MiR-4303 relieves chondrocyte inflammation by targeting ASPN in osteoarthritis

Background Osteoarthritis (OA) is a severe articular cartilage disease whose pathogenesis involves the inflammation of chondrocytes. MicroRNAs (miRNAs) are considered to be effective inflammation regulators. However, the regulatory mechanism of miRNAs in osteoarthritis needs to be further elucidated. In this paper, we aim to investigate the underlying mechanisms by which miR-4303 regulates osteoarthritis. Methods RT-qPCR is performed to detect the mRNA expression levels of miR-4303, ASPN, PDIA3, PIK3CA, and TRAF3. CCK-8 assay and EdU assay are carried to assess chondrocyte viability. The protein expression levels of ASPN, PCNA, Ki-67, CyclinA1, CyclinB1, CyclinD2, p27, Bax, Bcl-2, cleaved caspase-3, and Cleaved caspase-9 were measured by western blot. FACs is performed to detect the cell cycle and apoptosis of chondrocyte. ELISA is conducted to assess the levels of TNF-β, IL-1β and IL-6 in the supernatant of chondrocytes. The potential binding sites of miR-4303 and ASPN are predicted by the miRDB database and confirmed by the dual-luciferase reporter gene assay. Results Our findings illustrated that miR-4303 was down-regulated in arthritic tissues and LPS-induced chondrocytes; miR-4303 overexpression rescued the decrease in cell viability, cell cycle arrest and apoptosis induced by LPS. Furthermore, miR-4303 overexpression inhibited the release of inflammatory factors in LPS-induced chondrocytes, miR-4303 relieved chondrocyte inflammation via targeting ASPN. Conclusion MiR-4303 serves as a prognostic biomarker and relieves chondrocyte inflammation via targeting ASPN. Our findings provide novel prognostic biomarkers in predicting the progression and prognosis of osteoarthritis.

Identification of miR-338-5p and miR-4776-3p as mediators of articular cartilage degeneration

Purpose Osteoarthritis is one of the most common chronic diseases nowadays, and can cause serious physical illness and economic burden. Cartilage degeneration is one of primary reasons for osteoarthritis, but there were not thorough studies on cartilage lesions. Methods miR-338-5p and miR-4776-3p were overexpressed and knocked down in SW1353 cells. Dual luciferase assay is used to detect the binding of miR-338-5p, miR-4776-3p and SOX6. The expression of MMP13, TNFA, IL6, MCP1, PARP, Runx2, ALP, OPN, WNT4, WNT16, TRACP and CTSK was quantified by qPCR. And the expression of WNT4 at the protein level was quantified by WB. Results In this study, we found miR-338-5p and miR-4776-3p showed significantly high expression on cartilage in osteoarthritis through bioinformatics methods, and verified miR-338-5p and miR-4776-3p can improve catabolism, and trigger biological processes such as inflammation and apoptosis. Meanwhile we validated miR-338-5p and miR-4776-3p may play negative regulation in the process of bone biology. Conclusions Our study showed that miR-338-5p and miR-4776-3p can be regarded as medium of articular cartilage degeneration, and it provided new biomarker of early diagnosis in cartilage disease and osteoarthritis.

