ITRAQ-based proteomics analysis of human ectopic endometrial stromal cells treated by Maqian essential oil.

BACKGROUND: Endometriosis is a common and complex syndrome characterized by the presence of endometrial-like tissue outside the uterus. Chinese medicine has been recently found to show good efficacy in treating endometriosis. Our previous results revealed that Maqian fruit essential oil (MQEO) could inhibit the proliferation and induce apoptosis of ectopic endometrial stromal cells (EESCs), but the mechanisms remain unclear. In this study, we aim to explore the molecular mechanism of MQEO's specific effects in EESCs. METHODS: We conducted a quantitative proteomics analysis by iTRAQ on EESCs treated with MQEO or DMSO. Then deep analysis was performed based on differentially expressed proteins, including Gene Ontology enrichment analysis, pathway enrichment analysis and protein interaction analysis. Candidate protein targets were subsequently verified by western blotting. RESULTS: Among 6575 identified proteins, 435 proteins exhibited altered expression levels in MQEO-treated EESCs. Of these proteins, most were distributed in signal transduction as well as immune system and the most significantly altered pathway was complement and coagulation cascades. Moreover, two differentially expressed proteins (Heme oxygenase 1 and Acyl-CoA 6-desaturase) were verified and they can be potential biomarkers for endometriosis treatment. CONCLUSIONS: Our proteomic analysis revealed distinct protein expression patterns induced by MQEO treatment in EESCs, highlighting the potential of MQEO for endometriosis treatment and biomarker discovery.

In Vitro Inhibitory Effects of Maqian Essential Oil against Ectopic Endometrial Stromal Cells and LPS-Induced Endometrial Epithelial Cells.

Previous studies revealed that MQEO (Maqian fruits essential oil), which is extracted from the fruit of Maqian (Zanthoxylum myriacanthum var. Pubescens), had a good anti-inflammatory effect, but the effect on endometriosis in vitro remains unknown. In the present study, the inhibitory effects of MQEO against the EESCs (ectopic endometrial stromal cells) were investigated. Cells were treated with a concentration gradient (from 0.025 % to 0.15 %) of MQEO for 24 h and cell viability was detected by CCK-8. In addition, apoptotic rates were investigated using flow cytometry. The effect of MQEO on cell migration was determined by wound-healing and transwell assay. The expression of apoptosis-associated and cell adhesion-related proteins was assessed by western blotting. The transcriptional levels of IL-1, IL-6 and TNF-α were determined by Real-time qPCR. RNA-seq was used to identify the DEGs (differentially expressed genes) in MQEO-pretreated EESCs. We found that the MQEO condition dosage-dependently reduced the cell viability of EESCs. Based on flow cytometry results, the number of apoptotic cells increased significantly with dosage. The wound-healing and transwell results showed that MQEO group exhibited a significantly decreased cell motility and migration ability in comparison with the normal group. Western blotting results showed that MQEO down-regulated the expression of Bcl-2, ICAM-1 (intercellular adhesion molecule 1) and CD44, but up-regulated the cleaved caspase-3 expression in EESCs. What's more, MQEO also inhibited the LPS-induced inflammation in human EECs (endometrial epithelial cells). RNA-seq revealed that 221 DEGs were up-regulated genes and 284 DEGs were down-regulated in MQEO-pretreated EESCs. Our data uncovered the beneficial effects of MQEO in endometriosis and provided new insights into the mechanism of the effect of MQEO on EESCs, suggesting MQEO could be a promising new therapeutic agent for endometriosis.

An alternative surgical technique for varicoceles: a preliminary experience of the microsurgical spermatic (distal end)-inferior or superficial epigastric vein anastomosis in symptomatic varicoceles associated with perineal pain.

Many therapies are effective in treating varicoceles, including dilation of the pampiniform plexus in males. The most common method of treatment is varicocelectomy. We aimed to assess an alternative technique (microsurgical spermatic [distal end]-superficial or inferior epigastric vein anastomosis) that preserves the normal blood flow pattern for varicocele treatment. We retrospectively analyzed 27 men with varicocele between October 2019 and July 2020. All patients underwent microsurgical spermatic (distal end)-superficial or inferior epigastric vein anastomosis. The prognosis was reviewed retrospectively with an additional survey conducted 3 months after surgery. The mean ± standard deviation of the age was 26.1 ± 7.3 years in patients with microsurgical spermatic (distal end)-superficial or inferior epigastric vein anastomosis. The maximum diameter of the varicocele vein, perineal pain score, sperm density, and forward movement of sperm improved over 3 months after surgery. Microsurgical spermatic (distal end)-superficial or inferior epigastric vein anastomosis is a safe and efficient surgical treatment for varicoceles.

