Human Embryo Lung Research Articles

Establishment of a new equine embryo brain primary cell culture with long-term expansion

Primary cell cultures derived from human embryo lung play a crucial role in virology by aiding virus propagation and vaccine development. These cultures exhibit a notable ability to undergo multiple subcultures, often reaching up to 70 passages. However, finding alternative primary cell cultures with similar longevity and usefulness is challenging. In this study, we introduce a novel primary culture cells derived from equine embryo brain (FEB), which cells exhibited remarkable long-term cultivation potential. The FEB was established and maintained using Sumitomo Nerve-Cell Culture System Comparison studies were conducted with fetal equine kidney cell line (FEK-Tc13) to assess growth rates and subculture longevity. Immunological characterization was performed using neuronal markers to confirm the neural nature of FEB cells. Viral growth assessments were conducted using equine herpesviruses (EHV-1 and EHV-4) to evaluate infectivity and cytopathic effects in FEB cells. PCR analysis and real-time PCR assays were employed to detect viral genomic DNA and transcription activity of EHVs in infected FEB cells. FEB cells demonstrated faster growth rates compared to fetal equine kidney cell line (FEK-Tc13 cells) and exhibited sustained subculture capability exceeding 50 passages. Immunostaining confirmed the glial identity of FEB cells. Both equine herpesviruses 1 and 4 EHV-1 and EHV-4 viruses efficiently replicated in FEB cells, resulting in clear cytopathic effects. PCR analysis detected genomic DNA of EHVs in infected FEB cells, indicating successful viral infection. The establishment of FEB cells with extended subculture capability highlights their potential utility as a model system for studying neural cell biology and viral infections.

HOXA5-induced lncRNA DNM3OS promotes human embryo lung fibroblast fibrosis via recruiting EZH2 to epigenetically suppress TSC2 expression.

Idiopathic pulmonary fibrosis (IPF) is an unrepairable disease that results in lung dysfunction and decreased quality of life. Prevention of pulmonary fibrosis is challenging, while its pathogenesis remains largely unknown. Herein, we investigated the effect and mechanism of long non-coding RNA (lncRNA) DNM3OS/Antisense RNA in the pathogenesis of pulmonary fibrosis. EdU (5-ethynyl-2'-deoxyuridine) and wound healing assays were employed to evaluate the role of DNM3OS on cell proliferation and migration. Western blot detected the proteins expressions of alpha-smooth muscle actin (α-SMA), vimentin, and fibronectin. The interactions among genes were evaluated by RNA pull-down, luciferase reporter, RNA immunoprecipitation (RIP), chromatin immunoprecipitation (ChIP) and chromatin Isolation by RNA purification (ChIRP) assays. DNM3OS was upregulated by transforming growth factor beta 1 (TGF-β1) in a dose- and time-dependent manner. DNM3OS knockdown repressed the growth and migration of lung fibroblast, and fibrotic gene expression (CoL1α1, CoL3α1, α-SMA, vimentin, and fibronectin), while suppression of TSC2 accelerated the above process. DNM3OS recruited EZH2 to the promoter region of TSC2, increased the occupancy of EZH2 and H3K27me3, and thereby suppressed the expression of TSC2. HOXA5 promoted the transcription of DNM3OS. HOXA5-induced DNM3OS promoted the proliferation, migration, and expression of fibrosis-related genes in human embryo lung fibroblast via recruiting EZH2 to epigenetically suppress the expression of TSC2.

