Titanium dioxide nanoparticles induced the apoptosis of RAW264.7 macrophages through miR-29b-3p/NFAT5 pathway.

Titanium dioxide nanoparticles (TiO2 NPs) are widely found in consumer and industrial products, contributing to their prevalent presence in our surroundings. In this study, several miRNAs in the immuno-related pathways were found to be dysregulated in RAW264.7 cells after 24-h exposure to TiO2 NPs, including miR-29b-3p, which had not been previously found to be associated with the dysregulation of immunity after exposure to TiO2 NPs. The KEGG pathway and GO enrichment analysis suggested that miR-29b-3p functioned both in the T and B cell receptor signaling pathways. The NFAT5 gene was predicted to regulate miR-29b-3p using the MiRDB online database. The expression of miR-29b-3p and NFAT5 was found to be inversely correlated using qRT-PCR and western blotting analysis. Dual-luciferase reporter gene assays demonstrated the precise regulatory relationship between miR-29b-3p and NFAT5. The upregulation of miR-29b-3p was found to reinforce the apoptosis of cells, while no changes were found in terms of the cell cycle or cell proliferation, using MTT, cell apoptosis, and cycle detection experiments. Our results demonstrate that miR-29b-3p is involved in the response of RAW264.7 cells to exposure to TiO2, proving evidence for the further study of the toxicity and mechanisms of nano-TiO2 exposure.

Indian Hedgehog links obesity to development of hepatocellular carcinoma.

Obesity increases the risk of hepatocellular carcinoma (HCC), but precise identification and characterization of druggable oncogenic pathways that contribute to the progression of NAFLD to HCC, and hence to the increased incidence and aggressiveness of HCC in obese individuals is lacking. In this regard, we demonstrate that the Indian Hedgehog (Ihh) signaling pathway is upregulated in the fatty livers of mice consuming a high fat diet, and furthermore sustained in HCC tumors specifically within the context of a NAFLD microenvironment. Using a diet-induced mouse model of HCC wherein only obese mice develop HCC, targeted ablation of hepatocyte-secreted Ihh results in a decreased tumor burden and lower grade tumors. Ihh activation regulates the transdifferentiation of ciliated stellate cells and proliferation of Epcam+ ductal cells to promote fibrosis. Mechanistically, increased expression of hitherto uncharacterized effectors of Hh pathway, namely Myc and Tgf-ß2 is critical to the observed physiology. This pro-tumorigenic response is driven by increased expression of Wnt5a to effect a poorly-differentiated and invasive tumor phenotype. Wnt5a secreted from activated stellate cells act on Ror2-expressing hepatocytes. We further demonstrate that Wnt5a expression is also elevated in poorly-differentiated HCC cells, suggesting that these ligands are also able to function in an autocrine positive feedback manner to sustain poorly-differentiated tumors. Taken together, our study provides a mechanistic understanding for how Ihh signaling promotes HCC tumorigenesis specifically in obese mice. We propose that therapeutic targeting of the Hh pathway offers benefit for patients with dietary / NAFLD-driven steatotic HCC.

Molecular mechanism for miR-350 in regulating of titanium dioxide nanoparticles in macrophage RAW264.7 cells.

This study investigated the role of microRNA(miRNA) in regulating the cytotoxicity of TiO2 nanoparticles (nano-TiO2) to RAW264.7 cells. RAW264.7 cells were treated with 0 and 100 µg/ml nano-TiO2 for 24 h (for miRNA analysis). The differentially expressed miRNAs were detected using Illumina HiSeq™ 2000 sequencing. Through the bio-informatics analysis, miR-350 was found to play an important role in multiple signaling pathways, including MAPK signaling pathway, NF-kappa B signaling pathway and Apoptosis. To characterize the miR-350 function, miR-350 mimic was transfected into RAW264.7 cells for 24 h. MTT and Flow Cytometry were performed to detect cell proliferation, apoptosis and cell cycle (repetition), respectively. QRT-PCR, Western Blot methods and Luciferase assays were applied to detect expression of putative target gene PIK3R3. The results showed that miRNA profiles were differentially dysregulated. The apoptosis rate of miR-350 mimic group was significantly higher than negative control group (p < .05). Cell proliferation and cell cycle had no significant differences between treatment and negative control group. Compared with negative control, the level of protein of PIK3R3 was significantly decreased (p < .05), and the expression of 3'UTR constructs of PIK3R3 was significantly decreased (p < .05) in miR-350 mimic group. The expression of miRNAs was changed after exposed to nano-TiO2, and biological function and target gene results showed miR-350 may promote RAW264.7 cell apoptosis through the negative regulation of PIK3R3 gene. Our results could provide a basis for further understanding of toxicity and possible mechanisms of nano-TiO2 exposure.

