RAD54L may be a potential treatment target for OA.