Hypoxia exacerbates nonalcoholic fatty liver disease via the HIF-2α/PPARα pathway.

This study aimed to investigate whether hypoxia can affect nonalcoholic fatty liver disease (NAFLD) progression and the associated mechanisms, specifically regarding the hypoxia-inducible factor (HIF)-2α/peroxisome proliferator-activated receptor (PPAR)α pathway in vitro and in vivo. Recent studies have reported that, compared with HIF-1α, HIF-2α has different effects on lipid metabolism. We propose hypoxia may exacerbate NAFLD by the HIF-2α upregulation-induced suppression of PPARα in the liver. To verify this hypothesis, a steatotic human hepatocyte (L02) cell line treated with free fatty acids and a mouse model of NAFLD fed a high-fat diet were used. Steatotic hepatocytes were treated with hypoxia, HIF-2α siRNA, PPARα agonists, and inhibitors, respectively. Meanwhile, the NAFLD mice were exposed to intermittent hypoxia or intermittent hypoxia with PPARα agonists. The relative gene expression levels of HIF-1α, HIF-2α, mitochondrial function, fatty acid ß-oxidation and lipogenesis were examined. Evidence of lipid accumulation was observed, which demonstrated that, compared with normal hepatocytes, steatotic hepatocytes exhibited higher sensitivity to hypoxia. This phenomenon was closely associated with HIF-2α. Moreover, lipid accumulation in hepatocytes was ameliorated by HIF-2α silencing or a PPARα agonist, despite the hypoxia treatment. HIF-2α overexpression under hypoxic conditions suppressed PPARα, leading to PGC-1α, NRF-1, ESRRα downregulation, and mitochondrial impairment. Additionally, ß-oxidation genes such as CPT1α, CPT2α, ACOX1, and ACOX2 were downregulated and lipogenesis genes including LXRα, FAS, and SCD1 were upregulated by hypoxia. Therefore, we concluded that HIF-2α overexpression induced by hypoxia aggravated NAFLD progression by suppressing fatty acid ß-oxidation and inducing lipogenesis in the liver via PPARα.

Regulatory roles of Osteopontin in lung epithelial inflammation and epithelial-telocyte interaction.

BACKGROUND: Lung epithelial cells play important roles in lung inflammation and injury, although mechanisms remain unclear. Osteopontin (OPN) has essential roles in epithelial damage and repair and in lung cancer biological behaviours. Telocyte (TC) is a type of interstitial cell that interacts with epithelial cells to alleviate acute inflammation and lung injury. The present studies aim at exploring potential mechanisms by which OPN regulates the epithelial origin lung inflammation and the interaction of epithelial cells with TCs in acute and chronic lung injury. METHODS: The lung disease specificity of OPN and epithelial inflammation were defined by bioinformatics. We evaluated the regulatory roles of OPN in OPN-knockdown or over-expressed bronchial epithelia (HBEs) challenged with cigarette smoke extracts (CSE) or in animals with genome OPN knockout (gKO) or lung conditional OPN knockout (cKO). Acute lung injury and chronic obstructive pulmonary disease (COPD) were induced by smoking or lipopolysaccharide (LPS). Effects of OPN on PI3K subunits and ERK were assessed using the inhibitors. Spatialization and distribution of OPN, OPN-positive epithelial subtypes, and TCs were defined by spatial transcriptomics. The interaction between HBEs and TCs was assayed by the co-culture system. RESULTS: Levels of OPN expression increased in smokers, smokers with COPD, and smokers with COPD and lung cancer, as compared with healthy nonsmokers. LPS and/or CSE induced over-production of cytokines from HBEs, dependent upon the dysfunction of OPN. The severity of lung inflammation and injury was significantly lower in OPN-gKO or OPN-cKO mice. HBEs transferred with OPN enhanced the expression of phosphoinositide 3-kinase (PI3K)CA/p110α, PIK3CB/p110ß, PIK3CD/p110δ, PIK3CG/p110γ, PIK3R1, PIK3R2 or PIK3R3. Spatial locations of OPN and OPN-positive epithelial subtypes showed the tight contact of airway epithelia and TCs. Epithelial OPN regulated the epithelial communication with TCs, and the down-regulation of OPN induced more alterations in transcriptomic profiles than the up-regulation. CONCLUSION: Our data evidenced that OPN regulated lung epithelial inflammation, injury, and cell communication between epithelium and TCs in acute and chronic lung injury. The conditional control of lung epithelial OPN may be an alternative for preventing and treating epithelial-origin lung inflammation and injury.

