Human Bronchial Epithelial 16HBE Cells Research Articles

Bergenin-activated SIRT1 inhibits TNF-α-induced proinflammatory response by blocking the NF-κB signaling pathway

BackgroundBergenin, a type of polyphenol compound, exhibits antiulcerogenic, anti-inflammatory, antitussive, and burn wound-healing properties. However, its therapeutic effect on tumor necrosis factor α (TNF-α)-induced proinflammatory responses in the airway and potential mechanisms of actions are still unclear. This study aimed to investigate the anti-inflammatory effects and mechanism of bergenin in TNF-α-stimulated human bronchial epithelial (16-HBE) cells. MethodsCell Counting Kit-8 was used to evaluate cytotoxicity. Cytokine expression was analyzed by reverse transcription-quantitative PCR (RT-qPCR) and enzyme-linked immunosorbent assay. Immunofluorescence, western blot, and sirtuin-1 (SIRT1) activity assays were employed to investigate potential molecular mechanisms. ResultsBergenin obviously decreased both mRNA and protein expression levels of interleukins 6 and 8 (IL-6 and IL-8) in TNF-α-stimulated 16-HBE cells. Bergenin blocked TNF-α-mediated activation of nuclear factor κB (NF-κB) signaling and NF-κB nuclear translocation. Interestingly, RT-qPCR and western blotting results revealed that bergenin did not affect SIRT1 expression, but significantly increased its activity. Bergenin-mediated SIRT1 activation was further confirmed by results indicating decreased acetylation levels of NF-κB-p65 and p53. Moreover, the inhibitory effects of bergenin on mRNA and protein expression levels of IL-6 and IL-8 were reversed by a SIRT1 inhibitor. In addition, combining bergenin and dexamethasone (DEX) yielded additive effects on the reduction of IL-6 and IL-8 expression. ConclusionsThese findings demonstrate that bergenin could suppress TNF-α-induced proinflammatory responses by augmenting SIRT1 activity to block the NF-κB signaling pathway, which may provide beneficial effects for the treatment of airway inflammation associated with asthma.

Transient receptor potential canonical 1 channel mediates the mechanical stress‑induced epithelial‑mesenchymal transition of human bronchial epithelial (16HBE) cells.

Airway remodeling is a central event in the pathology of chronic obstructive pulmonary disease (COPD) that leads to airway narrowing and subsequently, to increased mechanical pressure. High mechanical pressure can exacerbate airway remodeling. Thus, a treatment regimen aimed at disrupting this high-pressure airway remodeling vicious cycle may improve the prognosis of patients with COPD. Recent studies have demonstrated that mechanical stress induces lung epithelial-mesenchymal transition (EMT), which is commonly present in airway epithelial cells of patients with COPD. As TRPC1 functions as a mechanosensitive channel that mediates non-selective cation entry in response to increased membrane stretch, the present study investigated the role of TRPC1 in the occurrence of EMT induced by mechanical stress. In the present study, the expression of TRPC1 in the bronchial epithelium was examined in vivo by immunohistochemistry. In vitro, human bronchial epithelial (16HBE) cells were subjected to mechanical stretching for up to 48 h, and TRPC1 expression was then examined by RT-qPCR and western blot analysis. In addition, TRPC1 receptor function was assessed by Ca2+ imaging and siRNA transfection. EMT was identified using immunofluorescence, western blot analysis and RT-qPCR. It was found that TRPC1 expression was upregulated in patients with COPD and in 16HBE cells subjected to mechanical stretch. The mechanical stress-induced activation of TRPC1 in 16HBE cells increased the intracellular calcium concentration and subsequently decreased the expression of cytokeratin 8 and E-cadherin, and increased the expression of α-smooth muscle actin, indicating the occurrence of EMT. On the whole, the findings of the present study demonstrate that TRPC1 plays a key role in the occurrence of EMT in human lung epithelial cells in response to mechanical stretch; thus, this protein may serve as a novel therapeutic target for progressive airway remodeling in COPD.

Interleukins 6/8 and cyclooxygenase-2 release and expressions are regulated by oxidative stress-JAK2/STAT3 signaling pathway in human bronchial epithelial cells exposed to particulate matter ≤2.5 μm.