Biological properties of domestic strain vRub-Ant of rubella virus

Introduction. Rubella is a mild infectious disease affecting mainly children and is caused by the rubella virus, part of the Matonoviridae family, genus Rubivirus. Rubella causes congenital rubella syndrome (CRS) and is the main cause of developmental abnormalities, especially blindness and deafness.
 There is no specific treatment for rubella and CRS. In order to avoid possible complications from rubella infection, a live attenuated rubella vaccine based on the foreign strain of Wistar RA 27/3 rubella virus is used. However, the actual, more effective and preferred vaccine strain the rubella virus for the Russian Federation is considered to be a viral strain of rubella circulating on its territory.
 The aim of the study was to study the biological properties of the developed domestic cold-adapted strain vRub-Ant circulating in the territory of the Russian Federation.
 Materials and methods. Following cell cultures were used in the study human embryo lung diploid cell strain LECH-3, transferable cell line from embryonic kidney cells of green monkeys Vero CCL-81 and Vero ECC, human mesenchymal stem cells, human peripheral blood mononuclear cells (PBMC). Cell cultures were grown on a DMEM/F12 nutrient medium with the addition of 5% fetal bovine serum. Swabs from the pharynx and nasal passages from a child with rubella were used as clinical virus-containing material. Monoclonal anti-idiotypic antibodies m(anti-ID)Ab were used to assess the expression level of alpha/beta and gamma interferon receptors (/ and IFN-R)Ab, imitating the biological effects of alpha/beta and gamma interferons (/ and IFN) of humans. The cultural, virological, immunochemical and serological research methods were applied in the study.
 Results. Attenuation of the vRub-Ant clinical isolate of rubella virus was carried out for 20 consecutive passages on LECH-3 diploid cells at a reduced temperature of 30C. The main biological markers of attenuation were determined to be ts and ca phenotypes. The avirulence of the attenuated viral strain (att-phenotype) was assessed by the level of expression of / and IFN-R. A lower level of / and IFN-R expression was found on the membranes of human PBMC induced by the vaccine strain vRub-Ant in comparison with the parent wild variant of the rubella virus. This trait,the att phenotype, is characteristic of attenuated viral strains. It has been shown that the vaccine strain vRub-Ant has lost neurotropism and was unable to bind to the membrane receptors of the brain (MRB) of guinea pig embryos, unlike its parent rubella virus strain. The high immunogenicity of the domestic cold-adapted strain vRub-Ant was confirmed by high titers of neutralizing rubella antibodies observed in guinea pigs immunized subcutaneously with one vaccination dose of the virus.
 Conclusion. A domestic attenuated vaccine strain vRub-Ant of the rubella virus that has the main biological markers of attenuation (ts-ca and att phenotypes) has been developed. The vaccine strain vRub-Ant induces a high levels of neutralizing antibodies in guinea pigs following the immunization with a single vaccination dose of the vaccine. The viral strain vRub-Ant has lost its tropism to the MRB of guinea pig embryos, unlike its parent variant.

Fenbendazole Attenuates Bleomycin-Induced Pulmonary Fibrosis in Mice via Suppression of Fibroblast-to-Myofibroblast Differentiation.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and fatal interstitial lung disease with unknown etiology. Despite substantial progress in understanding the pathogenesis of pulmonary fibrosis and drug development, there is still no cure for this devastating disease. Fenbendazole (FBZ) is a benzimidazole compound that is widely used as an anthelmintic agent and recent studies have expanded the scope of its pharmacological effects and application prospect. This study demonstrated that FBZ treatment blunted bleomycin-induced lung fibrosis in mice. In vitro studies showed that FBZ inhibited the proliferation and migration of human embryo lung fibroblasts. Further studies showed that FBZ significantly inhibited glucose consumption, moderated glycolytic metabolism in fibroblasts, thus activated adenosine monophosphate-activated protein kinase (AMPK), and reduced the activation of the mammalian target of rapamycin (mTOR) pathway, thereby inhibiting transforming growth factor-β (TGF-β1)-induced fibroblast-to-myofibroblast differentiation and collagen synthesis. In summary, our data suggested that FBZ has potential as a novel treatment for pulmonary fibrosis.