Hemangioma of the prostate with retrograde ejaculation - a case report.

INTRODUCTION AND IMPORTANCE: Hemangioma of the prostate is rarely reported. We here describe a hemangioma of the prostate in a 31-year-old man. CASE PRESENTATION: The history, imaging characteristics, treatment and one year follow-up results were well documented. The chief complaint was retrograde ejaculation. A 3.1 cm × 2.9 cm mass in the prostate was detected by ultrasound. Transurethral resection of the prostate (TURP) was performed. CLINICAL DISCUSSION: Pathological examination revealed the mass was hemangioma. Immunohistochemical study found the tissue was SMA, CD34, CD31 positive, but D2-40 negative. Imaging feature combined with pathological result suggests the diagnosis of hemangioma of the prostate. One year follow-up revealed the patient was infertile. CONCLUSION: We suggest TURP should be performed to remove the hemangioma. Combined treatment is necessary to resolve the patient's infertility.

Metabolomics Insights into Oleate-Induced Disorders of Phospholipid Metabolism in Macrophages.

BACKGROUND: Diet-induced disordered phospholipid metabolism and disturbed macrophage metabolism contribute to the pathogenesis of metabolic diseases. However, the effects of oleate, a main dietary fatty acid, on macrophage phospholipid metabolism are unclear. OBJECTIVES: We aimed to discover oleate-induced disorders of macrophage phospholipid metabolism and potential therapeutic targets for treating diet-related metabolic diseases. METHODS: RAW 264.7 cells were exposed to 65 µg oleate/mL, within the blood concentration range of humans and mice, to trigger disorders of phospholipid metabolism. Meanwhile, WY-14643 and pioglitazone, 2 drugs widely used for treating metabolic diseases, were employed to prevent oleate-induced disorders of macrophage phospholipid metabolism. Subsequently, an untargeted metabolomics approach based on liquid chromatography-mass spectrometry was used to discover relevant metabolic disorders and potential therapeutic targets. RESULTS: We showed that 196 metabolites involved in phospholipid metabolism were altered upon oleate treatment and interventions of WY-14643 and pioglitazone (P < 0.05, 2-tailed Mann-Whitney U test). Notably, most lysophospholipids were decreased, whereas most phospholipids were increased in oleate-treated macrophages. Phosphatidylethanolamines accumulated most among phospholipids, and their acyl chain polyunsaturation increased in oleate-treated macrophages. Additionally, saturated fatty acids were decreased, whereas polyunsaturated fatty acids were increased in oleate-treated macrophages. Furthermore, changes in phosphatidylglycerols, phosphatidylinositols, cardiolipins, phosphatidates, lysophosphatidylglycerols, and acylcarnitines in oleate-treated macrophages could be attenuated or even abolished by WY-14643 and/or pioglitazone treatment. CONCLUSIONS: Oleate induced accumulation of various phospholipids, increased acyl chain polyunsaturation of phosphatidylethanolamines, and decreased lysophospholipids in RAW 264.7 macrophages. This study suggests macrophage phospholipid and fatty acid metabolism as potential therapeutic targets for intervening diet-related metabolic diseases.

Simple Fabrication of Solid-Phase Microextraction with Surface-Coated Aluminum Foil for Enhanced Detection of Analytes in Biological and Clinical Samples by Mass Spectrometry.

Rapid detection of analytes in biological and clinical samples is highly desirable, and significant progress has been made with direct mass spectrometric (MS) analysis. Rapid and sensitive detection, however, remains a major challenge in direct analysis of raw samples. In this study, we described a simple, rapid, and efficient method for enhanced detection of analytes in complex samples, using surface-coated aluminum (Al) foil that was simply made with conductive resin for physical adhesion of functional particles. The surface-coated Al foils were used as a solid-phase microextraction (SPME) tip for rapid sampling of target analytes from raw samples and then applied as an electrospray ionization (ESI) tip to couple MS for sensitive detection. Our results show that surface-coated Al foil is highly effective for enhanced detection of analytes in complex samples with excellent analytical performances, including sensitivity, reproducibility, and linear ranges. Overall, this development enabled an extremely simplified protocol to integrate SPME and ESI that is expected to have a significant impact on rapid screening of raw samples.

