Alleviated NCOA4-mediated ferritinophagy protected RA FLSs from ferroptosis in lipopolysaccharide-induced inflammation under hypoxia.

OBJECTIVE: Ferroptosis is a reactive oxygen species (ROS)- and iron-dependent form of non-apoptotic cell death process. Previous studies have demonstrated that ferroptosis participates in the development of inflammatory arthritis. However, the role of ferroptosis in rheumatoid arthritis (RA) inflammatory hypoxic joints remains unclear. This study sought to explore the underlying mechanism of ferroptosis on lipopolysaccharide (LPS)-induced RA fibroblast-like synoviocytes (FLSs). METHODS: FLSs, isolated from patients with RA, were treated with LPS and ferroptosis inducer (erastin and RSL-3), and ferroptosis inhibitor (Fer-1 and DFO), respectively. The cell viability was measured by CCK-8. The cell death was detected by flow cytometer. The proteins level were tested by Western blot. The cytosolic ROS and lipid peroxidation were determined using DCFH-DA and C11-BODIPY581/591 fluorescence probes, respectively. The small interfering RNA (siRNA) was used to knock down related proteins. The levels of malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), iron, inflammatory cytokines (IL6 and IL8), and LDH were analyzed by commercial kits. RESULTS: Ferroptosis was activated by LPS in RA FLS with increased cellular damage, ROS and lipid peroxidation, intracellular Fe and IL8, which can be further amplified by ferroptosis inducer (erastin and RSL-3) and inhibited by ferroptosis inhibitor (Fer-1 and DFO). Mechanistically, LPS triggered ferroptosis via NCOA4-mediated ferritinophagy in RA FLSs, and knockdown of NCOA4 strikingly prevent the process of ferroptosis. Intriguingly, LPS-induced RA FLSs became insensitive to ferroptosis and NCOA4-mediated ferritinophagy under hypoxia compared with normoxia. Knockdown of HIF-1α reverted ferroptosis and ferritinophagy evoking by LPS-induced RA FLSs inflammation under hypoxia. In addition, low dose of auranofin (AUR) induced re-sensitization of ferroptosis and ferritinophagy through inhibiting the expression of HIF-1α under hypoxia. CONCLUSIONS: NCOA4-mediated ferritinophagy was a key driver of ferroptosis in inflammatory RA FLSs. The suppression of NCOA4-mediated ferritinophagy protected RA FLSs from ferroptosis in LPS-induced inflammation under hypoxia. Targeting HIF-1α/NCOA4 and ferroptosis could be an effective and valuable therapeutic strategy for synovium hyperplasia in the patients with RA.

A Microfluidic System for Detecting Tumor Cells Based on Biomarker Hexaminolevulinate (HAL): Applications in Pleural Effusion.

Malignant pleural effusion is a common clinical problem, which often occurs in cases of malignant tumors, especially in lung cancer. In this paper, a pleural effusion detection system based on a microfluidic chip, combined with specific tumor biomarker, hexaminolevulinate (HAL), used to concentrate and identify tumor cells in pleural effusion was reported. The lung adenocarcinoma cell line A549 and mesothelial cell line Met-5A were cultured as the tumor cells and non-tumor cells, respectively. The optimum enrichment effect was achieved in the microfluidic chip when the flow rates of cell suspension and phosphate-buffered saline achieved 2 mL/h and 4 mL/h, respectively. At the optimal flow rate, the proportion of A549 increased from 28.04% to 70.01% due to the concentration effect of the chip, indicating that tumor cells could be enriched by a factor of 2.5 times. In addition, HAL staining results revealed that HAL can be used to identify tumor cells and non-tumor cells in chip and clinical samples. Additionally, the tumor cells obtained from the patients diagnosed with lung cancer were confirmed to be captured in the microfluidic chip, proving the validity of the microfluidic detection system. This study preliminarily demonstrates the microfluidic system is a promising method with which to assist clinical detection in pleural effusion.

A deep learning model for detection of leukocytes under various interference factors.

