The homeostatic link between oxidative stress and autophagy plays an important role in cellular responses to a wide variety of physiological and pathological conditions. However, the regulatory ...pathway and outcomes remain incompletely understood. Here, we show that reactive oxygen species (ROS) function as signaling molecules that regulate autophagy through ataxia‐telangiectasia mutated (ATM) and cell cycle checkpoint kinase 2 (CHK2), a DNA damage response (DDR) pathway activated during metabolic and hypoxic stress. We report that CHK2 binds to and phosphorylates Beclin 1 at Ser90/Ser93, thereby impairing Beclin 1‐Bcl‐2 autophagy‐regulatory complex formation in a ROS‐dependent fashion. We further demonstrate that CHK2‐mediated autophagy has an unexpected role in reducing ROS levels via the removal of damaged mitochondria, which is required for cell survival under stress conditions. Finally, CHK2−/− mice display aggravated infarct phenotypes and reduced Beclin 1 p‐Ser90/Ser93 in a cerebral stroke model, suggesting an in vivo role of CHK2‐induced autophagy in cell survival. Taken together, these results indicate that the ROS‐ATM‐CHK2‐Beclin 1‐autophagy axis serves as a physiological adaptation pathway that protects cells exposed to pathological conditions from stress‐induced tissue damage.
Synopsis
Whether hypoxia and nutrient starvation are coupled to cellular autophagy remains unclear. Here, DNA damage response kinases ATM and CHK2 are shown to trigger autophagy in response to reactive oxygen species (ROS) accumulation, suggesting a novel physiological adaptation pathway toward metabolic stress.
Depletion of CHK2 or ATM impairs oxidative stress‐induced autophagy in MEFs.
CHK2 binds and phosphorylates Beclin1 at Ser90/Ser93, suppressing Beclin1‐Bcl‐2 autophagy regulatory complex formation.
CHK2‐induced autophagy limits intracellular ROS levels by clearing damaged mitochondria.
CHK2‐induced autophagy protects against cell death and tissue damage following cerebral ischemia.
ROS accumulation activates protective autophagy to prevent stress‐induced tissue damage.
Osteoarthritis (OA) is the most prevalent degenerative joint disease. In vitro experiments are an intuitive method used to investigate its early pathogenesis. Chondrocyte inflammation models in rats ...and mice are often used as in vitro models of OA. However, similarities and differences between them in the early stages of inflammation have not been reported.
This paper seeks to compare the chondrocyte phenotype of rats and mice in the early inflammatory state and identify chondrocytes suitable for the study of early OA.
Under similar conditions, chondrocytes from rats and mice were stimulated using the same IL-1β concentration for a short period of time. The phenotypic changes of chondrocytes were observed under a microscope. The treated chondrocytes were subjected to RNA-seq to identify similarities and differences in gene expression. Chondrocytes were labelled with EdU for proliferation analysis. Cell proliferation-associated proteins, including minichromosome maintenance 2 (MCM2), minichromosome maintenance 5 (MCM5), Lamin B1, proliferating cell nuclear antigen (PCNA), and Cyclin D1, were analysed by immunocytochemical staining, cell immunofluorescence, and Western blots to verify the RNA-seq results.
RNA-seq revealed that the expression patterns of cytokines, chemokines, matrix metalloproteinases, and collagen were similar between the rat and mouse chondrocyte inflammation models. Nonetheless, the expression of proliferation-related genes showed the opposite pattern. The RNA-seq results were further verified by subsequent experiments. The expression levels of MCM2, MCM5, Lamin B1, PCNA, and Cyclin D1 were significantly upregulated in rat chondrocytes (P < 0.05) and mouse chondrocytes (P < 0.05).
Based on the findings, the rat chondrocyte inflammation model may help in the study of the early pathological mechanism of OA.
Mice deficient in the Polycomb repressor Bmi1 develop numerous abnormalities including a severe defect in stem cell self-renewal, alterations in thymocyte maturation and a shortened lifespan. ...Previous work has implicated de-repression of the Ink4a/Arf (also known as Cdkn2a) locus as mediating many of the aspects of the Bmi1(-/-) phenotype. Here we demonstrate that cells derived from Bmi1(-/-) mice also have impaired mitochondrial function, a marked increase in the intracellular levels of reactive oxygen species and subsequent engagement of the DNA damage response pathway. Furthermore, many of the deficiencies normally observed in Bmi1(-/-) mice improve after either pharmacological treatment with the antioxidant N-acetylcysteine or genetic disruption of the DNA damage response pathway by Chk2 (also known as Chek2) deletion. These results demonstrate that Bmi1 has an unexpected role in maintaining mitochondrial function and redox homeostasis and indicate that the Polycomb family of proteins can coordinately regulate cellular metabolism with stem and progenitor cell function.
Nanodevices for deep cartilage penetration Xu, Xiao-Ling; Xue, Yan; Ding, Jia-Ying ...