Overexpression of mitofusion 2 inhibits acute respiratory distress syndrome pulmonary fibrosis and its mechanism

To study the inhibitory effect of overexpression of mitofusion 2 (Mfn2) protein on acute respiratory distress syndrome (ARDS) pulmonary fibrosis and its mechanism. Human embryo lung fibroblasts (HELF) were cultured in vitro, and digested and passaged when the adherent rate of HELF reached 80%, and then the cells in good condition were selected for experiment. The ARDS cell model was reproduced by 5 mg/L of lipopolysaccharide (LPS, LPS group); 75 mol/L adenovirus vector carrying mitofusion 2 (Adv-Mfn2) was transfected into HELF (Adv-Mfn2+LPS group); at the same time, blank control group (complete medium culture) and Adv-vector+LPS group were set as controls. The cell proliferation was observed by sulforhodamine B (SRB) method at 0, 12, 24, 36 and 48 hours. After Hoechst 33342 staining, the morphological changes were observed under confocal microscope. Western blotting was used to detect the protein expressions of Bcl-2 and caspase-3. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was used to detect the gene expressions of Bcl-2 and caspase-3. After LPS stimulation for 12-48 hours, the cell proliferation rates in the LPS group increased gradually, which were significantly higher than those in the blank control group [12 hours: (10.75±1.51)% vs. (0.73±1.22)%, 24 hours: (20.09±1.71)% vs. (1.15±1.12)%, 36 hours: (20.58±1.55)% vs. (1.20±1.12)%, 48 hours: (21.30±1.51)% vs. (1.23±1.10)%, all P < 0.01]. There was no statistically significant difference in the cell proliferation rate between the LPS group and the Adv-vector+LPS group. After overexpression of Mfn2, the cell proliferation rates at 12, 24, 36, 48 hours in the Adv-Mfn2+LPS group were (8.93±1.14)%, (10.52±1.24)%, (10.72±1.30)%, and (10.91±1.20)%, which were significantly lower than those in the LPS group (all P < 0.05). Confocal microscopy showed that some cells in the blank control group had nuclei of different sizes, and some nuclei fragmented or shrank to form apoptotic bodies. The nuclei of the cells in the LPS and Adv-vector+LPS groups were round or oval in size, and only a few apoptotic cells appeared. When Mfn2 was overexpressed, there were more apoptotic cells in the visual field in the Adv-Mfn2+LPS group than LPS group. Western blotting and RT-qPCR results showed that Bcl-2 expressions increased significantly after LPS stimulation in the LPS group as compared with the blank control group [Bcl-2 protein (Bcl-2/GAPDH): 0.68±0.01 vs. 0.29±0.01, Bcl-2 mRNA (2-ΔΔCT): 2.23±0.34 vs. 1.00±0.00, both P < 0.01], and caspase-3 expressions decreased significantly [caspase-3 protein (caspase-3/GAPDH): 0.37±0.02 vs. 0.66±0.02, caspase-3 mRNA (2-ΔΔCT): 0.31±0.05 vs. 1.00±0.00, both P < 0.01]. Compared with LPS group, the expressions of Bcl-2 after overexpression of Mfn2 in the Adv-Mfn2+LPS group were down-regulated [Bcl-2 protein (Bcl-2/GAPDH): 0.46±0.01 vs. 0.68±0.01, Bcl-2 mRNA (2-ΔΔCT): 1.45±0.14 vs. 2.23±0.34, both P < 0.01], and the expressions of caspase-3 were up-regulated [caspase-3 protein (caspase-3/GAPDH): 0.54±0.02 vs. 0.37±0.02, caspase-3 mRNA (2-ΔΔCT): 0.88±0.10 vs. 0.31±0.05, both P < 0.01]. Mfn2 protein is involved in ARDS pulmonary fibrosis, which may be related to mitochondrial mediated inhibition of cell proliferation.

The Potential Role of Cathepsin K in Non-Small Cell Lung Cancer.

(1) Background: Cathepsin K has been found overexpressed in several malignant tumors. However, there is little information regarding the involvement of Cathepsin K in non-small cell lung cancer (NSCLC). (2) Methods: Cathepsin K expression was tested in human NSCLC cell lines A549 and human embryo lung fibroblast MRC-5 cells using Western blot and immunofluorescence assay. Cathepsin K was transiently overexpressed or knocked down using transfection with a recombinant plasmid and siRNA, respectively, to test the effects on cell proliferation, migration, invasion, and on the mammalian target of rapamycin (mTOR) signaling pathway. (3) Results: Expression of Cathepsin K was increased significantly in A549 cells and diffused within the cytoplasm compared to the MRC-5 cells used as control. Cathepsin K overexpression promoted the proliferation, migration, and invasion of A549 cells, accompanied by mTOR activation. Cathepsin K knockdown reversed the above malignant behavior and inhibited the mTOR signaling activation, suggesting that Cathepsin K may promote the progression of NSCLC by activating the mTOR signaling pathway. (4) Conclusion: Cathepsin K may potentially represent a viable drug target for NSCLC treatment.

