Identification of aging-related genes in diagnosing osteoarthritis via integrating bioinformatics analysis and machine learning.

BACKGROUND: Osteoarthritis (OA) is one of the main causes of pain and disability in the world, it may be caused by many factors. Aging plays a significant role in the onset and progression of OA. However, the mechanisms underlying it remain unknown. Our research aimed to uncover the role of aging-related genes in the progression of OA. METHODS: In Human OA datasets and aging-related genes were obtained from the GEO database and the HAGR website, respectively. Bioinformatics methods including Gene Ontology (GO), Kyoto Encyclopedia of Genes Genomes (KEGG) pathway enrichment, and Protein-protein interaction (PPI) network analysis were used to analyze differentially expressed aging-related genes (DEARGs) in the normal control group and the OA group. And then weighted gene coexpression network analysis (WGCNA), the least absolute shrinkage and selection operator (LASSO) regression, and the Random Forest (RF) machine learning algorithms were used to find the hub genes. RESULTS: Four overlapping hub genes: HMGB2, CDKN1A, JUN, and DDIT3 were identified. According to the nomogram model and receiver operating characteristic (ROC) curve analysis, four hub DEARGs had good diagnostic value in distinguishing normal from OA. Furthermore, the qRT-PCR test demonstrated that HMGB2, CDKN1A, JUN, and DDIT3 mRNA expression levels were lower in OA group than in normal group. CONCLUSION: Finally, these four-hub aging-related genes may help us understand the underlying mechanism of aging in osteoarthritis and could be used as possible diagnostic and therapeutic targets.

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

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

SCP2 mediates the transport of lipid hydroperoxides to mitochondria in chondrocyte ferroptosis.

Sterol carrier protein 2 (SCP2) is highly expressed in human osteoarthritis (OA) cartilage, accompanied by ferroptosis hallmarks, especially the accumulation of lipid hydroperoxides (LPO). However, the role of SCP2 in chondrocyte ferroptosis remains unexplored. Here, we identify that SCP2 transports cytoplasmic LPO to mitochondria in RSL3-induced chondrocyte ferroptosis, resulting in mitochondrial membrane damage and release of reactive oxygen species (ROS). The localization of SCP2 on mitochondria is associated with mitochondrial membrane potential, but independent of microtubules transport or voltage-dependent anion channel. Moreover, SCP2 promotes lysosomal LPO increase and lysosomal membrane damage through elevating ROS. However, SCP2 is not directly involved in the cell membrane rupture caused by RSL3. Inhibition of SCP2 markedly protects mitochondria and reduces LPO levels, attenuating chondrocyte ferroptosis in vitro and alleviating the progression of OA in rats. Our study demonstrates that SCP2 mediates the transport of cytoplasmic LPO to mitochondria and the spread of intracellular LPO, accelerating chondrocyte ferroptosis.

Low-level laser selectively inhibiting colorectal cancer cell metabolic activity and inducing apoptosis for delaying the development of intestinal cancer.

Low-level laser irradiation (LLLI) is a novel approach that shows promise for the treatment of colorectal cancer (CRC). However, the molecular mechanisms underlying its biochemical effects and gene expression remain unclear. Here, LLLI (632.8 nm) was used to treat CRC RKO cells and normal small intestinal NCM460 cells. LLLI showed a significant dose- and time-dependent effect on cell viability, in which a single dose of irradiation at 15 J/cm2 selectively inhibited the growth of RKO cells but largely unaffected the activity of NCM460 cells. And then, LLLI produced an internal response, effectively reducing the level of H2O2 in tumor cells, downregulating the mitochondrial membrane potential, and improving the efficiency of apoptosis in CRC, but no internal response was observed in NCM460 cells under the same conditions. Furthermore, the expression of several important genes in the classical WNT pathway was significantly downregulated, and the pathway was inactivated after LLLI intervention, thereby inhibiting tumor cell growth. Simultaneously, TNF-α was effectively activated to stimulate the caspase family members of the death effector to initiate apoptosis led by the extrinsic pathway. LLLI successfully achieves tumor cell normalization while delivering a potent anticancer effect, expected to be a novel therapeutic modality for CRC.

Inhaled platelet vesicle-decoyed biomimetic nanoparticles attenuate inflammatory lung injury.