2-(3-Benzoylthioureido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylic acid ameliorates metabolic disorders in high-fat diet-fed mice.

AIM: Sterol-regulatory element binding proteins (SREBPs) are major transcription factors that regulate liver lipid biosynthesis. In this article we reported a novel synthetic compound 2-(3-benzoylthioureido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylic acid (ZJ001) that inhibited the SREBP-1c pathway, and effectively reduced hepatic lipid accumulation in diet-induced obesity (DIO) mice. METHODS: A luciferase reporter driven by an SRE-containing promoter transfected into HepG2 cells was used to discover the compound. Two approaches were used to evaluate the lipid-lowering effects of ZJ001: (1) diet-induced obesity (DIO) mice that were treated with ZJ001 (15 mg·kg(-1)·d(-1), po) for 7 weeks; and (2) HepG2 cells and primary hepatocytes used as in vitro models. RESULTS: ZJ001 (10, 20 µmol/L) dose-dependently inhibited the activity of SRE-containing promoter. ZJ001 administration ameliorated lipid metabolism and improved glucose tolerance in DIO mice, accompanied by significantly reduced mRNA levels of SREBP-1C and SREBP-2, and their downstream genes. In HepG2 cells and insulin-treated hepatocytes, ZJ001 (10-40 µmol/L) dose-dependently inhibited lipid synthesis, and reduced mRNA levels of SREBP-1C and SREBP-2, and their downstream genes. Furthermore, ZJ001 dose-dependently increased the phosphorylation of AMPK and regulatory-associated protein of mTOR (Raptor), and suppressed the phosphorylation of mTOR in insulin-treated hepatocytes. Moreover, ZJ001 increased the ADP/ATP ratio in insulin-treated hepatocytes. CONCLUSION: ZJ001 exerts multiple beneficial effects in diet-induced obesity mice. Its lipid-lowering effects may result from the suppression of mTORC1, which regulates SREBP-1c transcription. The results suggest that the SREBP-1c pathway may be a potential therapeutic target for the treatment of lipid metabolic disorders.

MicroRNA-125b may function as an oncogene in lung cancer cells.

The present study aimed to investigate the biofunctions of microRNA (miR)­125b on lung cancer cells. A miR genechip array was used to examine the differential expression of miRs between 95D lung cancer cells and 16 human bronchial epithelial (HBE) cells. Overexpression of miR­125b was observed in the cell lines and in the lung carcinoma tissues compared with the adjacent tissues, confirmed using reverse transcription quantitative polymerase chain reaction. Bioinformatic analysis of miR­125b was also performed, including target prediction, gene ontology and pathway analysis. MTT, flow cytometry and Transwell assays were also used to examine the effect of downregulated miR­125b on the proliferation, apoptosis, invasive ability and cell cycle of 95D cells. Significant differences were observed in the expression of 45 miRs in the 95D cells compared with those in 16HBE cells and the expression of miR­125b was significantly higher in 95D cells compared with that in 16HBE cells as well as in lung tumor tissues compared with that in adjacent tissues. In addition, inhibition of the expression of miR­125b in 95D cells induced apoptosis, G1/S phase arrest and reduction of their invasive ability. In addition, bioinformatics software predicted that miR­125b was involved in the regulation of several pathways associated with cancer, including the transforming growth factor­ß, Wnt and mitogen­activated protein kinase signaling pathways. These data indicated for the first time, to the best of our knowledge, that miR­125b may function as an oncogene in lung cancer.