The Expression of MBD6 Is Associated with Tumor Size in Uterine Leiomyomas.

Background: Uterine leiomyoma (UL) is the most common benign smooth muscle tumor of the uterus in reproductive women. Prior studies indicated that methyl-CpG-binding domain proteins (MBDs) may be involved in the pathogenesis of UL. Materials and Methods: In this study, UL tissues and paired adjacent myometrium were collected from a total of 51 patients. The expression of MBD mRNAs and their cognate proteins were analyzed via quantitative polymerase chain reaction assays and western blotting, respectively. The relationships between the MBD expression levels and the patients' clinicopathologic variables were assessed using Student's t test, nonparametric tests, or Pearson χ2 methods. Results: Our results show that both the mRNA and protein levels of MBD2 were significantly decreased in ULs compared to the adjacent myometrium. In addition, MBD6 protein expression was also decreased significantly in UL samples when compared to the adjacent myometrium. There was, however, no significant difference on the mRNA expression of MBD6 between these two groups. Neither the mRNA nor the protein levels of the other MBD members (MBD1, MBD3, MBD4, MBD5, and MeCP2) showed any significant differences between ULs and the adjacent myometria. The decreased expression of the MBD6 protein was correlated with the tumor size of ULs. Conclusions: These results suggest that the dysregulated expression of MBD2 and MBD6 in ULs may play a role in their development; however, a larger sample size together with cellular functional assays should be carried out to further elucidate the precise role of MBD6 in ULs.

Quantification of monohydroxylated polycyclic aromatic hydrocarbons in human urine samples using solid-phase microextraction coupled with glass-capillary nanoelectrospray ionization mass spectrometry.

Polycyclic aromatic hydrocarbons (PAHs) are environmental contaminants with carcinogenic effect raising worldwide concerns. Hydroxylated PAHs (OH-PAHs) could be formed in the body as metabolites of PAHs in human urine samples and thus considered as biomarkers of PAH exposure. In this study, five OH-PAHs including 3-phenanthrol, 1-naphthol, 2-hydroxy fluorene, 1-hydroxprene and 6-hydroxy chrysene in human urine samples were selectively enriched by C18 solid-phase microextraction (SPME), then SPME fiber was connected high voltage and then was inserted into a glass-capillary to elute and ionize the analytes for mass spectrometric (MS) detection. The coupling of SPME-MS showed excellent analytical performance for detection of urinary OH-PAHs under optimal conditions, providing an easy operation for rapid detection of a single sample within minutes. By use of internal standard (i.e., 2-hydroxy fluorene-d9), the limit of detection (LOD) and limit of quantitation (LOQ) of OH-PAHs were found to be less than 0.05 ng L-1 level (S/N > 3) and less than 0.1 ng L-1 level (S/N > 10), respectively. The dynamic ranges of five OH-PAHs were found to be a range at 0.1-5.0 ng L-1 with excellent coefficient (R2 > 0.99). This method also showed good precisions (intra-day: 3.4-5.5%, inter-day: 7.0-9.8%, n = 5) and good accuracy (85.3-95.3%, n = 5). Moreover, ion suppression and matrix effect in detection of OH-PAHs in urine were also investigated. Human urine samples collected from 12 volunteers including 6 non-smokers and 6 smokers have been successfully analyzed, it was found that individual OH-PAHs in smokers were higher than in non-smokers. This study demonstrated that SPME coupled with glass-capillary nanoESI-MS is a sensitive method for rapid detection of urinary OH-PAHs for health risk assessment of PAHs exposure.

Therapeutic effects of adipose-derived stem cells-based microtissues on erectile dysfunction in streptozotocin-induced diabetic rats.

