The role of ApoE in fatty acid transport from neurons to astrocytes under ischemia/hypoxia conditions.

BACKGROUND: The aim of this study was to investigate whether ischemia/hypoxia conditions induce fatty acid transport from neurons to astrocytes and whether this mechanism is affected by ApoE isoforms. METHODS AND RESULTS: A neonatal rat model of hypoxic-ischemic brain damage was established. Excessive accumulation of lipid droplets and upregulation of ApoE expression occurred in the hippocampus and cerebral cortex after hypoxia-ischemia, which implied the occurrence of abnormal fatty acid metabolism. Lipid peroxidation was induced in an oxygen-glucose deprivation and reperfusion (OGDR) model of ApoE-/- primary neurons. The number of BODIPY 558/568 C12-positive particles (fatty acid markers) transferred from neurons to astrocytes was significantly increased with the addition of human recombinant ApoE compared with that in the OGDR group, which significantly increased the efficiency of fatty acid transport from neurons to astrocytes and neuronal viability. However, ApoE4 was found to be associated with lower efficiency in fatty acid transport and less protective effects in OGDR-induced neuronal cell death than both ApoE2 and ApoE3. COG133, an ApoE-mimetic peptide, partially compensated for the adverse effects of ApoE4. FABP5 and SOD1 gene and protein expression levels were upregulated in astrocytes treated with BODIPY 558/568 C12 particles. CONCLUSIONS: In conclusion, ApoE plays an important role in mediating the transport of fatty acids from neurons to astrocytes under ischemia/hypoxia conditions, and this transport mechanism is ApoE isoform dependent. ApoE4 has a low transfer efficiency and may be a potential target for the clinical treatment of neonatal hypoxic-ischemic encephalopathy.

Circ-N4BP2L2 enhances mitochondrial function in non-small cell lung cancer cells through regulating the miR-135a-5p/ARL5B axis.

Non-small cell lung cancer (NSCLC) is the main histological subtype of lung cancer with a high incidence and mortality. Circular RNAs (circRNAs) exert vital functions in various cancers by acting as a sponge of miRNAs to abolish their inhibitory effect on target genes. This study aims to explore the biological function of circRNA NEDD4 binding protein 2 like 2 (circ-N4BP2L2) in NSCLC. We found that circ-N4BP2L2 was upregulated in NSCLC tissues and cells by using RT-qPCR. A549 cells were transfected with pcDNA-circN4BP2L2 or sh-circN4BP2L2 to obtain circN4BP2L2-overexpressed or -silenced cells, and then cell proliferation, invasion and apoptosis were determined. The results showed that knockdown of circ-N4BP2L2 repressed cell proliferation, invasion as well as mitochondrial function, and promoted cell apoptosis; while overexpression of circ-N4BP2L2 resulted in the opposite results. Mechanistically, the targeting correlations between miR-135a-5p and circ-N4BP2L2 or ADP-ribosylation factorlike 5B (ARL5B) were confirmed by using dual luciferase reporter, RNA pull-down and RNA immunoprecipitation assays. In addition, we found that circ-N4BP2L2 could promote the expression of ARL5B by serving as a sponge of miR-135a-5p. Moreover, rescue assays revealed that silencing miR-135a-5p or overexpressing ARL5B was able to abate the effects of circ-N4BP2L2 knockdown on malignant phenotypes and mitochondrial function of A549 cells. Finally, tumorigenicity assay demonstrated that circ-N4BP2L2 facilitated NSCLC tumor growth in vivo. Taken together, circ-N4BP2L2 enhanced NSCLC progression via the miR-135a-5p/ARL5B axis, which may provide a novel therapeutic target of NSCLC.

CN-3 increases TMZ sensitivity and induces ROS-dependent apoptosis and autophagy in TMZ-resistance glioblastoma.