The accurate detection of leukocytes is the basis for the diagnosis of blood system diseases. However, diagnosing leukocyte disorders by doctors is time-consuming and requires extensive experience. Automated detection methods with high accuracy can improve detection efficiency and provide recommendations to inexperienced doctors. Current methods and instruments either fail to automate the identification process fully or have low performance and need suitable leukocyte data sets for further study. To improve the current status, we need to develop more intelligent strategies. This paper investigates fulfilling high-performance automatic detection for leukocytes using a deep learning-based method. We established a new dataset more suitable for leukocyte detection, containing 6273 images (8595 leukocytes) and considering nine common clinical interference factors. Based on the dataset, the performance evaluation of six mainstream detection models is carried out, and a more robust ensemble model is proposed. The mean of average precision (mAP) @IoU = 0.50:0.95 and mean of average recall (mAR)@IoU = 0.50:0.95 of the ensemble model on the test set are 0.853 and 0.922, respectively. The detection performance of poor-quality images is robust. For the first time, it is found that the ensemble model yields an accuracy of 98.84% for detecting incomplete leukocytes. In addition, we also compared the test results of different models and found multiple identical false detections of the models, then provided correct suggestions for the clinic.

Synthetic-to-real: instance segmentation of clinical cluster cells with unlabeled synthetic training.

MOTIVATION: The presence of tumor cell clusters in pleural effusion may be a signal of cancer metastasis. The instance segmentation of single cell from cell clusters plays a pivotal role in cluster cell analysis. However, current cell segmentation methods perform poorly for cluster cells due to the overlapping/touching characters of clusters, multiple instance properties of cells, and the poor generalization ability of the models. RESULTS: In this article, we propose a contour constraint instance segmentation framework (CC framework) for cluster cells based on a cluster cell combination enhancement module. The framework can accurately locate each instance from cluster cells and realize high-precision contour segmentation under a few samples. Specifically, we propose the contour attention constraint module to alleviate over- and under-segmentation among individual cell-instance boundaries. In addition, to evaluate the framework, we construct a pleural effusion cluster cell dataset including 197 high-quality samples. The quantitative results show that the numeric result of APmask is > 90%, a more than 10% increase compared with state-of-the-art semantic segmentation algorithms. From the qualitative results, we can observe that our method rarely has segmentation errors.

Serological Phenotyping Analysis Uncovers a Unique Metabolomic Pattern Associated With Early Onset of Type 2 Diabetes Mellitus.

Background: Type 2 diabetes mellitus (T2DM) is a multifaceted disorder affecting epidemic proportion at global scope. Defective insulin secretion by pancreatic ß-cells and the inability of insulin-sensitive tissues to respond effectively to insulin are the underlying biology of T2DM. However, circulating biomarkers indicative of early diabetic onset at the asymptomatic stage have not been well described. We hypothesized that global and targeted mass spectrometry (MS) based metabolomic discovery can identify novel serological metabolic biomarkers specifically associated with T2DM. We further hypothesized that these markers can have a unique pattern associated with latent or early asymptomatic stage, promising an effective liquid biopsy approach for population T2DM risk stratification and screening. Methods: Four independent cohorts were assembled for the study. The T2DM cohort included sera from 25 patients with T2DM and 25 healthy individuals for the biomarker discovery and sera from 15 patients with T2DM and 15 healthy controls for the testing. The Pre-T2DM cohort included sera from 76 with prediabetes and 62 healthy controls for the model training and sera from 35 patients with prediabetes and 27 healthy controls for the model testing. Both global and targeted (amino acid, acylcarnitine, and fatty acid) approaches were used to deep phenotype the serological metabolome by high performance liquid chromatography-high resolution mass spectrometry. Different machine learning approaches (Random Forest, XGBoost, and ElasticNet) were applied to model the unique T2DM/Pre-T2DM metabolic patterns and contrasted with their effectiness to differentiate T2DM/Pre-T2DM from controls. Results: The univariate analysis identified unique panel of metabolites (n = 22) significantly associated with T2DM. Global metabolomics and subsequent structure determination led to the identification of 8 T2DM biomarkers while targeted LCMS profiling discovered 14 T2DM biomarkers. Our panel can effectively differentiate T2DM (ROC AUC = 1.00) or Pre-T2DM (ROC AUC = 0.84) from the controls in the respective testing cohort. Conclusion: Our serological metabolite panel can be utilized to identifiy asymptomatic population at risk of T2DM, which may provide utility in identifying population at risk at an early stage of diabetic development to allow for clinical intervention. This early detection would guide ehanced levels of care and accelerate development of clinical strategies to prevent T2DM.

