Human Embryonic Lung Fibroblast Cells Research Articles

[Mechanism of n-butanol fraction of Wenxia Formula combining with gefitinib in treating non-small cell lung cancer based on network pharmacology and in vitro experiment].

This study combined network pharmacology, molecular docking, and in vitro experiments to explore the potential mechanism of the active components of the n-butanol fraction of Wenxia Formula(NWXF) combined with gefitinib(GEF) in treating non-small cell lung cancer(NSCLC). Ultra-performance liquid chromatography-quadrupole Orbitrap mass spectrometry(UPLC-Q-Orbitrap MS) was employed to detect the main chemical components of NWXF. The active components of NWXF were retrieved from SwissADME, and the candidate targets of these active components were retrieved from SwissTargetPrediction. Online Mendelian Inheritance in Man(OMIM) and GeneCards were searched for the targets of NSCLC. Cytoscape 3.9.0 and STRING were employed to build the protein-protein interaction(PPI) network with the common targets shared by NWXF and NSCLC. Gene Ontology(GO) annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment were performed in DAVID to predict the potential mechanisms. Finally, molecular docking between the main active ingredients and key targets was conducted in SYBYL-X 2.0. The methyl thiazolyl tetrazolium(MTT) assay was employed to evaluate the inhibitory effects of NWXF and/or GEF on the proliferation of human non-small cell lung cancer cells(A549 and PC-9). Additionally, the impact of NWXF on human embryonic lung fibroblast cells(MRC-5) was assessed. The effectiveness of the drug combination was evaluated based on the Q value. The terminal-deoxynucleoitidyl transferase mediated nick-end labeling(TUNEL) assay was employed to examine the apoptosis of A549 and PC-9 cells treated with NWXF and/or GEF. Quantitative real-time PCR(qRT-PCR) was employed to measure the mRNA levels of epidermal growth factor receptor(EGFR), c-Jun N-terminal kinase(JNK), and Bcl2-associated X protein(Bax) in the A549 and PC-9 cells treated with NWXF and/or GEF. Western blot was employed to determine the protein levels of EGFR, p-EGFR, JNK, p-JNK, and Bax in the A549 and PC-9 cells treated with NWXF and/or GEF. A total of 77 active components, 488 potential targets, and 49 key targets involved in the treatment of NSCLC with NWXF were predicted. The results of GO annotation showed that NWXF may treat NSCLC by regulating the biological processes such as cell proliferation, apoptosis, and protein phosphorylation. KEGG enrichment revealed that the key targets of NWXF in treating NSCLC were enriched in the mitogen-activated protein kinase(MAPK), phosphatidylinositol 3-kinase(PI3K)-protein kinase B(AKT), hypoxia-inducible factor-1(HIF-1), and microRNA-related signaling pathways. Molecular docking results showed that 91.9% of the docking scores were greater than 5, indicating the strong binding capability between main active components and key targets. The cell experiments demonstrated that NWXF combined with GEF synergistically inhibited the proliferation, promoted the apoptosis, decreased p-EGFR/EGFR and p-JNK/JNK values, down-regulated the mRNA levels of EGFR and JNK, and up-regulated the mRNA and protein levels of Bax in A549 and PC-9 cells. In conclusion, NWXF combined with GEF can regulate the EGFR/JNK pathway to promote the apoptosis of NSCLC cells, thus treating NSCLC.

Relationship of human cytomegalovirus-infected endothelial cells and circulating leukocytes in the pathogenesis of disseminated human cytomegalovirus infection: A narrative review.

Among the leucocyte subpopulations circulating in peripheral blood of immune-compromised patients with disseminated Human cytomegalovirus (HCMV) infection, polymorphonuclear leuckocytes (PMNL) and M/M may carry infectious virus. While only in PMNL early HCMV replicative events do occur, monocytes are susceptible to complete virus replication when they enter human organs, where as macrophages become a site of active complete virus replication. In vivo leucocytes and endothelial cells interact continuously, as suggested by several in vitro experimental findings showing the bidirectional HCMV transmission from leucocytes to and from endothelial cells with the critical aid of adhesion molecules. Recently, the neutralising antibody response in sera from subjects with primary HCMV infection was reported to be much higher and earlier than in human embryonic lung fibroblasts (HELF) cells when measured in endothelial cells and epithelial cells, where virus entry is mediated mostly by the pentamer complex gH/gL/pUL128/pUL130/pUL131, whereas it was much lower and delayed when determined in HELF, where virus entry is mediated mostly by the trimer complex gH/gL/gO. Thus, these results suggested that products of UL128L were the molecules primary responsible for the differential neutralising antibody response. This conclusion was confirmed by a series of polyclonal and monoclonal antibodies directed to the components of pUL128L. Very recently, based on two sets of experiments including inhibition and immunoblotting assays, the pentamer complex/trimer complex ratio has been finally identified as the main factor of the neutralising antibody response. This ratio may change with the virus suspension producer and target cell system as well as number of cell culture passages.