Systemic immune effects of titanium dioxide nanoparticles after repeated intratracheal instillation in rat.

The potential immune effects of titanium dioxide nanoparticles (nano-TiO2) are raising concern. Our previous study verified that nano-TiO2 induce local immune response in lung tissue followed by intratracheal instillation administration. In this study, we aim to evaluate the systemic immune effects of nano-TiO2. Sprague Dawley rats were treated by intratracheal instillation with nano-TiO2 at doses of 0.5, 4, and 32 mg/kg body weight, micro-TiO2 with 32 mg/kg body weight and 0.9% NaCl, respectively. The exposure was conducted twice a week, for four consecutive weeks. Histopathological immune organs from exposed animals showed slight congestion in spleen, generally brown particulate deposition in cervical and axillary lymph node. Furthermore, immune function response was characterized by increased proliferation of T cells and B cells following mitogen stimulation and enhanced natural killer (NK) cell killing activity in spleen, accompanying by increased number of B cells in blood. No significant changes of Th1-type cytokines (IL-2 and INF-γ) and Th2-type cytokines (TNF-α and IL-6) were observed. Intratracheal exposure to nano-TiO2 may be one of triggers to be responsible for the systemic immune response. Further study is needed to confirm long-lasting lymphocyte responses and the potential mechanisms.

Azoxystrobin, a mitochondrial complex III Qo site inhibitor, exerts beneficial metabolic effects in vivo and in vitro.

BACKGROUND: Several anti-diabetes drugs exert beneficial effects against metabolic syndrome by inhibiting mitochondrial function. Although much progress has been made toward understanding the role of mitochondrial function inhibitors in treating metabolic diseases, the potential effects of these inhibitors on mitochondrial respiratory chain complex III remain unclear. METHODS: We investigated the metabolic effects of azoxystrobin (AZOX), a Qo inhibitor of complex III, in a high-fat diet-fed mouse model with insulin resistance in order to elucidate the mechanism by which AZOX improves glucose and lipid metabolism at the metabolic cellular level. RESULTS: Acute administration of AZOX in mice increased the respiratory exchange ratio. Chronic treatment with AZOX reduced body weight and significantly improved glucose tolerance and insulin sensitivity in high-fat diet-fed mice. AZOX treatment resulted in decreased triacylglycerol accumulation and down-regulated the expression of genes involved in liver lipogenesis. AZOX increased glucose uptake in L6 myotubes and 3T3-L1 adipocytes and inhibited de novo lipogenesis in HepG2 cells. The findings indicate that AZOX-mediated alterations to lipid and glucose metabolism may depend on AMP-activated protein kinase (AMPK) signaling. CONCLUSIONS: AZOX, a Qo inhibitor of mitochondrial respiratory complex III, exerts whole-body beneficial effects on the regulation of glucose and lipid homeostasis in high-fat diet-fed mice. GENERAL SIGNIFICANCE: These findings provide evidence that a Qo inhibitor of mitochondrial respiratory complex III could represent a novel approach for the treatment of obesity.

Effects of Th1 and Th2 cells balance in pulmonary injury induced by nano titanium dioxide.

To explore the potential immunoregulatory mechanisms linking nano TiO2 and pulmonary injury, Sprague Dawley rats were exposed by intra-tracheal instillation to nano TiO2 with the individual doses of 0.5, 4.0 and 32 mg/kgb.w., micro TiO2 with 32 mg/kgb.w. and 0.9% NaCl, respectively. The exposure was conducted twice a week, for four consecutive weeks. The results of lung histology demonstrated increased macrophages accumulation, extensive disruption of alveolar septa, slight alveolar thickness and expansion hyperemia. Mitochondria tumefaction organelles dissolution, endoplasmic reticulum expansion and the gap of nuclear broadening were shown. The changes of IFN-γ and IL-4 level showed no statistical difference. The mRNA expression of GATA-3 was up-regulated, whereas T-bet was significantly down-regulated. The protein expression of T-bet decreased and there were significant differences in nano 4 and 32 mg/kg groups. The imbalance of Th1/Th2 cytokines might be one of the mechanisms of immunotoxicity of respiratory system induced by nano TiO2 particles.