Human Lung Fibroblast Cell Line Research Articles

Aerosol inhalation of total ginsenosides repairs acute lung injury and inhibits pulmonary fibrosis through SMAD2 signaling-mediated mechanism

BackgroundPulmonary fibrosis (PF) is a progressive lung disease caused by previous acute lung injury (ALI), but there is currently no satisfactory therapy available. Aerosol inhalation of medicine is an effective way for treating PF. Total ginsenosides (TG) shows potential for the treatment of ALI and PF, but the effects of inhaled TG remain unclear. PurposeTo determine the therapeutic effects of TG in ALI and PF, to assess the superiority of the inhaled form of TG over the routine form, and to clarify the mechanism of action of inhaled TG. MethodsUltrahigh-performance liquid chromatography coupled with Q-Exactive Orbitrap mass spectrometry (UPLC-QE-MS) was applied to determine the chemoprofile of TG. A mouse model of ALI and PF was established to evaluate the effects of inhaled TG by using bronchoalveolar lavage fluid (BALF) analysis, histopathological observation, hydroxyproline assay, and immunohistochemical analysis. Primary mouse lung fibroblasts (MLF) and human lung fibroblast cell line (HFL1) were applied to determine the in vitro effects and mechanism of TG by using cell viability assay, quantitative real time PCR (qPCR) assay, and western blot (WB) analysis. ResultsThe UPLC-QE-MS results revealed the main types of ginsenosides in TG, including Re (14.15 ± 0.42%), Rd (8.42 ± 0.49%), Rg1 (6.22 ± 0.42%), Rb3 (3.28 ± 0.01%), Rb2 (3.09 ± 0.00%), Rc (2.33 ± 0.01%), Rg2 (2.09 ± 0.04%), Rb1 (1.43 ± 0.24%), and Rf (0.13 ± 0.06%). Inhaled TG, at dosages of 10, 20, and 30 mg/kg significantly alleviated both ALI and PF in mice. Analyses of BALF and HE staining revealed that TG modulated the levels of IFN-γ, IL-1β, and TGF-β1, reduced inflammatory cell infiltration, and restored the alveolar architecture of the lung tissues. Furthermore, HE and Masson's trichrome staining demonstrated that TG markedly decreased fibroblastic foci and collagen fiber deposition, evidenced by the reduction of blue-stained collagen fibers. Hydroxyproline assay and immunohistochemical analyses indicated that TG significantly decreased hydroxyproline level and down-regulated the expression of Col1a1, Col3a1, and α-sma. The inhaled administration of TG demonstrated enhanced efficacy over the oral route when comparable doses were used. Additionally, inhaled TG showed superior safety and therapeutic profiles compared to pirfenidone, as evidenced by a CCK8 assay, which confirmed that TG concentrations ranging from 20 to 120 μg/ml were non-cytotoxic. qPCR and WB analyses revealed that TG, at concentrations of 25, 50, and 100 μg/ml, significantly suppressed the phosphorylation of smad2 induced by TGF-β1 and down-regulated the expression of fibrotic genes and proteins, including α-sma, Col1a1, Col3a1, and FN1, suggesting an anti-fibrotic mechanism mediated by the smad2 signaling pathway. In vitro, TG's safety and efficacy were also found to be superior to those of pirfenidone. ConclusionsThis study demonstrates, for the first time, the therapeutic efficacy of inhaled TG in treating ALI and PF. Inhaled TG effectively inhibits inflammation and reduces collagen deposition, with a particular emphasis on its role in modulating the Smad2 signaling pathway, which is implicated in the anti-fibrotic mechanism of TG. The study also highlights the superiority of inhaled TG over the oral route and its favorable safety profile in comparison to pirfenidone, positioning it as an ideal alternative for ALI and PF therapy.