Many glioma patients develop resistance to temozolomide (TMZ) treatment, resulting in reduced efficacy and survival rates. TMZ-resistant cell lines SHG44R and U87R, which highly express O6 -methylguanine DNA methyltransferase (MGMT) and P-gp, were established. CN-3, a new asterosaponin, showed cytotoxic effects on TMZ-resistant cells in a dose- and time-dependent manner via reactive oxygen species (ROS)-mediated apoptosis and autophagy. Transmission electron microscopy and monodansylcadaverine (MDC) staining showed turgidity of the mitochondria and autophagosomes in CN-3-treated SHG44R and U87R cells. The autophagy inhibitor 3-methyladenine was used to confirm the important role of autophagy in CN-3 cytotoxicity in TMZ-resistant cells. The ROS scavenger N-acetyl- l-cysteine (NAC) attenuated the levels of ROS induced by CN-3 and, therefore, rescued the CN-3 cytotoxic effect on the viability of SHG44R and U87R cells by Cell Counting Kit-8 assays and JuLI-Stage videos. MDC staining also confirmed that NAC rescued an autophagosome increase in CN-3-treated SHG44R and U87R cells. Western blotting revealed that CN-3 increased Bax, cleaved-caspase 3, cytochrome C, PARP-1, LC3-Ⅱ, and Beclin1, and decreased P-AKT, Bcl-2, and p62. Further rescue experiments revealed that CN-3 induced apoptosis and autophagy through ROS-mediated cytochrome C, cleaved-caspase 3, Bcl-2, P-AKT, PARP-1, and LC3-Ⅱ. In addition, CN-3 promoted SHG44R and U87R cells sensitive to TMZ by reducing the expression of P-gp, MGMT, and nuclear factor kappa B p65, and it had a synergistic cytotoxic effect with TMZ. Moreover, CN-3 disrupted the natural cycle arrest and inhibited the migration of SHG44R and U87R cells by promoting cyclin E1 and D1, and by decreasing P21, P27, N-cadherin, ß-catenin, transforming growth factor beta 1, and Smad2.

CN-3 induces mitochondrial apoptosis in glioma via Ros-mediated PI3K/AKT pathway.

Recently we isolated CN-3, a new asterosaponin from starfish Culcita novaeguineae, and reported that asterosaponin arrests glioma cell cycle via SCUBE3. However, the multiple mechanisms underlying CN-3 anti-glioma action remains poorly known. Thus, the focus of this study was to evaluate the inhibitory effect of CN-3 on human glioma cells and its underlying molecular mechanisms. U87 and U251 cells were incubated with various concentrations of CN-3, and CCK-8, transmission electron microscopy, ICELLigence, TUNEL, flow cytometry, N-acetyl-L-cysteine, and western blot were conducted. As a result, it was found that CN-3 significantly inhibited U87 and U251 cell viability and proliferation in a time- and dose- dependent manner, and also induced mitochondrial apoptosis. Furthermore, we detected that CN-3 downregulated PI3K, P-Akt, AKT and BCL-2, and upregulated cytochrome C and BAX in U87 and U251 cells. Moreover, ROS triggered the inhibition and cell apoptosis for CN-3 via inactivation of P-Akt and activation of cytochrome C. In conclusion, these findings suggest that CN-3 may be a promising candidate for the development of a therapy of glioma.

PDLIM2 acts as a cancer suppressor gene in non-small cell lung cancer via the down regulation of NF-κB signaling.

