ANRIL regulating the secretion of Muc5ac induced by atmospheric PM2.5 via NF-κB pathway in Beas-2B cells.

PM2.5 can cause airway inflammation and promote the excessive secretion of mucin 5ac (Muc5ac), which can further induce many respiratory diseases. Antisense non-coding RNA in the INK4 locus (ANRIL) might regulate the inflammatory responses mediated by nuclear factor kappa-B (NF-κB) signaling pathway. Beas-2B cells were used to clarify the role of ANRIL in the secretion of Muc5ac induced by PM2.5 . The siRNA was used to silence ANRIL expression. Normal and gene silenced Beas-2B cells were respectively exposed to different doses of PM2.5 for 6, 12, and 24 h. The survival rate of Beas-2B cells was detected by methyl thiazolyl tetrazolium (MTT) assay. Tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and Muc5ac levels were determined by enzyme linked immunosorbent assay (ELISA). The expression levels of NF-κB family genes and ANRIL were detected by real time polymerase chain reaction (PCR). The levels of NF-κB family proteins and NF-κB family phosphorylated proteins were determined using Western blot. Immunofluorescence experiments were performed to observe the nuclear transposition of RelA. PM2.5 exposure increased the levels of Muc5ac, IL-1ß and TNF-α, and ANRIL gene expression (p < .05). With the dose and time of PM2.5 exposure increasing, the protein levels of inhibitory subunit of nuclear factor kappa-B alpha (IκB-α), RelA, and NF-κB1 decreased, the protein levels of phosphorylated RelA (p-RelA) and phosphorylated NF-κB1 (p-NF-κB1) increased, and RelA nuclear translocation increased, which indicated that the NF-κB signaling pathway was activated (p < .05). Silencing ANRIL could decrease the levels of Muc5ac, IL-1ß, TNF-α, decrease NF-κB family genes expression, inhibit the degradation of IκB-α and the activation of NF-κB pathway (p < .05). ANRIL played a regulatory role in the secretion of Muc5ac and the inflammation induced by atmospheric PM2.5 via NF-κB pathway in Beas-2B cells. ANRIL could be a target for prevention and treatment of the respiratory diseases caused by PM2.5 .

Effect of NF-κB signal pathway on mucus secretion induced by atmospheric PM2.5 in asthmatic rats.

BACKGROUND: Exposure to PM2.5 can stimulate the mucus secretion of airway, affecting the development of bronchial asthma. NF-κB signal pathway plays an important role in inflammation and dysimmunity, what may contribute to the mucus secretion. The present study was undertaken to explore the effect of NF-κB signal pathway on mucus secretion induced by PM2.5 in rats with bronchial asthma. METHODS: Fifty rats (25 males and 25 females) were divided randomly into the control group, ovalbumin asthmatic model group, asthma low-, middle- and high-dose groups (n = 10, 5 males and 5 females each group). The control group, ovalbumin asthmatic model group received physiological saline; the asthma low-, middle- and high-dose groups received 1.5, 7.5 and 37.5 mg/kg PM2.5 on saline, which instilled into the trachea at 2-day intervals for two doses. Lung histopathology was observed by HE staining. The mRNA levels of NF-κB family gens were detected with real time PCR. IκB-α protein expression levels were detected with Western blot. IL-1ß, TNF-α and Muc5ac levels were detected by ELISA. RESULTS: Respiratory mucus secretion increased with increasing dose of PM2.5. Compared with healthy rats, the protein expression levels of IκB-α were significantly lower in the lung of asthmatic rats (p < 0.05), while the relative mRNA expression levels of NF-κB family genes in tracheal tissue and in lung were significantly higher in the asthmatic rats (p < 0.05). Serum IL-1ß levels were significantly higher in the high-dose group than in the control group. Muc5ac protein levels in the trachea were higher in the high-dose compared with the low-and middle-dose groups. CONCLUSION: Short-term exposure to a high concentration of PM2.5 could up-regulate the mRNA expression levels of NF-κB family genes, activate the NF-κB signal pathway, stimulate more IL-1ß and mucus secretion in rats with bronchial asthma. NF-κB signal pathway may regulate the level of IL-1ß, which could influence the mucus secretion induced by PM2.5 in asthmatic rats.

Urinary levels of phthalate metabolites and their association with lifestyle behaviors in Chinese adolescents and young adults.