Plakevulin A induces apoptosis and suppresses IL-6-induced STAT3 activation in HL60 cells

(+)-Plakevulin A (1), an oxylipin isolated from an Okinawan sponge Plakortis sp. inhibits enzymatic inhibition of DNA polymerases (pols) α and δ and exhibits cytotoxicity against murine leukemia (L1210) and human cervix carcinoma (KB) cell lines. However, the half-maximal inhibitory concentration (IC50) value for cytotoxicity significantly differed from those observed for the enzymatic inhibition of pols α and β, indicating the presence of target protein(s) other than pols. This study demonstrated cytotoxicity against human promyelocytic leukemia (HL60), human cervix epithelioid carcinoma (HeLa), mouse calvaria-derived pre-osteoblast (MC3T3-E1), and human normal lung fibroblast (MRC-5) cell lines. This compound had selectivity to cancer cells over normal ones. Among these cell lines, HL60 exhibited the highest sensitivity to (+)-plakevulin A. (+)-Plakevulin A induced DNA fragmentation and caspase-3 activation in HL60 cells, indicating its role in apoptosis induction. Additionally, hydroxysteroid 17-β dehydrogenase 4 (HSD17B4) was isolated from the HL60 lysate as one of its binding proteins through pull-down experiments using its biotinylated derivative and neutravidin-coated beads. Moreover, (+)-plakevulin A suppressed the activation of interleukin 6 (IL-6)-induced signal transducer and activator of transcription 3 (STAT3). Because the knockdown or inhibition of STAT3 induces apoptosis and HSD17B4 regulates STAT3 activation, (+)-plakevulin A may induce apoptosis in HL60 cell lines by suppressing STAT3 activation, potentially by binding to HSD17B4. The present findings provide valuable information for the mechanism of its action.

Synthesis and Antiproliferative Effect of New Alkyne-Tethered Vindoline Hybrids Containing Pharmacophoric Fragments.

In the frame of our diversity-oriented research on multitarget small molecule anticancer agents, utilizing convergent synthetic sequences terminated by Sonogashira coupling reactions, a preliminary selection of representative alkyne-tethered vindoline hybrids was synthesized. The novel hybrids with additional pharmacophoric fragments of well-documented anticancer agents, including FDA-approved tyrosine-kinase inhibitors (imatinib and erlotinib) or ferrocene or chalcone units, were evaluated for their antiproliferative activity on malignant cell lines MDA-MB-231 (triple negative breast cancer), A2780 (ovarian cancer), HeLa (human cervical cancer), and SH-SY5Y (neuroblastoma) as well as on human embryonal lung fibroblast cell line MRC-5, which served as a reference non-malignant cell line for the assessment of the therapeutic window of the tested hybrids. The biological assays identified a trimethoxyphenyl-containing chalcone-vindoline hybrid (36) as a promising lead compound exhibiting submicromolar activity on A2780 cells with a marked therapeutic window.

Synthesis and Antiproliferative Effect of New Alkyne-Tethered Vindoline Hybrids Containing Pharmacophoric Fragments

In the frame of our diversity-oriented research on multitarget small molecule anticancer agents, utilizing convergent synthetic sequences terminated by Sonogashira coupling reactions, a preliminary selection of representative alkyne-tethered vindoline hybrids was synthesized. The novel hybrids with additional pharmacophoric fragments of well-documented anticancer agents, including FDA-approved tyrosine-kinase inhibitors (imatinib and erlotinib) or ferrocene or chalcone units, were evaluated for their antiproliferative activity on malignant cell lines MDA-MB-231 (triple negative breast cancer), A2780 (ovarian cancer), HeLa (human cervical cancer), and SH-SY5Y (neuroblastoma) as well as on human embryonal lung fibroblast cell line MRC-5, which served as a reference non-malignant cell line for the assessment of the therapeutic window of the tested hybrids. The biological assays identified a trimethoxyphenyl-containing chalcone-vindoline hybrid (36) as a promising lead compound exhibiting submicromolar activity on A2780 cells with a marked therapeutic window.

Synthesis and in vitro biological activity of chalcone derivatives as potential antiparasitic agents

Kinetoplastids are a group of flagellated protozoans including medically important parasites of the genus Trypanosoma and Leishmania. The corresponding diseases have afflicted humans for centuries. In an effort to combat kinetoplastid infections, a set of 21 chalcones was synthesized and evaluated for their in vitro anti-protozoal efficacy against Trypanosoma brucei, Trypanosoma brucei rhodesiense, Trypanosoma cruzi, and Leishmania infantum. To ensure safety, these compounds underwent a selectivity evaluation by assessing toxicity against a human lung fibroblast cell line. Compound K4 exhibited remarkable and selective trypanocidal activity against T. b. brucei with IC50 value of 0.31 ± 0.27 µM and T. b. rhodesiense with IC50 value of 0.96 ± 0.86 µM. Compound K9 also showed significant trypanocidal activity against T. b. brucei (IC50: 0.45 ± 0.14 µM) and T. b. rhodesiense (IC50: 0.93 ± 0.51 µM). In both compounds, electron withdrawing groups are appended to the styrenyl moiety.

