Bufotalin attenuates pulmonary fibrosis via inhibiting Akt/GSK-3ß/ß-catenin signaling pathway.

Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial lung disease with no cure. Bufotalin (BT), an active component extracted from Venenum Bufonis, has been prescribed as a treatment for chronic inflammatory diseases. However, whether BT has antifibrotic properties has never been investigated. In this study, we report on the potential therapeutic effect and mechanism of BT on IPF. BT was shown to attenuate lung injury, inflammation, and fibrosis as well as preserve pulmonary function in bleomycin (BLM)-induced pulmonary fibrosis model. We next confirmed BT's ability to inhibit TGF-ß1-induced epithelial-mesenchymal transition (EMT) and myofibroblast activation (including differentiation, proliferation, migration, and extracellular matrix production) in vitro. Furthermore, transcriptional profile analysis indicated the Wnt signaling pathway as a potential target of BT. Mechanistically, BT effectively prevented ß-catenin from translocating into the nucleus to activate transcription of profibrotic genes. This was achieved by blunting TGF-ß1-induced increases in phosphorylated Akt Ser437 (p-Akt S437) and phosphorylated glycogen synthase kinase (GSK)-3ß Ser9 (p-GSK-3ß S9), thereby reactivating GSK-3ß. Additionally, the antifibrotic effects of BT were further validated in another in vivo model of radiation-induced pulmonary fibrosis. Collectively, these data demonstrated the potent antifibrotic actions of BT through inhibition of Akt/GSK-3ß/ß-catenin axis downstream of TGF-ß1. Thus, BT could be a potential option to be further explored in IPF treatment.

CpG-oligodeoxynucleotides may be effective for preventing ionizing radiation induced pulmonary fibrosis.

Pulmonary fibrosis is a serious adverse effect of radiotherapy for thoracic tumor, which is believed to be a process that is tightly regulated by the phenotype of the developing Th response after radiation. Here, we will investigate whether CpG-oligodeoxynucleotides (ODN) prevent radiation-induced pulmonary fibrosis by shifting the imbalance of Th1 and Th2 response and summarizes the possible mechanism. In this study, female C57BL/6 mice were chosen to preform pulmonary fibrosis model, the whole-thorax of mice was exposed to a single radiation dose of 15 Gy. When irradiated mice were administrated with CpG-ODN, forming of pulmonary fibrosis was significantly prevented. Th2-related cytokines (IL-4 and IL-13) expression decreased, Th1 related-cytokine (IFN-γ and IL-12) expression increased. Alveolar macrophage accumulation was reduced in irradiated tissue. Profibrotic cytokine TGF-ß1 expression stayed at lower level. In TGF-ß1-Smad-dependent pathways, TGF-ß1, TßR and phosphor-Smad 2/3 were down regulated, and Smad 7 was up regulated. These suggested that CpG-ODN prevented pulmonary fibrosis after radiation. The mechanism might be associated with reduction of alveolar macrophages accumulation and profibrogenic cytokines secretion TGF-ß1 through stimulating the combination of Th1-promoting and Th2-limiting responses after radiation, and finally inhibited the fibrosis-related downstream TGF-ß1-Smad-dependent pathway.

Protective Effects of Myrtol Standardized Against Radiation-Induced Lung Injury.

BACKGROUND/AIMS: As a major complication after thoracic radiotherapy, radiation-induced lung injury (RILI) has great impact on long term quality of life and could result in fatal respiratory insufficiency The present study was aimed to evaluate the effects of Myrtol standardized on RILI, and to investigate the underlying mechanism. METHODS: A mouse model of radiation-induced lung injury was generated by using thoracic irradiation with a single dose of 16Gy. Mice were orally administrated with Myrtol (25 mg/kg/day) for 4 weeks after irradiation, while prednisone (5 mg/kg/day) was used as a positive control. After then, the body weight and lung coefficient were calculated. The severity of fibrosis was evaluated by observing pulmonary sections after radiation and collagen content in lung tissues was calculated following the hydroxyproline (HYP) assay. Pathological changes were observed in all the groups by using HE staining and Masson staining. The serum levels of TGF-ß1, TNF-α, IL-1ß, IL-6, and PGE2 were also measured with an ELISA assay. Western blot assay was used to measure the impact of Myrtol on AKT and its downstream signaling pathway, including MMP-2 and MMP-9. The levels of Vimentin and α-SMA were evaluated with an immunofluorescence assay. RESULTS: Treatment with Myrtol standardized, but not prednisone, reduced lung coefficient and collagen deposition in lung tissues, while attenuated histological damages induced by irradiation. Myrtol standardized also reduced the production of MDA, while increased the level of SOD. It was also observed that Myrtol standardized inhibited TGF-ß1 and a series of pro-inflammatory cytokines including TNF-α, IL-1ß, IL-6, PGE2. While in prednisone group, even though the early pneumonitis was ameliorated, the collagen disposition remained unchanged in latter times. Immunofluorescence analysis also revealed elevation of vimentin and α-SMA in the alveoli after a single dose of 16Gy. CONCLUSION: The present results suggest Myrtol standardized as an effective agent for attenuating the lung injury induced by irradiation.