BACKGROUND: Adolescence and young adulthood are critical periods of human growth and development. Phthalates are environmental endocrine disruptors, and their health hazards in adolescents and young adults cannot be ignored. This study was undertaken to assess phthalate exposure and determine the associations between lifestyle behaviors and phthalate metabolite levels in Chinese adolescents and young adults. METHODS: Four hundred and seventy-eight adolescents and young adults aged 16-20 years were included in this study. The levels of mono-ethyl phthalate (MEP), mono-butyl phthalate (MBP), mono-(2-ethylhexyl) phthalate (MEHP), mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP), mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono-(2-ethyl-5-carboxypentyl) phthalate (MECPP) and mono-(2-carboxmethyl)-hexyl phthalate (MCMHP) in the subjects' urine were measured by high-performance liquid chromatography-tandem mass spectrometry. The estimated daily intake (EDI) and hazard index (HI) of phthalates were calculated based on urinary metabolite levels. Relevant information on the subjects was collected via questionnaires. The associations between phthalate metabolite levels and lifestyle behaviors were examined using the independent-sample t-test, Mann-Whitney test and multiple linear regression. RESULTS: In this study, the detection rates of all seven metabolites were >98%. The highest median metabolite concentration was MBP, which was 43.00 µg/L (33.11 µg/g creatinine). The highest median EDI was for di-(2-ethylhexyl) phthalate (DEHP), which was 2.40 µg/kg-bw/day (volume-based) and 1.51 µg/kg-bw/day (creatinine-based). 2.7% (volume-based) and 1.0% (creatinine-based) of the subjects showed excessive HITDI (HI of the tolerable daily intake) values, which indicated the cumulative risk of anti-androgenic effects. Furthermore, factors significantly associated with phthalate metabolite levels included the use of plastic food packages (DEHP metabolites), physical exercise (MEOHP), the frequency of fast food consumption (MBP), and the frequency of skin care cosmetics and color cosmetics use (MEP). CONCLUSION: Our results suggest that Chinese adolescents and young adults are widely exposed to phthalates and their metabolite levels are influenced by lifestyle behaviors.

Over-expression of nuclear factor-κB family genes and inflammatory molecules is related to chronic obstructive pulmonary disease.

Background: Nuclear factor-κB (NF-κB) signaling plays essential roles in inflammatory responses. However, whether the expression levels of NF-κB family genes affect inflammatory responses is unclear. Moreover, little is known regarding the association between NF-κB family genes expression and the pathogenesis of chronic obstructive pulmonary disease (COPD). The present study was undertaken to assess the relationship between the expression levels of NF-κB family genes mRNA and of inflammatory markers relevant to COPD pathogenesis. Methods: A total of 186 unrelated patients with acute exacerbations of COPD and 180 healthy controls were recruited. Total RNA was extracted from the peripheral fasting blood of each subject using trizol reagent. The mRNA levels of NF-κB family genes (NF-κB1, NF-κB2 and c-REL) were measured by real-time quantitative polymerase chain reaction. The serum levels of cyclooxygenase-2 (COX-2), C-reactive protein, interleukin (IL)-1ß, IL-6, IL-8, IL-12 and tumor necrosis factor-α were measured with enzyme-linked immunosorbent assay kits. Results: The relative mRNA levels of the NF-κB family genes and the levels of inflammatory molecules were significantly higher in the COPD group than in the control group after adjustment for smoking. The IL-1ß, IL-8 and COX-2 levels were significantly lower in the NF-κB2 under-expression subgroup as compared to the NF-κB2 over-expression subgroup. The COX-2 level was significantly lower (P < 0.05) in the c-REL under-expression subgroup as compared to the c-REL over-expression subgroup. Conclusion: NF-κB2 over-expression was associated with IL-1ß, IL-8 and COX-2 levels, whereas c-REL overexpression was associated with COX-2 level. Over-expression of both NF-κB2 and c-REL was found to be related to COPD.

Effect of Atmospheric PM2.5 on Expression Levels of NF-κB Genes and Inflammatory Cytokines Regulated by NF-κB in Human Macrophage.

Exposure to PM2.5 induces systemic inflammation, and the NF-κB signaling pathway plays an important role in the inflammation process. We aim to clarify whether the expression of NF-κB gene family affects inflammation caused by PM2.5. Human monocytic cells (THP-1) were induced to differentiate into macrophages using phorbol myristate acetate. The macrophages were then treated with 100, 200, and 400 µg/ml of PM2.5 for 12, 24, and 48 h, respectively. Then, we determined the survival rate of macrophages through the MTT assay. The TNF-α and CRP levels in the cell culture medium were measured through enzyme-linked immunosorbent assay. The NF-κB1, NF-κB2, RelA, RelB, and Rel mRNA levels in macrophages were measured with reverse transcriptase-polymerase chain reaction. As a consequence, the survival rate of macrophages decreased with increasing PM2.5 exposure time and dose. The TNF-α levels in PM2.5-treated groups were lower as compared with the control group and in contrast to the NF-κB mRNA levels at all exposure times. The TNF-α level in the 400-µg/ml group and the NF-κB1, NF-κB2, RelB, and Rel mRNA levels in all PM2.5-treated groups were found to be higher at 24 h than at 12 h. Furthermore, the TNF-α, CRP, and NF-κB2 mRNA levels in the group treated with 400 µg/ml PM2.5 were higher at 48 h that at 12 and 24 h. On the other hand, the NF-κB1, RelA, RelB, and Rel mRNA levels in all PM2.5-treated groups were lower as compared to levels of TNF-α, CRP, and NF-κB2 mRNA. The levels of NF-κB genes and inflammatory cytokines demonstrated different correlations at different exposure times. Therefore, we conclude that PM2.5 reduces the survival rate of macrophages. As macrophages are exposed to PM2.5, the NF-κB gene family expression is increased, which subsequently affects inflammatory factor levels.