PDZ and LIM domain containing protein 2 (PDLIM2) has been identified as a vital tumor-associated gene that is aberrantly expressed in various types of tumors. Yet, the involvement of PDLIM2 in non-small cell lung cancer (NSCLC) is currently undetermined. The design of the current study was to evaluate whether PDLIM2 plays a role in NSCLC. We found that PDLIM2 expression was commonly decreased in NSCLC tissues. Moreover, low expression of PDLIM2 was also detected in NSCLC cell lines and demethylation treatment restored PDLIM2 expression. The re-expression of PDLIM2 impeded the proliferative, colony-forming, and invasive capabilities of NCLCL cells. In contrast, depletion of PDLIM2 markedly enhanced the malignant behaviors of NSCLC cells. Notably, PDLIM2 overexpression downregulated the expression of nuclear factor (NF)-κB p65 subunit and repressed NF-κB transcription reporter activity in NSCLC cells. The overexpression of p65 significantly reversed PDLIM2-mediated antitumor effects in NSCLC cells. Additionally, the Xenograft tumor formation assay revealed that the overexpression of PDLIM2 markedly restricted the tumor growth of NSCLC in vivo. Overall, our study confirms that PDLIM2 acts as a tumor-inhibitor in NSCLC through the inactivation of NF-κB, suggesting PDLIM2 as a candidate therapeutic target for NSCLC.

Circulating MiR-146a May be a Potential Biomarker of Coronary Heart Disease in Patients with Subclinical Hypothyroidism.

BACKGROUND/AIMS: Subclinical hypothyroidism (SCH) plays a crucial role in the development and progression of coronary heart disease (CHD). However, any associated changes in the circulating microRNAs (miRNAs) levels and slightly elevated thyroid stimulating hormone (TSH) levels in CHD patients are unknown. miR-146a is a well known miRNA associated with inflammatory autoimmune diseases. Here, we evaluated miR-146a expression in patients, with the goal of re-evaluating the effect of SCH on CHD. METHODS: A total of 192 study subjects who underwent coronary angiography for either suspected or confirmed CHD were enrolled in 3 groups: CHD with SCH, CHD alone, and healthy controls. The circulating levels of miR-146a were quantified using qRT-PCR. RESULTS: Levels of miR-146a were positively correlated with CHD severity, as indicated by the Gensini score (r=0.354). The relative expression of miR-146a in the CHD+SCH, CHD and healthy control groups was 2.223±0.827, 1.588±0.726 and 0.632±0.309, respectively. Plasma TSH levels were positively correlated with miR-146a levels (r=0.321). According to multivariate logistic regression analyses, miR-146a levels were associated with the incidence of CHD in patients with SCH. For diagnosing CHD, the area under the ROC curve (AUC) of miR-146a and TSH was 0.779 and 0.752, respectively. When the TSH and miR-146a levels were combined to form a composite panel, the AUC of the panel was 0.858. CONCLUSION: Plasma miR-146a levels correlated with the severity of coronary atherosclerosis and increased with TSH slightly elevated in patients with CHD. Thus, miR-146a may have good predictive value for CHD among individuals with elevated TSH levels.

Circ-ANAPC7 is Upregulated in Acute Myeloid Leukemia and Appears to Target the MiR-181 Family.

BACKGROUND/AIMS: Circular RNAs (circRNAs) are a family of novel non-coding RNAs associated with various diseases, especially cancer. Recent studies have demonstrated that circRNAs participate in pathogenesis mainly by acting as microRNA (miRNA) sponges. The expression profile of circRNAs in acute myeloid leukemia (AML) has rarely been reported. METHODS: Profiles of circRNAs were analyzed using an Arraystar human circRNA microarray with 5 bone marrow samples from patients with newly diagnosed AML and 5 from patients with iron-deficiency anemia. Quantitative reverse transcription PCR was used to validate the expression pattern of circRNAs. Furthermore, circRNA-miRNA network, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were applied. RESULTS: CircRNA microarray analysis revealed that 698 circRNAs were differentially expressed in AML patients, with 282 circRNAs found to be upregulated and 416 to be downregulated. Quantitative reverse transcription PCR showed that circ-ANAPC7 was significantly upregulated in AML. Bioinformatics analysis predicted that circ-ANAPC7 acts as a sponge for the miR-181 family, KEGG analysis revealed that it is associated with cancer-related pathways, and GO analysis indicated that most of its target genes are involved in biological processes. CONCLUSIONS: These findings show that circ-ANAPC7 is a promising biomarker for AML, and that it might participate in AML pathogenesis by acting as a sponge for the miR-181 family.

