Zhuxiang Zhao

Nicotine-induced epithelial-mesenchymal transition via Wnt/β-catenin signaling in human airway epithelial cells

Epithelial-mesenchymal transition (EMT) has been proposed to be a mechanism in airway remodeling, which is a characteristic of chronic obstructive pulmonary disease (COPD). Studies have shown that cigarette smoke and nicotine are factors that induce Wnt/β-catenin activation, which is a pathway that has also been implicated in EMT. The main aim of this study was to test whether human bronchial epithelial cells are able to undergo EMT in vitro following nicotine stimulation via the Wnt3a/β-catenin signaling pathway. We show that nicotine activates the Wnt3a signal pathway, which leads to the translocation of β-catenin into the nucleus and activation of β-catenin/Tcf-dependent transcription in the human bronchial epithelial cell (HBEC) line. This accumulation was accompanied by an increase in smooth muscle actin, vimentin, matrix metalloproteinases-9, and type I collagen expression as well as downregulation of E-cadherin, which are typical characteristics of EMT. We also noted that the release of TGF-β(1) from these cells was stimulated by nicotine. Knockdown of Wnt3a with small interfering RNA (siRNA) prevented these effects, implying that β-catenin activation in these responses is Wnt3a dependent. Furthermore, specific knockdown of TGF-β(1) with TGF-β(1) siRNA partially prevented nicotine-induced EMT, suggesting that TGF-β(1) has a role in nicotine-mediated EMT in HBECs. These results suggest that HBECs are able to undergo EMT in vitro upon nicotine stimulation via the Wnt3a/β-catenin signaling pathway.

Exon sequencing identifies a novel CHRNA3-CHRNA5-CHRNB4 variant that increases the risk for chronic obstructive pulmonary disease.

Recent genome‐wide association studies have established that single nucleotide polymorphisms in the CHRNA3‐CHRNA5‐CHRNB4 genes are susceptibility loci for chronic obstructive pulmonary disease COPD. However, further effort is still required to reveal their genetic contribution to COPD, considering the existence of ‘missing heritability’, which may be mediated by variants that are of a low frequency or rare. Here we aimed to identify genetic variants in the coding regions of the CHRNA3‐CHRNA5‐CHRNB4 genes and determine their associations with COPD risk in Chinese.

The pro-proliferative effects of nicotine and its underlying mechanism on rat airway smooth muscle cells.

Recent studies have shown that nicotine, a major component of cigarette smoke, can stimulate the proliferation of non-neuronal cells. Cigarette smoking can promote a variety of pulmonary and cardiovascular diseases, such as chronic obstructive pulmonary disease (COPD), atherosclerosis, and cancer. A predominant feature of COPD is airway remodeling, which includes increased airway smooth muscle (ASM) mass. The mechanisms underlying ASM remodeling in COPD have not yet been fully elucidated. Here, we show that nicotine induces a profound and time-dependent increase in DNA synthesis in rat airway smooth muscle cells (RASMCs) in vitro. Nicotine also significantly increased the number of RASMCs, which was associated with the increased expression of Cyclin D1, phosphorylation of the retinoblastoma protein (RB) and was dependent on the activation of Akt. The activation of Akt by nicotine occurred within minutes and depended upon the nicotinic acetylcholine receptors (nAchRs). Activated Akt increased the phosphorylation of downstream substrates such as GSK3β. Our data suggest that the binding of nicotine to the nAchRs on RASMCs can regulate cellular proliferation by activating the Akt pathway.

Small airway disease: A different phenotype of early stage COPD associated with biomass smoke exposure

Chronic exposure to biomass smoke (BS) can significantly compromise pulmonary function and lead to chronic obstructive pulmonary disease (COPD). To determine whether BS exposure induces a unique phenotype of COPD from an early stage, with different physiopathological features compared with COPD associated with smoking (cigarette‐smoke (CS) COPD), we assessed the physiopathology of early COPD associated with BS exposure (BS COPD) by incorporating spirometry, high‐resolution computed tomography (HRCT) imaging, bronchoscopy and pathological examinations.

Nicotine-Induced Airway Smooth Muscle Cell Proliferation Involves TRPC6-Dependent Calcium Influx Via α7 nAChR.