This study aimed to explore the therapeutic effects of adipose-derived stem cells (ADSCs)-based microtissues (MTs) on erectile dysfunction (ED) in streptozotocin (STZ)-induced diabetic rats. Fifty-six 8-week-old Sprague-Dawley rats received intraperitoneal injection of STZ (60 mg kg-1 ), and 8 weeks later, the determined diabetic rats randomly received intracavernous (IC) injection of phosphate buffer solution (PBS), ADSCs, or MTs. Another eight normal rats equally got IC injection of PBS. MTs were generated with a hanging drop method, and the injected cells were tracked in ADSC- and MT-injected rats. Four weeks after the treatments, intracavernous pressure (ICP), histopathological changes in corpus cavernosum (CC), and functional proteins were measured. Rat cytokine antibody array was used to detect ADSCs or MTs lysate. The results showed that MTs expressed vascular endothelial growth factor (VEGF), nerve growth factor (NGF), and tumor necrosis factor-stimulated gene-6 (TSG-6). MTs injection had a higher retention than ADSCs injection and MTs treatment improved ICP, neuronal nitric oxide synthase (nNOS) expression, smooth muscle, and endothelial contents in diabetic rats, ameliorated local inflammation in CC better. Thus, our findings demonstrate that IC injection of MTs improves erectile function and histopathological changes in STZ-induced diabetic rats and appears to be more promising than traditional ADSCs. The underlying mechanisms involve increased cell retention accompanied with neuroprotection and anti-inflammatory behaviors of the paracrine factors.

Downregulation of DNA methyltransferase 3 alpha promotes cell proliferation and invasion of ectopic endometrial stromal cells in adenomyosis.

Adenomyosis is a common benign gynecological condition in female reproductive tract and the detailed molecular etiology remains largely elusive. Previous studies implicated that deregulated expression of DNA (cytosine-5)-methyltransferase 3A (DNMT3A), a de novo DNA methyltransferase, might be involved in the pathogenesis of adenomyosis. Meanwhile, ectopic endometrial stromal cells (EESCs) were suggested to play crucial roles in adenomyosis. Herein, we evaluated the expression of DNMT3A protein in 36 ectopic endometriums with adenomyosis and 37 eutopic endometriums in controls with Western blotting (WB) or immunohistochemistry (IHC), we found that the expression of DNMT3A was significantly decreased in the ectopic endometriums and EESCs in adenomyosis relative to that of eutopic endometriums and EESCs in control samples, respectively. In addition, our functional assays revealed that overexpression of DNMT3A suppressed cell proliferation and invasion, while knockdown of DNMT3A enhanced cell proliferation and invasion in EESCs. Taken together, our results suggested that DNMT3A expression was decreased in ectopic endometriums and EESCs in adenomyosis, and we provided the first evidence that decreased DNMT3A expression in EESCs facilitated the development of adenomyosis via enhanced cell growth and invasion.

Medical swab touch spray-mass spectrometry for newborn screening of nicotine and cotinine in meconium.

Newborn screening is one of public health concerns designed to screen infants shortly after birth. Prenatal exposure to tobacco smoke such as nicotine has been reported to affect babies. Levels of nicotine and cotinine in meconium were widely used to evaluate the tobacco exposure of foetuses during pregnancy in a polluted environment. In this study, medical swabs were applied by using touch spray-mass spectrometry (TS-MS) to collect meconium from newborn infants for detection of nicotine and cotinine. Parameters such as choice of spray solvents, solvent volume and collision energy for screening of nicotine and cotinine were optimized. The limits of detection, reproducibility and matrix effect for analysis of meconium were also investigated. In this study, the levels of nicotine and cotinine in 54 puerpera volunteers were screened by TS-MS and were validated by using traditional liquid chromatography-mass spectrometry. These results showed that medical swab TS-MS would be useful for newborn screening of nicotine and cotinine in meconium with high reproducibility, speed, sensitivity and specificity. The use of disposable medical swabs involves no sample preparation and no chromatographic separation, significantly reducing the cost and time required for screening a large number of clinical sample. Copyright © 2016 John Wiley & Sons, Ltd.

A novel somatic MAPK1 mutation in primary ovarian mixed germ cell tumors.