MiR-135a inhibits migration and invasion and regulates EMT-related marker genes by targeting KLF8 in lung cancer cells.

Epithelial-mesenchymal transition (EMT) has been shown to be related to the pathogenesis of various diseases. Recently, microRNAs (miRNA) have been recognized as a new class of genes involved in human tumorigenesis. In this study, we found that the expression levels of miR-135a were dramatically decreased in NSCLC cell lines and clinical NSCLC tissue samples. Then, we demonstrated that miR-135a significantly suppressed the migration and invasion of lung cancer cells in vitro, suggesting that miR-135a may be a novel tumor suppressor. Further studies revealed that the transcription factor KLF8 was a target gene of miR-135a in NSCLC cells, as miR-135a bound directly to the 3'-untranslated region (3'-UTR) of KLF8, thus reducing both the expression of KLF8 at the mRNA and protein levels. In addition, the EMT marker E-cadherin or vimentin was also down-regulated or up-regulated on miR-135a treatment. Moreover, silencing KLF8 was able to inhibit the migration and invasion of lung cancer cells. In conclusion, these findings indicate that miR-135a suppresses the migration and invasion of NSCLC cells through targeting KLF8, which is involved in the EMT process. This finding provides new insight into the mechanism of NSCLC progression.

Coxsackievirus B3 induces viral myocarditis by upregulating toll-like receptor 4 expression.

In the present study, we investigated the potential pathogenesis of coxsackievirus B3 (CVB3)-induced viral myocarditis and the promising protective effect of silencing RNA (small interfering RNA, siRNA). One hundred and twenty mice were included in the study, and 30 mice were intraperitoneally inoculated with CVB3 to establish an acute viral myocarditis model. The survival rate was observed for the CVB3-infected mouse model (MOD), and myocardial injury was examined by HE (hematoxylin and eosin) staining assay. Real-time PCR (RT-PCR) and Western blot assay were selected to detect the toll-like receptor 4 (TLR4) expression in myocardial tissues. The TLR4 gene was silenced for the MOD mice, and the effects of this treatment were observed. The results indicate that the expression of TLR4 mRNA and the protein significantly and persistently increased during the progression of CVB3-induced myocarditis. The activities of cardiac enzymes including CK, LDH, AST, and CK-MB were also enhanced in CVB3-induced myocardial tissues. Interestingly, when the TLR4 gene was silenced, the CVB3-induced TLR4 production was significantly decreased and the severity of myocarditis was significantly lessened. In conclusion, CVB3 may induce viral myocarditis by upregulating toll-like receptor 4 expression. The viral myocarditis can be ameliorated by silencing the TLR4 gene in the CVB3 viral myocarditis model, which may be a feasible therapeutic method for treatment of viral myocarditis.

Potential actions of capsaicin for preventing vascular calcification of vascular smooth muscle cells in vitro and in vivo.

Vascular calcification (VC) is an accurate risk factor and predictor of adverse cardiovascular events; however, there is currently no effective therapy to specifically prevent VC progression. Capsaicin (Cap) is a bioactive alkaloid isolated from Capsicum annuum L., a traditional medicinal and edible plant that is beneficial for preventing cardiovascular diseases. However, the effect of Cap on VC remains unclear. This study aimed to explore the effects and related mechanisms of Cap on aortic calcification in a mouse and on Pi-induced calcification in vascular smooth muscle cells (VSMCs). First, we established a calcification mouse model with vitamin D3 and evaluated the effects of Cap on calcification mice using von Kossa staining, calcium content, and alkaline phosphatase activity tests. The results showed that Cap significantly improved calcification in mice. VSMCs were then cultured in 2.6 mM Na2HPO4 and 50 µg/mL ascorbic acid for 7 days to obtain a calcification model, and we investigated the effects and mechanisms of Cap on VSMCs calcification by assessing the changes of calcium deposition, calcium content, and subsequent VC biomarkers. These results showed that Cap alleviated VSMCs calcification by upregulating the expressions of TRPV1. Moreover, Cap reduced the expression of Wnt3a and ß-catenin, whereas DKK1 antagonised the inhibitory effect of Cap on VSMC calcification. This study is the first to offer direct evidence that Cap inhibits the Wnt/ß-catenin signaling pathway by upregulating the expression of the TRPV1 receptor, resulting in the decreased expression of Runx2 and BMP-2, thereby reducing VSMC calcification. Our study may provide novel strategies for preventing the progression of VC. This could serve as a theoretical basis for clinically treating VC with spicy foods.