Acute lung injury (ALI) is an inflammatory response which causes serious damages to alveolar epithelia and vasculature, and it still remains high lethality and mortality with no effective treatment. Based on the inflammatory homing of platelets and cell membrane cloaking nanotechnology, in this study we developed a biomimetic anti-inflammation nanoparticle delivery system for ALI treatment. PM@Cur-RV NPs were designed by combining the poly (lactic-co-glycolic acid) nanoparticles (NPs) coated with platelet membrane vesicles (PM) for the purpose of highly targeting delivery of curcumin (Cur) and resveratrol (RV) to inflammatory lungs. PM@Cur-RV NPs showed good biocompatibility and biosafety both in vitro and in vivo. Accumulation of NPs into lung tract was observed after inhaled NPs. Remarkably, the inhalation of PM@Cur-RV NPs effectively inhibited lung vascular injury evidenced by the decreased lung vascular permeability, and the reduced proinflammatory cytokine burden in an ALI mouse model. The analysis of infiltrated macrophages in the lungs showed that the Cur-RV-modulated macrophage polarized towards M2 phenotype and the decreased histone lactylation might contribute to their anti-inflammation effects. Together, this work highlights the potential of inhalation of biomimetic nanoparticle delivery of curcumin and resveratrol for the treatment of pulmonary diseases.

Cross-Linked Versus Conventional Polyethylene for Long-Term Clinical Outcomes After Total Hip Arthroplasty: A Systematic Review and Meta-Analysis.

Background: Cross-linked polyethylene (HXLPE) liners have been used for total hip arthroplasty (THA) to address the problem of osteolysis and revision surgery associated with conventional polyethylene (CPE) liners. This systematic review and meta-analysis investigated the long-term efficacy of HXLPE in preventing revision surgery and radiological osteolysis in comparison to CPE. Materials and Methods: A comprehensive search of PubMed, Embase, and the Cochrane Library from their respective inception to September 2018 was conducted to identify potential candidate articles. Data were pooled using Stata software 14.0. The quality of randomized controlled trials (RCTs) and observational studies was assessed by two different authors using the Cochrane risk-of-bias tool and the Newcastle-Ottawa scale (NOS), respectively. Results: Eight RCTs and six observational studies were included in this review. The pooled results significantly favored HXLPE over CPE in terms of total number of revisions and radiological osteolysis, with a risk reduction of 78% (95% confidence interval [CI] 0.13-0.36; p < 0.001) and 80% (95% CI 0.13-0.29; p < 0.001), respectively. Additionally, subgroup analyses of pooled data from RCTs and observational studies both showed the efficacy of HXLPE in the prevention of revision and osteolysis. Polyethylene wear in the HXLPE group was significantly less than that in the CPE group in terms of linear wear rates and head penetration rates (both p < 0.001). No significant differences were observed with regard to functional outcomes. Conclusions: The current evidence shows that HXLPE significantly improved the clinical and radiographic outcomes, but not the functional outcomes, in comparison to CPE in long-term follow-up.

The involvement of cytokine-like 1 (Cytl1) in chondrogenesis and cartilage metabolism.

The Cytokine-like 1 (Cytl1) is first identified in bone marrow cells and preferentially expressed in cartilaginous tissue, and showed chondrogenic effects in mesenchymal cells, not essential for cartilage or bone development as in Cytl1 knock-out mice but associated with cartilage inflammatory and destruction. Here, we show the involvement of Cytl1 in chondrogenesis. Using specified chondrogenic embryonic skeleton and adult cartilage, the Cytl1 gene expression was investigated with associated chondrogenic factors by quantitative RT-PCR. The effect of Cytl1 protein (rCytl1) on cultured chondrocytes to regulate expression of key factors and phenotypic markers was studied. The results revealed that Cytl1 was highly expressed in chondrogenic process in embryos and adult cartilage. The rCytl1 increased the expression of Sox9 and Col2α1 with stabilized Col1α1 in cultured chondrocytes (redifferentiation). The Cytl1 was expressed and involved at all stages of cartilage development. Furthermore, Cytl1 expression shared similar patterns as other chondrogenic factors, implying interactions with other factors in chondrogenic process. Cytl1 is involved in cartilage development and matrix homeostasis, which defines the dedifferentiation phenotype of chondrocytes, essential to forming of functional cartilage in both physiologic remodeling and pathologic regeneration.

Promoted Viability and Differentiated Phenotype of Cultured Chondrocytes With Low Level Laser Irradiation Potentiate Efficacious Cells for Therapeutics.