Protective effect of CpG-oligodeoxynucleotides against low- and high-LET irradiation.

BACKGROUND/AIMS: CpG-oligodeoxynucleotides (ODNs) are synthetic DNA sequences containing unmethylated cytosine-guanine motifs with potent immunomodulatory effects. Previous reports showed a powerful protective effect of CpG-ODN against the damage induced by low-LET γ-rays. In this study, we explored whether CpG-ODN also protects against the damage induced by high-LET irradiation. Parallel experiments were performed with low-LET irradiation. METHODS: RAW264.7 cells were incubated with 1 µM of CpG-ODN after γ-ray or carbon-beam irradiation. Cell death was then measured by PI/DAPI double staining, cell survival was assessed by colony-formation assays, DNA damage was evaluated by comet assays, cell cycle was monitored by flow cytometry, and the levels of apoptosis-related proteins were detected by western blots. RESULTS: When irradiated cells were treated with the CpG-ODN, cell viability decreased, cell survival increased, DNA damage and G2/M-phase arrest were ameliorated, and apoptosis was inhibited. CONCLUSIONS: The CpG-ODN showed protective effects against low-LET γ-ray and high-LET carbon-beam irradiation. These effects might be associated with the repair of DNA damage and inhibition of apoptosis.

Inhibition of TBK1 attenuates radiation-induced epithelial-mesenchymal transition of A549 human lung cancer cells via activation of GSK-3ß and repression of ZEB1.

Radiotherapy is an effective treatment method for lung cancer, particularly when the disease is at an advanced stage. However, previous researchers have observed that the majority of patients with conventional radiation therapy develop distant metastases and succumb to the disease. Thus, identifying and understanding novel pathways for the development of new therapeutic targets is a major goal in research on pulmonary neoplasms. Recent studies suggest that epithelial-mesenchymal transition (EMT) is the most important contributor to cancer metastasis. Induction of this complex process requires endogenously produced microRNAs; specifically, downregulation of the miRNA-200c causes an induction of EMT. We recently identified the tank-binding kinase-1 (TBK1) as a downstream effector of the miR-200c-driven pathway, but the biological function of TBK1 in EMT remains unknown. In this study, we tested whether TBK1 has a role in radiation-induced EMT and identified associated potential mechanisms. Human alveolar type II epithelial carcinoma A549 cells were irradiated with (60)Co γ-rays. Western blotting revealed a time- and dose-dependent decrease in E-cadherin with a concomitant increase in vimentin after radiation, suggesting that the epithelial cells acquired a mesenchymal-like morphology. TBK1 siRNA significantly inhibited radiation-induced suppression of the epithelial marker E-cadherin and upregulation of the mesenchymal marker vimentin. The invasion and migratory potential of lung cancer cells upon radiation treatment was also reduced by TBK1 knockdown. Furthermore, radiation-induced EMT attenuated by TBK1 depletion was partially dependent on transcriptional factor ZEB1 expression. Finally, we found glycogen synthase kinase-3ß (GSK-3ß) is involved in regulation of radiation-induced EMT by TBK1. Thus, our findings reveal that TBK1 signaling regulates radiation-induced EMT by controlling GSK-3ß phosphorylation and ZEB1 expression. TBK1 may therefore constitute a useful target for treatment of radiotherapy-induced metastasis diseases.

CpG-Oligodeoxynucleotide Treatment Protects against Ionizing Radiation-Induced Intestine Injury.