L-PTMI-co-L-PEI copolymers obtained through copolymerization of cyclic imino ethers and their chain modification: Antibacterial properties and cytotoxicity

Polymeric antimicrobial agents are intensively investigated as potential novel antibiotics. In this work we use copolymerization of 2-propyl-2-oxazine (PrOzi) and 2-methyl-2-oxazoline (MeOx) combined with hydrolysis of the polymer side chains as a tool to obtain various linear polymeric derivatives of polytrimethylenimines (L-PTMI) and polyethylenimine (L-PEI). To obtain well-defined polymers, initially we determined copolymerization rates, and subsequently, we investigated the hydrolytic rates of various repeating units, namely PrOzi, MeOx, and 2-methyl-2-oxazine (MeOzi) in both homo- and copolymers. We observed that the structure of the polymeric main chain significantly influences the rate of acidic hydrolysis. In particular, the protonated L–PEI repeating unit (r.u.) accelerates the hydrolytic cleavage of the amide bond more than the protonated L-PTMI r.u., due to the shorter distance between amine centers.The biological activity of the polymers was assessed against three model microorganisms, namely Escherichia coli, Staphylococcus aureus and Candida albicans, and benchmarked in toxicity test against human lung fibroblast cell line. The antibacterial activity of L-PTMI-co-L–PEI increases monotonically with the increasing content of PTMI r.u., indicating an additive effect of covalently bonded L-PTMI and L-PEI. The antimicrobial activity of the copolymers is almost identical to that of the tested polymer blends in the corresponding ratios.The introduction of up to 40 % of PrOzi r.u. into the polyamine structure of PTMI (PPrOzi–co-L-PTMI copolymer) does not decrease the antimicrobial activity, compared to L–PTMI. The molecule remains active, particularly against Staphylococcus aureus (MIC = 4 μg/mL). At the same time, the presence of amide r.u. reduces toxicity. The highest observable selectivity index can reach values as high as 20 (PPrOzi40%–co-L-PTMI60%).

Factors Affecting the Evaluation of Collagen Deposition and Fibrosis In Vitro.

Immune responses to biomedical implants, wound healing, and diseased tissues often involve collagen deposition by fibroblasts and other stromal cells. Dysregulated collagen deposition can lead to complications, such as biomaterial fibrosis, cardiac fibrosis, desmoplasia, liver fibrosis, and pulmonary fibrosis, which can ultimately result in losses of organ function or failure of biomedical implants. Current in vitro methods to induce collagen deposition include growing the cells under macromolecular crowding conditions or on fibronectin-coated surfaces. However, the majority of these methods have been demonstrated with a single cell line, and the combined impacts of culture conditions and postculture processing on collagen deposition have not been explored in detail. In this work, the effects of macromolecular crowding versus fibronectin coating, fixation with methanol versus fixation with paraformaldehyde, and use of plastic substrates versus glass substrates were evaluated using the WI-38 human lung fibroblast cell line. Fibronectin coating was found to provide enhanced collagen deposition under macromolecular crowding conditions, while a higher plating density led to improved collagen I deposition compared with macromolecular crowding. Collagen deposition was found to be more apparent on plastic substrates than on glass substrates. The effects of primary cells versus cell lines, and mouse cells versus human cells, were evaluated using WI-38 cells, primary human lung fibroblasts, primary human dermal fibroblasts, primary mouse lung fibroblasts, primary mouse dermal fibroblasts, and the L929 mouse fibroblast cell line. Cell lines exhibited enhanced collagen I deposition compared with primary cells. Furthermore, collagen deposition was quantified with picrosirius red staining, and plate-based drug screening through picrosirius red staining of decellularized extracellular matrices was demonstrated. The results of this study provide detailed conditions under which collagen deposition can be induced in vitro in multiple cell types, with applications including material development, development of potential antifibrotic therapies, and mechanistic investigation of disease pathways. Impact Statement This study demonstrated the effects of cell type, biological conditions, fixative, culture substrate, and staining method on in vitro collagen deposition and visualization. Further the utility of plate-based picrosirius red staining of decellularized extracellular matrices for drug screening through collagen quantification was demonstrated. These results should provide clarity and a path forward for researchers who aim to conduct in vitro experiments on collagen deposition.

