A universal orthogonal imaging platform for living-cell RNA detection using fluorogenic RNA aptamers.

MicroRNAs (miRNAs) are crucial regulators of gene expression at the post-transcriptional level, offering valuable insights into disease mechanisms and prospects for targeted therapeutic interventions. Herein, we present a class of miRNA-induced light-up RNA sensors (miLS) that are founded on the toehold mediated principle and employ the fluorogenic RNA aptamers Pepper and Squash as imaging modules. By incorporating a sensor switch to disrupt the stabilizing stem of these aptamers, our design offers enhanced flexibility and convertibility for different target miRNAs and aptamers. These sensors detect multiple miRNA targets (miR-21 and miR-122) with detection limits of 0.48 and 0.2 nM, respectively, while achieving a robust signal-to-noise ratio of up to 44 times. Capitalizing on the distinct fluorescence imaging channels afforded by Pepper-HBC620 (red) and Squash-DFHBI-1T (green), we establish an orthogonal miRNA activation imaging platform, enabling the simultaneous visualization of different intracellular miRNAs in living cells. Our dual-color orthogonal miLS imaging platform provides a powerful tool for sequence-specific miRNA imaging in different cells, opening up new avenues for studying the intricate functions of RNA in living cells.

Bioluminescence Imaging with Functional Amyloid Reservoirs in Alzheimer's Disease Models.

Bioluminescence imaging has changed the daily practice of preclinical research on cancer and other diseases over the last few decades; however, it has rarely been applied in preclinical research on Alzheimer's disease (AD). In this Article, we demonstrated that bioluminescence imaging could be used to report the levels of amyloid beta (Aß) species in vivo. We hypothesized that AkaLumine, a newly discovered substrate for luciferase, could bind to Aß aggregates and plaques. We further speculated that the Aß aggregates/fibrils/plaques could be considered as "functional amyloids", which have a reservoir function to sequester and release AkaLumine to control the bioluminescence intensity, which could be used to report the levels of Aßs. Our hypotheses have been validated via in vitro solution tests, mimic studies with brain tissues and mice, two-photon imaging with AD mice, and in vivo bioluminescence imaging using transgenic AD mice that were virally transduced with AkaLuciferase (AkaLuc), a new luciferase that generates bioluminescence in the near-infrared window. As expected, compared to the control group, we observed that the Aß group showed lower bioluminescence intensity due to AkaLumine sequestering at early time points, while higher intensity was due to AkaLumine releasing at later time points. Lastly, we demonstrated that this method could be used to monitor AD progression and the therapeutic effectiveness of avagacestat, a well-studied gamma-secretase inhibitor. Importantly, a good correlation (R2 = 0.81) was established between in vivo bioluminescence signals and Aß burdens of the tested AD mice. We believe that our approach can be easily implemented into daily imaging experiments and has tremendous potential to change the daily practice of preclinical AD research.

A Photolabile Curcumin-Diazirine Analogue Enables Phototherapy with Physically and Molecularly Produced Light for Alzheimer's Disease Treatment.

The development of Alzheimer's disease (AD) drugs has recently witnessed substantial achievement. To further enhance the pool of drug candidates, it is crucial to explore non-traditional therapeutic avenues. In this study, we present the use of a photolabile curcumin-diazirine analogue, CRANAD-147, to induce changes in properties, structures (sequences), and neurotoxicity of amyloid beta (Aß) species both in cells and in vivo. This manipulation was achieved through irradiation with LED light or molecularly generated light, dubbed as "molecular light", emitted by the chemiluminescence probe ADLumin-4. Next, aided by molecular chemiluminescence imaging, we demonstrated that the combination of CRANAD-147/LED or CRANAD-147/ADLumin-4 (molecular light) could effectively slow down the accumulation of Aßs in transgenic 5xFAD mice in vivo. Leveraging the remarkable tissue penetration capacity of molecular light, phototherapy employing the synergistic effect of a photolabile Aß ligand and molecular light emerges as a promising alternative to conventional AD treatment interventions.