Single-nucleotide gene polymorphisms involving cell death pathways: a study of Chinese patients with lumbar disc herniation.

OBJECTIVE: To evaluate the effect of polymorphisms of death pathway genes FAS and FASL on the risk of developing lumbar disc herniation (LDH) in a Northern Chinese population. BACKGROUND DATA: The FAS receptor-ligand system plays a key role to regulate apoptosis of cell. There is evidence that the apoptosis-mediated FAS receptor-ligand system is involved in the pathogenesis of disc degeneration. Some research considered single-nucleotide polymorphisms of FAS-1377G/A, FAS-670A/G, FASL-844T/C, and FASL INV2nt-124A/G may increase the risk of developing cancer. We therefore assess these four single-nucleotide polymorphisms as candidate susceptibility for LDH. METHODS: A total of 475 patients with LDH and 533 control subjects were selected. Genotypes were determined by polymerase chain reaction-based restriction fragment length polymorphism and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Associations with the risk of LDH were estimated by logistic regression model. RESULTS: Significant differences were found in genotypic distributions between cases and controls for FASL-844T/C, but not for other three polymorphisms. When compared with CC genotype, subjects with the TT genotype had a higher risk to develop LDH (odds ratio = 3.12; 95% confidence interval: 1.73-5.40). Moreover, an association was found between this genotype of FASL-844TT and more severe grades of disc degeneration. We observed statistically significant interactions between polymorphisms of FASL-844T/C and lumbar load, tobacco smoking, and age. CONCLUSION: Genetic polymorphisms of FASL-844T/C may be associated with an increased risk of developing disc degeneration and LDH.

Correlation Analysis of CML, sRAGE, and esRAGE and the Measure of Atherosclerosis of Coronary Heart Disease.

Objective: To investigate the correlation between CML, sRAGE, and esRAGE and the measure of atherosclerosis of coronary heart disease. Methods: From June 2019 to December 2021, there were 100 patients in all suffering from coronary heart disease (CHD) selected as the observation group. On the basis of Gensini score, they were divided into mild group (Gensini score < 12 points), moderate group (12 points ≤ Gensini score ≤60 points), and severe group (Gensini score > 60). Apart from that, 50 normal people staying in our hospital for physical examination were chosen as the control group in the meantime. N in each group was detected and compared ε-Carboxymethyl lysine (CML), soluble advanced glycation end product receptor (sRAGE), and endogenous secretory advanced glycation end product receptor (esRAGE). Pearson correlation coefficient was adapted to assay the relevance between CML, sRAGE, and esRAGE, as well as the degree of atherosclerosis in CHD. Receiver operator characteristic (ROC) curve was applied to during the evaluation of the diagnosis of CML, sRAGE, and esRAGE, as well as their combined detection of severe atherosclerosis in CHD. Results: In contrast with the control group, the level of serum CML together with sRAGE in the observation group was considerably elevated, while the level of esRAGE appeared in a downward trend (P < 0.05). The level of serum CML and sRAGE was directly proportional to the measure of atherosclerosis in CHD, while the level of esRAGE was inversely proportional to the measure of atherosclerosis in CHD (P < 0.05). That is to say that serum CML and sRAGE were positive in matter of the measure of atherosclerosis in CHD, while esRAGE negatively appertains to the measure of atherosclerosis in CHD (P < 0.05). Serum CML, sRAGE, and esRAGE could effectively diagnose severe atherosclerosis in CHD, and the combined detection sensitivity (89.79%), specificity (77.16%), accuracy (86.12%), positive predictive value (86.63%), negative predictive value (88.59%), and area under ROC curve (AUC) (0.924) were higher (P < 0.05). Conclusion: CML and sRAGE, as well as esRAGE, are bound up with the degree of atherosclerosis in CHD, which is conducive to clinical diagnosis and treatment.