Acta biomaterialia,
December 2022, 2022-Dec, 2022-12-00, Letnik:
154
Journal Article
Recenzirano
Osteoarthritis (OA) is a degenerative joint disease and is the main cause of chronic pain and functional disability in adults. Articular cartilage is a hydrated soft tissue that is composed of ...normally quiescent chondrocytes at a low density, a dense network of collagen fibrils with a pore size of 60–200 nm, and aggrecan proteoglycans with high-density negative charge. Although certain drugs, nucleic acids, and proteins have the potential to slow the progression of OA and restore the joints, these treatments have not been clinically applied owing to the lack of an effective delivery system capable of breaking through the cartilage barrier. Recently, the development of nanotechnology for delivery systems renders new ideas and treatment methods viable in overcoming the limited penetration. In this review, we focus on current research on such applications of nanotechnology, including exosomes, protein-based cationic nanocarriers, cationic liposomes/solid lipid nanoparticles, amino acid-based nanocarriers, polyamide derivatives-based nanocarriers, manganese dioxide, and carbon nanotubes. Exosomes are the smallest known nanoscale extracellular vesicles, and they can quickly deliver nucleic acids or proteins to the required depth. Through electrostatic interactions, nanocarriers with appropriate balance in cationic property and particle size have a strong ability to penetrate cartilage. Although substantial preclinical evidence has been obtained, further optimization is necessary for clinical transformation.
The dense cartilage matrix with high-negative charge was associated with reduced therapeutic effect in osteoarthritis patients with deep pathological changes. However, a systematic review in nanodevices for deep cartilage penetration is still lacking.
Current approaches to assure penetration of nanosystems into the depth of cartilage were reviewed, including nanoscale extracellular vesicles from different cell lines and nanocarriers with appropriate balance in cationic property and size particle. Moreover, nanodevices entering clinical trials and further optimization were also discussed, providing important guiding significance to future research.
Display omitted
Background: Osteosarcoma is the most common primary bone malignancy of adolescents and young adults. Methods: We analyzed liver X receptor α (LXRα) mRNA expression in 16 pairs of human osteosarcoma ...tissues and adjacent noncancerous tissues. Moreover, we investigated LXRα's potential role in regulating cell proliferation in Saos-2 and U2OS cells. Results: We found that activation of LXRα, a member of nuclear receptor, was able to inhibit cell proliferation in Saos-2 and U2OS cells. At the molecular level, our results further revealed that expression of tumor suppressor gene, FoxO1, was up-regulated by LXRα activation. LXRα activates FoxO1 transcription through a direct binding on its promoter region. Conclusion: LXRα acts as a tumor suppressor for osteosarcoma, which may offer a new way in molecular targeting cancer treatment.
•ER-staining and RNA-seq analyses indicated increased protein catabolism in chondrocytes after thrombin treatment, reflected in the downregulation of many collagens, proteoglycans, and Sox family ...transcription factors.•Thrombin induced proliferation and the development of fibrotic phenotypes in chondrocytes.•The levels of a variety of catabolic factors, including inflammatory factors (IL-6, IL-6st), chemokines (Cxcll6, Cxcl6, Cxcl3, Ccl20, Ccl2, Ccl7), and matrix metalloenzymes (MMP13, MMP3, MMP9) increased in the presence of thrombin.
The present study was designed to explore the relationship between thrombin and catabolic activity in chondrocytes. Primary rat chondrocytes were cultured for 24 h with rat serum (RS), rat plasma (RP), or rat plasma supplemented with thrombin (RPT). RNA-sequencing was then performed. Cell proliferation was analyzed by EdU uptake, CCK-8 assays and protein–protein interaction (PPI) network of proliferation-related genes. Heatmaps were used to visualize differences in gene expression. Gene Ontology (GO) enrichment analyses of up- and down-regulated differentially expressed genes were conducted. Molecular probes were used to label the endoplasmic reticulum in chondrocytes from three treatment groups. Immunofluorescence and Safranin O staining were used to assess type II collagen (Col2a1) expression and proteoglycan synthesis, whereas Lox expression was assessed by immunocytochemistry. The expression of enzymes involved in the synthesis and maturation of extracellular matrix (ECM) components and chemokines were measured by qPCR while matrix metalloproteinases (MMPs) levels were evaluated by Western blotting. Relevant nodules were selected through further PPI network analyses. A total of 727 and 1162 genes were up- and down-regulated based on the Venn diagrams comparison among groups. Thrombin was thus able to promote chondrocyte proliferation and a shift towards fibrotic morphology, while upregulating MMPs and chemokines linked to ECM degradation. In addition, thrombin decreased the enzyme expression involved in the synthesis and maturation of ECM.
To explore influence of external factors of wind, cold and dampness on clinical symptoms in knee osteoarthritis (KOA) patients with different constitutions of traditional Chinese medicine.