Atmospheric particulate matter with a diameter ≤2.5 μm (PM2.5) can induce inflammation of the respiratory system, which is the pathological basis of asthma or other respiratory diseases; however, the underlying regulation mechanism has not been clearly addressed. The aim of this study was to explore the potential role of the oxidative stress-JAK/STAT signaling pathway in the inflammation of human bronchial epithelial cells induced by PM2.5. The human bronchial epithelial cell line 16HBE cells were stimulated with PM2.5 at 50 and 100 μg/mL doses for 12 or 24 hours. Intracellular reactive oxygen species (ROS) was detected using flow cytometry. Gene and protein expressions of JAK2, STAT3 and cyclooxygenase 2 (COX-2) were determined using reverse transcription-polymerase chain reaction and western blotting, respectively. The ratio of intracellular glutathione/glutathione disulfide (GSH/GSSG) and the levels of interleukin (IL)-6 and IL-8 in cellular supernatant were analyzed using enzyme-linked immunosorbent assay. The results indicated that PM2.5 treatment significantly increased gene expressions of JAK2/STAT3 and protein levels of p-JAK2/p-STAT3, accompanied by increased intracellular ROS levels, decreased GSH/GSSG ratio at 50 and 100 μg/mL of PM2.5, and significantly enhanced levels of IL-6, IL-8 and COX-2 at a dose of 100 μg/mL. Pretreatment with N-acetyl-l-cysteine (NAC) attenuated the oxidative stress induced by PM2.5; similarly, pretreatment with AG490 (an inhibitor of JAK) decreased the cytokine levels stimulated by PM2.5. Therefore, we concluded that PM2.5 exposure could activate oxidative stress-JAK2/STAT3 signaling pathway, elevate the levels of IL-6, IL-8 and COX-2 in 16HBE cells, which can be inhibited by the NAC or AG490.

Circ_0000638 inhibits neodymium oxide-induced bronchial epithelial cell inflammation through the miR-498-5p/NF-κB axis.

Inhalation of neodymium oxide, a type of rare earth metal, can induce various respiratory diseases, such as lung tissue inflammation, but the associated mechanisms underlying this induction are still unclear. In this study, we explored the role and mechanisms of circular RNA in neodymium oxide-induced airway inflammation. Our study showed that treating human bronchial epithelial (16HBE) cells with neodymium oxide caused an inflammatory response by upregulating the expression of interleukin-8 (IL-8) and interleukin-1 beta (IL-1β). Quantitative real-time polymerase chain reaction (qRT-PCR) analyses revealed significant downregulation of circRNA_0000638 in neodymium oxide-treated 16HBE cells. Data from functional analyses further showed that circ_0000638 inhibited inflammation induced by neodymium oxide in 16HBE cells. circ_0000638 targeted miR-498-5p and further increased the expression of NKRF (NF-κB repressing factor), which inhibited the activation of NF-κB (nuclear factor κB). Moreover, circ_0000638 reduced the expression of IL-8 and IL-1β by inhibiting NF-κB activation in neodymium oxide-treated 16HBE cells. These results suggest that circ_0000638 can inhibit NF-κB activation by competitively binding to miR-498-5p, further downregulating the expression of IL-8 and IL-1β in neodymium oxide-treated 16HBE cells.

Sirtuin 6 attenuates epithelial–mesenchymal transition by suppressing the TGF-β1/Smad3 pathway and c-Jun in asthma models