Polydatin relieves paraquat-induced human MRC-5 fibroblast injury through inhibiting the activation of the NLRP3 inflammasome.

BackgroundParaquat (PQ) is a herbicide that is highly toxic to the lungs and kidneys. When it enters the body, it will disrupt the balance of the microenvironment in the body, induce a large number of inflammatory factors and cause cell damage. Polydatin (PD), resveratrol glycoside, has multiple pharmacological effects. However, the protective effect of PD on human embryo lung fibroblast damage caused by PQ poisoning has not been reported. The purpose of this study was to investigate the regulatory effect of PD on human embryo lung fibroblast damage caused by PQ poisoning.MethodThe optimal experimental concentration of PQ for human embryonic lung fibroblast MRC-5 was 100 µmol/L, and then the cells of 100 µmol/L PQ group were treated with different concentrations of PD for 24 h. MTT assay to detect MRC-5 cell viability and flow cytometry to detect apoptosis. The corresponding kit was used to detect the contents of glutathione peroxidase (GSH-PX), malondialdehyde (MDA) and superoxide dismutase (SOD). Enzyme-linked immunosorbent assay (ELISA) to detect the levels of related inflammatory factors tumor necrosis factor alpha (TNF-α), transforming growth factor beta (TGF-β), interleukin 1 beta (IL-1β), and interleukin 6 (IL-6). Western blot detection of NLRP3 inflammatory body activation-related protein expression.ResultsCompared with the PQ group, cell activity, GSH-Px content, and SOD content in PD intervention group were significantly increased, while apoptosis, MDA content, inflammatory factor level, and activation-related proteins of the NLRP3 inflammasome were significantly reduced and were dose-dependent.ConclusionsPD can relieve PQ-induced human MRC-5 fibroblasts injury by reducing the inflammatory response, improving the antioxidant stress capacity, and inhibiting the activation of the NLRP3 inflammasome.

Activation of MAPK and Cyclin D1/CDK4 in Malignant Transformation of Human Embryonic Lung Fibroblasts Induced by Silica and Benzopyrene.

Objective:Silica and Benzo(a)pyrene are listed as carcinogens. This study aims to explore Cyclin D1, CDK4 and difference of cell cycle adjusted by MAPK signal transduction pathway in silica and B(a)P-induced malignant transformation of human embryonic lung fibroblasts. Methods:Activity of the subfamily (ERK, p38 and JNK) of mitogen-activated protein kinase (MAPK), cyclin D1 and CDK4 (cyclin dependent kinase) were evaluated using Human embryonic lung fibroblast (HELF) purchased from the cell room, basic research institute, Chinese Academy of Medical Sciences. The expression of cyclin D1 and CDK4 (cyclin dependent kinase) were measured in silica and B(a)P induced malignant using Western blot (WB) assay. Results: P-ERK and P-JNK expression increased significantly (P<0.01), while CDK4 and P-p38 expression decreased (P<0.01, P<0.05) in silica-induced malignant transformation cells compared with the control group. P-ERK, P-JNK and Cyclin D1 expression increased (P<0.01, P<0.01, P<0.05) in B(a)P-induced group compared with the control group. P-ERK and P-JNK expression decreased (P<0.01), while P-p38, Cyclin D1 and CDK4 expression increased (P<0.05, P<0.05, P<0.01) in B(a)P-induced group compared with the silica-induced group. Conclusion:MAPK and cyclin D1/CDK4 activation expressed differently in human embryo lung fibroblasts malignant transformation induced by silica and benzopyrene.

