Gossypol Acetic Acid alleviates the Ferroptosis of Chondrocytes in Osteoarthritis by Inhibiting GPX4 Methylation.

BACKGROUND: Osteoarthritis (OA) is a chronic joint disease, usually accompanied by degeneration of the articular cartilage, fibrosis, bone hyperplasia around the joint, and damage to the entire articular surface. Gossypol is a natural phenolic compound isolated from the seed of cotton plants, and gossypol acetic acid (GAA) is a medicinal form of Gossypol. Recently, various biological activities of GAA, including anti-inflammatory and anti-tumor effects, have been widely reported. However, its effect on chondrocytes in OA has yet to be determined. METHODS: In this study, we investigated the effect of GAA on ferroptosis in OA chondrocytes. The effect of GAA on the cell viability and cytotoxicity of chondrocytes in rat cells was investigated using CCK8. Western blotting, Reverse-transcription PCR (RT-PCR), and immunofluorescence staining were used to elucidate the molecular mechanisms and signaling pathways of GAA inhibition of ferroptosis in OA chondrocytes. The effect of GAA on reactive oxygen species (ROS) production and lipid peroxidation levels in chondrocytes was examined using dihydroethidium (DHE) staining and fluorescent dye BODIPY581/591 C11. in vivo, micro-CT imaging, hematoxylin and eosin staining, Safranin O-Fast staining, and immunohistochemistry were performed to evaluate the effects of GAA on OA cartilage. RESULTS: The results showed that GAA treatment regulated the expression of chondrocyte extracellular matrix (ECM) related factors, including ADAMTS5, MMP13, SOX9, Aggrecan, and COL1A2 and reduced the ROS and lipid peroxidation levels. Besides, Erastin could reverse the effects of GAA on chondrocytes. Similar to GAA, 5-AZA caused the reduction of ROS and lipid peroxidation levels and reversed the effect of IL-1ß on the expression of ECM-related factors in OA chondrocytes. The above results clarified that GAA alleviated the ferroptosis of chondrocytes in OA by inhibiting GPX4 methylation. CONCLUSION: Our findings revealed that GAA might be developed as a drug for treating OA clinically.

Microenvironment-responsive bilayer hydrogel microspheres with gelatin-shell for osteoarthritis treatment.

Osteoarthritis is a long-term degenerative condition of the joints that is characterized by the breakdown of cartilage and inflammation of the synovial membrane. The presence of an inflammatory microenvironment and the degradation of the extracellular matrix produced by chondrocytes leads to the aggravation of cartilage injury, hindering the treatment of osteoarthritis. A promising approach to address this issue is to apply a combined strategy that is sensitive to the specific conditions in osteoarthritic joints and possesses properties that can reduce inflammation and promote cartilage healing. Here, inspired by the structure of chocolate-covered peanuts, we developed an injectable, environment-responsive bilayer hydrogel microsphere using microfluidics technology. The microsphere applied chondroitin sulfate methacryloyl (ChsMA) as its core and was coated with a methacryloyl gelatin (GelMA) shell that was loaded with celecoxib (CLX) liposomes (ChsMA+CLX@Lipo@GelMA). CLX was released from the liposomes when the GelMA shell rapidly degraded in response to the osteoarthritic microenvironment and suppressed the generation of inflammatory agents, demonstrating a beneficial impact of the outer shell in reducing inflammation. While the inner methacryloyl microsphere core degraded, chondroitin sulfate was released to promote chondrocyte anabolism and facilitate cartilage repair. Thus, the synthesized bilayer hydrogel microspheres hold great potential for treating osteoarthritis.

Adipokines regulate mesenchymal stem cell osteogenic differentiation.

Mesenchymal stem cells (MSCs) can differentiate into various tissue cell types including bone, adipose, cartilage, and muscle. Among those, osteogenic differentiation of MSCs has been widely explored in many bone tissue engineering studies. Moreover, the conditions and methods of inducing osteogenic differentiation of MSCs are continuously advancing. Recently, with the gradual recognition of adipokines, the research on their involvement in different pathophysiological processes of the body is also deepening including lipid metabolism, inflammation, immune regulation, energy disorders, and bone homeostasis. At the same time, the role of adipokines in the osteogenic differentiation of MSCs has been gradually described more completely. Therefore, this paper reviewed the evidence of the role of adipokines in the osteogenic differentiation of MSCs, emphasizing bone formation and bone regeneration.