Expression of VLA-4 molecule in PBMC from patients with hemorrhagic fever with renal syndrome.

OBJECTIVE AND DESIGN: The very late antigen-4 (VLA-4) bound to vascular cell adhesion molecule-1 could provide co-stimulatory signals for the activation of T lymphocytes, and these adhesion molecules play key roles in leukocyte adherence and propagation of inflammatory responses. We examined the levels of VLA-4 in the peripheral blood mononuclear cells (PBMC) of patients with hemorrhagic fever with renal syndrome (HFRS). MATERIALS AND METHODS: The levels of VLA-4 in PBMC samples collected from 53 patients by immunohistochemical staining were detected. RESULTS: The expression of VLA-4 in PBMC of HFRS patients at different stages were significantly higher than those in normal controls (P < 0.05), except recovery stage (P > 0.05). The expression of VLA-4 in PBMC of HFRS patients at different types were significantly higher than those in healthy controls (P < 0.05). The levels of VLA-4 in patients with HFRS were positively correlated with serum levels of blood urea nitrogen (BUN) and creatinine (Cr). CONCLUSIONS: VLA-4 might play an important role in the immunopathological lesions of HFRS. We found that VLA-4 levels were closely correlated to the severity of the HFRS and the degree of kidney damage.

Erratum: Overexpression of Notch1 is associated with the progression of cervical cancer.

[This corrects the article DOI: 10.3892/ol.2015.3143.].

Comparison of healing patterns of different side-cut angulations after FS-LASIK.

AIM: To investigate and evaluate healing patterns around flaps made with different side-cut angulations after femtosecond laser in situ keratomileusis (FS-LASIK). METHODS: Thirty-four patients (68 eyes) received a 90° side-cut (n=34) or a 120° side-cut flaps (n=34) made with a femtosecond laser. One day, 1wk, 1 and 3mo postoperatively, side-cut scar was evaluated under slit-lamp photography according to a new grading system (Grade 0=transparent scar, 1=faint healing opacity, and 2=evident healing opacity). In vivo corneal confocal microscopy and anterior segment optical coherence tomography (AS-OCT) were used to observe wound-healing patterns around flap margin in the two groups. Sirius Scheimpflug Analyzer was also used to analyze higher order aberrations 3mo after surgery. RESULTS: There were no significant differences in flap wound-healing patterns at each follow up between the two groups (P>0.05). Three months after surgery, the flap edge scar classified as Grade 0 had excellent apposition and rapid nerve regeneration. At 3 mm and 5 mm pupil diameters, there were significant differences in trefoil aberrations between the two groups (P<0.05), but no statistically significant differences were found in total higher order aberrations (HOAs), spherical aberrations or coma in any of the pupil size conditions (P>0.05). CONCLUSION: Flap edge scars classified as Grade 0 have excellent apposition and rapid nerve regeneration, and 120° side-cut angle flaps induce less trefoil aberrations after FS-LASIK.

Promotion of Ros-mediated Bax/Cyt-c apoptosis by polyphyllin II leads to suppress growth and aggression of glioma cells.