Engineering fluorescent protein chromophores with an internal reference for high-fidelity ratiometric G4 imaging in living cells.

G-quadruplexes (G4s) are significant nucleic acid secondary structures formed by guanine-rich sequences. Many single-emission G4 fluorescent probes that are lit up by inhibiting intramolecular rotation have been reported. However, they are non-fluorescent unless structurally rigidified, making them sensitive to other intracellular crowding and confinement environments in the cell, like viscosity. Ratiometric measurements provide built-in self-calibration for signal correction, enabling more sensitive and reliable detection. Herein, we structurally modulate green fluorescent protein (GFP)-like chromophores by integrating the imidazolidinone scaffold of the GFP chromophore and coumarin 6H, obtaining a G4 responsive dual-emission chromophore, called NHCouI. The red emission signal of NHCouI can specifically respond to parallel G4s, while its green emission signal is inert and acts as an internal reference signal. NHCouI-G4 complexes feature high fluorescence quantum yield and excellent anti-photobleaching properties. NHCouI can self-calibrate the signal and avoid viscosity disturbances within the range of major subcellular organelles during G4 imaging in living cells. It is also applied to reflect the difference between apoptosis and ferroptosis via tracking G4s. To the best of our knowledge, NHCouI is the first small molecule G4 probe enabled by internal reference correction capability, opening up new avenues for dual-emission chromophore development and high-fidelity and reliable analysis in G4 imaging research.

[Effect of Polygonati Rhizoma in improving pyroptosis injury of diabetic macroangiopathy via NLRP3/caspase-1/GSDMD pathway].

This study aims to explore the influence of Polygonati Rhizoma on the pyroptosis in the rat model of diabetic macroangiopathy via the NOD-like receptor thermal protein domain associated protein 3(NLRP3)/cysteinyl aspartate specific proteinase-1(caspase-1)/gasdermin D(GSDMD) pathway. The rat model of diabetes was established by intraperitoneal injection of streptozotocin(STZ) combined with a high-fat, high-sugar diet. The blood glucose meter, fully automated biochemical analyzer, hematoxylin-eosin(HE) staining, enzyme-linked immunosorbent assay, immunofluorescence, immunohistochemistry, and Western blot were employed to measure blood glucose levels, lipid levels, vascular thickness, inflammatory cytokine levels, and expression levels of pyroptosis-related proteins. The mechanism of pharmacological interventions against the injury in the context of diabetes was thus explored. The results demonstrated the successful establishment of the model of diabetes. Compared with the control group, the model group showed elevated levels of fasting blood glucose, total cholesterol(TC), triglycerides(TG) and low-density lipoprotein cholesterol(LDL-c), lowered level of high-density lipoprotein cholesterol(HDL-c), thickened vascular intima, and elevated serum and aorta levels of tumor necrosis factor-α(TNF-α), interleukin-1ß(IL-1ß) and interleukin-18(IL-18). Moreover, the model group showed increased NLRP3 inflammasomes and up-regulated levels of caspase-1 and GSDMD in aortic vascular cells. Polygonati Rhizoma intervention reduced blood glucose and lipid levels, inhibited vascular thickening, lowered the levels of TNF-α, IL-1ß, IL-18 in the serum and aorta, attenuated NLRP3 inflammasome expression, and down-regulated the expression levels of caspase-1 and GSDMD, compared with the model group. In summary, Polygonati Rhizoma can slow down the progression of diabetic macroangiopathy by inhibiting pyroptosis and alleviating local vascular inflammation.

Multifunctional stimuli-responsive chemogenetic platform for conditional multicolor cell-selective labeling.