E2F1/IGF-1R Loop Contributes to BRAF Inhibitor Resistance in Melanoma

E2F1/IGF-1R Loop Contributes to BRAF Inhibitor Resistance in Melanoma

Expression and role of HEPIS in breast cancer.

Human embryo lung cellular protein interacting with severe acute respiratory syndrome-coronavirus nonstructural protein-10 (HEPIS) is expressed at varying levels in multiple organs and breast cancer cell lines. However, its expression and function in breast cancer cells has yet to be studied. Therefore, RNA in situ hybridization was used to detect the expression of HEPIS in breast cancer and cancer-adjacent normal breast tissue. HEPIS was expressed at lower levels in breast cancer compared with that in adjacent normal tissue. Subcellular localization of HEPIS was mainly found in the cytoplasm of HeLa cells. Cell Counting Kit-8 and 5-ethynyl-2′-deoxyuridine cell proliferation assays were used to investigate the role of HEPIS in cancer cell proliferation. Ectopic expression of HEPIS in MCF-7 cells was found to significantly inhibit cell proliferation. In contrast, knockdown of HEPIS by RNA interference exhibited the opposite effect. Furthermore, a dual-luciferase reporter assay was performed and HEPIS overexpression specifically inhibited the activity of the NF-κB reporter gene. Results of the gene chip assay revealed that 2,231 genes were differentially expressed in HEPIS-overexpressing cells. Results of the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses indicated that these genes were enriched in the ‘mitogen-activated protein kinase signaling pathway’, ‘JAK-STAT signaling pathway’ and ‘focal adhesion’. Reverse transcription-quantitative PCR was used to confirm the expression levels of the differentially expressed genes interleukin 2 receptor subunit α (IL2RA), interferon α and β receptor subunit 2 (IFNAR2) and IFα8 (IFNA8). In conclusion, the results of the present study indicated that HEPIS may function as a potential repressor of breast cancer.

High-content analysis of particulate matters-induced oxidative stress and organelle dysfunction in vitro

Oxidative stress is usually considered to be a common mechanism by which particulate matter (PM) exposure induces adverse effects. However, the further biological events such as organelle dysfunction following oxidative stress remain to be explored. In this study, we applied high-content screening (HCS) technique to investigate the toxicological effects of carbon black (CB), diesel exhaust particle (DEP) and PM2.5 on oxidative stress and organelle function in human bronchial epithelial cell (16HBE), human embryo lung fibroblast cell (HELF) and human umbilical vein endothelial cell (HUVEC) which were used to represent distinct regions of the lung, and compared the toxicity impacts of different PMs and the sensitiveness of cell lines. We found three types of PMs induced mitochondrial dysfunction in three cell lines and lysosomal alkalinization in HUVEC while only CB triggered endoplasmic reticulum (ER) stress in 16HBE and HUVEC, and oxidative stress might mediate these processes. Moreover, CB basically exhibited more potent toxicity compared with DEP and PM2.5, which might be attributed to its less oxygen content. Finally, the finding that PMs-induced toxicity impacts exhibited a cell-type dependent manner might provide some information to help to understand the sensitivity of different tissue in the lung.

Sonochemical-assisted green synthesis of nitrogen-doped carbon dots from crab shell as targeted nanoprobes for cell imaging

A one-pot sonochemical method was developed for the green synthesis of highly fluorescent carbon dots with nitrogen dopants ([email protected]) derived from crab shells. The synthesized [email protected] exhibited excellent water solubility with a fluorescence quantum yield of 14.5%. The [email protected] then conjugated with folic acid (FA) to construct the [email protected] nanoprobe with the ability to selectively target folate-receptor positive cancer cells. Moreover, in vitro cytotoxicity studies showed excellent biocompatibility. Cellular uptake of the [email protected] was further studied via in vitro confocal microscopy studies. Results showed that [email protected] nanoprobes were mostly localized in the cytoplasm of the folate receptor-overexpressing Human cervical cancer cell (HeLa) while cellular uptake by human embryo lung cell (HEL) cells (folate receptor-deficient control) was negligible. Based on their improved diagnostic imaging and enhanced targeting effect, the [email protected] probe has high potential in the theranostic applications. This study demonstrates an economically viable approach to synthesize [email protected] probe using seafood waste.

