[Effect of irradiated human lung fibroblasts on activation of canonical Wnt/ß-catenin signaling pathway in mesenchymal stem cells].

OBJECTIVE: To investigate the effect of irradiated human lung fibroblasts (HLFs) on the canonical Wnt/ß-catenin signaling pathway in human umbilical cord mesenchymal stem cells (HUMSCs). METHODS: HUMSCs were cultured alone (single group) or co-cultured with HLFs exposed to 5Gy X-rays (co-culture group). The protein levels of GSK-3ß, p-GSK-3ß, FRAT1 and ß-catenin in HUMSCs were examined by Western blotting 3 days after culture or co-culture. WISP-1 protein levels in conditioned medium were examined by ELISA. RESULTS: The levels of p-GSK3ß/GSK3ß (0.15 ± 0.05), FRAT1 (0.48 ± 0.07) and ß-catenin (0.50 ± 0.07) in co-cultured HUMSCs significantly decreased compared to those in single group (0.55 ± 0.05, 1.16 ± 0.13 and 2.39 ± 0.15, all P<0.05). The supernatant level of WISP-1 in co-culture group was significantly decreased [(602.23 ± 161.47) ng/mL], compared to the single group [(977.77 ± 110.56) ng/mL, P<0.05]. CONCLUSION: Irradiated HLFs attenuate the activation of canonical Wnt/ß-catenin signaling pathway in HUMSCs in vitro.

Facile Entry to Pharmaceutically Important 3-Difluoromethyl-quinoxalin-2-ones Enabled by Visible-Light-Driven Difluoromethylation of Quinoxalin-2-ones.

CF2H moiety has a significant potential utility in drug design and discovery, and the incorporation of CF2H into biologically active molecules represents an important and efficient strategy for seeking lead compounds and drug candidates. On the other hand, quinoxalin-2-one is of great interest to pharmaceutical chemists as a common skeleton frequently occurring in plenty of natural products and bioactive compounds. Herein, we reported a practical and efficient protocol for the synthesis of 3-CF2H-quinoxalin-2-ones. Thus, in the presence of 3 mol% of photocatalyst and S-(difluoromethyl)sulfonium salt as difluoromethyl radical sources, a wide range of quinoxalin-2-ones readily underwent a visible-light redox-catalyzed difluoromethylation reaction, to deliver structurally diverse 3-difluoromethyl-quinoxalin-2-ones. We believe that this would facilitate increasing chances and possibilities for seeking potential lead compounds and drug candidates and further boost the development of fluorine-containing pharmaceuticals.

Coculture with bone marrow­derived mesenchymal stem cells attenuates inflammation and apoptosis in lipopolysaccharide­stimulated alveolar epithelial cells via enhanced secretion of keratinocyte growth factor and angiopoietin­1 modulating the Toll­like receptor­4 signal pathway.

Acute lung injury (ALI) is a common, costly and potentially lethal disease with characteristics of alveolar­capillary membrane disruption, pulmonary edema and impaired gas exchange due to increased apoptosis and pulmonary inflammation. There is no effective and specific therapy for ALI; however, mesenchymal stem cells (MSCs) have been demonstrated to be a potential option. Lipopolysaccharide (LPS) is a highly proinflammatory molecule that is used to mimic an in vivo inflammatory and damaged state in vitro. The present study investigated the effect of bone marrow­derived MSCs on an LPS­induced alveolar epithelial cell (A549 cell line) injury and its underlying mechanisms by a Transwell system. It was identified that a high LPS concentration caused a decrease in cell viability, increases in apoptosis, inflammatory cytokine release and NF­κB activity, disruption of the caspase­3/Bcl­2 ratio, upregulation of Toll­like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88) and toll­interleukin­1 receptor domain­containing adaptor inducing interferon (TRIF) expression, and facilitation of TLR4/MyD88 and TLR4/TRIF complex formation in A549 cells. Coculture with MSCs attenuated all of these activities induced by LPS in A549 cells. In addition, an increased level of keratinocyte growth factor (KGF) and angiopoietin­1 (ANGPT1) secretion from MSCs was observed under inflammatory stimulation. KGF and/or ANGPT1 neutralizing antibodies diminished the beneficial effect of MSC conditioned medium. These data suggest that MSCs alleviate inflammatory damage and cellular apoptosis induced by LPS in A549 cells by modulating TLR4 signals. These changes may be partly associated with an increased secretion of KGF and ANGPT1 from MSCs under inflammatory conditions. These data provide the basis for development of MSC­based therapies for ALI.