Effect of Silver-Graphene Oxide-Cobalt Oxide Nanocomposite on Cytotoxic Levels in MRC-5 and HepG2 Cell Lines and Molecular Docking Studies

The cytotoxic properties of cobalt oxide (Co3O4) nanoparticles (NPs), in addition to graphene oxide (GO)-Co3O4 and silver (Ag)-GO-Co3O4 nanocomposites (NCs), were evaluated against both human healthy lung fibroblast (MRC-5) and hepatocellular carcinoma (HepG2) cell lines utilizing the XTT assay. The investigation revealed that synthesized Co3O4 NPs and NCs (GO-Co3O4 and Ag-GO-Co3O4) elicited significant cytotoxic responses in MRC-5 and HepG2 cell lines in a concentration-dependent manner. Through molecular docking analyses, it was observed that all fabricated nanomaterials exhibited DNA recognition via minor groove binding, with molecular affinities ranging from − 4.82 to -11.66 kcal/mol. Furthermore, the docking outcomes illustrated that the angular conformations of GO-Co3O4 and Ag-GO-Co3O4 conferred ‘shape-selective’ characteristics as DNA minor groove binders, leading to heightened cytotoxicity, particularly in the HepG2 cell line compared to the normal MRC-5 cell line.

Design, synthesis, molecular modelling and biological evaluation of novel 6-amino-5-cyano-2-thiopyrimidine derivatives as potent anticancer agents against leukemia and apoptotic inducers

Herein, a novel series of 6-amino-5-cyano-2-thiopyrimidines and condensed pyrimidines analogues were prepared. All the synthesized compounds (1a-c, 2a-c, 3a-c, 4a-r and 5a-c) were evaluated for in vitro anticancer activity by the National Cancer Institute (NCI; MD, USA) against 60 cell lines. Compound 1c showed promising anticancer activity and was selected for the five-dose testing. Results demonstrated that compound 1c possessed broad spectrum anti-cancer activity against the nine cancerous subpanels tested with selectivity ratio ranging from 0.7 to 39 at the GI50 level with high selectivity towards leukaemia. Mechanistic studies showed that Compound 1c showed comparable activity to Duvelisib against PI3Kδ (IC50 = 0.0034 and 0.0025 μM, respectively) and arrested cell cycle at the S phase and displayed significant increase in the early and late apoptosis in HL60 and leukaemia SR cells. The necrosis percentage showed a significant increase from 1.13% to 3.41% in compound 1c treated HL60 cells as well as from 1.51% to 4.72% in compound 1c treated leukaemia SR cells. Also, compound 1c triggered apoptosis by activating caspase 3, Bax, P53 and suppressing Bcl2. Moreover, 1c revealed a good safety profile against human normal lung fibroblast cell line (WI-38 cells). Molecular analysis of Duvelisib and compound 1c in PI3K was performed. Finally, these results suggest that 2-thiopyrimidine derivative 1c might serve as a model for designing novel anticancer drugs in the future.

In Vitro and In Silico Studies on Benzimidazole‐Based Compounds

AbstractColon cancer remains a significant health concern, necessitating the synthesis of novel therapeutic agents. Benzimidazole derivatives have shown promising anticancer potential. In this study, two benzimidazole derivatives were synthesized, and their antiproliferative activities were investigated in vitro against the human colon colorectal cancer cell line and the human normal lung fibroblast cell line. The cell viability revealed that molecule 3 had high antiproliferative activity and inhibited the growth of DLD‐1 cells for 48 h. Furthermore, molecule 3 was found to have more selectivity against the human normal cell line. Computational calculations were performed to predict the molecular properties of molecules using the B3LYP/6‐311++(2d,2p) density functional theory level. The quantum theory of atoms in molecules (QTAIM) and energy decomposition analysis (EDA) were utilized to investigate the nature of the interatomic bonding existing among the studied molecules. The ultraviolet‐visible (UV–vis) results for molecules were calculated using time‐dependent density‐functional theory (TD‐DFT). The chemical reactivity and molecular stability were also investigated by DFT computational analysis. The molecular docking and dynamics simulations were conducted, and their results reveal their concordance with the experimental results. Molecule 3 has better anti‐cancer activity against colon cancer targets (PDB ID: 2HQ6) compared to molecule 2.