Effective clinical treatments of cartilage lesions in affected joints require large numbers of viable chondrogenic cells generated through in vivo stimulation or ex vivo expansion of chondrocytes isolated from small biopsy specimens. Conventional passaging of chondrocytes in culture provides sufficient cells for treatments but these cells usually lose their differentiated phenotype. This leads to the formation of fibrocartilaginous tissue due to a malfunctioning repair process. Biostimulation of passaging chondrocytes with low level laser irradiation (LLLI) may theoretically produce more functional chondrocytes for cell-based repair of cartilage defects. Molecular and cellular analyses, cytochemistry, cell cultivation, and microscopy showed that LLLI treatments were found to (1) increase chondrocyte viability, (2) promote secretion of matrix proteins, (3) upregulate expression of chondrogenic genes, and (4) downregulate gene expression of cell destructive proteases and genes coding for mediators involved in the extrinsic apoptosis signaling pathway. Furthermore, LLLI attenuated induction of genes associated with cell death and matrix breakdown induced by IL-1ß, some of which was seen at the protein level, with verification of effects on gene expression in the C28/I2 human chondrocyte line. LLLI treatments during culture generated larger numbers of viable chondrocytes compared to untreated cultures. Moreover, LLLI-treated chondrocytes in culture also rectified and simultaneously maintained their differentiated phenotype. Cultured chondrocytes treated with LLLI are a promising cell source for repairing cartilage lesions in vivo and restoration of articular function using tissue engineering strategies.

Characterization of balofloxacin-stressed proteomics and identification of balofloxacin-binding proteins pre-peptidase and integration host factor in Edwardsiella tarda.

The overuse of antibiotics to control bacterial pathogens leads to the generation of their antibiotic-resistant strains including Edwardsiella tarda. Understanding of mechanisms of the antibiotic resistance is crucial to develop novel methods to manage the infection. Here, two-dimensional electrophoresis-based proteomics was used to characterize balofloxacin-responsive proteins. The altered proteome consisted of 19 proteins with differential abundance, where six metabolic pathways were enriched. The metabolic modulation activated the central carbon metabolism with elevation of NADH, PMF, and ATP. Among the 19 proteins, ETAE_1987 (pre-peptidase) and ETAE_2174 (integration host factor beta subunit) were bound with balofloxacin directly. This was further confirmed by the binding of balofloxacin with recombinant ETAE_1987 and ETAE_2174 using Oxford cup method. Compared with bovine serum albumin, a known balofloxacin-binding protein, ETAE_1987 and ETAE_2174 increased the binding capability by 3.3- and 22-fold, respectively. The combination was validated by microscale thermophoresis. These data characterize the balofloxacin-stressed proteome as a result of the increased central carbon metabolism and energy metabolism and determine ETAE_1987 and ETAE_2174 as balofloxacin-binding proteins. These findings have significant implications in understanding bacterial antibiotic-resistant and drug action mechanisms based on balofloxacin-binding proteins. SIGNIFICANCE: Antibiotic-resistant Edwardsiella tarda strains are frequently isolated and cause a great loss in aquaculture since these bacterial strains are insensitivity to antibiotics. The present study showed that the increased central carbon metabolism forms a characteristic feature of the balofloxacin-stressed proteomics. Furthermore, two proteins, ETAE_1987 (pre-peptidase) and ETAE_2174, of the balofloxacin-stressed proteome were identified as balofloxacin-binding proteins. The binding capability is 0.39 ±â€¯0.017 and 2.67 ±â€¯0.066 ng/µg proteins for ETAE_1987 and ETAE_2174, respectively. These results reveal the elevated central carbon metabolism as a key feature of the balofloxacin-stressed proteomics and pre-peptidase and integration host factor as balofloxacin-binding proteins in E. tarda. These findings are useful in the understanding of bacterial balofloxacin-stressed mechanisms and providing new targets for controlling antibiotic-resistant bacteria.

Catalase Enhances Viability of Human Chondrocytes in Culture by Reducing Reactive Oxygen Species and Counteracting Tumor Necrosis Factor-α-Induced Apoptosis.

BACKGROUND/AIMS: Both physiologic remodeling and pathologic regeneration of cartilage tissue rely upon chondrocyte functions and are benefited from factors that promote viability and inhibit apoptosis of the cell, and associated mechanisms. High level of reactive oxygen species (ROS) and proinflammatory cytokines activate apoptosis signaling and initiate cell death, which can be attenuated by antioxidants. This study examined the effect of catalase (CAT) on ROS and tumor necrosis factor-α (TNF-α)-induced apoptosis in human C28/I2 chondrocytes cultured in monolayer. METHODS: Chondrocytes were treated with diluted CAT in the presence or absence of TNF-α and compared to untreated cells. Levels of hydrogen peroxide (H2O2) and mitochondrial membrane potential (Δψm) were measured using fluorescent labeling, cell apoptosis was assayed by flow cytometry using Annexin V/propidium iodide (PI) staining, gene expression was detected by quantitative real time polymerase chain reaction (qRT-PCR) and the proteins were investigated by Western blotting. RESULTS: CAT effectively reduced the intracellular ROS caused by the monolayer culture system, enhanced the Δψm depending on the presence of TNF-α and promoted morphological features at sub-cellular level. CAT also attenuated the TNF-α-upregulated expression of factors/mediators of extrinsic cell death cascade and apoptotic caspases, ultimately resulted in promoted cellular viability. CONCLUSION: The anti-apoptotic effect of CAT on chondrocytes via scavenging ROS and suppressing TNF-α-induced cell apoptosis by TNF/TNF receptor (TNFR) mediated death signaling pathway and potentiate CAT as a complementary agent beneficial to cartilage remodeling and regeneration in vivo, and cell-based therapies of cartilage repair demanding viable cells expanded ex vivo.