The lncRNA H19/miR-29a-3p/SNIP1/c-myc regulatory axis is involved in pulmonary fibrosis induced by Nd2O3.

Some rare earth elements are occupational and environmental toxicants and can cause organ and systemic damage; therefore, they have attracted global attention. Neodymium oxide (Nd2O3) is a rare earth element that is refined and significantly utilized in China. The long noncoding RNA (lncRNA) H19 is encoded by the H19/IGF2 imprinted gene cluster located on human chromosome 11p15.5. H19 has become a research focus due to its ectopic expression leading to the promotion of fibrosis. However, the mechanisms by which it causes pulmonary fibrosis are elusive. This investigation indicates that biologically active Nd2O3 increases H19, SNIP1, and c-myc, decreases miR-29a-3p, accelerates macrophage M2 polarization, and causes pulmonary fibrosis in mice lung tissues. In macrophage-differentiated THP-1 cells, Nd2O3 (25 μg/ml) enhanced H19, SNIP1, and c-myc, reduced miR-29a-3p, accelerated macrophages M2 polarization, and stimulated fibrogenic cytokine (TGF-β1) secretion. Furthermore, the coculturing of Nd2O3-treated macrophage-differentiated THP-1 cells. And human embryonic lung fibroblast cells activated lung fibroblast, which increases the levels of collagen I, α-SMA, p-Smad2/3, and Smad4, whereas H19 knockdown or miR-29a-3p upregulation in macrophages had opposite effects. Moreover, it was revealed that H19/miR-29a-3p/SNIP1/c-myc regulatory axis is involved in pulmonary fibrosis induced by Nd2O3. Therefore, this study provides new molecular insights into the mechanism of pulmonary fibrosis by Nd2O3.

Structural modification of naturally occurring phenolics as a strategy for developing cytotoxic molecules towards cancer cells.

Natural products belonging to different chemical classes have been established as a promising source of novel anticancer drugs. Several low-molecular-weight compounds from the classes of monoterpenes, phenylpropanoids, and flavonoids were shown to possess anticancer activities in previous studies. In this work, over 20 semisynthetic derivatives of molecules belonging to these classes, namely thymol, eugenol, and 6-hydroxyflavanone were synthesized and tested for their cytotoxicity against two human cancer cell lines, namely AGS cells (gastric adenocarcinoma) and A549 cells (human lung carcinoma). An initial screening based on viability assessment was performed to identify the most cytotoxic compounds at 100 μM. The results evidenced that two 6-hydroxyflavanone derivatives were the most cytotoxic among the compounds tested, being selected for further studies. These derivatives displayed enhanced toxicity when compared with their natural counterparts. Moreover, the lactate dehydrogenase (LDH) assay showed that the loss of cell viability was not accompanied by a loss of membrane integrity, thus ruling out a necrotic process. Morphological studies with AGS cells demonstrated chromatin condensation compatible with apoptosis, confirmed by the activation of caspase 3/7. Furthermore, a viability assay on a noncancer human embryonic lung fibroblast cell line (MRC-5) confirmed that these two derivatives possess selective anticancer activity.

Baricitinib improves pulmonary fibrosis in mice with rheumatoid arthritis-associated interstitial lung disease by inhibiting the Jak2/Stat3 signaling pathway

ObjectiveThe study explored improvements in pulmonary inflammation and fibrosis in a bovine type II collagen-induced rheumatoid arthritis-associated interstitial lung disease mouse model after treatment with baricitinib and the possible mechanism of action.MethodsA rheumatoid arthritis-associated interstitial lung disease mouse model was established, siRNA Jak2 and lentiviral vectors were transfected with human embryonic lung fibroblast cells. And the levels of relevant proteins in mouse lung tissue and human embryonic lung fibroblasts were detected by Western blotting.ResultsThe levels of JAK2, p-JAK2, p-STAT3, p-SMAD3, SMA, TGFβR2, FN and COL4 were increased in the lung tissues of model mice (P < 0.5) and decreased after baricitinib intervention (P < 0.05). The expression levels of p-STAT3, p-SMAD3, SMA, TGFβR2, FN and COL4 were reduced after siRNA downregulation of the JAK2 gene (P < 0.01) and increased after lentiviral overexpression of the JAK2 gene (P < 0.01).ConclusionBaricitinib alleviated fibrosis in the lung tissue of rheumatoid arthritis-associated interstitial lung disease mice, and the mechanism of action may involve the downregulation of Smad3 expression via inhibition of the Jak2/Stat3 signaling pathway, with consequent inhibition of the profibrotic effect of transforming growth factor-β1.