Allergic asthma is a chronic inflammatory airway disease involving airway remodeling. The histone deacetylase sirtuin6 (SIRT6) has protective effects in cardiac and liver fibrosis; however, its role in airway remodeling is unclear. In this study, we investigated the expression of SIRT6 in a rat model of airway remodeling and observed its effects on the epithelial–mesenchymal transition (EMT) in human bronchial epithelial 16HBE cells. Sprague–Dawley rats were sensitized and challenged with ovalbumin to induce airway remodeling or with phosphate-buffered saline as a control for different periods. Morphological changes, cell counts in the bronchoalveolar lavage fluid, and SIRT6 expression were assessed. 16HBE cells were transfected with plasmids to silence or overexpress SIRT6. Western blotting, quantitative polymerase chain reaction, Transwell assays, and cell proliferation assays were performed to examine the transforming growth factor (TGF)-β1-induced changes in EMT indicators and EMT-related cell behaviors. SIRT6 expression was upregulated in bronchial epithelial cells from rats with airway remodeling and in TGF-β1-treated 16HBE cells. SIRT6 overexpression affected TGF-β1-induced changes in EMT markers and EMT-like cell behaviors. In particular, SIRT6 overexpression alleviated the reduction in E-cadherin and the increases in N-cadherin, vimentin, alpha-smooth muscle actin, and metalloproteinase-9 levels in TGF-β1-treated 16HBE cells. Forced expression of SIRT6 also decreased the rates of cell migration and proliferation, reduced activation of phosphorylated Smad3 induced by TGF-β1 treatment, suppressed the acetylation level at histone H3K9, and inhibited the transcriptional activity of the c-Jun promotor. These results suggested that SIRT6 expression is upregulated during airway remodeling and modulates EMT in bronchial epithelial cells targeting Smad3 and c-Jun, highlighting a new therapeutic candidate for improving airway remodeling in asthma.

Down expression of lnc-BMP1-1 decreases that of Caveolin-1 is associated with the lung cancer susceptibility and cigarette smoking history.

Lnc-BMP1-1 is a lncRNA transcribed from SFTPC (surfactant associated protein C), a lung tissue specific gene encoding pulmonary-associated surfactant protein C (SPC) that is solely secreted by alveolar typeⅡ epithelial cells, among which the ones with SFTPC+ might be transformed into lung adenocarcinoma cells. Caveolin-1 (Cav-1) is a candidate tumor suppressor gene and is vital for coping with oxidative stress induced by cigarette smoke. When comparing lung cancer tissues with their adjacent normal tissues, the expression of lnc-BMP1-1 were decreased, especially in patients with cigarette smoking history (P=0.027), and positively associated with the expression of Cav-1 (P<0.001). When comparing to A549 cells transfected with empty vector (A549-NC cells), the expression level of Cav-1 in A549 cells with over-expressed lnc-BMP1-1 (A549-BMP cells) was increased along with the decreased level of HDAC2 protein. The drug sensitivity of A549-BMP cells to Doxorubicin hydrochloride (DOX) was increased; the growth and migration capability of A549-BMP cells were inhibited along with the decreased protein level of Bcl-2 and DNMT3a; the growth of tumor in nude mice injected with A549-BMP cells were inhibited, too. Furthermore, the lnc-BMP1-1 and Cav-1 expression was also down-regulated in the human bronchial epithelial (16HBE) cells treated with cigarette smoke extract (CSE).

A novel peptide binding to the C-terminal domain of connective tissue growth factor for the treatment of bleomycin-induced pulmonary fibrosis

Using phage display technology, 810A was developed as a novel peptide, which binds to the C-terminal domain of connective tissue growth factor (CTGF). The present study investigated whether this peptide could neutralize CTGF and block its biological activity as a specific strategy to treat pulmonary fibrosis in vitro and in vivo. Human bronchial epithelial (16HBE) cells were treated with CTGF to evaluate the anti-CTGF capability of 810A using MTT testing, cell migration assays, and Western blotting. The action of 810A in mouse models of bleomycin (BLM)-induced pulmonary fibrosis was evaluated according to leukocyte counts in bronchoalveolar lavage fluid (BALF), hydroxyproline content, and pathological analysis. Treatment with 810A resulted in inhibition of cell proliferation, migration, and increases in transforming growth factor-beta (TGF-β) and α-Smooth muscle actin (α-SMA) caused by CTGF treatment in vitro. In vivo, 810A significantly alleviated fibrosis in the pulmonary index. Inflammation was inhibited, as inferred by the reduced number of leukocytes in BALF. Pathophysiological analysis indicated that hydroxyproline content, alveolar enlargement, interstitial inflammatory infiltration, and collagen deposition were reduced. Collectively, these data suggest that 810A may potentially be a CTGF-specific, small-molecule antagonist, providing a new method for prevention and treatment of pulmonary fibrosis.