Salvianolic acid B regulates gene expression and promotes cell viability in chondrocytes.

Articular chondrocytes reside in lacunae distributed in cartilage responsible for the remodelling of the tissue with limited ability of damage repairing. The in vitro expanded chondrocytes enhanced by factors/agents to obtain large numbers of cells with strengthened phenotype are essential for successful repair of cartilage lesions by clinical cell implantation therapies. Because the salvianolic acid B (Sal B), a major hydrophilic therapeutic agent isolated from Salvia miltiorrhiza, has been widely used to treat diseases and able to stimulate activity of cells, this study examines the effects of Sal B on passaged chondrocytes. Chondrocytes were treated with various concentrations of Sal B in monolayer culture, their morphological properties and changes, and mitochondrial membrane potential were analysed using microscopic analyses, including cellular biochemical staining and confocal laser scanning microscopy. The proteins were quantified by BCA and Western blotting, and the transcription of genes was detected by qRT-PCR. The passaged chondrocytes treated with Sal B showed strengthened cellular synthesis and stabilized mitochondrial membrane potential with upregulated expression of the marker genes for chondrocyte phenotype, Col2-α1, Acan and Sox9, the key Wnt signalling molecule ß-catenin and paracrine cytokine Cytl-1. The treatments using CYTL-1 protein significantly increased expression of Col2-α1 and Acan with no effect on Sox9, indicating the paracrine cytokine acts on chondrocytes independent of SOX9. Sal B has ultimately promoted cell growth and enhanced chondrocyte phenotype. The chondrocytes treated with pharmaceutical agent and cytokine in the formulated medium for generating large number of differentiated chondrocytes would facilitate the cell-based therapies for cartilage repair.

Characterization of ampicillin-stressed proteomics and development of a direct method for detecting drug-binding proteins in Edwardsiella tarda.

Antibiotic-resistant Edwardsiella tarda poses a severe challenge to aquaculture. An understanding for antibiotic-resistant mechanisms is crucial to control the disease. The present study characterizes E. tarda ampicillin-stressed proteome and shows the importance of energy metabolism including the TCA cycle and glycolysis/gluconeogenesis in the antibiotic resistance. Further combination with antibiotic measurement develops a new method for identification of antibiotic-binding proteins out of differential abundances of proteins and results in determination of ETAE_0175 and ETAE_3367 as ampicillin-binding proteins in E. tarda. Genes of the two proteins are cloned and recombinant proteins are purified for validation of antibiotic-binding capability. Results show that higher binding capability is detected in ETAE_3367 than ETAE_0175. Out of the two proteins, ETAE_3367 is first reported here to be an antibiotic-binding protein, while ETAE_0175 homology in other bacteria has been shown to bind with other antibiotics. Bioinformatics analysis shows that ETAE_3367 may closely interact with aceF and sucA belonging to the TCA cycle and glycolysis/gluconeogenesis, respectively. These results indicate that energy metabolism contributes to ampicillin resistance in E. tarda and a new method to identify antibiotic-binding proteins is developed. These findings highlight the way to an understanding of antibiotic-resistant mechanisms in content of antibiotic-binding proteins. BIOLOGICAL SIGNIFICANCE: Our data characterizes Edwardsiella tarda ampicillin-stressed proteome and shows the importance of energy metabolism including the TCA cycle and glycolysis/gluconeogenesis in the antibiotic resistance. Furthermore, a new method based 2-DE proteomics is developed for identification of antibiotic-binding proteins, which results in determination of ETAE_0175 and ETAE_3367 as ampicillin-binding proteins in E. tarda. ETAE_3367 is closely interacted with proteins of the TCA cycle and glycolysis/gluconeogenesis, suggesting the drug-resistant mechanism.