Circ_0136666 aggravates osteosarcoma development through mediating miR-1244/CEP55 axis.

BACKGROUND: Accumulating articles demonstrate that circular RNAs play pivotal functions in tumorigenesis. However, the working mechanism of circ_0136666 in osteosarcoma (OS) progression remains to be further clarified. METHODS: Real time-quantitative polymerase chain reaction and western blot assay were applied to determine RNA and protein expression, respectively. Cell proliferation was assessed by 5-Ethynyl-2'-deoxyuridine assay and colony formation assay. Transwell assays were carried out to assess cell migration and invasion abilities. Flow cytometry was performed to analyze cell apoptosis. Cell glycolysis was evaluated by analyzing the uptake of glucose and the production of lactate using the corresponding kits. Dual-luciferase reporter assay and biotinylated RNA-pull down assay were performed to confirm the target interaction between microRNA-1244 (miR-1244) and circ_0136666 or centrosomal protein 55 (CEP55). Xenograft tumor model was utilized to explore the role of circ_0136666 in tumor growth in vivo. RESULTS: Circ_0136666 expression was prominently elevated in OS tissues and cell lines. Circ_0136666 absence restrained the proliferation, migration, invasion and glycolytic metabolism and promoted the apoptosis of OS cells. Circ_0136666 negatively regulated miR-1244 expression by binding to it in OS cells. MiR-1244 overexpression suppressed the malignant behaviors of OS cells. CEP55 was a target of miR-1244 in OS cells. Circ_0136666 positively regulated CEP55 expression partly by sequestering miR-1244 in OS cells. CEP55 overexpression largely reversed circ_0136666 silencing-mediated influences in OS cells. Circ_0136666 silencing significantly suppressed tumor growth in vivo. CONCLUSION: Circ_0136666 silencing inhibited OS progression partly by targeting miR-1244/CEP55 signaling. Silencing circ_0136666 and CEP55 or restoring miR-1244 level might be a potential therapeutic strategy for OS.

PPARγ Attenuates Interleukin-1ß-Induced Cell Apoptosis by Inhibiting NOX2/ROS/p38MAPK Activation in Osteoarthritis Chondrocytes.

INTRODUCTION: Reactive oxygen species (ROS) induced by extracellular cytokines trigger the expression of inflammatory mediators in osteoarthritis (OA) chondrocyte. Peroxisome proliferator-activated receptor gamma (PPARγ) exerts an anti-inflammatory effect. The aim of this study was to elucidate the role of PPARγ in interleukin-1ß- (IL-1ß-) induced cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) expression through ROS generation in OA chondrocytes. METHODS: IL-1ß-induced ROS generation and chondrocyte apoptosis were determined by flow cytometry. Contents of NADPH oxidase (NOX), caspase-3, and caspase-9 were evaluated by biochemical detection. The involvement of NOX2 and mitogen-activated protein kinases (MAPKs) in IL-1ß-induced COX-2 and PGE2 expression was investigated using pharmacologic inhibitors and further analyzed by western blotting. Activation of PPARγ was performed by using a pharmacologic agonist and was analyzed by western blotting. RESULTS: IL-1ß-induced COX-2 and PGE2 expression was mediated through NOX2 activation/ROS production, which could be attenuated by N-acetylcysteine (NAC; a scavenger of ROS), GW1929 (PPARγ agonist), DPI (diphenyleneiodonium chloride, NOX2 inhibitor), SB203580 (p38MAPK inhibitor), PD98059 (extracellular signal-regulated kinase, ERK inhibitor), and SP600125 (c-Jun N-terminal kinase, JNK inhibitor). ROS activated p38MAPK to enter the nucleus, which was attenuated by PPARγ. CONCLUSION: In OA chondrocytes, IL-1ß induced COX-2 and PGE2 expression via activation of NOX2, which led to ROS production and MAPK activation. The activation of PPARγ exerted protective roles in the pathogenesis of OA.

LncRNA PART1/miR-185-5p/RUNX3 feedback loop modulates osteogenic differentiation of bone marrow mesenchymal stem cells.