STAT3/HIF-1α/fascin-1 axis promotes RA FLSs migration and invasion ability under hypoxia.

Rheumatoid arthritis (RA) synovium was identified as "tumor-like" tissues because of the hypoxic microenvironment, significant cell proliferation, and invasion phenotypes. It was reported that hypoxia promoted tumor aggressiveness via up-regulated expression of fascin-1 in cancer. However, the role of fascin-1 in RA synovial hyperplasia and joint injury progression remains unknown. In the current study, we first identified that both fascin-1 and HIF-1α were highly expressed in the RA synovium, in which they were widely colocalized, compared to osteoarthritis(OA). As well, levels of fascin-1 in RA fibroblast-like synoviocytes(FLSs) were found significantly higher than those in OA FLSs. Further, it was demonstrated that the mRNA and protein levels of fascin-1 in RA FLSs were up-regulated in hypoxia (3 % O2) and experimental hypoxia induced by cobalt chloride. Mechanistically, the HIF-1α-mediated hypoxia environment activated the gene expression of the fascin-1 protein, which in turn promoted the migration and invasion of RA FLSs. Accordingly, the restoration of FLSs migration and invasion was observed following siRNA-mediated silencing of fascin-1 and HIF-1α expression. Notably, under the experimental hypoxia, we found that the expression levels of fascin-1, HIF-1α, and p-STAT3 were increased in a time-dependent manner, and fascin-1and HIF-1α expressions were dependent on p-STAT3. Our results indicated that hypoxia-induced fascin-1 up-regulation promoted RA FLSs migration and invasion through the STAT3/HIF-1α/fascin-1 axis, which might represent a novel therapeutic target for the treatment of RA.

Exploring the value of pleural fluid biomarkers for complementary pleural effusion disease examination.

OBJECTIVE: Pleural fluid biomarkers are beneficial for the complementary diagnosis of pleural effusion etiologies. This study focuses on the multidimensional evaluation of deep learning to investigate the pleural effusion biomarkers value and the diagnostic utility of combining these markers, in distinguishing pleural effusion etiologies. METHODS: Pleural effusion were divided into three groups according to the diagnosis and treatment guidelines: malignant pleural effusion (MPE), parapneumonic effusion (PPE), and congestive heart failure (CHF). First, the value of the biomarker was analyzed by a receiver operating characteristic (ROC) curve. Then by utilizing deep learning and entropy weight method (EWM), the clinical value of biomarkers was computed multidimensionally for complementary diagnosis of pleural effusion diseases. RESULTS: There were significant differences in the six biomarkers, TP, ADA, CEA, CYFRA211, NSE, MNC% (p < 0.05) and no significant differences in three physical characteristics including color, transparency, specific gravity and six other biomarkers such as WBC, PNC%, MTC%, pH level, GLU, LDH (p > 0.05) among the three pleural effusion groups. The comprehensive test of pleural fluid biomarkers based on deep learning is of high accuracy. The clinical value of cytomorphology biomarkers WBC, MNC %, PNC %, MTC % was higher among pleural fluid biomarkers. CONCLUSION: The clinical value of multi-dimensional analysis of biomarkers by deep learning and entropy weight method is different from the ROC curve analysis. It is suggested that during the clinical examination process, more attention should be paid to the cell morphology biomarkers, but the physical properties of the pleural fluid are less clinical significance.

Improved efficiency of urine cell image segmentation using droplet microfluidics technology.