MiR-4303 relieves chondrocyte inflammation by targeting ASPN in osteoarthritis

Background Osteoarthritis (OA) is a severe articular cartilage disease whose pathogenesis involves the inflammation of chondrocytes. MicroRNAs (miRNAs) are considered to be effective inflammation regulators. However, the regulatory mechanism of miRNAs in osteoarthritis needs to be further elucidated. In this paper, we aim to investigate the underlying mechanisms by which miR-4303 regulates osteoarthritis. Methods RT-qPCR is performed to detect the levels of miR-4303, ASPN, PDIA3, PIK3CA, and TRAF3; CCK-8 assay is conducted to evaluate chondrocyte viability; EdU assay was carried to assess chondrocyte proliferation; western blot is conducted to measure the levels of ASPN, PCNA, Ki-67, CyclinA1, CyclinB1, CyclinD2, p27, Bax, Bcl-2, cleaved caspase-3, and Cleaved caspase-9 proteins; FACs is performed to detect the distribution of chondrocytes in each cell cycle and chondrocyte apoptosis; ELISA is conducted to assess the levels of TNF-β, IL-1β and IL-6 in the supernatant of chondrocytes; the potential binding sites of miR-4303 and ASPN are predicted by miRDB software, which is confirmed by the dual-luciferase reporter gene assay. Results Our findings illustrated that miR-4303 was down-regulated in arthritic tissues and LPS-induced chondrocytes; miR-4303 overexpression rescued LPS-induced chondrocyte viability, proliferation and cell cycle and alleviated LPS-induced chondrocyte apoptosis; miR-4303 overexpression inhibited the release of inflammatory factors in LPS-induced chondrocytes; miR-4303 targeted ASPN and miR-4303 relieved chondrocyte inflammation via targeting ASPN. Conclusion MiR-4303 serves as a prognostic biomarker and relieves chondrocyte inflammation via targeting ASPN. Our findings provide novel prognostic biomarkers in predicting the progression and prognosis of osteoarthritis.

Effect of Glucose on Morphofunctional Properties Chondrocytes in Vitro

Articular cartilage is a highly specialized dense connective tissue, and can be considered as a composite gel with a relatively low content (5%) of cells, chondrocytes, embedded in the extracellular matrix. Chondrocytes are the only cell type in articular cartilage and are responsible for the biosynthesis and catabolism of the extracellular matrix. Osteoarthritis, the most common cartilage disease, has many independent risk factors, among which is diabetes mellitus, which allows us to hypothesize that different glucose concentrations have a huge effect on the morfunctional properties of chondrocytes in general and on the formation of osteoarthritis in particular. Despite numerous studies, the question of the effect of glucose on cartilage function is still open. In this regard, the study of morphofunctional changes in chondrocytes under the influence of various glucose concentrations is an urgent problem. The following results were obtained: an increase in the concentration of glucose in cell culture has a positive effect on cell viability and proteoglycan synthesis, but at an external glucose concentration of 25 мМ, cells die, while the synthesis of proteoglycans remains at a high level. The higher the concentration of glucose in the nutrient medium, the larger the cell size, which is probably due to hypertrophy of chondrocytes. In the future, the results obtained will be useful for understanding the process of hypertrophy and identifying ways to control it, as well as for a detailed study of other biochemical processes.

Therapeutic Potential of Bioactive Compounds in Honey for Treating Osteoarthritis

Dysregulation of joint tissue homeostasis induces articular degenerative changes and musculoskeletal diseases such as osteoarthritis. This pathology represents the first cause of motor disability in individuals over 60 years of age, impacting their quality of life and the costs of health systems. Nowadays, pharmacological treatments for cartilage disease have failed to achieve full tissue regeneration, resulting in a functional loss of the joint; therefore, joint arthroplasty is the gold standard procedure to cure this pathology in severe cases of Osteoarthritis. A different treatment is the use of anti-inflammatory drugs which mitigate pain and inflammation in some degree, but without significant inhibition of disease progression. In this sense, new therapeutic alternatives based on natural compounds have been proposed to delay osteoarthritis progression, particularly those agents that regulate articular homeostasis. Preclinical studies have shown a therapeutic application of honey and its bioactive compounds, ranging from treating wounds, coughs, skin infections, and are also used as a biological stimulant by exerting antioxidant and anti-inflammatory properties. In this article, we reviewed the current medicinal applications of honey with particular emphasis on its use regulating articular homeostasis by inhibiting inflammation and oxidative stress.