[Identification the key factor of pulmonary fibrosis following silica nanoparticles exposure based on bioinformatics analysis].

Objective: To investigate the main mechanisms of pulmonary fibrosis following silica nanoparticles (SiNPs) exposure through constructing the macrophage-fibroblast model in vitro, which simulated the process of pulmonary fibrosis. Methods: In January 2021, human mononuclear leukemia cells (THP-1) were treated with 0, 25, 50, 100 μg/ml SiNPs for 24 h. The supernatant of THP-1 cells was collected and applied to human embryonic lung fibroblast cells (MRC-5) which divided into control and low, medium and high dose groups at the logarithmic growth stage for 24 h. MRC-5 cell viability was detected by CCK8. The hydroxyproline (Hyp), interleukin 6 (IL-6), interleukin 1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) expression were detected in the supernatants of MRC-5. The changed proteins were detected by liquid-phase mass spectrometry in high dose group. GeneCard database were applied to identity the differential pulmonary fibrosis proteins in high dose group. Gene Ontology (GO) was performed to identity the key biological process in differential pulmonary fibrosis proteins of high dose group. The String database was used to construct the protein-protein interactions (PPI) network of differential pulmonary fibrosis proteins. The APP of CytoHubba was applied to calculate the key protein of differential pulmonary fibrosis proteins in PPI network. Correlation coefficients between key differential pulmonary fibrosis proteins were calculated using Pearson correlation analysis. Western blotting was applied to detect the expression of key proteins of differential pulmonary fibrosis proteins in different groups. Results: CCK8 results showed that MRC-5 cell viability was increasing in low, medium and high dose groups compared with control group (P<0.05). The expression levels of Hyp and IL-1β in different group were increased compared with control group, the expression levels of IL-6 and TNF-α were increased in high dose group compared with control group (P<0.05). GeneCard database identified 26 differential pulmonary fibrosis proteins, which were mainly involved in extracellular matrix hydrolysis, cell inflammatory response, tissue repair, cell proliferation, inflammation response by GO analysis. The APP of CytoHubba was calculated that matrix metalloproteinase 9 (MMP9) and tissue inhibitor metalloproteinase 1 (TIMP1) played an important role in PPI network. The results of correlation analysis showed that MMP9 was correlated with the expression of matrix metalloproteinase 1 (MMP1), matrix metalloproteinase 3 (MMP3), TIMP1 and epidermal growth factor receptor (EGFR) (r=0.97, 0.98, 0.94, 0.93, P<0.05). Western blotting results showed that TIMP1 protein expression was increased in low, medium and high dose groups, while MMP9 protein expression was increased only in high dose group (P<0.05) . Conclusion: Differential expression proteins related with pulmonary fibrosis in MRC-5 cells mainly regulate biological processes of extracellular matrix hydrolysis, tissue repair, and cellular inflammation response following SiNPs exposure. MMP9 and TIMP1 may be the key proteins, which affected the fibrosis process in vitro pulmonary fibrosis model.

Isolation of the Lanostane Triterpenes Pholiols L-S from Pholiota populnea and Evaluation of Their Antiproliferative and Cytotoxic Activities.