The expression of LINC00052 during glycidyl methacrylate-induced malignant transformation of 16HBE cells

Objective: To investigate the expression and role of LINC00052 during glycidyl methacrylate (GMA) -induced malignant transformation of 16HBE cells. Methods: Human bronchial epithelial (16HBE) cells were divided into GMA transformation group and corresponding DMSO control group, and the 10th, 20th and 30th generation cells of each group were collected LncRNA microarrays were used to analysis expression of LINC00052 in different stage of malignant transformation. Bioinformatics analysis was applied and the relative expression of LINC00052 and its potentially target genes was detected by real-time quantification PCR (qPCR) . Results: The results of microarray analysis showed that LINC00052 was up-regulated by 1.32-fold, down-regulated by 1.64-fold and down-regulated by 4.92-fold in the malignant transformation early (P10) , middle term (P20) and late (P30) , respectively, The results of qPCR showed that compared with the DMSO control group, the expression of LINC00052 was up-regulated by 1.55 times, down-regulated by 1.20 times and down-regulated by 2.35 times in P10, P20 and P30, respectively, and the difference was statistically significant (P<0.05) . There was a statistically significant difference in the relative expression of NTRK3 between the GMA transformation group of P10 and P30 generations with the corresponding DMSO control group (P<0.05) . Conclusion: LINC00052 is highly expressed in early time of GMA-induced malignant transformation of 16HBE, and down-regulated in the middle and last stage of malignant transformation and may play a protective role in GMA-induced malignant transformation of 16HBE by influencing the expression of its target gene NTRK3.

Toxic effects of the Emamectin Benzoate exposure on cultured human bronchial epithelial (16HBE) cells

Pesticides pollution has caused serious environmental problems in recent years, and mounting evidence has shown that more and more insecticides have serious risk in human health. Emamectin Benzoate formally regarded as a highly safety insecticide based on its exclusive targets, but the cytotoxicity to human lung was ignored for a long time. In the present study, bioassay experiments were used to assess the toxicity of the Emamectin Benzoatein on human non-target cells including cell viability assay, DNA damage assay, flow cytometer assay and western blotting assay. The results indicated that Emamectin Benzoatecan cause the inhibition of the proliferation, cytochrome c release, activation of caspase-3/9 and increase Bax/Bcl-2 ratio, which means it induced the cytotoxicity on 16HBE cells associated with the mitochondrial apoptosis. Besides, the DNA damge caused by the Emamectin Benzoate suggest it has a potential genotoxic effect on human lung cells.

Hydrogen sulfide attenuates cigarette smoke-induced airway remodeling by upregulating SIRT1 signaling pathway

Airway remodeling is one of the characteristics for chronic obstructive pulmonary disease (COPD). The mechanism underlying airway remodeling is associated with epithelial-mesenchymal transition (EMT) in the small airways of smokers and patients with COPD. Sirtuin 1 (SIRT1) is able to reduce oxidative stress, and to modulate EMT. Here, we investigated the effects and mechanisms of hydrogen sulfide (H2S) on pulmonary EMT in vitro and in vivo. We found that H2S donor NaHS inhibited cigarette smoke (CS)-induced airway remodeling, EMT and collagen deposition in mouse lungs. In human bronchial epithelial 16HBE cells, NaHS treatment also reduced CS extract (CSE)-induced EMT, collagen deposition and oxidative stress. Mechanistically, NaHS upregulated SIRT1 expression, but inhibited activation of TGF-β1/Smad3 signaling in vivo and in vitro. SIRT1 inhibition by a specific inhibitor EX527 significantly attenuated or abolished the ability of NaHS to reverse the CSE-induced oxidative stress. SIRT1 inhibition also abolished the protection of NaHS against CSE-induced EMT. Moreover, SIRT1 activation attenuated CSE-induced EMT by modifying TGF-β1-mediated Smad3 transactivation. In conclusion, H2S prevented CS-induced airway remodeling in mice by reversing oxidative stress and EMT, which was partially ameliorated by SIRT1 activation. These findings suggest that H2S may have therapeutic potential for the prevention and treatment of COPD.

Carbon black nanoparticles induce pulmonary fibrosis through NLRP3 inflammasome pathway modulated by miR-96 targeted FOXO3a.