Recent advances in the recognition of biological samples using machine vision have made this technology increasingly important in research and detection. Image segmentation is an important step in this process. This study focuses on how to reduce the interference factors such as the overlap between different types (or within the same type) of urine cells according to microfluidics and improve the machine vision segmentation accuracy for cell images. In this study, we demonstrate that the platform can realize this hypothesis using urine cell image segmentation as an example application. We first discuss the reported urine cell droplet microfluidic chip system, which can realize the test conditions in which urine cells are encapsulated in the droplet and isolated from salt crystallization and/or bacteria and other urine-formed elements. Then, based on the analysis conditions set in the aforementioned experiment, the proportions of red blood cells, white blood cells, and squamous epithelial cells covered by various formed elements in the total urine cells in the same urine sample are measured. We simultaneously analyze the percentage of urine cells covered by salt crystallization and the incidence of overlapping between urine cells. Finally, the Otsu algorithm is used to segment the urine cell images encapsulated by the droplet and the urine cell images not encapsulated by the droplet, and the Dice, Jaccard, precision, and recall values are calculated. The results suggest that the method of encapsulating single cells based on droplets can improve the image segmentation effect without optimizing the algorithm.

LKB1 up-regulation inhibits hypothalamic inflammation and attenuates diet-induced obesity in mice.

BACKGROUND: Diet-induced obesity (DIO) is associated with chronic, low-grade inflammation in the hypothalamus. The inflammatory pathway of the hypothalamus is activated during obesity, and inhibition of activation of the inflammatory pathway can partially reverse obesity. Therefore, exploring new targets for inhibiting hypothalamic inflammation will provide new ideas for the prevention and treatment of obesity. Liver kinase B1 (LKB1), a serine/threonine kinase, is a tumor suppressor and metabolic regulator. Recent studies have shown that LKB1 has a certain anti-inflammatory effect. However, a role of LKB1 in the regulation of hypothalamic inflammation remains unclear. Therefore, we examined whether LKB1 overexpression in the hypothalamus could weaken the hypothalamic inflammation and inhibit the development of obesity. METHODS: LKB1 overexpressing adeno-associated virus (AAV) particles were injected stereotactically into the third ventricle (3 V) of C57BL/6 mice fed with HFD. We assessed changes in body mass and adiposity, food intake, hypothalamic inflammatory markers, and energy and glucose metabolism. RESULTS: LKB1 up-regulation in hypothalamus attenuated diet-induced hypothalamic inflammation, reduced food intake and body weight gain. In addition, the overexpression of hypothalamic LKB1 increased the insulin sensitivity and improved whole-body lipid metabolism, which attenuated hepatic fat accumulation and serum lipid levels. CONCLUSION: Hypothalamic LKB1 up-regulation attenuates hypothalamic inflammation, and protects against hypothalamic inflammation induced damage to melanocortin system, resulting in lower food intake and lower fat mass accumulation, which consequently protects mice from the development of obesity. Our data suggest LKB1 as a novel negative regulator of hypothalamic inflammation, and also a potentially important target for treating other inflammatory diseases.

Deletion of liver kinase B1 in POMC neurons predisposes to diet-induced obesity.

AIMS: Liver kinase B1 (LKB1) is a serine/threonine kinase. Although many biological functions of LKB1 have been identified, the role of hypothalamic LKB1 in the regulation of central energy metabolism and susceptibility to obesity is unknown. Therefore, we constructed POMC neuron-specific LKB1 knockout mice (PomcLkb1 KO) and studied it at the physiological, morphological, and molecular biology levels. MAIN METHODS: Eight-week-old male PomcLkb1 KO mice and their littermates were fed a standard chow fat diet (CFD) or a high-fat diet (HFD) for 3 months. Body weight and food intake were monitored. Dual-energy X-ray absorptiometry was used to measure the fat mass and lean mass. Glucose and insulin tolerance tests and serum biochemical markers were evaluated in the experimental mice. In addition, the levels of peripheral lipogenesis genes and central energy metabolism were measured. KEY FINDINGS: PomcLkb1 KO mice did not exhibit impairments under normal physiological conditions. After HFD intervention, the metabolic phenotype of the PomcLkb1 KO mice changed, manifesting as increased food intake and an enhanced obesity phenotype. More seriously, PomcLkb1 KO mice showed increased leptin resistance, worsened hypothalamic inflammation and reduced POMC neuronal expression. SIGNIFICANCE: We provide evidence that LKB1 in POMC neurons plays a significant role in regulating energy homeostasis. LKB1 in POMC neurons emerges as a target for therapeutic intervention against HFD-induced obesity and metabolic diseases.