Pholiols L-S (1−8), eight undescribed triterpenes were isolated from the sporocarps of the mushroom Pholiota populnea. Various chromatographic techniques, such as open column chromatography, flash chromatography, gel filtration, preparative thin layer chromatography, and HPLC, were applied to purify the compounds. The structure elucidation was carried out by spectroscopic analysis, including 1D (1H NMR and 13C JMOD) and 2D NMR (1H-1H COSY, HSQC, HMBC and NOESY) and HRESIMS experiments. The isolated compounds had lanostane (1−7) or trinorlanostane (8) skeletons; all of them were substituted with 3-hydroxy-3-methylglutaroyl group or its 6-methyl ester. Five compounds (1, 2, 4, 6, and 8) were investigated for their antiproliferative and cytotoxic activity in vitro by MTT assay on breast cancer (MCF-7), human colon adenocarcinoma (sensitive Colo 205, and resistant Colo 320), non-small cell lung cancer (A549), and human embryonic lung fibroblast (MRC-5) cell lines. Pholiols M (2) and O (4) showed antiproliferative activity against the MCF-7 cell line with IC50 of 2.48 and 9.95 µM, respectively. These compounds displayed tumor cell selectivity on MCF-7 cells with SI values of >40 (2) and 4.3 (4), but they did not show a cytotoxic effect, proving their action exclusively on tumor cell proliferation. Pholiols L (1) and Q (8) were found to have selective cytotoxicity on drug resistant cells in comparison to their effects on Colo320 and Colo205 cells [relative resistance values 0.84 (1) and 0.62 (8)].

ETS1 acts as a regulator of human healthy aging via decreasing ribosomal activity.

Adaptation to reduced energy production during aging is a fundamental issue for maintaining healthspan or prolonging life span. Currently, however, the underlying mechanism in long-lived people remains poorly understood. Here, we analyzed transcriptomes of 193 long-lived individuals (LLIs) and 86 spouses of their children from two independent Chinese longevity cohorts and found that the ribosome pathway was significantly down-regulated in LLIs. We found that the down-regulation is likely controlled by ETS1 (ETS proto-oncogene 1), a transcription factor down-regulated in LLIs and positively coexpressed with most ribosomal protein genes (RPGs). Functional assays showed that ETS1 can bind to RPG promoters, while ETS1 knockdown reduces RPG expression and alleviates cellular senescence in human dermal fibroblast (HDF) and embryonic lung fibroblast (IMR-90) cells. As protein synthesis/turnover in ribosomes is an energy-intensive cellular process, the decline in ribosomal biogenesis governed by ETS1 in certain female LLIs may serve as an alternative mechanism to achieve energy-saving and healthy aging.

Human cytomegalovirus autophagy is related to the interferon synthesis and mTOR signal pathway

Introduction: Human cytomegalovirus (HCMV) is reported to be involved in the occurrence of many human diseases. To further investigate the biological changes of HCMV, we analyzed the relevant factors that affect the autophagy caused by HCMV infection. Methods: Firstly, we cultured human embryonic lung fibroblasts (HELF) cells with HCMV infection, and evaluated the effects of HELF cells infected with different viruses through Enzyme-linked immunoabsorbent assay (ELISA), Real-time quantitative Polymerase Chain Reaction (RT-qPCR), Acridine orange (AO) staining and Western blotting (WB) experiments. Results: Through the above experiments, we found that the combined treatment of HCMV infection and carbamazepine, rapamycin and si-mTOR promoted the increase of interferon (IFN)-α/β expression and protein level, and caused the increase of LC3B protein level in HELF cells. In addition, HCMV infection could also affect the biological activities of HELF cells by regulating signal pathways like the JAK/STAT. Conclusion: Autophagy induced by HCMV is affected by the changes in the biological behavior of HELF cells, especially IFN-α/β synthesis and mTOR signal pathway. These findings might shed new light on HCMV-related disease treatment.

In Vitro Evaluation of DNA Damage Effect Markers toward Five Nitrogen Mustards Based on Liquid Chromatography-Tandem Mass Spectrometry.