In vitro expression of human cytomegalovirus UL148 RNA and prediction of its functional sites

Objective To express UL148 RNA of human cytomegalovirus (HCMV) clinical strains in vitro and to study its functions. Methods Urine of a newborn with HCMV infection was inoculated into human embryo lung cells. HCMV clinical strain was isolated and identified by multiplex PCR. UL148 gene was amplified and cloned into pGEM-T-Easy plasmid after double enzyme digestion. A recombinant plasmid was constructed and located at the downstream of the T7 promoter. The recombinant plasmid was identified by electrophoresis of the recombinant plasmid, PCR product and double enzyme product. Sequencing analysis was used for final confirmation. UL148 was transcribed into RNA by 32P labeling. Post-translational modification sites were analyzed by bioinformatics method based on UL148 sequence characteristics. Results The clinical strain of HCMV was obtained in vitro. Electrophoresis and sequencing analysis confirmed the successful construction of the recombinant plasmid. UL148 RNA was transcribed in vitro by T7RNA polymerase. Post-translational modification sites showed that UL148 gene contained one cell adhesion sequence, one legume lectins beta-chain signature, two N-myristoylation sites, one casein kinase Ⅱ phosphorylation site, seven protein kinase C phosphorylation sitse, one cAMP/cGMP-dependent protein kinase phosphorylation site, two N-glycosylation sites and one transmembrane region. Conclusion UL148 gene might encode a viral adhesion molecule involving in the signal transduction in host cells. Key words: Human cytomegalovirus; Clinical virus strain; UL148; Signal transduction

Screening and identification of mRNAs targeted by human cytomegalovirus (HCMV)-encoded miR-US33-1-5p in HCMV-infected human embryo lung fibroblast cells

Objective To screen and identify mRNAs targeted by human cytomegalovirus-encoded miR-US33-1-5p (HCMV miR-US33-1-5p) for understanding the biological functions of HCMV miR-US33-1-5p. Methods Potential target mRNAs of HCMV miR-US33-1-5p were screened out by using hybrid-PCR, a simple and effective method. Dual-Luciferase Reporter Assay System was used to identify the binding abilities of HCMV miR-US33-1-5p to 3′-UTRs of these potential target mRNAs. Results Twelve potential mRNAs targeted by HCMV miR-US33-1-5p were screened out in human embryo lung fibroblast cells infected with HCMV. Luciferase activities of 3′-UTRs of seven target mRNAs were significantly down-regulated. Conclusion Proteins encoded by these target mRNAs are involved in virus replication, immune system regulation, protein synthesis, cell cycle regulation, energy metabolism and so on. Identification of mRNAs targeted by HCMV miR-US33-1-5p is helpful for further studies on biological functions of miR-US33-1-5p. Key words: Human cytomegalovirus; miRNA; Target mRNA

Expression profile and promoter analysis of HEPIS.

Human embryo lung cellular protein interacting with severe acute respiratory syndrome-coronavirus nonstructural protein-10 (HEPIS) is a novel transcriptional repressor, the expression profile and promoter activity of which have not been well studied. In the present study, in situ hybridization of RNA was used to study differential HEPIS expression levels in different types of cancer and normal tissues. A total of six truncated lengths of the HEPIS promoter regulatory sequences were cloned into the pGL3-basic vector, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and dual luciferase reporter assays were performed. The results of RT-qPCR demonstrated that HEPIS expression levels differed across four breast cancer cell lines. The results of the dual luciferase reporter assays revealed that the activities of the reporter gene fragments spanning -1334/+373, -1203/+373, -1060/+373 and -899/+373 bp were higher compared with the reporter gene fragments spanning -759/+373 and -279/+373 bp. A search of the transcription factor database TRANSFAC identified numerous octamer transcription factor-1 (OCT-1), nuclear factor (NF)-κB and C-JUN transcription factor binding sites located on the HEPIS promoter (pHEPIS). Furthermore, the results revealed that mutations of the OCT-1 (-1236/-1223 bp), NF-κB (-1186/-1176 bp) and C-JUN (-856/-846 bp) sites on the human pHEPIS resulted in a decrease in luciferase activity. A chromatin immunoprecipitation assay revealed that OCT-1, NF-κB and C-JUN bound to pHEPIS in a site-dependent manner at the basal state. The TRANSFAC database was used to analyze the pHEPIS of multiple species and several activator protein-1, NF-κB and OCT-1 transcription factor binding sites were predicted. In conclusion, the results of the present study suggest that HEPIS is expressed at different levels in multiple organs and breast cancer cell lines. Furthermore, these findings indicate that OCT-1, NF-κB and C-JUN transcription factors are associated with transcriptional regulation of the HEPIS gene.