A recent exome-sequencing study revealed prevalent mitogen-activated protein kinase 1 (MAPK1) p.E322K mutation in cervical carcinoma. It remains largely unknown whether ovarian carcinomas also harbor MAPK1 mutations. As paralogous gene mutations co­occur frequently in human malignancies, we analyzed here a total of 263 ovarian carcinomas for the presence of MAPK1 and paralogous MAPK3 mutations by DNA sequencing. A previously unreported MAPK1 p.D321N somatic mutation was identified in 2 out of 18 (11.1%) ovarian mixed germ cell tumors, while no other MAPK1 or MAPK3 mutation was detected in our samples. Of note, OCC­115, the MAPK1­mutated sample with bilateral cancerous ovaries affected, harbored MAPK1 mutation in the right ovary while retained the left ovary intact, implicating that the genetic alterations underlying ovarian mixed germ cell tumor may be different, even in patients with similar genetic backgrounds and tumor microenvironments. The results of evolutionary conservation and protein structure modeling analysis implicated that MAPK1 p.D321N mutation may be pathogenic. Additionally, mutations in protein phosphatase 2 regulatory subunit α (PPP2R1A), ring finger protein 43 (RNF43), DNA directed polymerase ε (POLE1), ribonuclease type III (DICER1), CCCTC­binding factor (CTCF), ribosomal protein L22 (RPL22), DNA methyltransferase 3α (DNMT3A), transformation/transcription domain­associated protein (TRRAP), isocitrate dehydrogenase (IDH)1 and IDH2 were not detected in ovarian mixed germ cell tumors, implicating these genetic alterations may be not associated with MAPK1 mutation in the development of this malignancy. The present study identified a previously unreported MAPK1 mutation in ovarian mixed germ cell tumors for the first time, and this mutation may be actively involved in the tumorigenesis of this disease.

Absence of DICER1, CTCF, RPL22, DNMT3A, TRRAP, IDH1 and IDH2 hotspot mutations in patients with various subtypes of ovarian carcinomas.

Cancer is caused by multiple genetic alterations within cells. Recently, large-scale sequencing has identified frequent ribonuclease type III (DICER1), CCCTC-binding factor (CTCF), ribosomal protein L22 (RPL22), DNA (cytosine-5-)-methyltransferase 3α (DNMT3A), transformation/transcription domain-associated protein (TRRAP), isocitrate dehydrogenase (IDH)1 and IDH2 hotspot mutations in diverse types of cancer. However, it remains largely unknown whether these mutations also exist in ovarian carcinomas. In the present study, a collection of 251 patients with distinct subtypes of ovarian carcinomas were recruited and sequenced for the presence of these hotspot mutations. However, no mutations in the seven genes were detected in the samples. These negative results, together with certain recent reports, indicate that the hotspot mutations in the CTCF, RPL22, DNMT3A, TRRAP, IDH1 and IDH2 genes may not be actively involved in the carcinogenesis of ovarian carcinoma. Of note, the DICER1 mutation frequency in Sertoli-Leydig cell tumor in the present study was significantly lower compared to prior observation, and therefore, it is speculated that this discrepancy may be mainly due to the small sample size analyzed in the study. In addition, among these samples, frequent polymerase (DNA directed) ε, catalytic subunit (POLE1) and ring finger protein 43 (RNF43) mutations were identified in endometrioid and mucinous ovarian carcinomas, respectively; thus DICER1, CTCF, RPL22, DNMT3A, TRRAP, IDH1 and IDH2 hotspot mutations may not play synergistic roles with POLE1 or RNF43 mutations in the carcinogenesis of endometrioid or mucinous ovarian carcinomas.

Transcript profiling analysis of in vitro cultured THP-1 cells after exposure to crotonaldehyde.

Crotonaldehyde, a highly toxic α, ß-unsaturated aldehyde, is a major component of cigarette smoke and a ubiquitous environmental pollutant. Crotonaldehyde exposure is known to have adverse effects on respiratory health, but the underlying mechanisms remain obscure. As alveolar macrophages display important immunological and inflammatory properties in response to extraneous substances in the lung, we aimed at gaining more insight in changes of human macrophage-like cells transcriptome in response to crotonaldehyde. In vitro cultures of human THP-1 cells (a human monocytic leukemia cell line) were differentiated into macrophage-like cells treated by PMA (phorbol 12-myristate 13-acetate) and be exposed crotonaldehyde. Using RNA-seq technology such as digital gene expression, the global changes in transcriptional level were analyzed. Real-time quantitative polymerase chain reaction (qPCR) was performed to validate RNA-seq data. The differential regulated genes in many biological processes were dysregulated, including in antigen processing and presentation, oxidative stress, inflammation, cytokine signaling, and apoptosis. Collectively, our study demonstrated that crotonaldehyde altered gene expression profile in the genome-wide transcriptional level in human macrophage-like cells, and many of them may represent potential mechanisms of crotonaldehyde causing cytotoxicity and tissue injury in the human lung.

Crotonaldehyde-exposed macrophages induce IL-8 release from airway epithelial cells through NF-κB and AP-1 pathways.