Endogenous DNA lesions frequently occur due to internal effects such as oxidative stress, inflammation, endogenous alkylation, and epigenetic modifications. However, exposure to chemical toxicants from the environment, diet, or drugs can also induce significant endogenous DNA damage. The quantification of endogenous DNA damage effect markers might reflect the actual DNA damage level of chemical toxicants. Herein, we report a liquid chromatography-triple quadrupole tandem mass spectrometry (LC-QqQ MS/MS) method for simultaneous determination of eight representative endogenous DNA damage biomarkers, including five endogenous DNA damage effect markers (oxidative damage, 8-oxo-dG; lipid peroxidation, εdA and N2-Et-dG; inflammation, 5-Cl-dC; and endogenous alkylation, O6-Me-dG), and three epigenetic modifications (5-m-dC, 5-hm-dC, and N6-Me-dA). The method validation was performed, and the linear range was 0.05 pg to 2 ng (on-column), the limit of detection was 0.02 pg (on-column), and the precision, accuracy, matrix effect, and recovery were all between 85 and 115%. We then applied this method to evaluate endogenous DNA damage to human embryonic lung fibroblast cells exposed to five nitrogen mustards [NMs, i.e., HN1, HN2, HN3, chlorambucil (CB), and cyclophosphamide (CTX)], where curcumin exposure was used as a control due to its inability to induce the formation of endogenous DNA adducts. The amounts of eight DNA adducts in the low-, middle-, and high-concentration exposure groups of five NMs were almost all significantly different from those in the blank group (P < 0.05). We obtained a positive correlation between the contents of eight DNA damage biomarkers and the inhibition dose of five NMs, except for N2-Et-dG and 5-Cl-dC. Via further principal component analysis and partial least squares discriminant analysis, we clustered all NMs into three units with different cytotoxicity levels, that is, HN2 and HN1 (highly toxic), HN3 and CB (moderately toxic), and CTX (less toxic). Moreover, for the same concentration of HN1/2/3 exposure groups, as the cytotoxicity increased according to the order of HN3 < HN1 < HN2, the contents of 8-oxo-dG, 5-m-dC, 5-hm-dC, and N6-Me-dA increased, whereas the content of O6-Me-dG decreased. Therefore, the contents of these DNA damage effect markers were somewhat related to the cytotoxicity and concentration of NMs. We hope that this method will provide an alternative evaluation approach for the toxicological effects of NMs and the safety of the medication.

Approach to the Connection between Meconium Consistency and Adverse Neonatal Outcomes: A Retrospective Clinical Review and Prospective In Vitro Study.

Whether meconium-stained amniotic fluid (MSAF) serves as an indicator of fetal distress is under debate; however, the presence of MSAF concerns both obstetricians and pediatricians because meconium aspiration is a major contributor to neonatal morbidity and mortality, even with appropriate treatment. The present study suggested that thick meconium in infants might be associated with poor outcomes compared with thin meconium based on chart reviews. In addition, cell survival assays following the incubation of various meconium concentrations with monolayers of human epithelial and embryonic lung fibroblast cell lines were consistent with the results obtained from chart reviews. Exposure to meconium resulted in the significant release of nitrite from A549 and HEL299 cells. Medicinal agents, including dexamethasone, L-Nω-nitro-arginine methylester (L-NAME), and NS-398 significantly reduced the meconium-induced release of nitrite. These results support the hypothesis that thick meconium is a risk factor for neonates who require resuscitation, and inflammation appears to serve as the primary mechanism for meconium-associated lung injury. A better understanding of the relationship between nitrite and inflammation could result in the development of promising treatments for meconium aspiration syndrome (MAS).

DNA aptamer S11e recognizes fibrosarcoma and acts as a tumor suppressor

Fibrosarcoma is a serious malignant mesenchymal tumor with strong invasiveness, high recurrence, and poor prognosis. Currently, surgical resection is the main treatment for fibrosarcoma. However, due to the lack of specific biomarkers, the inability to accurately diagnose fibrosarcoma can lead to sub-optimal surgical outcomes and decreased survival. Here, we seek to address this translational barrier and we show that DNA aptamer S11e was able to recognize fibrosarcoma cells (HT1080) but not human embryonic lung fibroblast cells with Kd values in the nanomolar range. In addition, we found that S11e discerned tumors in HT1080 xenograft mouse models and tumor tissues from fibrosarcoma patients. Furthermore, we demonstrated that S11e internalized into HT1080 cells independent of the lysosome pathway and located in mitochondria. Moreover, we revealed that S11e promoted the apoptosis of HT1080 cells and inhibited HT1080 cell migration. Finally, we investigated the biologically functional cellular target of S11e using a mass spectrometry approach, and identified that Diablo/SMAC protein is a cellular binding protein of S11e, by interacting to which S11e inhibited HT1080 cell migration and invasion. Taken together, these results provide the evidence that S11e may be useful for early diagnosis, targeted therapy, and prognostication of fibrosarcoma.