The antibacterial and antifungal activity of six essential oils and their cyto/genotoxicity to human HEL 12469 cells

Six essential oils (from oregano, thyme, clove, lavender, clary sage, and arborvitae) exhibited different antibacterial and antifungal properties. Antimicrobial activity was shown against pathogenic (Escherichia coli, Salmonella typhimurium, Yersinia enterocolitica, Staphylococcus aureus, Listeria monocytogenes, and Enterococcus faecalis) and environmental bacteria (Bacillus cereus, Arthrobacter protophormiae, Pseudomonas fragi) and fungi (Chaetomium globosum, Penicillium chrysogenum, Cladosporium cladosporoides, Alternaria alternata, and Aspergillus fumigatus). Oregano, thyme, clove and arborvitae showed very strong antibacterial activity against all tested strains at both full strength and reduced concentrations. These essential oils showed different fungistatic and fungicidal activities when tested by direct application and in the vapor phase. The genotoxic effects of these oils on HEL 12469 human embryo lung cells were evaluated using an alkaline comet assay for the first time, revealing that none of the oils induced significant DNA damage in vitro after 24 h. This study provides novel approaches for assessing the antimicrobial potential of essential oils in both direct contact and the vapor phase and also demonstrates the valuable properties of the phenol-free arborvitae oil. These results suggest that all the tested essential oils might be used as broad-spectrum anti-microbial agents for decontaminating an indoor environment.

Repression of TSC1/TSC2 mediated by MeCP2 regulates human embryo lung fibroblast cell differentiation and proliferation

Pulmonary fibrosis (PF) is a severe inflammatory disease with limited effective treatments. It is known that the transdifferentiation of human embryo lung fibroblast (HELF) cells from pulmonary fibroblasts into myofibroblasts, contributes to the progression of pulmonary fibrogenesis. The tuberous sclerosis proteins TSC1 and TSC2 are two key signaling factors which can suppress cell growth and proliferation. However, the roles of TSC1 and TSC2 in lung fibroblast are unclear. Here, we developed a PF model with bleomycin (BLM) in mice and conducted several simulation experiments in HELF cells. Our study shows that the expression of TSC1 and TSC2 in fibrotic mice lung was reduced and stimulation of HELF cells with TGF-β1 resulted in a down-regulation of TSC1 and TSC2. In addition, overexpression of TSC1 or TSC2 decreased cell proliferation and differentiation. Furthermore, we found that reduced expression of TSC1 and TSC2 caused by TGF-β1 is associated with the promoter methylation status of TSC1 and TSC2. MeCP2, controls an epigenetic pathway that promotes myofibroblast transdifferentiation and fibrosis. We found that expression of TSC1 and TSC2 can be repressed by MeCP2, which regulates HELF cell differentiation and proliferation as myofibroblasts and lead to PF ultimately.