Carbon black nanoparticle (CBNP) is a core constituent of air pollutants like fine particulate matter (PM2.5) as well as a common manufactural material. It was proved to pose adverse effects on lung function and even provoke pulmonary fibrosis. However, the underlying mechanisms of CBNPs-induced pulmonary fibrosis remain unclear. The present study aimed to investigate the mechanism of fibrotic effects caused by CBNPs in rat lung and human bronchial epithelial (16HBE) cells. Forty-nine male rats were randomly subjected to 7 groups, means the 14-day exposure group (30 mg/m3), the 28-day exposure groups (5 mg/m3 and 30 mg/m3), the 90-day exposure group (30 mg/m3) and their respective controls. Rats were nose-only-inhaled CBNPs. 16HBE cells were treated with 0, 50, 100 and 200 μg/mL CBNPs respectively for 24 h. Besides, Forkhead transcription factor class O (FOXO)3a and miR-96 overexpression or suppression 16HBE cells were established to reveal relative mechanisms. Our results suggested CBNPs induced pulmonary fibrosis in time- and dose-dependent manners. CBNPs induced persisting inflammation in rat lung as observed by histopathology and cytology analyses in whole lung lavage fluid (WLL). Both invivo and invitro, CBNPs exposure significantly increased the expression of NLRP3 inflammasome, accompanied by the increased reactive oxygen species (ROS), decreased miR-96 and increased FOXO3a expressions dose -and time-dependently. MiR-96 overexpression or FOXO3a suppression could partially rescue the fibrotic effects through inhibiting NLRP3 inflammasome. Conclusively, our research show that CBNPs-induced pulmonary fibrosis was at least partially depended on activation of NLRP3 inflammasome which modulated by miR-96 targeting FOXO3a.

Role of B-Cell Lymphoma 2 Ovarian Killer (BOK) in Acute Toxicity of Human Lung Epithelial Cells Caused by Cadmium Chloride.

BackgroundB-cell lymphoma 2 (BCL-2) ovarian killer (BOK) is a Bcl-2 family member with sequence homology to pro-apoptotic BAX and BAK, but its physiological and pathological roles remain largely unclear. Exposure of cells to cadmium may cause DNA damage, decrease DNA repair capacity, and increase genomic instability.Material/MethodsThe present study investigated the effects of BOK on the toxicity of cadmium chloride (CdCl2) to human bronchial epithelial (16HBE) cells. We constructed BOK over-expressing (16HBE-BOK) cells and BOK knockdown (16HBE-shBOK) cells using the BOK-ORF plasmid and BOK-siRNA. qRT-PCR for BOK mRNA expression. We used Trypan blue exclusion assay for cell growth, MTT colorimetric assays for cells inhibition rate, and Comet assays for detecting damaged DNA.ResultsCdCl2, at various concentrations and exposure times, increased BOK mRNA. 16HBE-BOK cells (BOK over-expressing) proliferated more than 16HBE cells after 72 h; 16HBE-shBOK (BOK knockdown) cells proliferated less. In addition, BOK deficiency enhanced cell death induced by CdCl2. Similarly, CdCl2- and H2O2-induced DNA damage was greater in BOK-deficient cells.ConclusionsThese findings support a role for BOK in CdCl2-induced DNA damage and cell death.

Investigating the regulatory role of ORMDL3 in airway barrier dysfunction using in vivo and in vitro models.