Antiviral and cytotoxic activities and chemical profiles of two species of Abies nordmanniana from Türkiye.

Abies is an important genus of the family Pinaceae, with about 50 species found in the highlands of Asia, Europe, North Africa, and North and Central America. The principal aim of the present work was to investigate the chemical content and biological potential of the resin and cone from Abies nordmanniana subsp. bornmulleriana and Abies nordmanniana subsp. equi-trojani, respectively. The flavonoid and phenolic contents of the resin and cones were evaluated using liquid chromatography-high resolution mass spectrometry (LC-HRMS). Additionally, the essential oil and fatty acid compositions were analyzed using gas chromatography-mass spectrometry (GC-MS) and gas chromatography-flame ionization detector (GC-FID), respectively. Cytotoxicity of the extracts and essential oils were screened against certain cancer cell lines, namely, human prostate adenocarcinoma cell line (PC3), human lung adenocarcinoma cell line (A549), human pancreatic cancer cell line (PANC-1), human hepatocellular carcinoma cell line (HepG2), human breast cancer cell line (MDA-MB231), and normal human lung fibroblast cell line (CCD-34-LU), with MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay. According to the MTT results, hexane extracts of both cone (CH) and resin (RH), ethanol-water (CEW), dichloromethane (CD), and acetone (CA) extracts of the cone mostly inflict cytotoxicity in HepG2 cell line. Antiviral activities of Abies nordmanniana subsp. extracts at doses of 5 μg/g and 10 μg/g were also evaluated in ovo for their virucidal activity against avian coronavirus. Abies nordmanniana subsp. extracts exhibited concentration-dependent antiviral activity on specific pathogen-free embryonated chicken eggs. Significantly, cone acetone extract (CA), cone ethanol extract (CE), and cone dichloromethane extract (CD) of Abies nordmanniana subsp. exhibited strong inhibition of the virus at a concentration of 10 μg/g. The most potent virucidal activity was observed with ethanol-water extract of conifer form (CEW). According to these results, it was proved that Abies nordmanniana species could be a potential, sustainable, and renewable drug source, especially considering the impressive antiviral and significant cytotoxic activity potentials.

Copper(II) coordination compounds based on bis-hydrazones of 2,6-diacetylpyridine: synthesis, structure, and cytotoxic activity.

By reacting a series of 2,6-diacetylpyridine bis-hydrazones containing pyrimidine (H2L1), benzimidazole (H2L2) and phthalazine (H2L3) heterocyclic fragments with copper(II) chloride and bromide, a variety of pentacoordinated complexes of the composition [Cu(H2L1)X]X, [Cu(HL2)X] and [Cu(HL3)X], where X = Cl-, Br-, are formed. The properties and structure of the compounds were studied by means of NMR, IR, UV-vis, ESR, and X-ray absorption spectroscopy, cyclic voltammetry and X-Ray single crystal diffraction methods. It was shown that complexes of the cationic type [Cu(H2L1)X]X have an asymmetric structure with a distorted square-pyramidal geometry of the coordination unit. The coordination polyhedron of metal chelates [Cu(HL2)X] and [Cu(HL3)X] is an almost ideal square pyramid. Investigations of the cytotoxic activity of the obtained compounds in vitro on human hepatocellular carcinoma (HepG2) and non-tumor human lung fibroblast (MRC-5) cell lines demonstrated that complexes show higher activity compared with the well-known anticancer agent cisplatin. In addition, metal chelates [Cu(H2L1)Cl]Cl, [Cu(HL2)Cl], [Cu(HL2)Br] and [Cu(HL3)Cl] were less toxic to non-tumor cells MRC-5. A study of the binding of complexes to bovine serum albumin (BSA) protein using fluorescence spectroscopy showed that copper complexes are strongly bound to BSA. To study the mechanism of interaction of the complexes with the DNA of cancer cells, molecular dynamics simulation of the compound [Cu(HL3)Cl] was carried out. It was shown that the complex enters into π-stacking interactions predominantly with adenine and thymine bases.