Multicolor conditional labeling is a powerful tool that can simultaneously and selectively visualize multiple targets for bioimaging analysis of complex biological processes and cellular features. We herein report a multifunctional stimuli-responsive Fluorescence-Activating and absorption-Shifting Tag (srFAST) chemogenetic platform for multicolor cell-selective labeling. This platform comprises stimuli-responsive fluorogenic ligands and the organelle-localizable FAST. The physicochemical properties of the srFAST ligands can be tailored by modifying the optical-tunable hydroxyl group with diverse reactive groups, and their chemical decaging process caused by cell-specific stimuli induces a conditionally activatable fluorescent labeling upon binding with the FAST. Thus, the resulting switch-on srFASTs were designed for on-demand labeling of cells of interest by spatiotemporally precise photo-stimulation or unique cellular feature-dependent activation, including specific endogenous metabolites or enzyme profiles. Furthermore, diverse enzyme-activatable srFAST ligands with distinct colors were constructed and simultaneously exploited for multicolor cell-selective labeling, which allow discriminating and orthogonal labeling of three different cell types with the same protein tag. Our method provides a promising strategy for designing a stimuli-responsive chemogenetic labeling platform via facile molecular engineering of the synthetic ligands, which has great potential for conditional multicolor cell-selective labeling and cellular heterogeneity evaluation.

Advances in G-quadruplexes-based fluorescent imaging.

G-quadruplexes (G4s), the noncanonical nucleic acid secondary structure, form within guanine-rich DNA or RNA sequences. G4s formation can affect chromatin architecture and gene regulation and has been associated with various cellular functions, including DNA replication, transcription, and genome maintenance. Visualizing and detecting G4s precisely in such processes is essential to increasing our understanding of G4s biology. Considerable attention has focused on the G4s targeting molecular imaging studies. Besides, fluorescent light-up aptamers (FLAPs, also referred to as fluorogenic aptamers) have gained momentum, which commonly have a G4 scaffolding for imaging intracellular RNAs and metabolites. In this review, we first introduce several representative fluorescent imaging approaches for tracking G4s in cells and in vivo. We also discuss the potential of G4-containing FLAPs in bioimaging and summarize current developments in this field from the standpoint of fluorescent molecules. Finally, we discuss the present challenges and future potential of G4 imaging and G4-containing FLAPs development.

Visualization of Deep Tissue G-quadruplexes with a Novel Large Stokes-Shifted Red Fluorescent Benzothiazole Derivative.

G-quadruplex (G4) is a noncanonical nucleic acid secondary structure that has implications for various physiological and pathological processes and is thus essential to exploring new approaches to G4 detection in live cells. However, the deficiency of molecular imaging tools makes it challenging to visualize the G4 in ex vivo tissue samples. In this study, we established a G4 probe design strategy and presented a red fluorescent benzothiazole derivative, ThT-NA, to detect and image G4 structures in living cells and tissue samples. By enhancing the electron-donating group of thioflavin T (ThT) and optimizing molecular structure, ThT-NA shows excellent photophysical properties, including red emission (610 nm), a large Stokes shift (>100 nm), high sensitivity selectivity toward G4s (1600-fold fluorescence turn-on ratio) and robust two-photon fluorescence emission. Therefore, these features enable ThT-NA to reveal the endogenous RNA G4 distribution in living cells and differentiate the cell cycle by monitoring the changes of RNA G4 folding. Significantly, to the best of our knowledge, ThT-NA is the first benzothiazole-derived G4 probe that has been developed for imaging G4s in ex vivo cancer tissue samples by two-photon microscopy techniques.

Arthroscopic Intra-Articular Repair of the Torn Subscapularis Tendon with Single Anterior Portal, Single Suture Anchor, and X-Shaped Fixation Technique.

Subscapular tendon plays an important role in shoulder joint function. With the advance of magnetic resonance imaging technology and the popularization of arthroscopic shoulder surgery, subscapularis tears have been increasingly detected. However, reduction and fixation of subscapular tendon tears appears to be technically challenging. This study aims to describe an arthroscopic intra-articular X-shaped fixation technique: a procedure of subscapularis tendon repair performed with the aid of a suture passer using only a single anterior portal and a single suture anchor. By incorporating the advantages of a single anterior working portal for anchor placement and tear repair, this technique provides an easier way to use suture lasso and make knots in a limited working space, and the whole procedure is minimally invasive with a short learning curve. This technique has been applied in patients with subscapularis tears involving no intraoperative or postoperative complications. Our technology offers a valuable new treatment option for subscapularis tears.