A ...cross-sectional stratified study was performed to select 108 patients with GradeⅡKOA in Kellgren & Lawrence (K-L) classification, including 22 males and 86 females, aged from 47 to 75 years old with an average of (60.7±6.0) years old;body mass index(BMI) ranged from 17.87 to 31.22 kg·m
with an average of (23.80±2.86) kg·m
. According to Classification and Judgment of TCM Physique (ZYYXH/T157-2009), the types of TCM physique were determined and divided into 4 layers according to the deficiency and actual physique. Among them, there were 24 patients without biased physique, 12 males and 12 females, aged from 51 to 73 years old with an average of(62.8±6.0) years old, BMI ranged from 17.87 to 31.14 kg·m
with an average of (24.32±3.25) kg·m-2;there were 46 patients with virtual bias constitution, including 7 males and 39 femal
Objective：Although chondroprotective activities have been documented for polysaccharides,the potential target of different polysaccharide may differ.The study was aimed to explore the effect of ...glucan HBP-A in chondrocyte monolayer culture and chondrocytes-alginate hydrogel constructs in vivo,especially on the expression of type Ⅱ collagen.Methods：Chondrocytes isolated from rabbit articular cartilage were cultured and verified by immunocytochemical staining of type Ⅱ collagen.Chondrocyte viability was assessed after being treated with HBP-A in different concentrations.Morphological status of chondrocytes-alginate hydrogel constructs in vitro was observed by scanning electron microscope（SEM）.The constructs were treated with HBP-A and then injected to nude mice subcutaneously.Six weeks after transplantation,the specimens were observed through transmission electron microscopy（TEM）.The mRNA expressions of disintegrin and metalloproteinase with thrombospondin motifs 5（ADAMTs-5）,aggrecan and type Ⅱ collagen in both monolayer culture and constructs were determined by real time polymerase chain reaction（PCR）.The expression of typeⅡ collagen and matrix metalloproteinases-3（MMP-3） in chondrocyte monolayer culture was also tested through Western blot and enzyme linked immunosorbent assay（ELISA）,respectively.Results：MMP-3 secretion and ADAMTs-5 mRNA expression in vitro were inhibited by HBP-A at 0.3 mg/mL concentration.In morphological study,there were significant appearance of collagen in those constructs treated by HBP-A.Accordingly,in both chondrocyte monolayer culture and chondrocytes-alginate hydrogel constructs,the expression of type Ⅱcollagen was increased significantly in HBP-A group when compared with control group（P〈0.001）.Conclusions：The study documented that the potential pharmacological target of glucan HBP-A in chondrocytes monolayer culture and tissue engineered cartilage in vivo may be concerned with the inhibition of catabolic enzymes MMP-3,ADAMTs-5,and increasing of type Ⅱ collagen expression.
Rheumatoid arthritis fibroblast-like synoviocytes (RAFLSs) proliferate abnormally and resist apoptosis. Bufalin inhibits cell proliferation and induces apoptosis in human cancer cells. In this study, ...we explored the effects of bufalin on interleukin-1beta (IL-1β)-induced proliferation and apoptosis of RAFLSs. The cell proliferation and apoptosis were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay and annexin V/propidium iodide staining, respectively. Bufalin dose-dependently inhibited IL-1β-induced RAFLS proliferation. Mechanistically, bufalin decreased the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB), both of which are involved in IL-1β-mediated RAFLS proliferation. Moreover, bufalin induced apoptosis and mitochondrial damage of RAFLSs, which was associated with Bcl-2 downregulation, Bax upregulation, mitochondrial cytochrome c release, and enhanced cleavages of caspase-3 and poly-(ADP-ribose) polymerase. Collectively, our results reveal that bufalin suppresses IL-1β-induced proliferation of RAFLSs through MAPK and NF-κB signaling pathways and induces RAFLS apoptosis via the mitochondria-dependent pathway.
The aim of this cross-sectional study was to examine the relationship among pain and other symptoms intensity, and health-related quality of life (HRQoL) in Chinese patients with knee osteoarthritis ...(OA).
The study was cross-sectional, descriptive, and correlational. A convenience sample of 466 patients with knee OA was recruited in the study. Age, gender, body mass index (BMI), duration of disease, and Kellgren- Lawrence (KL) scores were recorded. HRQoL and symptoms were assessed using the 36-item Short Form Health Survey (SF-36) and the Western Ontario and McMaster (WOMAC) index in participants.
The sample was predominantly female (82%) with mean age 56.56 years and mean BMI 24.53 kg/m(2). We found that WOMAC subscale scores significantly negative correlated with the majority of SF-36 subscale scores in knee OA patients (P < 0.05). There were no correlations between BMI, duration of disease, KL score and the vast majority of SF-36 subscale scores in patients (P > 0.05). In addition, there was a significant correlation between age and PCS, gender and MCS in patients (P < 0.05). Regression analysis showed, WOMAC subscale scores significantly negative correlated with the vast majority of SF-36 subscale scores. WOMAC-pain score had the strongest relationship with SF-36 PCS and MCS scores.
In summary, pain severity has a greater impact on HRQoL than patient characteristics, other joint symptoms and radiographic severity in Chinese knee OA patients. Relieving of knee symptoms may help to improve patients' HRQOL. The study provided the evidence that relieving pain should be the first choice of therapy for knee osteoarthritis.
PDF