Synergistic impacts of rifampicin and doxorubicin against thioacetamide-induced hepatocellular carcinoma in rats

Background and aimsCombination therapy is a promising new strategy that has been proposed to increase the efficacy of cancer treatment. We aimed to investigate the anti-cancer activity of rifampicin monotherapy and its combination with doxorubicin against hepatocellular carcinoma (HCC). Materials and methodsThe in vitro half maximal inhibitory concentration (IC50) and selectivity index (SI) of the drugs under investigation against HepG2 and human lung fibroblast (WI38) cell lines were determined. For the in vivo experiment, male Sprague-Dawley albino rats were injected with thioacetamide at 200 mg/kg twice a week for 90 days; HCC development was confirmed histopathologically. Following HCC induction, the rats were treated with intraperitoneal doxorubicin, rifampicin, or their combination for 45 or 90 days. After sacrifice, the livers were examined histopathologically. The levels of aminotransferases, albumin, bilirubin, malondialdehyde, superoxide dismutase (SOD), catalase (CAT), total antioxidant capacity (TAC), and nitric oxide were measured by spectrophotometry. Alpha-fetoprotein, cancer antigen 19-9, tumor necrosis factor-alpha, interleukin-6, Bcl-2-associated X protein, caspase 3, caspase 8, and p53 were estimated using ELISA. ResultsIn vitro, the combination of doxorubicin and rifampicin showed the highest SI of 3.43. In vivo, among the measured markers, the levels of TAC, CAT, SOD, and p53 decreased (P < 0.001) and the rest of the measured marker levels increased (P < 0.001) in the HCC-bearing rats; after treatment in all groups, all these changes improved toward normal in a time-dependent manner. The combination of doxorubicin and rifampicin optimized the effects of the two individual drugs and exerted the best antioxidant effects. ConclusionsIn general, compared with rifampicin or doxorubicin alone, combination therapy has favorable outcomes. Based on our results, the combination of rifampicin and doxorubicin might be applicable for HCC chemotherapy.

A Pilot Study to Investigate the Antimicrobial Activity of Pulsed UVA and UVC

UV irradiation has shown potential in reducing bacterial and viral loadings. This is a pilot study aimed at investigating the antimicrobial effect of a novel pulsed UVA and UVC technology on bacteria and human coronavirus 229E. The selection of these microorganisms is based on their relevance and significance in real-world scenarios. This study consists of independent experiments for the assessment of antibacterial and antiviral activities by using a lawn plate approach, measuring levels of adenine triphosphate (ATP) in three bacterial strains, Escherichia coli, Staphylococcus epidermidis and Bacillus subtilis, and performing Median Tissue Culture Infectious Dose (TCID50) of HCoV-229E on MRC-5 human lung fibroblast cell line. The results demonstrated the ability of UVA and UVC irradiation to reduce levels of adenine triphosphate (ATP) over a 12 h exposure period in all three bacterial strains, comparative to dark and artificial/natural light conditions using non-pulsing experiments. In addition to this, there was a reduction in colonies exposed to UVA and UVC pulsing experiments for E. coli K12 and S. epidermis compared to bacteria stored in artificial/natural and dark conditions. Furthermore, using dose-dependent modelling, it was demonstrated that the cross-contamination risk was reduced by 50% using E. coli as a typical model. Regarding the antiviral assay, the results showed that TCID50 of HCoV-229E was reduced after the first cycle of UV engagement. No cytopathic effect (CPE) was detected after three cycles using Protocol 1. The findings showed that UVA and UVC were effective under the conditions outlined in this paper for a reduction in the number of bacteria with additional applications to viruses.

MicroRNA-29a-3p Accelerates Inflammatory Damage in Neonatal Pneumonia Via Targeting Krüppel-like Factor 4.