Photodecaging of a Mitochondria-Localized Iridium(III) Endoperoxide Complex for Two-Photon Photoactivated Therapy under Hypoxia.

Despite the clinical success of photodynamic therapy (PDT), the application of this medical technique is intrinsically limited by the low oxygen concentrations found in cancer tumors, hampering the production of therapeutically necessary singlet oxygen (1O2). To overcome this limitation, we report on a novel mitochondria-localized iridium(III) endoperoxide prodrug (2-O-IrAn), which, upon two-photon irradiation in NIR, synergistically releases a highly cytotoxic iridium(III) complex (2-IrAn), singlet oxygen, and an alkoxy radical. 2-O-IrAn was found to be highly (photo-)toxic in hypoxic tumor cells and multicellular tumor spheroids (MCTS) in the nanomolar range. To provide cancer selectivity and improve the pharmacological properties of 2-O-IrAn, it was encapsulated into a biotin-functionalized polymer. The generated nanoparticles were found to nearly fully eradicate the tumor inside a mouse model within a single treatment. This study presents, to the best of our knowledge, the first example of an iridium(III)-based endoperoxide prodrug for synergistic photodynamic therapy/photoactivated chemotherapy, opening up new avenues for the treatment of hypoxic tumors.

Development of a triazolobenzodiazepine-based PET probe for subtype-selective vasopressin 1A receptor imaging.

OBJECTIVES: To enable non-invasive real-time quantification of vasopressin 1A (V1A) receptors in peripheral organs, we sought to develop a suitable PET probe that would allow specific and selective V1A receptor imaging in vitro and in vivo. METHODS: We synthesized a high-affinity and -selectivity ligand, designated compound 17. The target structure was labeled with carbon-11 and tested for its utility as a V1A-targeted PET tracer by cell uptake studies, autoradiography, in vivo PET imaging and ex vivo biodistribution experiments. RESULTS: Compound 17 (PF-184563) and the respective precursor for radiolabeling were synthesized in an overall yield of 49% (over 7 steps) and 40% (over 8 steps), respectively. An inhibitory constant of 0.9 nM towards the V1A receptors was measured, while excellent selectivity over the related V1B, V2 and OT receptor (IC50 >10,000 nM) were obtained. Cell uptake studies revealed considerable V1A binding, which was significantly reduced in the presence of V1A antagonists. Conversely, there was no significant blockade in the presence of V1B and V2 antagonists. In vitro autoradiography and PET imaging studies in rodents indicated specific tracer binding mainly in the liver. Further, the pancreas, spleen and the heart exhibited specific binding of [11C]17 ([11C]PF-184563) by ex vivo biodistribution experiments. CONCLUSION: We have developed the first V1A-targeted PET ligand that is suitable for subtype-selective receptor imaging in peripheral organs including the liver, heart, pancreas and spleen. Our findings suggest that [11C]PF-184563 can be a valuable tool to study the role of V1A receptors in liver diseases, as well as in cardiovascular pathologies.

A mitochondrion-targeted BODIPY-Ir(III) conjugate as a photoinduced ROS generator for the oxidative destruction of triple-negative breast cancer cells.