The airway epithelium (AE) is the main protective barrier between the host and external environmental factors causing asthma. Allergens or pathogens induce AE dysfunction, including epithelial permeability alteration, trans-epithelial electrical resistance (TEER) reduction, upregulation of inflammatory mediators and downregulation of junctional complex molecules. Orosomucoid-like protein isoform 3 (ORMDL3), a gene closely associated with childhood onset asthma, is involved in airway inflammation and remodeling. It was hypothesized that ORMDL3 plays an important role in regulating AE barrier function. In vivo [chronic asthma induced by ovalbumin-respiratory syncytial virus (OVA-RSV)] in mice) and in vitro (human bronchial epithelial cells and 16HBE cells) models were used to assess ORMDL3′s role in AE function regulation, evaluating paracellular permeability, TEER and the expression levels of junctional complex molecules. The effects of ORMDL3 on the extracellular signal-regulated protein kinase (ERK) pathway were determined. In mice with OVA-RSV induced chronic asthma, ORMDL3 and sphingosine kinase 1 (SPHK1) were upregulated whereas the junction related proteins Claudin-18 and E-cadherin were downregulated. Overexpression of ORMDL3 resulted in decreased TEER, downregulation of junctional complex molecules and induced epithelial permeability. In contrast, ORMDL3 inhibition showed the opposite effects. In 16HBE cells, ORMDL3 overexpression induced SPHK1 distribution and activity, while SPHK1 inhibition resulted in increased TEER upon administration of an ORMDL3 agonist or ORMDL3 overexpression. In addition, ERK activation occurred downstream of SPHK1 activation in 16HBE cells. High levels of ORMDL3 result in damaged AE barrier function by inducing the SPHK1/ERK pathway.

Short-term and long-term exposure to hexavalent chromium alters 53BP1 via H3K18ac and H3K27ac

Hexavalent chromium (Cr(VI)) is a well-known human carcinogen and a strong oxidizer that causes severe DNA damage. However, the associations between epigenetic dysregulation and DNA damage have not been well-characterized. In this study, we evaluated the effects of short-term and long-term exposure to Cr(VI) in human bronchial epithelial (16HBE) cells. Then, we explored the role of epigenetic modification in Cr(VI)-induced DNA damage. We found that short- and long-term exposure to Cr(VI) induced DNA damage and reduced the expression 53BP1, but increased the expression of other DNA repair mediators. Short- and long-term exposure to Cr(VI) reduced the levels of H3K18ac and H3K27ac and reduced their enrichment at the promoter of 53BP1. Long-term Cr(VI) exposure resulted in multiple malignant characteristics including cell invasion, migration, and tumorgenicity. These data demonstrated that reduced H3K18ac and H3K27ac following Cr(VI) treatment contributed to the suppression of 53BP1. Our study demonstrated that epigenetic changes and DNA damage responses are involved in short-term toxicity and long-term carcinogenesis induced by Cr(VI).

Transient Receptor Potential Melastatin 8 (TRPM8)-Based Mechanisms Underlie Both the Cold Temperature-Induced Inflammatory Reactions and the Synergistic Effect of Cigarette Smoke in Human Bronchial Epithelial (16HBE) Cells.

Transient receptor potential melastatin 8 (TRPM8) is a major receptor of cold environment. Recently, we found that cigarette smoke extract (CSE) upregulated TRPM8 mRNA and protein expression in bronchial tissues that made them more sensitive to cold stimuli. In our present study, we found that cold temperature (18°C)-induced activation of TRPM8 in 16HBE (human bronchial epithelial) cells facilitated Ca2+ influx and subsequently led to the increased expression of interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α via the upregulation of p-extracellular signal-regulated kinase (ERK) and the activation of NF-κB. In addition, 16HBE cells that co-stimulated with 18°C and CSE were used to explore the synergistic effect of CSE on cold temperature-induced inflammatory cytokine production as well as the possible involved signaling pathway. RT-PCR and western blot analysis revealed that CSE upregulated TRPM8 mRNA and protein level in 16HBE cells. Ca2+ imaging, western blot, and luciferase assay showed more robust increase in intracellular Ca2+ and promoted phosphorylated ERK, P38, and NF-κB activity, respectively, in 16HBE cells co-stimulated with CSE and cold temperature, and such alteration was attenuated by TRPM8 short hairpin RNA (shRNA) transfection and BCTC pretreatment. Furthermore, enhanced levels of IL-6, IL-8, and TNF-α showed by enzyme-linked immunosorbent assay (ELISA) were reduced by specific inhibitors of ERK and NF-κB. Collectively, our results suggest that mitogen-activated protein kinase (MAPK)/NF-κB signaling is involved in TRPM8-mediated cold temperature-induced inflammatory cytokine expression. In addition, CSE synergistically amplifies cold temperature-induced inflammatory factors release via upregulating TRPM8 expression and enhancing MAPK/NF-κB signaling pathway.