Study on the Effects of miR-490-3p on the Malignant Biological Behavior of Lung Tumor Cell A549 and Its Molecular Mechanism

The objective of this research was to prove whether miR-490-3p regulated the propagation, migration, and invasion of lung tumor cells using targeting sequence similarity as family 83F (FAM83F). The gene expression of miR-490-3p and FAM83F of human embryonic lung fibroblasts (HELF) and lung cancer cells (A549, H1299, 95D) were analyzed using real-time quantitative reverse transcription-polymerase chain reaction PCR (RT-qPCR) and western blot. Analysis of the targeted relationship of miR-490-3P with FAM83F was performed through double luciferase reporting assays with the addition of western blotting. The impacts of miR-490-3p and FAM83F gene expression were tested by cell counting kits (CCK-8), a scraping healing test, and a transwell test on the proliferation activity, scratch healing rate, and the numbers of migrating and invading A549 cells. Impacts of miR-490-3P and FAM83F expressions on the phosphorylated protein kinase B’ (p-AKT) expression were tested with western blotting. miR-490-3P gene expressions in A549 were observably less expressed compared to that of HELF cells, but the mRNA and protein expressions of FAM83F were observably greater than those in HELF (P &lt; 0.05), which indicates that miR-490-3p-bound FAM83F regulates the expression of FAM83F immediately and negatively. After up-regulating miR-490-3p expression or down-regulating FAM83F expression, the proliferation activity, rate of wound-healing, the numbers of migrating and invading cells, and p-AKT protein expression of A549 cells were observably reduced (P &lt; 0.05). Compared to upregulation of miR-490-3p, the propagation activity, wound-healing rate, numbers of migrating and invading cells, and expression of the P-Akt protein of A549 cells were significantly decreased after the upregulation of miR-490-3p and FAM83F (P &lt; 0.05). MiR-490-3p observably inhibited migration and invasion in lung cancer cells with negatively regulating expression of FAM83F, and the mechanisms might be relevant to restraining the AKT pathway.

Increased antibacterial properties of indoline-derived phenolic Mannich bases.

The search for antibacterial agents for the combat of nosocomial infections is a timely problem, as antibiotic-resistant bacteria continue to thrive. The effect of indoline substituents on the antibacterial properties of aminoalkylphenols was studied, leading to the development of a library of compounds with minimum inhibitory concentrations (MICs) as low as 1.18μM. Two novel aminoalkylphenols were identified as particularly promising, after MIC and minimum bactericidal concentrations (MBC) determination against a panel of reference strain Gram-positive bacteria, and further confirmed against 40 clinical isolates (Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Enterococcus faecium, and Listeria monocytogenes). The same two aminoalkylphenols displayed low toxicity against two invivo models (Artemia salina brine shrimp and Saccharomyces cerevisiae). The invitro cytotoxicity evaluation (on human keratinocytes and human embryonic lung fibroblast cell lines) of the same compounds was also carried out. They demonstrated a particularly toxic effect on the fibroblast cell lines, with IC50 in the 1.7-5.1μM range, thus narrowing their clinical use. The desired increase in the antibacterial properties of the aminoalkylphenols, particularly indoline-derived phenolic Mannich bases, was reached by introducing an additional nitro group in the indolinyl substituent or by the replacement of a methyl by a bioisosteric trifluoromethyl substituent in the benzyl group introduced through use of boronic acids in the Petasis borono-Mannich reaction. Notably, the introduction of an additional nitro moiety did not confer added toxicity to the aminoalkylphenols.

[Role of PTEN in FAK and α-SMA protein levels changes in human embryonic lung fibroblasts induced by silica].

To investigate the roles of phosphatase and tensin homolog deleted on chromosome 10(PTEN) in focal adhesion kinase(FAK) and α-smooth muscle actin(α-SMA) protein levels changes in human embryonic lung fibroblasts(HELFs) induced by silica. The HELF cells were cultured in low serum medium containing 0, 25, 50, 100 and 200 μg/cm~2 silica for 24 hours, and the cell counting kit-8(CCK8) experiment was used to determine the appropriate dose of silica for stimulation. Meanwhile, the effect of different doses of silica on the morphology of HELFs was observed under inverted microscope. 50 μg/cm~2 silica solution was used to culture HELFs for 0, 1, 2, 4, 8, 12 and 24 hours(h), and the control group were used as the control group. In addition, Western blot was used to detect HELFs PTEN, p-PTEN, FAK, p-FAK and α-SMA protein levels at each culture time. Besides, HELFs were cultured with 2×10~(-3) mol/L PTEN inhibitor(VO-Ohpic) and/or silica for 24 h, including HELFs group, HELFs plus silica group, and HELFs plus silica plus VO-Ohpic group, and the FAK, p-FAK and α-SMA in each group were also detected by Western blot. With the increase of silica dose, HELFs viability firstly increased and then decreased, and the cell viability of 50 μg/cm~2 group(144. 91±5. 10) was significantly higher than that of 0 μg/cm~2 group(101. 23±6. 57)(P&lt;0. 05). Compared with the control group of silica treated HELFs, the expression levels of PTEN and p-PTEN at 12 h and 24 h were significantly decreased(PTEN: 0. 44±0. 08 at 12 h, 0. 25±0. 02 at 24 h; p-PTEN: 0. 09±0. 01 at 12 h, 0. 01±0. 00 at 24 h; all P values&lt;0. 05); whereas, FAK at 12 h(0. 92±0. 05) and 24 h(0. 89±0. 01), and p-FAK(0. 77±0. 02) and α-SMA at 24 h(1. 32±0. 01) were significantly increased(all P values&lt;0. 05). The expression levels of FAK(0. 25±0. 03), p-FAK(0. 40±0. 02) and α-SMA(0. 36±0. 01) of HELFs plus silica plus VO-Ohpic group were significantly higher than those of HELFs plus silica group(P&lt;0. 05). While silica induces HELFs FAK, p-FAK, and α-SMA increase, PTEN may downregulate FAK, p-FAK and α-SMA expression levels.