Background/Aims: The proliferation of human bronchial smooth muscle cells (HBSMCs) is a key pathophysiological component of airway remodeling in chronic obstructive pulmonary disease (COPD) for which pharmacotherapy is limited, and only slight improvements in survival have been achieved in recent decades. Cigarette smoke is a well-recognized risk factor for COPD; however, the pathogenesis of cigarette smoke-induced COPD remains incompletely understood. This study aimed to investigate the mechanisms by which nicotine affects HBSMC proliferation. Methods: Cell viability was assessed with a CCK-8 assay. Proliferation was measured by cell counting and EdU immunostaining. Fluorescence calcium imaging was performed to measure intracellular Ca2+ concentration ([Ca2+]i). Results: The results showed that nicotine promotes HBSMC proliferation, which is accompanied by elevated store-operated calcium entry (SOCE), receptor-operated calcium entry (ROCE) and basal [Ca2+]i in HBSMCs. Moreover, we also confirmed that canonical transient receptor potential protein 6 (TRPC6) and α7 nicotinic acetylcholine receptor (α7 nAChR) are involved in nicotine-induced upregulation of cell proliferation. Furthermore, we verified that activation of the PI3K/Akt signaling pathway plays a pivotal role in nicotine-enhanced proliferation and calcium influx in HBSMCs. Inhibition of α7 nAChR significantly decreased Akt phosphorylation levels, and LY294002 inhibited the protein expression levels of TRPC6. Conclusion: Herein, these data provide compelling evidence that calcium entry via the α7 nAChR-PI3K/Akt-TRPC6 signaling pathway plays an important role in the physiological regulation of airway smooth muscle cell proliferation, representing an important target for augmenting airway remodeling.

PM2.5 promotes human bronchial smooth muscle cell migration via the sonic hedgehog signaling pathway

BackgroundThe contribution of airway remodeling in chronic obstructive pulmonary disease (COPD) has been well documented, with airway smooth muscle cell proliferation and migration playing a role in the remodeling process. Here, we aimed to verify the effects of fine particulate matter (PM2.5) on human bronchial smooth muscle cell (HBSMC) migration and to explore the underlying signaling pathways.MethodsHBSMC apoptosis, proliferation and migration were measured using flow cytometry, cell counting and transwell migration assays, respectively. The role of the hedgehog pathway in cell migration was assessed by western blotting to measure the expression of Sonic hedgehog (Shh), Gli1 and Snail. Furthermore, siRNA was used to knock down Gli1 or Snail expression.ResultsPM2.5 induced HBSMC apoptosis in a dose-dependent manner, although certain concentrations of PM2.5 did not induce HBSMC proliferation or apoptosis. Interestingly, cell migration was stimulated by PM2.5 doses far below those that induced apoptosis. Additional experiments revealed that these PM2.5 doses enhanced the expression of Shh, Gli1 and Snail in HBSMCs. Furthermore, PM2.5-induced cell migration and protein expression were enhanced by recombinant Shh and attenuated by cyclopamine. Similar results were obtained by knocking down Gli1 or Snail.ConclusionsThese findings suggest that PM2.5, which may exert its effects through the Shh signaling pathway, is necessary for the migration of HBSMCs. These data define a novel role for PM2.5 in airway remodeling in COPD.

In vitro synergistic effect of amlodipine and imipenem on the expression of the AdeABC efflux pump in multidrug-resistant Acinetobacter baumannii

Introduction Multidrug-resistant Acinetobacter baumannii (A. baumannii) has become one of the greatest threats worldwide to the therapeutic management of infections. Our previous research confirmed an in vitro synergistic effect of amlodipine and imipenem against A. baumannii, and this study is designed to understand its mechanism. Methods Sixty-four non-duplicate A. baumannii isolates were collected and tested for antimicrobial susceptibility by the disk diffusion method. PCR amplification and sequencing were used to identify the presence of the adeB, adeE, adeH, adeJ, abeM and abeS efflux pump genes. The minimal inhibitory concentrations of imipenem, imipenem+amlodipine and imipenem+carbonyl cyanide m–chlorophenyl-hydrazone against these isolates were also determined by the broth microdilution method before and after siRNA silencing of the expression of the adeABC efflux pump. Results In this study, the combination of amlodipine with imipenem showed synergistic antimicrobial activity against sixty-four A. baumannii isolates when compared with the activity of imipenem alone (p<0.025). In the multidrug-resistant group, AML was more effective than carbonyl cyanide m–chlorophenyl-hydrazone (p<0.001). The efflux pump genes adeB, adeE, adeH, adeJ, abeM and abeS were detected in 100% (4/64), 75% (48/64), 0% (0/64), 100% (64/64), 96.9% (62/64) and 96.9% (62/64) of the sixty-four A. baumannii isolates, respectively. The expression of the adeABC efflux pump genes in the multidrug-resistant group (5.05±19.25) is clearly higher than in the non-multidrug-resistant group (0.17±0.20), (p = 0.01). A gene silencing test verified that the mRNA expression levels of adeABC were decreased at 12 h and increased at 24 h, while the reversal of imipenem resistance by amlodipine disappeared at 12 h and reappeared at 24 h. Conclusions The combination of amlodipine with imipenem exhibits an in vitro synergistic antimicrobial effect on multidrug-resistant A. baumannii, which may be due to the inhibition of the AdeABC efflux pump.