Neonatal pneumonia (NP) is a frequently occurring illness during the neonatal phase. The study investigated the molecular process and the role of microRNA (miR)-29a-3p in NP. Peripheral blood was collected from NP patients and healthy newborns. Human lung fibroblasts cell line (WI-38) were treated with lipopolysaccharide (LPS)) to establish a cellular model for NP. Then, miR-29a-3p and Krüppel-like Factor 4 (KLF4) levels were detected by RT-qPCR or Western blot. The relationship between miR-29a-3p and KLF4 was confirmed by dual luciferase reporter gene assay. Cell survival was assessed using the CCK-8 assay, whereas the levels of interleukin-6, tumor necrosis factor-α, and IL-1β were quantified using ELISA. Additionally, apoptosis was evaluated through flow cytometry. Meanwhile, Bax and Bcl-2 were detected by RT-qPCR. Neonatal rats were administered LPS intraperitoneally (3 mg/kg) to induce NP, and pathological injury and inflammatory reaction were analyzed. MiR-29a-3p was elevated but KLF4 was silenced in NP patient's serum, LPS-treated WI-38 cell line, and LPS-treated newborn rats. Silence of miR-29a-3p or elevation of KLF4 constrained cell proliferation with inflammation of LPS-treated WI-38 cell line. MiR-29a-3p immediately targeted KLF4. Additionally, silence of miR-29a-3p alleviated LPS-stimulated lung injury and inflammation in neonatal rats. The protective action of silenced miR-29a-3p in LPS-treated WI-38 cell line and newborn rats was turned around by silencing KLF4. This study demonstrates originally that miR-29a-3p boosts inflammatory damage in NP via targeting KLF4, offering a basis for clinically diagnosing and treating NP.

SENP1 knockdown-mediated CTCF SUMOylation enhanced its stability and alleviated lipopolysaccharide-evoked inflammatory injury in human lung fibroblasts via regulation of FOXA2 transcription

BackgroundExcessive inflammation is the main cause of treatment failure in neonatal pneumonia (NP). CCCTC-binding factor (CTCF) represents an important node in various inflammatory diseases. In the present study, we tried to clarify the function and underlying molecular mechanism of CTCF on an in vitro cellular model of NP, which was generated by simulating the human lung fibroblast cell line WI-38 with lipopolysaccharide (LPS). MethodsThe SUMOylation level and protein interaction were verified by Co-immunoprecipitation assay. Cell viability was measured by Cell Counting Kit-8 assay. Inflammatory factors were examined by Enzyme-linked immunosorbent assay. Cell apoptosis was evaluated by TUNEL assay. The binding activity of CTCF to target promoter was tested by chromatin immunoprecipitation and luciferase reporter assay. ResultsLPS treatment restrained cell viability, promoted the production of inflammatory factors, and enhanced cell apoptosis. CTCF overexpression played anti-inflammatory and anti-apoptotic roles. Furthermore, CTCF was modified by SUMOylation with small ubiquitin-like modifier protein 1 (SUMO1). Interfering with sumo-specific protease 1 (SENP1) facilitated CTCF SUMOylation and protein stability, thus suppressing LPS-evoked inflammatory and apoptotic injuries. Moreover, CTCF could bind to the forkhead box protein A2 (FOXA2) promoter region to promote FOXA2 expression. The anti-inflammatory and anti-apoptotic roles of CTCF are associated with FOXA2 activation. In addition, SENP1 knockdown increased FOXA2 expression by enhancing the abundance and binding ability of CTCF. ConclusionsSUMOylation of CTCF by SENP1 knockdown enhanced its protein stability and binding ability and it further alleviated LPS-evoked inflammatory injury in human lung fibroblasts by positively regulating FOXA2 transcription.

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.

Eotaxin-1/CCL11 promotes cellular senescence in human-derived fibroblasts through pro-oxidant and pro-inflammatory pathways.

Eotaxin-1/CCL11 is a pivotal chemokine crucial for eosinophil homing to the lungs of asthmatic patients. Recent studies also suggest that CCL11 is involved in the aging process, as it is upregulated in elderly, and correlated with shorter telomere length in leukocytes from asthmatic children. Despite its potential pro-aging effects, the precise contribution of CCL11 and the underlying mechanisms involved in the promotion of cellular senescence remains unclear. Therefore, the primary goal of this study was to explore the role of CCL11 on senescence development and the signaling pathways activated by this chemokine in lung fibroblasts. To investigate the targets potentially modulated by CCL11, we performed an in silico analysis using PseudoCell. We validated in vitro the activation of these targets in the human lung fibroblast cell line MRC-5 following rhCCL11 exposure. Finally, we performed differential gene expression analysis in human airway epithelial cells of asthmatic patients to assess CCL11 signaling and activation of additional senescent markers. Our study revealed that eotaxin-1/CCL11 promote reactive oxygen secretion (ROS) production in lung fibroblasts, accompanied by increased activation of the DNA damage response (DDR) and p-TP53 and γH2AX. These modifications were accompanied by cellular senescence promotion and increased secretion of senescence-associated secretory phenotype inflammatory cytokines IL-6 and IL-8. Furthermore, our data show that airway epithelial lung cells from atopic asthmatic patients overexpress CCL11 along with aging markers such as CDKN2A (p16INK4a) and SERPINE1. These findings provide new insights into the mechanisms underlying the pro-aging effects of CCL11 in the lungs of asthmatic patients. Understanding the role of CCL11 on senescence development may have important implications for the treatment of age-related lung diseases, such as asthma.