New focuses on roles of communications between endoplasmic reticulum and mitochondria in identification of biomarkers and targets.

The communication between endoplasmic reticulum (ER) and mitochondria (Mt) plays important roles in maintenance of intra- and extra-cellular microenvironment, metabolisms, signaling activities and cell-cell communication. The present review aims to overview the advanced understanding about roles of ER-Mt structural contacts, molecular interactions and chemical exchanges, signal transmissions and inter-organelle regulations in ER-Mt communication. We address how the ER-Mt communication contributes to the regulation of lipid, amino acid and glucose metabolisms by enzymes, transporters and regulators in the process of biosynthesis. We specially emphasize the importance of deep understanding about molecular mechanisms of ER-Mt communication for identification and development of biology-specific, disease-specific and metabolism-specific biomarkers and therapeutic targets for human diseases. The inhibitors and modulators of the ER-Mt communication are categorized according to therapeutic targets. Rapid development of biotechnologies will provide new insights for spatiotemporally understanding the molecular mechanisms of ER-Mt communication.

Regulatory roles of osteopontin in human lung cancer cell epithelial-to-mesenchymal transitions and responses.

BACKGROUND: Lung cancer is still the main cause of death in patients with cancer, due to poor understanding of intracellular regulations. Of those, osteopontin (OPN) may induce the epithelial-to-mesenchymal transition (EMT) to promote tumor cell metastasis. The present study aims to evaluate the regulatory mechanism of internal and external OPN in the development of lung cancer. METHODS: We evaluated genetic variations and different bioinformatics of genes in chromosome 4 among subtypes of lung cancer using global databases. We validated the expression of OPN and EMT-related proteins (e.g., E-cadherin, vimentin) in 208 non-small-cell lung cancer (NSCLC) tumors and the adjacent nontumorous tissues, further to explore the function of OPN in the progression of lung cancer, with a focus on a potential communication between OPN and EMT in the lung cancer. RESULTS: We found that OPN might act as a target molecule in lung cancer, which is associated with lymph node metastasis, postresection recurrence/metastasis, and prognosis of patients with lung cancer. Biological behaviors and pathological responses of OPN varied among diseases, challenges, and severities. Overexpression of OPN was correlated with the existence of EMT in lung cancer tissues. Internal and external OPN plays the decisive roles in lung cancer cell movement, proliferation, and EMT formation, through the upregulation of OPN-PI3K and OPN-MEK pathways. PI3K and MEK inhibitors downregulated the process of EMT and biological behaviors of lung cancer cells, probably through altering vimentin-associated cytoskeletons. CONCLUSION: OPN can be a metastasis-associated or specific biomarker for lung cancer and a potential target for antimetastatic treatment.

A cellular census of human peripheral immune cells identifies novel cell states in lung diseases.

Increasing evidence supports a central role of the immune system in lung diseases. Understanding how immunological alterations between lung diseases provide opportunities for immunotherapy. Exhausted T cells play a key role of immune suppression in lung cancer and chronic obstructive pulmonary disease was proved in our previous study. The present study aims to furthermore define molecular landscapes and heterogeneity of systemic immune cell target proteomic and transcriptomic profiles and interactions between circulating immune cells and lung residential cells in various lung diseases. We firstly measured target proteomic profiles of circulating immune cells from healthy volunteers and patients with stable pneumonia, stable asthma, acute asthma, acute exacerbation of chronic obstructive pulmonary disease, chronic obstructive pulmonary disease and lung cancer, using single-cell analysis by cytometry by time-of-flight with 42 antibodies. The nine immune cells landscape was mapped among those respiratory system diseases, including CD4+ T cells, CD8+ T cells, dendritic cells, B cells, eosinophil, γδT cells, monocytes, neutrophil and natural killer cells. The double-negative T cells and exhausted CD4+ central memory T cells subset were identified in patients with acute pneumonia. This T subset expressed higher levels of T-cell immunoglobulin and mucin domain-containing protein 3 (Tim3) and T-cell immunoreceptor with Ig and ITIM domains (TIGIT) in patients with acute pneumonia and stable pneumonia. Biological processes and pathways of immune cells including immune response activation, regulation of cell cycle and pathways in cancer in peripheral blood immune cells were defined by bulk RNA sequencing (RNA-seq). The heterogeneity among immune cells including CD4+ , CD8+ T cells and NK T cells by single immune cell RNA-seq with significant difference was found by single-cell sequencing. The effect of interstitial telocytes on the immune cell types and immune function was finally studied and the expressions of CD8a and chemokine C-C motif receptor 7 (CCR7) were increased significantly in co-cultured groups. Our data indicate that proteomic and transcriptomic profiles and heterogeneity of circulating immune cells provides new insights for understanding new molecular mechanisms of immune cell function, interaction and modulation as a source to identify and develop biomarkers and targets for lung diseases.