BACKGROUND: Osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) is essential for bone formation, and its dysfunction is reported to be associated with osteoporosis (OP). Recent researches have determined that lncRNA PART1 participates in the pathogenesis of multiple diseases. However, its role in modulating osteogenic differentiation of hBMSCs is unclear. METHODS: PART1, miR-185-5p, and RUNX3 levels were assessed via RT-qPCR. The protein levels of OCN, OSN, and COL1A1 were measured by western blotting. The osteoblastic phenotype was evaluated via ALP activity and ARS staining. The relationship between miR-185-5p and PART1 or RUNX3 was validated by luciferase reporter, RIP assays. RESULTS: PART1 and RUNX3 expression were enhanced during hBMSC osteogenic differentiation. PART1 deletion decreased OCN, OSN, and COL1A1 levels and weakened ALP activity, but promoted the apoptosis of hBMSCs. Moreover, PART1 served as a ceRNA to influence the RUNX3 level via targeting miR-185-5p. In addition, RUNX3 was verified to activate the transcription of PART1 in hBMSCs. Finally, rescue assays indicated that suppression of miR-185-5p or addition of RUNX3 partially abolished the effects of PART1 knockdown on the levels of OCN, OSX, and COL1A1 levels, ALP activity, and apoptosis. CONCLUSION: Our study elaborated that PART1/miR-185-5p/RUNX3 feedback contributed to osteogenic differentiation and inhibited the hBMSCs apoptosis, suggesting that PART1 might be a novel target for OP treatment.

PI3K/Akt and caspase pathways mediate oxidative stress-induced chondrocyte apoptosis.

Chondrocyte apoptosis is closely related to the development and progression of osteoarthritis (OA); however, the underlying mechanisms remain enigmatic. Previous studies have confirmed that cell apoptosis is one of the main pathological alterations during oxidative stress, and chondrocyte apoptosis induced by oxidative stress plays an important role in the development of OA. Rat chondrocytes exposed to hydrogen peroxide (H2O2) were used as the experimental oxidative stress model. We assessed cell viability, cell apoptosis, levels of intracellular reactive oxygen species (ROS), nitric oxide (NO) production, gene relative expression level of inducible nitric oxide synthase (iNOS), and expressions of iNOS, PI3K, phospho-Akt, caspase-9, and caspase-3. With the rising of intracellular ROS and increasing iNOS synthesis, producing a large amount of NO in chondrocytes, H2O2 decreased the cell viability and induced cell apoptosis of chondrocytes. Furthermore, the levels of caspase-9 and caspase-3 protein expression were significantly elevated as well as the level of p-Akt protein expression when induced by oxidative stress. These findings suggest that oxidative stress-induced chondrocyte apoptosis occurred via activating both PI3K/Akt and caspase pathways in the early stage in these processes.

Role of matrix metalloproteases 1/3 gene polymorphisms in patients with rotator cuff tear.

An association of Matrix Metalloproteinases-1/3 (MMP-1/3) rs1799750/rs3025058 polymorphism with increased risk of rotator cuff tear (RCT) has been reported in a Brazilian population. However, this significant association has not been confirmed in the Chinese population. Genotyping was conducted by polymerase chain reaction (PCR)-restriction fragment length polymorphism and direct sequencing. Our results demonstrated that individuals with the TT genotype had a significantly higher risk of RCT compared with those with the CC genotype. The increased risk of RCT progression was associated with the 2G allele of the rs1799750 polymorphism. No significant association was observed for genotypic and allelic frequencies of the rs3025058 polymorphism. A significant association of the MMP-1 rs1799750 polymorphism was observed with smokers, drinkers and people aged ≥60 years and non-diabetic people. Additionally, the MMP-1 rs1799750 polymorphism was associated with pre-operative stiffness in RCT patients. In conclusion, a significant correlation was identified between the MMP-1 rs1799750 polymorphism and RCT. The MMP-1 rs1799750 polymorphism might be considered as a biomarker of genetically high-risk RCT, helping to clarify the mechanism of RCT.

Polysaccharide from Angelica sinensis protects chondrocytes from H2O2-induced apoptosis through its antioxidant effects in vitro.