Full wave 3D inverse scattering transmission ultrasound tomography in the presence of high contrast

We present here a quantitative ultrasound tomographic method yielding a sub-mm resolution, quantitative 3D representation of tissue characteristics in the presence of high contrast media. This result is a generalization of previous work where high impedance contrast was not present and may provide a clinically and laboratory relevant, relatively inexpensive, high resolution imaging method for imaging in the presence of bone. This allows tumor, muscle, tendon, ligament or cartilage disease monitoring for therapy and general laboratory or clinical settings. The method has proven useful in breast imaging and is generalized here to high-resolution quantitative imaging in the presence of bone. The laboratory data are acquired in ~ 12 min and the reconstruction in ~ 24 min—approximately 200 times faster than previously reported simulations in the literature. Such fast reconstructions with real data require careful calibration, adequate data redundancy from a 2D array of 2048 elements and a paraxial approximation. The imaging results show that tissue surrounding the high impedance region is artifact free and has correct speed of sound at sub-mm resolution.

Roles of TRPV4 and piezo channels in stretch-evoked Ca2+ response in chondrocytes

Chondrocyte mechanotransduction is not well understood, but recently, it has been proposed that mechanically activated ion channels such as transient receptor potential vanilloid 4 (TRPV4), Piezo1, and Piezo2 are of functional importance in chondrocyte mechanotransduction. The aim of this study was to distinguish the potential contributions of TRPV4, Piezo1, and Piezo2 in transducing different intensities of repetitive mechanical stimulus in chondrocytes. To study this, TRPV4-, Piezo1-, or Piezo2-specific siRNAs were transfected into cultured primary chondrocytes to knock down (KD) TRPV4, Piezo1, or Piezo2 expression, designated TRPV4-KD, Piezo1-KD, or Piezo2-KD cells. Then we used Flexcell® Tension System to apply cyclic tensile strains (CTS) of 3% to 18% at 0.5 Hz for 8 h to the knockdown and control siRNA-treated cells. Finally, using a Ca2+ imaging system, stretch-evoked intracellular Ca2+ ([Ca2+] i) influx in chondrocytes was examined to investigate the roles of TRPV4, Piezo1, and Piezo2 in Ca2+ signaling in response to different intensities of repetitive mechanical stretch stimulation. The characteristics of [Ca2+] i in chondrocytes evoked by stretch stimulation were stretch intensity dependent when comparing unstretched cells. In addition, stretch-evoked [Ca2+] i changes were significantly suppressed in TRPV4-KD, Piezo1-KD, or Piezo2-KD cells compared with control siRNA-treated cells, indicating that any channel essential for Ca2+ signaling induced by stretch stimulation in chondrocytes. Of note, they played different roles in calcium oscillation induced by different intensities of stretch stimulation. More specifically, TRPV4-mediated Ca2+ signaling played a central role in the response of chondrocytes to physiologic levels of strain (3% and 8% of strain), while Piezo2-mediated Ca2+ signaling played a central role in the response of chondrocytes to injurious levels of strain (18% of strain). These results provide a basis for further examination of mechanotransduction in cartilage and raise a possibility of therapeutically targeting Piezo2-mediated mechanotransduction for the treatment of cartilage disease induced by repetitive mechanical forces. Impact statement Chondrocytes in cartilage are constantly subjected to load-induced stimuli and regulate their metabolic activities in order to maintain cartilage homeostasis. Therefore, mechanotransduction is important in chondrocytes and is vital for their role in cartilage function. Our results indicate that chondrocytes might sense and distinguish the different intensities of repetitive mechanical stimulus by using different mechanosensitive ion channels. Specifically, TRPV4 is mainly responsible for sensing physiologic levels of repetitive CTS stimulus, while Piezo2 mainly contributes to chondrocyte sensing noxious levels of repetitive CTS loading. These results provide a basis for further examination of mechanotransduction in cartilage and raise the possibility of therapeutically targeting Piezo2-mediated mechanotransduction for the treatment of OA which is induced by injurious and repetitive mechanical stimulation.