Identification of Lysosome-Associated Protein Transmembrane-4 as a Novel Therapeutic Target for Osteosarcoma Treatment.

OBJECTIVE: The aim of the study is to evaluate the expression of lysosome-associated protein transmembrane-4 (LAPTM4B) in human osteosarcoma tissue samples collected in our hospital, and to explore the possible correlations between the clinical pathological features of osteosarcoma patients and LAPTM4B expression. METHODS: Immunohistochemical (IHC) assays were performed to detect the expression levels of LAPTM4B in 62 tissue samples of osteosarcoma tissues and corresponding non-tumor tissues. According to LAPTM4B staining intensity in tumor tissues, osteosarcoma patients were classified into LAPTM4B high expression and low expression groups. In addition, the potential correlations between LAPTM4B expression levels and clinical pathological features were evaluated. In addition, we detected the effects of LAPTM4B on the proliferation and invasion of esteosarcoma cells through colony formation assay and transwell assay, respectively. We further explored the potential effects of LAPTM4B on tumor growth and metastasis using in vivo animal model. RESULTS: We revealed that LAPTM4B was highly expressed in human osteosarcoma tissues. We determined the significance between LAPTM4B and clinical features, including the tumor size (P = 0.004*) and the clinical stage (P = 0.035*) of osteosarcoma patients. Our results further demonstrated that ablation of LAPTM4B obviously blocked the proliferation and invasion of osteosarcoma cells in vitro and restrained tumor growth and metastasis in mice. CONCLUSION: We investigated the potential involvement of LAPTM4B in osteosarcoma progression and confirmed LAPTM4B as a novel therapeutic target for osteosarcoma.

Plasma exosomal miRNAs involved in endothelial injury in microscopic polyangiitis patients.

Microscopic polyangiitis (MPA) is a systemic autoimmune disease that primarily affects the small and medium blood vessels. Endothelial injury is one of the pathological hallmarks of MPA. However, the pathogenesis for this has not yet been fully elucidated. Exosomal microRNAs (miRNAs) have recently emerged as a new molecular pattern involved in the endothelial injury in other diseases. Hence, we speculated that MPA plasma-derived exosomes (MPA-exo) could induce the endothelial injury, which was likely to be aroused by the dysregulated exosomal miRNAs in MPA. In the present study, plasma-derived exosomes were isolated and identified. MPA-exo could be internalized by human renal glomerular endothelial cells (HRGECs) in vitro and induced HRGECs injury. Subsequently, a series of differentially expressed miRNAs in MPA-exo were identified by high-throughput sequencing analysis. Further bioinformatics analysis for the target genes of these differentially expressed miRNAs showed a potential mechanism for their possible role in MPA endothelial injury. Notably, we revealed a considerable correlation between miR-185-3p, miR-125a-3p, and clinical parameters. In conclusion, the current study revealed that differentially expressed miRNAs in MPA-exo are associated with the endothelial injury. Our results suggested that these miRNAs and their target genes might be involved in the inflammation process of MPA.

A Microfluidic Detection System for Bladder Cancer Tumor Cells.