BACKGROUND: Gliomas remain among the most difficult cancers to treat, with a 5-year overall survival no greater than 5%. Many saponins showed a wide spectrum of anti-cancer activities at low concentration. Polyphyllin II is one of the common saponins from Paris polyphylla. However, the effect of Polyphyllin II on glioma cells has not been evaluated. Objective of the present study was to investigate whether Polyphyllin II have inhibition on glioma cells, and the possible mechanisms. METHODS: The viability of U87 and U251 cells was detected by cell counting kit-8, cell counting real time cellular analysis and cell clone formation methods. Transwell was used to estimate the aggression of U87 and U251. The cell apoptosis rate was tested by flow cytometry. The morphological change was determined by transmission electron microscopy. The levels of AKT, phosphorylation of AKT, Bax, Bcl-2, cytochrome c, and cleaved caspase 3 proteins were assessed by Western blot. N-acetyl-L-cysteine was used to check the role of ROS in polyphyllin II inhibition to glioma cells. RESULTS: Polyphyllin II showed significant suppress to proliferation and aggression of U87 and U251 in a dose- and time- dependent manner. Result of flow cytometry confirmed that Polyphyllin II induced apoptosis to U87 and U251 cells. Transmission electron microscopy observation revealed majority of the glioma cells treated with Polyphyllin II had turgidity of mitochondrion, disarrangement, diminution and vacuolization, those refer to mitochondrial apoptosis. Western blot indicated that Polyphyllin II promoted cyt-c, Bax, caspase 3 and cleaved-caspase 3, and decreased Bcl-2, AKT and p-AKT. Rescue experiments using N-acetyl-L-cysteine, a reactive oxygen species scavenger, reversed the levels of Bax and cyt-c, and the inhibition in Polyphyllin II-treated U87 and U251 cells. CONCLUSIONS: The present findings revealed that polyphyllin II may be a potential drug against glioma.

Effects of CpG oligodeoxynucleotides on the differentiation of Treg/Th17 cells.

AIM: The balance between Th17 cells and T regulatory (Treg) cells has emerged as a prominent factor in regulating cancer development. However, the effect of CpG oligodeoxynucleotides (ODNs) on the differentiation of Treg/Th17 cells has not been well studied. We sought here to explore the function of CpG ODNs in the differentiation of Tregs and Th17 cells in vitro and in vivo. METHODS: Mouse spleen cells were cultured with anti-CD3 monoclonal antibodies in vitro. Tregs and Th17 cell differentiation was induced by transforming growth factor (TGF)-ß and interleukin (IL)-2, or TGF-ß, IL-6, and IL-23, respectively. Then cells were treated with two CpG ODNs, CpG 1982, or CpG 1826. FBL-3-inoculated C57Bl/6 mice were treated with CpG 1826, tumor vaccine, or combination of CpG 1826 and tumor vaccine. After treatment, spleen cells and serum were isolated, and Tregs/Th17 cells were detected by flow cytometry. The expression of forkhead box P3 (Foxp3), retinoid-related orphan receptor gamma-t (RORγt), IL-10, and IL-17 mRNA was measured by real-time PCR, and protein levels were measured by Western blot and enzyme-linked immunosorbent assay. RESULTS: The frequency of Treg cells increased significantly (p < 0.05) in the FBL-3-inoculated leukemia mouse model compared with control mice, whereas the frequency of Th17 cells did not change. Median survival of mice after treatment with CpG 1826 and tumor vaccine was significantly prolonged compared with that of control mice (p < 0.05). The frequency of induced Treg cells decreased after treatment with CpG 1826, whereas the frequency of Th17 cells induced by cytokines in vitro and in the murine leukemia model increased following treatment with CpG 1826. Furthermore, after treatment with CpG 1826, the mRNA and protein levels of Foxp3 and IL-10 decreased significantly both in vitro and in vivo (p < 0.05), whereas those of RORγt and IL-17 increased significantly (p < 0.05). CONCLUSION: CpG 1826 may inhibit the differentiation of Treg cells induced by cytokines, promote the differentiation of Th17 cells in vitro and in murine leukemia models, and prolong the median survival of mice with leukemia.

LINGO-1 regulates Wnt5a signaling during neural stem and progenitor cell differentiation by modulating miR-15b-3p levels.