Photodynamic therapy (PDT) provides an alternative option to root out localized triple-negative breast cancer (TNBC) and has been experiencing a surge of research interest over recent years. In this study, we put forward a paradigm of designing novel transition metal-based PSs with the following characteristics: favorable cell-permeability, significant light-harvesting ability and prominent ROS yield. A novel BODIPY-Ir(III) conjugate has been designed as a photoinduced ROS (1O2, ˙OH and ˙O2-) generator. BODIPY-Ir is highly photoactive in subduing cancer cells in the PDT regimen with PI values ranging from 172 to 519 and EC50 in the nanomolar regime. Among various cancerous cell lines, TNBC was especially sensitive to BODIPY-Ir-mediated PDT, with a stunning EC50 value of 4.32 nM (PI = 519) under a moderate flux of visible-light irradiation (500 nm, 10.5 mW cm-2). BODIPY-Ir mainly accumulates in mitochondria and induces cell apoptosis under irradiation. Furthermore, the nanomolar antiproliferative activity of BODIPY-Ir is retained under hypoxia (2.5% O2). This work sheds light on instilling the O2-independent type I mechanism and conferring a red-shift absorption to metal-based PSs which fundamentally facilitate the clinical translation of PSs.

Ruthenium(II) complexes coordinated to graphitic carbon nitride: Oxygen self-sufficient photosensitizers which produce multiple ROS for photodynamic therapy in hypoxia.

The photodynamic therapy (PDT) of cancer is limited by tumor hypoxia as PDT efficiency depends on O2 concentration. A novel oxygen self-sufficient photosensitizer (Ru-g-C3N4) was therefore designed and synthesized via a facile one-pot method in order to overcome tumor hypoxia-induced PDT resistance. The photosensitizer is based on [Ru(bpy)2]2+ coordinated to g-C3N4 nanosheets by Ru-N bonding. Compared to pure g-C3N4, the resulting nanosheets exhibit increased water solubility, stronger visible light absorption, and enhanced biocompatibility. Once Ru-g-C3N4 is taken up by hypoxic tumor cells and exposed to visible light, the nanosheets not only catalyze the decomposition of H2O2 and H2O to generate O2, but also catalyze H2O2 and O2 concurrently to produce multiple ROS (•OH, •O2-, and 1O2). In addition, Ru-g-C3N4 affords luminescence imaging, while continuously generating O2 to alleviate hypoxia greatly improving PDT efficacy. To the best of our knowledge, this oxygen self-sufficient photosensitizer produced via grafting a metal complex onto g-C3N4 is the first of its type to be reported.

ß-Amyloid Peptides Manipulate Switching Behaviors of Donor-Acceptor Stenhouse Adducts.

Molecular switching plays a critical role in biological and displaying systems. Donor-acceptor Stenhouse adducts (DASAs) is a newly re-discovered series of switchable photochromes, and light is the most used approach to control its switching behavior. In this report, we speculated that hydrophobic binding pockets of biologically relevant peptides/proteins could be harnessed to alter its switching behavior without the assistance of light. We designed and synthesized a DASA compound SHA-2, and we demonstrated that the Aß40 species could stabilize SHA-2 in the linear conformation and decrease the rate of molecular switching via fluorescence spectral studies. Moreover, molecular dynamics simulation revealed that SHA-2 could bind to the hydrophobic fragment of the peptide and resulted in substantial changes in the tertiary structure of Aß40 monomer. This structural change is likely to impede the aggregation of Aß40, as evidenced by the results from thioflavin T fluorescence and ProteoStat aggregation detection experiments. We believe that our study opens a new window to alter the switching behavior of DASA via DASA-peptide/protein interactions.

Nano-assembly of ruthenium(II) photosensitizers for endogenous glutathione depletion and enhanced two-photon photodynamic therapy.

Photodynamic therapy (PDT) is a promising noninvasive cancer treatment. PDT in the clinic faces several hurdles due to the unique tumor environment, a feature of which is high levels of glutathione (GSH). An excess amount of GSH consumes reactive oxygen species (ROS) generated by photosensitizers (PSs), reducing PDT efficiency. Herein, nano-photosensitizers (RuS1 NPs and RuS2 NPs) are reported. These consist of ruthenium complexes joined by disulfide bonds forming GSH sensitive polymer nanoparticles. The NPs achieve enhanced uptake compared to their constituent monomers. Inside cancer cells, high levels of GSH break the S-S bonds releasing PS molecules in the cell. The level of GSH is also then reduced leading to excellent PDT activity. Furthermore, RuS2 NPs functionalized with tumor targeting hyaluronic acid (HA@RuS2 NPs) assessed in vivo were highly effective with minimal side effects. To the best of our knowledge, RuS NPs are the first metal complex-based nano-assembled photosensitizers which exhibit enhanced specificity and consume endogenous GSH simultaneously, thus achieving excellent two-photon PDT efficiency in vitro and in vivo.