The clinical characteristics of excreted tumor cells can be found in the urine of bladder cancer patients, meaning the identification of tumor cells in urine can assist in bladder cancer diagnosis. The presence of white blood cells and epithelial cells in the urine interferes with the recognition of tumor cells. In this paper, a technique for detecting cancer cells in urine based on microfluidics provides a novel approach to bladder cancer diagnosis. The bladder cancer cell line (T24) and MeT-5A were used as positive bladder tumor cells and non-tumor cells, respectively. The practicality of the tumor cell detection system based on microfluidic cell chip detection technology is discussed. The tumor cell (T24) concentration was around 1 × 104 to 300 × 104 cells/mL. When phosphate buffer saline (PBS) was the diluted solution, the tumor cell detected rate was 63-71% and the detection of tumor cell number stability (coefficient of variation, CV%) was 6.7-4.1%, while when urine was the diluted solution, the tumor cell detected rate was 64-72% and the detection of tumor cell number stability (CV%) was 6.3-3.9%. In addition, both PBS and urine are tumor cell dilution fluid solutions. The sample was analyzed at a speed of 750 microns per hour. Based on the above experiments, a system for detecting bladder cancer cells in urine by microfluidic analysis chip technology was reported. The rate of recognizing bladder cancer cells reached 68.4%, and the speed reached 2 mL/h.

TNF-α/calreticulin dual signaling induced NLRP3 inflammasome activation associated with HuR nucleocytoplasmic shuttling in rheumatoid arthritis.

OBJECTIVE: The present study was undertaken to validate whether TNF-α and calreticulin (CRT) serve as dual signaling to activate nucleotide-binding oligomerization domain-, leucine-rich repeat- and pyrin domain-containing 3 (NLRP3) inflammasome in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) and HUVECs. The effect of human antigen R (HuR) in NLRP3 inflammasome activation was also explored in RA FLS. METHODS: Immunofluorescence was used to determine the expression of NLRP3 and adaptor protein apoptosis associated speck-like protein containing a CARD (ASC) in RA synovial tissue and HuR location in RA FLS. Western blot and quantitative real-time PCR were employed to measure the priming effect of NLRP3 inflammasome in cells and HuR expression in synovial tissue. The concentrations of IL-1ß and IL-18 were detected by enzyme linked immunosorbent assay. Immunohistochemistry was used to visualize the expression of HuR in synovial tissue. HuR knockdown in RA FLS was achieved by siRNA-mediated gene silencing. RESULTS: Higher expression of NLRP3 and ASC in RA synovial tissue than those in osteoarthritis was detected. The staining of NLRP3, ASC and cleaved IL-1ß were observed in FLS and vascular endothelial cells in RA synovium. Expression of NLRP3 and pro-IL-1ß in RA FLS and HUVECs treated with TNF-α was increased. The pro-IL-18 expression was also enhanced in HUVECs, but not in RA FLS. TNF-α/CRT dual stimulation of cells gave rise to caspase-1 p20 expression and the secretion of IL-1ß. The secreted IL-18 was also elevated in HUVECs but not in RA FLS. HuR expression was significantly elevated in RA synovial tissue. TNF-α initiated the nucleocytoplasmic shuttling of HuR in both FLS and HUVECs. The knockdown of HuR in FLS incubated with TNF-α led to reduced caspase-1 p20 protein expression and further resulted in decreased secretion of IL-1ß in the presence of CRT. CONCLUSIONS: TNF-α/CRT dual signaling induced NLRP3 inflammasome activation, which could be suppressed by HuR knockdown presumably due to the block of HuR translocating from nucleus to cytoplasma.

Label-free microfluidic chip for the identification of mesothelial cell clusters in pleural effusion.

The detection of tumor cells and clusters in pleural effusion assists in the diagnosis of lung cancer. The proportion of tumor cells and clusters to the total number of cells in each patient varies substantially due to individual differences and the severity of the disease. The identification of one tumor cell or cluster from a large number of pleural effusions is the main challenge for hydrothorax tumor cell detection techniques. In the present study, by using A549 lung cancer and Met-5A mesothelial cell lines, a label-free microfluidic chip based on cell cluster size was designed. By setting the parameters of the chip, individual cells and clusters were able to enter different microfluidic channels. Subsequent to non-specific staining, the recovered components were stained using acridine orange (AO). A charge-coupled device camera was used to captured images of the cell, and the features of these cells were analyzed in their R and G channels using Matlab software to establish the characteristics and finally differentiate between the tumor and non-tumor cell or clusters. According to the results, when inlet A and B were under a velocity of 10 and 8.5 ml/h, respectively, the tumor cell clusters were successfully collected through microfluidic channels III-V, with a recovery rate of ~80%. Subsequent to staining with AO, the feature values in the R and G channels were identified, and initial differentiation was achieved. The present study combined the microfluidic chip, which is based on cluster size, with a computer identification method for pleural effusion. The successful differentiation of tumor cell clusters from non-tumor clusters provides the basis for the identification of tumor clusters in hydrothorax.