Paralemmin-3 contributes to lipopolysaccharide-induced inflammatory response and is involved in lipopolysaccharide-Toll-like receptor-4 signaling in alveolar macrophages.

Alveolar macrophages (AMs) are the first line of defense against foreign stimulation in alveoli, and they participate in inflammatory responses during acute lung injury (ALI). Previous studies indicated that paralemmin-3 (PALM3) expression is induced by lipopolysaccharides (LPS) and may be involved in LPS-Toll-like receptor 4 (TLR4) signaling in alveolar epithelial cells. The aim of the present study was to investigate the effect of PALM3 on LPS-induced inflammation and its underlying mechanisms in rat AMs. For this purpose, the authors detected the expression of PALM3 in AMs by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting following LPS stimulation. Following this, a recombinant adenovirus expressing short hairpin RNA (shRNA) for PALM3 was constructed, as well as a recombinant adenovirus carrying the rat PALM3 gene to modulate the expression of PALM3 in rat AMs. At 48 h after transfection, the PALM3 expression in AMs was detected by RT-qPCR and western blotting. The levels of several cytokines and the activity of nuclear factor-κB and interferon regulatory factor 3 in AMs were measured after LPS stimulation. The localization of PALM3 and LPS-TLR4 signaling adaptor molecules in AMs was analyzed by confocal microscopy, and the physical interactions of PALM3 with these adaptors were assessed by co-immunoprecipitation assays. LPS induced PALM3 expression in AMs and that PALM3 expression promoted the LPS-induced inflammatory response, while PALM3 downregulation suppressed the LPS-induced inflammatory response in AMs. In addition, the results demonstrated that PALM3 could interact with TLR4, myeloid differentiation factor 88, interleukin (IL)-1 receptor associated kinase-1, tumor necrosis factor receptor associated factor-6, and Toll-IL-1 receptor containing adapter molecule-2 in AMs after LPS stimulation. These results suggested that PALM3 contributes to the LPS-induced inflammatory response and participates in LPS-TLR4 signaling in AMs. These data may provide the basis for the development of novel targeted therapeutic strategies of treating ALI.

[CIAPIN1 expression in human lung cancer tissues and inhibitory effects of the gene on human pulmonary carcinoma NCI-H446 cells].

AIM: To explore CIAPIN1 gene expression in lung carcinoma tissues and its regulatory in the growth of NCI-H446. METHODS: Fifty-one formalin-fixed paraffin-embedded specimens of primary lung cancer and their non-cancerous counterparts were detected by SABC immunohistochemistry method. Adenoviral vector construction was recombined and the gene was transduced into NCI-H446 cells. The expression of CIAPIN1 protein was identified by Western blot. Trypan blue staining was used to count the alive cells and to draw a cellular growth curve. The changes of cell cycle and apoptosis were analyzed by flow cytometry. RESULTS: The positive rate of CIAPIN1 expression in cancer tissues (39.2%)was much lower than that in non-cancerous counterparts (100%, P<0.05). In Ad-CIAPIN1 group, the growth of transfected NCI-H446 cells in vitro was significantly inhibited. In addition the Ad-CIAPIN1-induced cell apoptosis and a predominant arrest in the G1/S phase (P<0.01) were observed. CONCLUSION: The down-regulation of CIAPIN1 expression in tumor is associated with the development of lung carcinoma. Transduction of NCI-H446 CIAPIN1-negative cell, with Ad-CIAPIN1 can inhibit cell growth, suggesting that CIAPIN1 can be a new tumor-related suppressor gene.