SiO2 stimulates macrophage stress to induce the transformation of lung fibroblasts into myofibroblasts and its relationship with the sphingomyelin metabolic pathway.

Silicosis is a serious occupational disease with the highest incidence in China. However, its pathogenesis has not been fully elucidated. Studies have shown that the sphingomyelin signaling pathway may play an important role in different fibrotic diseases but its role in silicosis-mediated fibrosis is still unclear. In this study, the supernatant of human peripheral blood mononuclear cell line (THP-1)-derived macrophages exposed to silica (SiO2 ) was used to stimulate the transformation of human embryonic lung fibroblast cell line (HFL-1) into myofibroblasts, and the intervention effect of recombinant human acid ceramidase (rAC) was observed. The results showed that SiO2 stimulated the production of reactive oxygen species and malondialdehyde in the supernatant of THP-1-derived macrophages and increased the secretion of TGF-β1, TNF-α, and IL-8. In addition, we found that the expression levels of α-SMA, FN, Col I, and Col III in HFL-1 cells increased. Meanwhile, the activities of ASMase and ACase and the expression levels of Cer, Sph, and S1P were increased. Intervention by rAC can suppress these changes to different degrees. In conclusion, the present study shows that SiO2 dust poisoning may stimulate HFL-1 cell differentiation into myofibroblasts by inducing oxidative stress in THP-1-derived macrophages, thereby promoting the secretion of a variety of inflammatory factors and activating the sphingolipid signaling pathway in HFL-1 cells. Exogenous rAC can effectively interfere with the stimulation of HFL-1 cells by silica in vitro.

Neoagarotetraose extends the lifespan of Caenorhabditis elegans through AMPK mediated signaling pathways and activation of autophagy

Neoagarotetraose (NAT) has been reported to ameliorate aging-associated diseases. The present study found that NAT extended the lifespan of Caenorhabditis (C.) elegans up to 21.9% and improved resistance to stresses of heat and oxidation. Moreover, using a series of C. elegans mutants, insulin/insulin-like signaling (IIS) was found to be involved in NAT mediated lifespan extension. Meanwhile, the extended lifespan by NAT was in part via AMP-activated protein kinase (AMPK), which further enhanced the activity of DAF-16. NAT could lower ATP levels and act as a mimic of dietary restriction (DR) as evidenced by the involvement of nutrient sensing pathways, such as IIS, AMPK, and target of rapamycin (TOR). Moreover, NAT induced autophagy both in worms and in human embryonic lung fibroblast cells. Taken together, NAT could be potentially used as a DR mimic centered on AMPK. The results from this study provide new evidence for anti-aging effect of NAT in C. elegnas and insights into implication of anti-aging activity of NAT in higher organisms.

Apigenin Alleviates Oxidative Stress-Induced Cellular Senescence via Modulation of the SIRT1-NAD+-CD38 Axis