Two CHRN susceptibility variants for COPD are genetic determinants of emphysema and chest computed tomography manifestations in Chinese patients

Quantitative computed tomography (CT) measures of emphysema have been shown to be associated with increased mortality in humans, but genetic variants affecting the quantitative parameters of chest CT that measure degree of emphysema have not yet been examined. In this study, using available chest CT data from a total of 344 emphysema patients, we assessed the correlations between five chronic obstructive pulmonary disease (COPD) susceptibility variants in the cholinergic receptor nicotinic (CHRN) genes and the degree of emphysema and chest CT manifestations. We verified that most of the parameters were significantly correlated with the degree of emphysema. Compared to rs76071148AA and TT genotype carriers, the rs76071148AT genotype carriers exhibited a decreased probability of having severe emphysema (odds ratio [OR] =0.63, 95% confidence interval [CI] =0.40–0.99), whereas the variant rs8040868C allele was negatively correlated with the emphysema index (P=0.002). Interestingly, further stratification analysis grouped by spirometry-diagnosed COPD status revealed that the variant rs8040868C (CT + CC) genotypes exerted a protective effect against severe emphysema with borderline significance (OR =0.41, 95% CI =0.16–1.05) and affected the mean lung density, emphysema index, ratio of airway wall thickness to airway dimensions (AWT/AD), and AWT grade in spirometry-diagnosed non-COPD subjects. The rs76071148 variant was also significantly associated with AWT/AD and AWT grade in those individuals. In summary, we determined that rs8040868 and rs76071148 are promising indicators of the degree of emphysema and chest CT manifestations, especially in spirometry-diagnosed non-COPD subjects.

An efficient method to genotype the polymorphisms of cholinergic nicotinic receptor subunit genes and their associations with COPD onset risk

ABSTRACT Background: Single-nucleotide polymorphisms (SNPs) in the cholinergic nicotinic receptor subunit genes on chromosome 15q25.1, including CHRNA3, CHRNB4 and CHRNA5, are well-established biomarkers of chronic obstructive pulmonary disease (COPD) and lung cancer. Thus, there is great demand for a rapid, easy and inexpensive method to detect these variations for purpose of risk prediction in large populations. Aim of the Study: The aim of this study was to establish an accurate and efficient method for genotyping CHRN SNPs and testing their association with age at onset of COPD in Chinese population as well as the clinical stage in COPD patients. Materials and Methods: We designed a method to specifically genotype 5 SNPs of CHRN genes based on a modified high-resolution melt (HRM) method and then validated the genotyping results by direct sequencing of 120 samples. We further used the HRM method to genotype these 5 SNPs in 1,013 COPD patients. Results: Requiring little time, few material costs and only a simplified protocol, the modified HRM method could accurately distinguish the genotypes of CHRN SNPs, demonstrating kappa coefficients >0.96 based on the results from direct sequencing. Furthermore, the data showed that the GG genotype of SNP rs56218866 was associated with a significantly earlier age of COPD onset than A (AA+AG) genotypes (61.0 ± 8.93 vs. 67.8 ± 9.88; P = 0.031), which was not found for the other SNPs. No significant association was observed between the COPD stages and any of the above SNPs. Conclusion: A simple, rapid and efficient HRM method was introduced for CHRN SNP genotyping and a suggestion that the SNP rs56218866A>G is associated with early-onset COPD in a Chinese population was found.