Measurements of cardiac efficiency and internal work of the left ventricle via reconstructed impedance cardiography.

Objective. The aim of this study was to investigate methods for measuring the cardiac efficiency (CE) and internal work (IW) of the left ventricle via reconstructed impedance cardiography (RICG).Approach.On the basis of the physiological context and Bernoulli's equation in physics, methods of measuring the CE and IW were proposed. The CE, IW, internal work index (IWI), and other data from 180 healthy adults and 144 patients with cardiovascular disease were measured.Main results. The CE of 180 healthy adults was 22.5 ± 2.2%, and the IWI was 22.3 ± 5.2 J l-1m-2. CE decreased with age, and the CE of the younger group (23.5 ± 1.9%) was larger than that of the older group (21.5 ± 1.9%),P < 0.01. The IWI increased with age, and the IWI of the younger group (19.0 ± 3.8 J l-1m-2) was smaller than that of the older group (24.8 ± 4.3 J l-1m-2),P < 0.01. There were no significant difference in CE (22.4 ± 2.2% and 22.6 ± 2.2%) or in the IWI (21.9 ± 5.1 J l-1m-2and 22.6 ± 5.2 J l-1m-2) between the male and female groups. The CEs and IWIs of patients with hypertension, coronary heart disease, and heart failure were 17.4 ± 2.4% and 41.8 ± 15.6 J l-1m-2, 17.6 ± 3.0% and 35.1 ± 10.4 J l-1m-2, and 15.8 ± 3.5% and 42.1 ± 15.6 J l-1m-2, respectively. These CEs were all smaller than that (21.6 ± 2.0%) of the healthy contrast groupP < 0.01, while the IWIs were all larger than that (24.6 ± 4.8 J l-1m-2) of the healthy contrast group,P < 0.01.Significance.The CEs and IWIs measured in this study may reflect physiological changes in healthy humans and pathogenic conditions in patients with cardiovascular disease.

Clinical results of arthroscopic tight fibrous band release for adult moderate-to-severe gluteal fibrosis using anterior and posterior portals: a retrospective analysis of 118 consecutive cases.

BACKGROUND: To evaluate the clinical outcomes of arthroscopic tight fibrous band release in the treatment of adult moderate-to-severe gluteal fibrosis using anterior and posterior portals during mid-term follow-up. METHODS: The data of 138 patients (58 males, 80 females) aged between 18 and 42 years (mean, 28.6 years), presenting with bilateral moderate-to-severe gluteal fibrosis (GF) from October 2013 to August 2019, was retrospectively analyzed. All patients underwent arthroscopic tight fibrous band release using anterior and posterior portals with radiofrequency energy. Under arthroscopic guidance through the posterior portal, we debrided the fatty tissue overlying the contracted band of the gluteal muscle and excised the contracted bands using a radiofrequency device introduced through the anterior portal. The pre- and post-operative gluteal muscle contracture disability (GD) scale and the patient satisfaction rate were compared to evaluate the curative effect of the operation. RESULTS: The average operation time was 18 min (range, 10-30 min) and the average blood loss was 4 ml (range, 2-10 ml) for unilateral arthroscopic release. Two cases of post-operative minimal hematomas, 2 cases of bruising and 2 cases of local subcutaneous edema were observed as early complications and were cured by conservative treatment. After surgery, all incisions healed in stage I, and no other complications such as wound infection, nerve and blood vessel injury were detected. One hundred eighteen patients were followed up for 6 to 72 months (mean, 36 months). No lateral instability of the hip was observed and all patients returned to normal gait. The degree of adduction of the hip joint in all these 118 patients was significantly improved relative to their pre-operative conditions. One hundred fifteen patients (97.5%) were able to crouch with knees close to each other after surgery. One hundred fourteen patients (96.6%) were able to cross the affected leg completely without any support. The GD scale was improved from 55.5 ± 10.6 before operation to 90.1 ± 5.2 at the last follow-up (p < 0.05). The patient satisfaction rate was 95.8%. CONCLUSION: Arthroscopic tight fibrous band release using anterior and posterior portals is minimally invasive for adult moderate-to-severe gluteal fibrosis, with a high success rate, quick recovery after surgery and reliable medium-term effect.