BACKGROUND: Manipulation of neural stem and progenitor cells (NSPCs) is critical for the successful treatment of spinal cord injury (SCI) by NSPC transplantation, since their differentiation into neurons and oligodendrocytes can be inhibited by factors present in inflamed myelin. In this study, we examined the effects of LINGO-1 on spinal cord-derived NSPC (sp-NSPC) differentiation, the underlying mechanisms of action, and the functional recovery of mice after transplantation of manipulated cells. METHODS: sp-NSPCs were harvested from female adult C57/BL6 mice after SCI induced with an NYU impactor. These cells were infected with lentiviral vectors containing LINGO-1 shRNA sequence or a scrambled control and transplanted into SCI mice. Tuj-1- and GFAP-positive cells were assessed by immunofluorescence staining. Wnt5a, p-JNK, JNK, and ß-catenin expression was determined by Western blot and RT-qPCR. miRNAs were sequenced to detect changes in miRNA expression. Motor function was evaluated 0-35 days post-surgery by means of the Basso Mouse Scale (BMS) and by the rotarod performance test. RESULTS: We discovered that LINGO-1 shRNA increased neuronal differentiation of sp-NSPCs while decreasing astrocyte differentiation. These effects were accompanied by elevated Wnt5a protein expression, but unexpectedly, no changes in Wnt5a mRNA levels. miRNA-sequence analysis demonstrated that miR-15b-3p was a downstream mediator of LINGO-1 which suppressed Wnt5a expression. Transplantation of LINGO-1 shRNA-treated sp-NSPCs into SCI mice promoted neural differentiation, wound compaction, and motor function recovery. CONCLUSIONS: LINGO-1 shRNA promotes neural differentiation of sp-NSPCs and Wnt5a expression, probably by downregulating miR-15b-3p. Transplantation of LINGO-1 shRNA-treated NSPCs promotes recovery of motor function after SCI, highlighting its potential as a target for SCI treatment.

Th17 cells: positive or negative role in tumor?

Th17 cells have been recently identified as a distinct Th cell lineage and found in an experimental animal model of cancer and in human cancers, but whether these cells promote tumor growth or regulate antitumor responses remains controversial. This review provides a summary of the current literature regarding interleukin (IL)-17/IL-23 and Th17 cells in cancer and discusses their potential roles in cancer development. Finally, we note several issues in this research area that must be resolved before the design of novel therapeutic approaches specifically targeting Th17 cells in cancer become feasible.

SFRP5 mediates downregulation of the wnt5a/caveolin-1/JNK signaling pathway.

This study investigated the effects of Wnt5a/caveolin/JNK signaling pathway and SFRP5 protein on ox-LDL-induced apoptosis of HUVEC cells. The difference of serological indexes between healthy average weight and obese children and the expression of Wnt 5a and SFRP5 was detected by clinical examination, and the correlation between serum SFRP5, Wnt 5a and the vascular endothelial injury was detected. HUVEC cells were induced by ox-LDL to construct an endothelial injury model, siRNA-transfected cells were used to construct downregulated SFRP5 and Wnt 5a expression groups, and recombination methods were used to construct upregulated Wnt5a and SFRP5 expression groups. The expression of Wnt 5a, caveolin-1, JNK and apoptosis-related proteins under different treatments were detected by the Western blot method, and apoptosis was detected by flow cytometry. Serological results showed that the level of Sfrp5 in obese children was significantly lower than that in healthy children, and the level of Wnt5a was significantly higher than that in healthy children. Moreover, Ln Sfrp5 was significantly negatively correlated with Ang-2 in blood circulation, ICAM-1 and E-selectin selectin, but not with VCAM-1. When Wnt5a was upregulated, the expression of caveolin-1 and JNK increased significantly, Bcl-2 decreased significantly, and the apoptotic rate increased significantly. Nevertheless, when Sfrp5 expression was upregulated, the result was the opposite. SFRP5 and Wnt5a are involved in the vascular endothelial injury. Wnt5a can promote apoptosis of HUVEC cells through Wnt5a/JNK/Caveolin-1 pathway, while SFRP5 can inhibit apoptosis by interfering with this pathway.