GLP-1 promotes osteogenic differentiation of human ADSCs via the Wnt/GSK-3ß/ß-catenin pathway.

Glucagon-like peptide-1 (GLP-1) analogues are promising anti-diabetic drugs which had been shown to have beneficial effects on bone metabolism in clinical practice, but the molecular mechanism remains unclear. In this study, we evaluated whether GLP-1 can affect the "intestine-fat-bone axis" via the Wnt/GSK-3ß/ß-catenin pathway. We established a diabetic mouse model and then treated mice with GLP-1 analogue liraglutide. The results showed that after liraglutide treatment, glucose tolerance and insulin tolerance were significantly improved in diabetic mice as expected. Moreover, osteogenic markers such as collagenⅠ, Runx2 and OCN were upregulated; and the adipogenic differentiation markers C/EBP-α and PPAR-γ were downregulated, these results indicated that liraglutide could ameliorate the osteogenic metabolism in diabetic mice. In the cell model, human ADSCs (hADSCs) were cultured and induced to undergo osteogenic and adipogenic differentiation under high glucose conditions in vitro and then treated with GLP-1. The results showed that GLP-1 repressed the induction of adipocyte differentiation biomarkers and the secretion of GSK-3ß in a dose-dependent manner. In addition, GLP-1 enhanced the expression of osteoblastogenic biomarkers, such as OCN, Runx2 and collagenⅠ, and promoted osteoblastic mineralization. These effects were substantially suppressed by the Wnt signal recombinant human DKK-1 or activated by Wnt pathway agonist LiCl. Silencing of GSK-3ß showed that the levels of ß-catenin, GSK-3ß and Runx2 were significantly increased by 2.46-, 2.05-, 4.44-fold after GLP-1 treatment compared to that observed in the GSK-3ß lentiviral group, respectively. We conclude that GLP-1 promotes the osteogenic differentiation of hADSCs via the Wnt/GSK-3ß/ß-catenin pathway.

[Simultaneous determination of sudan red dyes in foods by high performance liquid chromatography with a clean-up procedure by gel column].

A method was developed for the simultaneous determination of Sudan Red I, II, III and IV in foods by high performance liquid chromatography (HPLC) with a clean-up procedure by gel column. Sample was extracted from foods with ethanol. The extract was cleaned up with a Bio-Beads SX3 gel column (200 mm x 10 mm i. d. ) and eluted with cyclohexane-ethyl acetate (1:1, v/v). The analysis was performed on a Symmetry Shield RP18 column (250 mm x 4. 6 mm i. d. , 5 microm) with 100% methanol as the mobile phase at a flow rate of 1.5 mL/min, detection at 478 nm and confirmation by diode-array spectra. All of the four compounds demonstrated good linear relationship (r > 0.999) in the range of 0.1 - 10.0 mg/L. The limits of detection (LOD) were from 7 to 14 microg/kg. The average recoveries for all four dyes (spiked at the levels of 0.25 and 2. 5 mg/kg) in chili sauce and sausage ranged from 80.7% to 96.3%, and the relative standard deviations were from 2.4% to 5.9%. The method is sensitive, reliable and can be applied for the analysis of four Sudan Red dyes in foods.