One- and Two-Photon Phototherapeutic Effects of RuII Polypyridine Complexes in the Hypoxic Centre of Large Multicellular Tumor Spheroids and Tumor-Bearing Mice*.

During the last decades, photodynamic therapy (PDT), an approved medical technique, has received increasing attention to treat certain types of cancer. Despite recent improvements, the treatment of large tumors remains a major clinical challenge due to the low ability of the photosensitizer (PS) to penetrate a 3D cellular architecture and the low oxygen concentrations present in the tumor center. To mimic the conditions found in clinical tumors, exceptionally large 3D multicellular tumor spheroids (MCTSs) with a diameter of 800 µm were used in this work to test a series of new RuII polypyridine complexes as one-photon and two-photon PSs. These metal complexes were found to fully penetrate the 3D cellular architecture and to generate singlet oxygen in the hypoxic center upon light irradiation. While having no observed dark toxicity, the lead compound of this study showed an impressive phototoxicity upon clinically relevant one-photon (595 nm) or two-photon (800 nm) excitation with a full eradication of the hypoxic center of the MCTSs. Importantly, this efficacy was also demonstrated on mice bearing an adenocarcinomic human alveolar basal epithelial tumor.

OPN Deficiency Increases the Severity of Osteoarthritis Associated with Aberrant Chondrocyte Senescence and Apoptosis and Upregulates the Expression of Osteoarthritis-Associated Genes.

Objectives: A recent work has reported that the elevated osteopontin (OPN) levels in the articular cartilage and synovial fluid are correlated with the progressive osteoarthritis (OA) joint damage, and OPN has a protective effect against OA by suppressing the expressions of OA-associated genes. The present study examined whether the OPN deficiency was susceptible to OA through the regulation of chondrocyte senescence and apoptosis and the expressions of OA-associated genes. Methods: The mRNA levels of COL2A1 and OPN were compared between human OA chondrocytes and normal chondrocytes. The effects of OPN siRNA on the SA-ß-Gal expressions and the percentage of apoptotic chondrocytes were examined by using SA-ß-Gal staining and apoptosis assay, and the effects on the expressions of COL2A1 and OA-associated genes (COL10A1, IL-1ß, TNF-ɑ, MMP-13, and ADAMTS5) were examined by western blot analysis and quantitative real-time RT-PCR. Furthermore, an in vivo OA model was established to examine the effects of OPN siRNA on the senescence and apoptosis of OA chondrocytes and the expressions of OA-associated genes. Results: The mRNA levels of COL2A1 and OPN were decreased in knee OA chondrocytes in comparison with those in normal chondrocytes. The OPN deficiency enhanced the senescence and apoptosis of OA chondrocytes and increased the expressions of COL10A1, IL-1ß, TNF-ɑ, MMP-13, and ADAMTS5 but decreased the expression of COL2A1. Meanwhile, OPN deficiency could result in severe, accelerated OA in vivo, which was also associated with enhanced senescence and apoptosis of chondrocytes and elevated expressions of OA-associated genes. Conclusions: The findings of this study suggest that the OPN deficiency can result in accelerated OA, which is associated with enhanced senescence and apoptosis of OA chondrocytes and the upregulated expressions of OA-associated genes.