This study aimed to explore the protective effects of Angelica sinensis polysaccharide (ASP) on rat chondrocyte injury induced by hydrogen peroxide (H2O2). Rat chondrocytes were cultured and treated with different concentrations of ASP alone or in combination with H2O2, and they were measured with cell viability, apoptosis, release of inflammatory cytokines, such as interleukin-1beta (IL-1ß) and tumor necrosis factor-alpha (TNF-α), activity of superoxide dismutase (SOD), and catalase (CAT), and levels of malondialdehyde (MDA) production, respectively. In addition, quantitative real-time reverse transcription polymerase chain reaction was used to estimate the relative expression levels of osteoarthritis (OA)-associated genes, such as collagen type II (Col2a1), aggrecan, SOX9, matrix metalloproteinase (MMP)-1, -3, and -9, as well as tissue inhibitor of matrix metalloproteinase (TIMP)-1, respectively. Results indicated that ASP protected chondrocytes from H2O2-induced oxidative stress and subsequent cell injury through its antioxidant, antiapoptotic and anti-inflammatory effects in vitro. Our study suggests that ASP could become a therapeutic supplementation for the treatment of OA.

The involvement of follistatin-like protein 1 in osteoarthritis by elevating NF-κB-mediated inflammatory cytokines and enhancing fibroblast like synoviocyte proliferation.

INTRODUCTION: Our previous work has revealed that expression of follistatin-like protein 1 (FSTL1) is elevated in the synovial tissues from osteoarthritis (OA) patients. The aim of this study was to elucidate the underlying molecular mechanisms by which FSTL1 plays a role in the pathogenesis of OA. METHODS: Cultured fibroblast-like synoviocytes (FLSs) from synovial tissues of OA patients were stimulated with human recombinant FSTL1, and then the expression of inflammatory cytokines in FLS and their concentrations in the cell supernatants were measured by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Nuclear factor kappa B (NF-κB) activation was examined by western blot and chromatin immunoprecipitation (ChIP) assay at the p65 binding site. Finally, the proliferation of FLSs and the expression level of the proliferation-related tumor suppressors (p53 and p21) were determined by MTS assay kit and western blot in the presence or absence of FSTL1, respectively. RESULTS: FSTL1 remarkably promoted expression levels of several inflammatory cytokines (tumor necrosis factor alpha (TNF-α), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6)) in vitro. Western blot analysis showed that FSTL1 activated the inflammatory-related NF-κB signaling pathway, as validated by ChIP assay detecting p65-binding level on the cytokine promoter region. Moreover, FSTL1 promoted the proliferation of OA FLS by downregulating the expression of p53 and p21. Interestingly, the concentration of synovial fluid IL-6 was remarkably elevated in OA patients, and was correlated with synovial fluid and serum FSTL1 levels. CONCLUSIONS: These findings show that FSTL1 functions as an important proinflammatory factor in the pathogenesis of OA by activating the canonical NF-κB pathway and enhancing synoviocytes proliferation, suggesting that FSTL1 may be a promising target for the treatment of OA.

Hidden blood loss and its influential factors after total hip arthroplasty.

BACKGROUND: Total hip arthroplasty (THA) is a vital therapy for various hip joint diseases. However, patients have lower hemoglobin level post-operatively, remarkably inconsistent with the measured blood loss. The inconsistence is majorly attributed to hidden blood loss (HBL). In this study, we investigated the HBL and its influential factors among patients after THA. METHODS: From January 2008 to June 2014, 322 patients (99 males and 223 females) undergoing THA were enrolled in this study. All patients were assessed comprehensively before the operation. The demographic information of the patients was collected. Intra-operative and post-operative blood loss was recorded, and then, the total perioperative blood loss and the HBL were calculated. Influential factors were further analyzed by multiple and stepwise regression. RESULTS: The HBL was 429 ± 223 mL, with a percentage of 35.4% ± 11.0% in the total perioperative blood loss (1,155 ± 377 mL). Multiple and stepwise regression analysis revealed that HBL was positively associated with body mass index (BMI), blood transfusion volume, length of incision, change of hematocrit (HCT) between pre-operation and post-operation but negatively associated with age. As compared to male patients, female patients had a risk of increased HBL. Development displasia hip (DDH) patients had a less risk of HBL in all patients. CONCLUSION: HBL is a significant portion of total blood loss in the patients after THA. Gender, age, BMI, blood transfusion, length of incision, change of HCT, and diagnosis are influential factors of HBL.