Emodin alleviates bleomycin-induced pulmonary fibrosis in rats

Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease with few treatment options and poor prognosis. Emodin, extracted from Chinese rhubarb, was found to be able to alleviate bleomycin (BLM)-induced pulmonary fibrosis, yet the underlying mechanism remains largely unknown. This study aimed to further investigate the effects of emodin on the inflammation and fibrosis of BLM-induced pulmonary fibrosis and the mechanism involved in rats. Our results showed that emodin improved pulmonary function, reduced weight loss and prevented death in BLM-treated rats. Emodin significantly relieved lung edema and fibrotic changes, decreased collagen deposition, and suppressed the infiltration of myofibroblasts [characterized by expression of α-smooth muscle actin (α-SMA)] and inflammatory cells (mainly macrophages and lymphocytes). Moreover, emodin reduced levels of TNF-α, IL-6, TGF-β1 and heat shock protein (HSP)-47 in the lungs of BLM-treated rats. In vitro, emodin profoundly inhibited TGF-β1-induced α-SMA, collagen IV and fibronectin expression in human embryo lung fibroblasts (HELFs). Emodin also inhibited TGF-β1-induced Smad2/3 and STAT3 activation, indicating that Smad2/3 and STAT3 inactivation mediates emodin-induced effects on TGF-β1-induced myofibroblast differentiation. These results suggest that emodin can exert its anti-fibrotic effect via suppression of TGF-β1 signaling and subsequently inhibition of inflammation, HSP-47 expression, myofibroblast differentiation and extracellular matrix (ECM) deposition.

Global DNA methylation and PTEN hypermethylation alterations in lung tissues from human silicosis.

Silicosis is a respiratory disease caused by long-term silica dust exposure. Our previous study has demonstrated that silica mediates the activation of phosphatidylinositol 3-kinase (PI3K)/phosphatase and tensin homolog deleted on chromosome 10 (PTEN)/serine or threonine kinase (AKT)/mitogen-activated protein kinases (MAPK)/AP-1 pathway in human embryo lung fibroblasts (HELFs). The purpose of this study is to identify genome-wide aberrant DNA methylation profiling in lung tissues from silicosis patients. We performed Illumina Human Methylation 450K Beadchip arrays to investigate the methylation alteration in formalin-fixed, paraffin-embedded (FFPE) lung specimens, immunohistochemistry to detect the level of c-Jun and PTEN proteins; methylation specific PCR (MS-PCR) to identify PTEN and c-Jun promoter methylation in HELFs. We found 86,770 CpG sites and 79,660 CpG sites significantly differed in methylation status in early-stage and advanced-stage compared with GEO normal lung methylation data. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed the methylated status of MAPK signaling pathway was considered changed. The number of PTEN and c-Jun CpG promoter methylated-sites were increased in advanced-stage. Early-stage showed the positive expression of c-Jun and PTEN protein and negative or mild expression in advanced-stage. PTEN promoter was no differentially methylated and c-Jun promoter differed at 12 and 24 h in HELFs. Abnormal DNA methylation on genome-scale was implicated in silicosis, and PTEN promoter hypermethylation might be associated with decrease of PTEN protein.

Study of the synergistic effects of all-transretinoic acid and C-phycocyanin on the growth and apoptosis of A549 cells.

In the present study, we investigated the effects of the combination of all-transretinoic acid (ATRA) and natural nontoxic C-phycocyanin (C-PC) on the growth of A549 lung cancer cells in vitro and in vivo. Furthermore, the anticancer mechanism of the drug combination was revealed. Results showed both C-PC and ATRA could inhibit the growth of A549 cells in vivo. The combination of ATRA+C-PC could yield a higher inhibition rate. C-PC exerted a major effect on the proliferation of human embryo lung cells, but ATRA at a high concentration exerted an inhibitory effect. In addition, ATRA+C-PC could decrease the CDK4 mRNA level, but upregulated caspase-3 protein expression and induced cell apoptosis. A mouse model with tumor was constructed by a subcutaneous injection to the left axilla of nu nude (NU/NU) mice. Compared with the control group, the tumor weight was decreased in the single-drug treatment group and was the lowest in the combination group. C-PC+ATRA could upregulate tumor necrosis factor levels and downregulate Bcl-2 expression and the cyclin D1 gene in the tumor. C-PC could promote T cells' activities and spleen weight, but a single use of ATRA exerted an opposite effect. The dosage of ATRA could be reduced when combined with C-PC to reduce the toxic side-effects. In summary, the antitumor effects of the C-PC+ATRA combination were more significant than a single drug in vivo and in vitro.