Crotonaldehyde, a highly toxic α, ß-unsaturated aldehyde, is a major component of cigarette smoke and a ubiquitous environmental pollutant. Crotonaldehyde exposure is known to have adverse effects on respiratory health, but the underlying mechanisms remain obscure. To examine the interaction between macrophages and airway epithelial cells after exposure to crotonaldehyde, BEAS-2B and A549 cells were treated with conditioned media from a human monocytic leukemia cell line (THP-1) cells stimulated with crotonaldehyde. We demonstrate that conditioned media from THP-1 cells stimulated with crotonaldehyde increased interleukin (IL)-8 production, enhanced nuclear factor (NF)-κB and AP-1 DNA-binding activity in BEAS-2B and A549 cells. Analysis of these conditioned media revealed marked increases in tumor necrosis factor (TNF)-α, IL-1ß and IL-8 levels. Preincubation of conditioned media with either TNF-α- or IL-1ß-neutralizing antibodies reduced IL-8 production. Furthermore, BEAS-2B and A549 cells directly treated with crotonaldehyde induced increase in IL-8 production. These data suggest that crotonaldehyde is capable of directly stimulating the production of IL-8 in both macrophages and airway epithelial cells. Crotonaldehyde-stimulated macrophages also amplify the inflammatory response by enhancing IL-8 release from airway epithelial cells mediated by NF-κB and AP-1 pathways through a mechanism involving TNF-α and IL-1ß. These findings indicate that crotonaldehyde can cause lung inflammatory response via multiple mechanisms, and may contribute to chronic airway inflammation in smokers.

Crotonaldehyde induces apoptosis in alveolar macrophages through intracellular calcium, mitochondria and p53 signaling pathways.

Crotonaldehyde, a highly electrophilic α, ß-unsaturated aldehyde, is a ubiquitous environmental pollutant and a risk factor for multiple respiratory diseases. Crotonaldehyde is highly volatile and hydrophilic, so it is efficiently absorbed in the respiratory tract. Alveolar macrophages are major effector cells of the nonspecific host defence in the lung. The aim of this study was to investigate the molecular mechanisms and signaling pathways responsible for cell death of alveolar macrophage induced by crotonaldehyde. Our results show that crotonaldehyde induces apoptosis in alveolar macrophages, as indicated by phosphatidylserine externalization and DNA fragmentation. Pretreatment of alveolar macrophages with N-acetylcysteine, ascorbic acid, α-tocopherol, superoxide dismutase inhibited crotonaldehyde-induced apoptosis. Crotonaldehyde-induced apoptosis was characterized by ROS generation, GSH depletion, loss of mitochondrial membrane potential (ΔΨm), the release of cytochrome c from mitochondria, caspase-3/7 and caspase-9 activation, elevation of intracellular Ca(2+) concentration and the increase of p53 expression. Furthermore, pretreatment with either p53 inhibitor pifithrin-α or calcium chelator BAPTA-AM effectively attenuated apoptosis induced by crotonaldehyde. Taken together, our results showed that crotonaldehyde induce apoptosis in alveolar macrophages through intracellular calcium, mitochondria and p53 signaling pathways. These results would help to illustrate the mechanism of toxicity induced by crotonaldehyde and to look for a novel treatment for diseases induced by exposure to crotonaldehyde-rich pollutants such as cigarette smoke.

Crotonaldehyde induces apoptosis and immunosuppression in alveolar macrophages.

Crotonaldehyde, a highly toxic α, ß-unsaturated aldehyde, is a major component of cigarette smoke (CS) and a ubiquitous environmental pollutant. Exposure to crotonaldehyde-rich pollutants such as CS is associated with suppression of respiratory host defense against infections. The aim of this study was to evaluate the apoptotic and immunological effects of crotonaldehyde exposure in a rat alveolar macrophage (AM) cell line, NR8383. Our studies showed that crotonaldehyde induced AM cell death mainly via the apoptotic process. Crotonaldehyde also decreased the phagocytic activity of AMs. Crotonaldehyde caused inhibition of NO, TNF-α, IL-1ß and IL-12 production in AMs treated with lipopolysaccharide (LPS), which is probably related to inhibition of NF-κB activation. These results indicate that crotonaldehyde can cause adverse effects in AMs via multiple mechanisms, and may contribute to compromised lung immunological response in smokers.