Synthesis, spectroscopic characterization and anticancer potential studies of organoruthenium(II) arene dithiocarbamate complexes

Four organoruthenium(II) arene dithiocarbamate complexes: RuCl(piperidine dithiocarbamate)(p-cymene), C1; RuCl(1-phenylpiperazine dithiocarbamate)(p-cymene), C2, RuCl(ethylphenyl dithiocarbamate)(p-cymene), C3; RuCl(4-benzylpiperidine dithiocarbamate)(p-cymene), C4 were synthesized and characterized by elemental analysis, UV-Vis, FTIR, NMR and mass spectroscopic techniques. Spectroscopic data indicate that the complexes are four coordinate tetrahedral geometry consisting of one dithiocarbamato anion, p-cymene and a chlorido ligand around the ruthenium(II) ion. The dithiocarbamato anions coordinate the ruthenium(II) ions isobidentately. The cytotoxic effects of the compounds were assessed against human cervical cancer (HeLa) and human lung fibroblast (MRC5-SV2) cell lines using the 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyl tetrazolium bromide (MTT) assay. Only two compounds, C1 and C3 showed potent cytotoxicity after 48 h treatment. C1 was the most potent, with IC50 values of 6.7 ± 2.3 µM and 8.1 ± 0.8 µM against the HeLa and MRC5-SV2 cells, respectively, while C3 had IC50 values of 11.5 ± 3.1 µM and 10.3 ± 1.3 µM, respectively. Remarkably, compared to cisplatin (used as the reference anticancer drug), C1 was twice more potent against HeLa cells and more than five times more potent against MRC5-SV2 cells, while C3 was nearly equipotent with cisplatin against HeLa cells but five times more potent against MRC5-SV2 cells. These two compounds exhibited good potential as anticancer agents, thus warranting further studies.

Enhanced immune responses in dexamethasone immunosuppressed male rats supplemented with herbal extracts, chitosan nanoparticles, and their conjugates

Nowadays, the world is challenged with highly contagious diseases, one of their preliminary virulence mechanisms is the suppression of innate immunity. Therefore, promoting natural immunity is a good precautionary strategy. we investigated and compared the effects of several natural herbal extracts -Moringa oleifera, Ziziphus spina christi, and Saussurea costus, and chitosan nanoparticles (CS NPs)- as well as conjugated extracts with CS NPs on the immunological parameters of dexamethasone immunosuppressed (IS) male rats. The plant extracts were assessed for total flavonoids, phenolics, and antioxidant activity. The CS NPs and their conjugates were characterized using particles size, zeta potentials, and Fourier-transform infrared spectroscopy analyses. The chemical analysis of the plant extracts, CS NPs, and their conjugates was performed using energy dispersive X-ray fluorescence, and their cytotoxicity was evaluated in human lung fibroblast (WI-38) and human embryonic kidney (HEK-293) cell lines. For in vivo evaluations, 72 adult male rats were divided into 9 groups: control, IS, three plant extracts, CS, and conjugates of the three plant extracts and CS NPs. Oral supplementation (day after day) lasted for 28 days. Liver, kidney, and spleen tissue samples were collected for histopathology and Ki-67 expression analyses. The results revealed that the plant extracts and CS improved the total leukocyte counts, complement 3, complement 4, interferon-gamma, and tumor necrosis factor levels at day 28. However, the plant extract-CS NPs conjugates faster and have higher immunostimulatory effects at day 14. Furthermore, the atrophied white pulp of the spleen induced by dexamethasone was alleviated, and Ki-67 expression was elevated in all the treated groups. Conclusively, the conjugates of Moringa oleifera, Ziziphus spina christi, and Saussurea costus extract with CS NPs demonstrated more potent and rapid immune responses at lower doses and concentrations compared to the plant extracts or CS NPs alone, without causing liver or kidney injuries. Thus, supplementation of these conjugated plant extracts at lower doses and concentrations is recommended to improve immunity while considering safety considerations.