Oxidative stress-induced cellular senescence is now regarded as an important driving mechanism in chronic lung diseases-particularly chronic obstructive pulmonary disease (COPD). 4',5,7-trihydroxyflavone (Apigenin) is a natural flavonoid product abundantly present in fruits, vegetables, and Chinese medicinal herbs. It has been known that apigenin has anti-oxidant, anti-inflammatory and liver-protecting effects. The efficacy of apigenin for lung aging, however, has not been reported. In this study, we selected the hydrogen peroxide (H2O[Formula: see text]- or doxorubicin (DOXO)-induced senescence model in WI-38 human embryonic lung fibroblast cells to determine the potential anti-aging effects of apigenin in vitro and associated molecular mechanisms. We found that apigenin reduced senescence-associated [Formula: see text]-galactosidase (SA-[Formula: see text]-gal) activity and promoted cell growth, concomitant with a decrease in levels of Acetyl (ac)-p53, p21[Formula: see text], and p16[Formula: see text] and an increase in phospho (p)-Rb. Apigenin also increased the activation ratio of silent information regulator 1 (SIRT1), nicotinamide adenine dinucleotide (NAD[Formula: see text], and NAD[Formula: see text]/NADH and inhibited cluster of differentiation 38 (CD38) activity in a concentration-dependent manner. SIRT1 inhibition by SIRT1 siRNA abolished the anti-aging effect of apigenin. In addition, CD38 inhibition by CD38 siRNA or apigenin increased the SIRT1 level and reduced H2O2-induced senescence. Our findings suggest that apigenin is a promising phytochemical for reducing the impact of senescent cells in age-related lung diseases such as COPD.

In vitro anticancer activity of Eclipta alba whole plant extract on colon cancer cell HCT-116

BackgroundsColon cancer is the third most deadly and one of the most diagnosed diseases in the world. Although routine screening and early detection during last decades has improved the survival, colon cancer still claims hundreds of thousands lives each year worldwide. Surgery and chemotherapy is mainstay of current treatment, nevertheless toxicity associated with this treatment underscores the urgency of demand of a better therapeutics. Close to 50% of current chemotherapeutic drugs are direct or indirect descendants compounds isolated from medicinal plants, which indicate plants are great potential sources of novel therapeutics. In our literature review we found Eclipta alba to posses many pharmacological activities, including those with anticancer potentials. However, no study on anticancer activity of this kind has been reported.MethodsPhytochemicals were extracted by maceration method from shade dried whole plant of Eclipta alba using methanol as a solvent. The anticancer effect of extract was investigated on various cancer cell lines like human colorectal carcinoma (HCT-116), human prostate cancer (PC-3), Michigan cancer foundation-breast cancer (MCF-7) and renal cell carcinoma (RCC-45). We have also studied the effects on normal human embryonic lung fibroblast cell (WI-38) using MTT (methyl thiazoldiphenyltetrazolium bromide) assay, clonogenic (colony formation) and migration assay. Finally obtained results were analyzed using ANNOVA and Dunnett’s test.ResultsResults obtained from MTT assay revealed that the methanolic extract of Eclipta alba carried significant (p < 0.005) specificity against HCT-116 cells as compared to the other cancer cells. This extract also showed minimal or nontoxicity to WI-38 cells. Migration as well as clonogenic assays also confirmed the anticancer potential of the extract against HCT-116 cells.ConclusionThis is a unique finding of its kind because the specific anticancer effect with minimal toxicity on normal cells has not been reported on Eclipta alba extract. Finally this finding opens up a great possibility to develop a novel antitumor drug candidate against deadly colon cancer in the future.

Thalidomide inhibits the gene promoter of connective tissue growth factor in human embryonic lung fibroblasts.

The etiology and pathogenesis of idiopathic pulmonary fibrosis (IPF) remain unclear, and the early detection and treatment are vital to the prognosis of IPF patients. It's necessary to investigate the effect of thalidomide on the gene promoter activation of connective tissue growth factor (CTGF) induced by transforming growth factor-β1 (TGF-β1) in human embryonic lung fibroblast (HELF). The gene vector of pGL3-CTGFP containing human CTGF gene promoter was constructed and transfected into HELF cells. We used different TGF-β1 (0, 2.5, 5, 10 and 20 µg/L respectively) to stimulate HELF to identify the optimal concentration for gene promoter of CTGF. The activity of luciferase was measured to observe the effect of TGF-β1 and THALIDOMIDE on the activity of CTGF gene promoter. The relative luciferase activity increased significantly with the stimulation of TGF-β1, and the relative luciferase activity peaked in the 5 µg/L TGF-β1 group (all P<0.01). The thalidomide inhibited the TGF-β1-induced activation of CTGF gene promoter in HELF, and the effect peaked in the 25 µg/L group (all P<0.001). Thalidomide produces a significant inhibitory effect on the gene promoter activation of CTGF induced by TGF-β1 in HELF, it may be a potentially effective drug for the treatment of pulmonary fibrosis.