Vitexin induces apoptosis through mitochondrial pathway and PI3K/Akt/mTOR signaling in human non-small cell lung cancer A549 cells

BackgroundCurrently, the prognosis of patients with non-small cell lung cancer (NSCLC) remains dismal; hence, it is critical to identify effective anti-NSCLC agents with limited side effects. This study aimed to evaluate the therapeutic potential of flavonoid compound vitexin in human NSCLC cells and the underlying mechanisms.ResultsThe experimental results indicated that vitexin reduced the viability of A549 cells in a dose-dependent manner with nearly no toxicity against normal human bronchial epithelial 16HBE cells. Vitexin also dose-dependently increased A549 cell apoptosis, accompanied by the decreased Bcl-2/Bax ratio and the increased expression of cleaved caspase-3. Moreover, the in vivo anticancer activity of vitexin was further determined in nude mice bearing A549 cells. In addition, vitexin induced the release of cytochrome c from the mitochondria to the cytosol and the loss of mitochondrial membrane potential. Vitexin also significantly reduced the levels of p-PI3K, p-Akt and p-mTOR, and the pro-apoptotic effect of vitexin on A549 cells was partly blocked by SC79, an Akt activator.ConclusionsAccordingly, we believed that vitexin could be used as a potential therapeutic agent for the treatment of NSCLC in the future.

IL-37 alleviates house dust mite-induced chronic allergic asthma by targeting TSLP through the NF-κB and ERK1/2 signaling pathways.

Interleukin (IL)-37 has been described as a negative regulator of immune responses and is critical for asthma pathogenesis, but the mechanisms behind the protective role of IL-37 against allergic asthma are less well understood. We show here that IL-37 administered intranasally inhibited house dust mite (HDM)-induced chronic airway eosinophilic inflammation, goblet cell hyperplasia, peribronchial collagen deposition and airway hyperresponsiveness (AHR) to methacholine. In contrast to a weakened Th2 response in the lung that was characterized by the downregulation of Th2-associated cytokines and chemokines in IL-37-treated mice, IL-37 has no effect on relevant markers of systemic Th2 immune including serum immunoglobulins expression and invitro production of Th2-associated cytokines by splenocytes on HDM recall. We demonstrated that the production of thymic stromal lymphopoietin (TSLP) in the lung tissue was associated with IL-37. Importantly, compared with IL-37 alone, TSLP coadministration with IL-37 restored HDM-induced airway inflammation and structural alterations, increased AHR to methacholine and promoted Th2-associated cytokine production. We further found that IL-37 inhibited the induction of TSLP expression by the main antigen of house dust mite, Der p1, by suppressing NF-κB and extracellular signal regulated kinase 1/2 (ERK1/2) activation in human bronchial epithelial (16-HBE) cells invitro. These data highlight the importance of TSLP in IL-37-mediated protective role in asthma. IL-37 might represent a useful innovative and alternative therapy to control TSLP production in the airway.

MUC1 downregulation promotes TNF-α-induced necroptosis in human bronchial epithelial cells via regulation of the RIPK1/RIPK3 pathway.

MUC1 (mucin 1), a membrane‐tethered mucin glycoprotein, is highly expressed on the surface of respiratory epithelial cells and plays a key role in anti‐inflammatory and antiapoptotic responses against infections. However, little is known about the link between MUC1 and necroptosis in asthma. This study aimed to investigate the effects of MUC1 on TNF‐α‐induced necroptosis in human bronchial epithelial (16HBE) cells and the underlying molecular mechanism. Negative control and MUC1‐siRNA cells were treated with TNF‐α in the presence or absence of necrostatin‐1 (Nec‐1). Necroptosis was investigated using flow cytometry analyses, and the protein expression levels of MUC1, receptor‐interacting protein kinase‐1 (RIPK1), RIPK3, and phosphorylated RIPK1 were detected by western blot analysis. In addition, the interactions between RIPK and MUC1 were analyzed by coimmunoprecipitation. The results demonstrated that TNF‐α could induce necroptosis of 16HBE cells, and MUC1 expression was increased upon treatment with TNF‐α. The coimmunoprecipitation outcomes showed that MUC1 interacted with RIPK1 but not with RIPK3 in 16HBE cells, and the interaction was augmented by TNF‐α. Furthermore, MUC1 downregulation obviously increased the TNF‐α‐induced necroptosis of 16HBE cells and enhanced the expression of p‐RIPK1‐Ser166 and RIPK3, whereas these phenomena were partially attenuated by Nec‐1. These results may provide a new insight into the mechanism of severe asthma‐related necroptosis and lay a foundation for the future development of new anti‐inflammatory drugs for asthma.