Calreticulin induced endothelial ICAM-1 up-regulation associated with tristetraprolin expression alteration through PI3K/Akt/eNOS/p38 MAPK signaling pathway in rheumatoid arthritis.

The present study was undertaken to determine whether extracellular calreticulin (CRT) participates in the regulation of ICAM-1in rheumatoid arthritis (RA) and further explore the potential mechanism. Our results showed that ICAM-1 and VCAM-1 levels were positively correlated with CRT levels in RA serum and synovial fluid, respectively. In RA synovial tissue, increased co-expressions of CRT and ICAM-1 in vascular endothelium and perivascular areas and elevated co-location of CRT and VCAM-1 localized predominantly to lining layer were observed compared to those in OA. In in vitro HUVECs model, enhanced ICAM-1expression and increased phosphorylation levels of Akt and eNOS were detected in the presence of CRT. Increased phosphorylated eNOS was significantly inhibited by a PI3K inhibitor LY294002 and elevated ICAM-1expression was partially blocked by the inhibitors of both PI3K and eNOS (L-NAME). It has been certified that the RNA-binding protein TTP targets AU-rich elements in the ICAM-1 3'-UTR and suppresses ICAM-1 expression. Knocking down TTP in HUVECs led to an increased induction of ICAM-1 by CRT. We have currently known that activation of p38 downstream kinase MK-2 leads to phosphorylation and inactivation of human TTP. The block of p38 MAPK/MK-2 signaling led to decreased protein expression and mRNA stability of TTP and ICAM-1. Furthermore, L-NAME and/or LY294002 pre-treated HUVECs manifested decreased p38 and MK-2 phosphorylation, which was accompanied by reduced TTP and ICAM-1 protein expression as well as decreased mRNA stability. Our results suggested that CRT could promote ICAM-1 expression in endothelial cells through PI3K/Akt/eNOS/p38 MAPK signaling mediated TTP accumulation, probably in an inactive form, which may provide a possible proinflammatory mechanism of CRT in RA.

Therapeutic evaluation of acupoint stimulation with needle-scapelon on rat model of degenerative cervical intervertebral discs.

Cervical spondylosis (CS), which is resulted from degeneration of cervical intervertebral disc, is a common disease seriously threatening human health and quality of life. However, there is still no effective clinic strategies for the treatment of this disease. The acupoint stimulation with needle-scalpel is a widely used approach to treat orthopedic diseases. In the present study, we evaluated the therapeutic effects of acupoint stimulation around neck with needle-scalpel on delaying the degeneration of cervical intervertebral discs and hopefully provided an approach for the precaution and early intervention of CS. We firstly established a rat model of CS by cervical static-dynamic imbalance to mimics disc degeneration and then stimulated the acupoints around neck with needle-scalpel. The cervical intervertebral disc samples were collected to measure type I and II collagen by quantitative PCR (qPCR), immunohistochemistry, and western blot. The changes in micro-structure and ultra-structure of nucleus pulposus were analyzed under the optical microscope and electron microscope respectively. Acupoint stimulation with needle-scapelon increased type I collagen production and decreased type II collagen production, and improved the micro-structure and ultra-structure of nucleus pulposus. Our results suggest that acupoint stimulation around neck with needle-scapelon could inhibit intervertebral disc degeneration through modulating the extracellular matrix collagen system and improving the changed structure of nucleus pulposus.

Knockdown of lncRNA H19 inhibits abnormal differentiation of small intestinal epithelial cells in diabetic mice.