BACKGROUND: the bone marrow and the intestine are the major sites of ionizing radiation (IR)-induced injury. Our previous study demonstrated that CpG-oligodeoxynucleotide (ODN) treatment mitigated IR-induced bone marrow injury, but its effect on the intestine is not known. In this study, we sought to determine if CpG-ODN have protective effect on IR-induced intestine injury, and if so, to determine the mechanism of its effect. METHODS AND FINDINGS: Mice were treated with CpG-ODN after IR. The body weight and survival were daily monitored for 30 days consecutively after exposure. The number of surviving intestinal crypt was assessed by the microcolony survival assay. The number and the distribution of proliferating cell in crypt were evaluated by TUNEL assay and BrdU assay. The expression of Bcl-2, Bax and caspase-3 in crypt were analyzed by Immunohistochemistry assay. The findings showed that the treatment for irradiated mice with CpG-ODN diminished body weight loss, improved 30 days survival, enhanced intestinal crypts survival and maintained proliferating cell population and regeneration in crypt. The reason might involve that CpG-ODN up-regulated the expression of Bcl-2 protein and down-regulated the expression of Bax protein and caspase-3 protein. CONCLUSION: CpG-ODN was effective in protection of IR-induced intestine injury by enhancing intestinal crypts survival and maintaining proliferating cell population and regeneration in crypt. The mechanism might be that CpG-ODN inhibits proliferating cell apoptosis through regulating the expression of apoptosis-related protein, such as Bax, Bcl-2 and caspase-3.

[Proteomic analysis of proteins related to radiation-induced carcinogenesis].

BACKGROUND & OBJECTIVE: Carcinogenesis is the most serious late effect of radiation, but the mechanism of radiation-induced carcinogenesis remains unknown. This study was to compare the protein expression profiles between radiation-induced cancer cells and normal cells. METHODS: Immortalized human bronchia epithelial cell line BEAS-2B was irradiated by gamma-ray to prepare malignant BR22P50 cells. Protein profiles of BR22P50 cells and normal cell line BNP50 were detected by two-dimensional (2D) electrophoresis. The differentially expressed proteins were identified by mass spectrometry. The protein levels of ENO1, Prx I, Dyrk2, and GPX1 in different phases of radiation-induced carcinogenesis were detected by Western blot. RESULTS: A total of 59 proteins were differentially expressed between BR22 P50 and BNP50 cells. Of the 59 proteins, 14 were only expressed in BR22P50 cells, 15 were only expressed in BNP50, 7 were overexpressed and 23 were lowly expressed in BR22P50 cells. Using MALDI-TOF MS technology, 26 proteins were identified, including enzymes, structure proteins, cell signal proteins, binding proteins, metabolism-related proteins, some unknown functional proteins, and poly-peptides. The expression of ENO1 and Prx I was up-regulated and that of Dyrk2 and GPX1 was down-regulated with the advancement of radiation-induced carcinogenesis. CONCLUSIONS: The proteins related to radiation-induced carcinogenesis are identified by 2D electrophoresis. This study may provide a novel clue to probe the mechanism of radiation-induced carcinogenesis.

[Effects of STAT3 antisense oligonucleotide on proliferation and apoptosis of non-small cell lung cancer cell line A549].

BACKGROUND & OBJECTIVE: Signal transducer and activator of transcription 3 (STAT3) is highly expressed in various human tumor tissues and tumor cell lines, and may be involved in tumor genesis and development. This study was to design effective antisense oligonucleotide targeting STAT3 mRNA, and explore its effect on the proliferation and apoptosis of human non-small cell lung cancer cell line A549. METHODS: Ten sets of antisense sequences targeting STAT3 were designed with RNAstructure4.2 software and STAT3 mRNA total sequences, and transfected respectively into A549 cells (AS group). Cell proliferation inhibition was measured by Cell Count Kit (CCK-8) assay. Cell proliferation and apoptosis were observed under inverted phase contrast microscope. Cell apoptosis was determined by flow cytometry (FCM) with Hoechst33258 staining and Annexin V/PI double staining. The expression of STAT3, p-STAT3, and Bcl-x(L) were detected by Western blot. Cell cycle was detected by FCM. RESULTS: The 10 sets of designed sequences inhibited the proliferation of A549 cells. The inhibition rate of A549 cell proliferation reached 75.46% after transfection of AS10; the higher the concentration of the antisense oligonucleotide was, the heavier the inhibitory effect was displayed (P<0.01). Apoptotic cells were increased after transfection of antisense oligonucleotide. Antisense oligonucleotide induced early apoptosis in A549 cells: the early apoptosis rate was significantly higher in AS group than in control group (11.51% vs. 5.18%, P<0.01). The expression of STAT3, p-STAT3, and Bcl-x(L) were down-regulated after transfection of antisense oligonucleotide. The G1 phase proportion of A549 cells was significantly higher in AS group than in control group (63.96% vs. 44.47%, P<0.01). CONCLUSION: The antisense oligonucleotide sequences targeting STAT3 designed with computer could inhibit the proliferation and induce the apoptosis of A549 cells.