Protective effects of metformin on lipopolysaccharide‑induced airway epithelial cell injury via NF‑κB signaling inhibition.

Asthma is a heterogeneous disease characterized by chronic airway inflammation. It has been demonstrated that metformin, an extensively used drug for the treatment of type2 diabetes, improves airway inflammation and remodeling. However, the mechanism by which this occurs remains poorly understood. The present study investigated the protective effects of metformin in lipopolysaccharide (LPS)‑induced human bronchial epithelial (16HBE) cells injury and the associated mechanisms. 16HBE cells were preincubated with metformin for 1h and subsequently exposed to LPS for 12h. A lactate dehydrogenase (LDH) leakage assay was used to determine the extent of injury to 16HBE cells. The expression of tumor necrosis factor‑α (TNF‑α) and interleukin‑6 (IL‑6) was measured by ELISA. The protein expression of intercellular adhesion molecule‑1 (ICAM‑1) and vascular cell adhesion molecule‑1 (VCAM‑1), as well as proteins associated with nuclear factor (NF)‑κB signaling, was measured by western blotting. Immunofluorescence assays confirmed the nuclear translocation of NF‑κB p65. The LDH leakage assays suggested that metformin significantly reduced LPS‑induced 16HBE cell injury. Furthermore, it was confirmed that metformin suppressed the LPS‑induced secretion of TNF‑α, IL‑6, ICAM‑1 and VCAM‑1. The mechanism occurred at least partially via inhibition of NF‑κB signaling. The results demonstrated that metformin inhibited NF‑κB mRNA expression and the nuclear translocation of NF‑κB p65. To the best of our knowledge, the present study was the first to demonstrate that metformin ameliorated LPS‑induced bronchial epithelial cell injury via NF‑κB signaling suppression.

Inhibition of C-X-C Motif Chemokine 10 (CXCL10) Protects Mice from Cigarette Smoke-Induced Chronic Obstructive Pulmonary Disease.

BackgroundChronic obstructive pulmonary disease (COPD) is a type of obstructive lung disease characterized by long-term breathing problems and poor airflow. COPD can progress to persistent decline of pulmonary function. This study explored the effect of CXCL10 on COPD induced by cigarette smoke (CS) and its underlying mechanism.Material/MethodsWild-type (WT) mice were randomly assigned into 3 groups: the control group, the CS group, and the intervention group. Mice in the CS group were exposed to CS and mice in the CXCL10 group were exposed to CS and CXCL10 neutralizing antibody. At 24 h after the last CS exposure, body weight and lung functions of each mouse were recorded. Mice were then anesthetized for collecting bronchoalveolar lavage fluid (BALF) and lung tissues. Levels of interleukin-6 (IL-6), keratinocyte chemotactic factor (KC), and monocyte chemoattractant protein-1 (MCP-1) in supernatant and lung homogenate were detected by ELISA and real-time PCR (RT-PCR), respectively. For in vitro experiments, human bronchial epithelial cells 16HBE were stimulated with different concentrations of cigarette smoke extract (CSE) and CXCL10. Cell viability and levels of inflammatory cytokines in the cell supernatant were detected by Cell Counting Kit-8 (CCK-8) and ELISA assay, respectively.ResultsOur data showed significant weight loss and reduction of lung functions in mice in the CS group compared with those in the control group and intervention group. Increased levels of IL-6, KC, and MCP-1 in BALF and lung homogenate were observed in mice in the model group compared to those in the control group and intervention group. In vitro experiments also confirmed that CXCL10-neutralizing antibody can inhibit CSE-induced cell necrosis and activation of inflammatory cytokines.ConclusionsInhibited CXCL10 protects against COPD progression by decreasing secretion of inflammatory factors, which provides a new direction for the clinical prevention and treatment of COPD.