Diabetes mellitus (DM) comprises a group of metabolic diseases characterized by insulin deficiency or resistance and hyperglycemia. We previously reported the presence of abnormal differentiation of small intestinal epithelial cells (IECs) in diabetic mice, but the exact mechanism of this phenomenon has not been thoroughly elucidated to date. In this study, we found that H19 was markedly upregulated in IECs of DM mice. H19 knockdown significantly inhibited abnormal differentiation of IECs in DM mice. Bioinformatics analysis identified miR-141-3p as a candidate for H19. Based on luciferase reporter assays, we found that miR-141-3p directly targeted H19. Luciferase reporter assays also showed that miR-141-3p could directly target ß-catenin. Furthermore, H19 might act as an endogenous "sponge" by competing for miR-141-3p binding to regulate miRNA targets in vitro and in vivo. In summary, our findings provide the first evidence supporting the role of H19 in IECs of DM mice, and miR-141-3p targets not only protein-coding genes but also the lncRNA H19.

Bcl-XL and Mcl-1 upregulation by calreticulin promotes apoptosis resistance of fibroblast-like synoviocytes via activation of PI3K/Akt and STAT3 pathways in rheumatoid arthritis.

OBJECTIVES: Fibroblast-like synoviocytes (FLS) play key roles in synovium hyperplasia and pannus formation in rheumatoid arthritis (RA). The present study was undertaken to explore the mechanisms that calreticulin (CRT) promoted anti-apoptosis of RA FLS. METHODS: The expression of CRT and anti-apoptotic proteins Bcl-XL and Mcl-1 in RA synovium were detected by immunohistochemistry. The expression of Bcl-XL and Mcl-1 in RA FLS by CRT were determined. The phosphorylation of Akt and STAT3 was detected by western blot. The effect of CRT on proliferation of RA FLS was examined by MTT assay. The ability of CRT to inhibit RA FLS apoptosis was assessed by flow cytometry. RESULTS: Increased expressions of CRT, Bcl-XL and Mcl-1 were detected in RA synovium compared with osteoarthritis (OA). Moreover, CRT expression correlated positively with Bcl-XL and Mcl-1 in RA, respectively. In vitro, CRT induced upregulation of Bcl-XL and Mcl-1 protein levels in RA FLS, in dose/time dependent manners. Upregulated expression of Bcl-XL and Mcl-1 induced by CRT were inhibited by PI3K/Akt or STAT3 pathways inhibitors in RA FLS, respectively. The increased phosphorylation levels of Akt and STAT3 were also detected with CRT incubation, in dose/time dependent manners. Additionally, CRT rescued apoptosis of RA FLS mediated by FasL. CONCLUSIONS: This study showed that upregulation of Bcl-XL and Mcl-1 expression in RA FLS by CRT were PI3K/Akt and STAT3 signal pathways dependent, and promoted the anti-apoptosis of RA FLS. Therefore, this may represent a therapeutic target for the treatment of RA.

Two-way detection of image features and immunolabeling of lymphoma cells with one-step microarray analysis.

Detecting the number of pathological lymphoma cells and lymphocyte subtypes in blood is helpful for clinical diagnosis and typing of lymphoma. In the current study, cell type is identified by cell morphological features and immunolabeled lymphocyte subtypes. Red blood cells and leukocytes were separated using a microfluidic cell chip based on physical blood cell parameters, and leukocytes were identified using five characteristic parameters: energy variance, entropy variance, moment of inertia variance, color mean, and cell area individually. The number of red blood cells that could come into contact with the leukocyte membrane was ≤2 based on the microfluidic injection flow rate of microfluidic chips. Anti-CD3 and anti-CD19 antibodies were used for immunofluorescence staining of T-lymphocyte and B-lymphocyte surface antigens, respectively. The results suggested that the microfluidic assay could detect lymphocyte surface antigen markers and intact leukocytes. Therefore, we report a one-step microfluidic chip for classifying hematological lymphoma cells based on the physical parameters of cells, which can simultaneously measure the overall morphology of blood cells and immunolabeling of lymphocyte surface antigens in one step, solving the current problem of detecting subtypes of hematological lymphoma cells based on multiple methods and multi-step detection.