Role of thioredoxin nitration in bleomycin-induced pulmonary fibrosis in rats.

Oxidant stimulation has been suggested to play an important role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Our study aimed to investigate the role and mechanisms of thioredoxin (Trx) nitration during the development of IPF. A rat model of IPF was established by intratracheal instillation of bleomycin (BLM). Male Wistar rats were randomly distributed among the control group and BLM-treated group, in which rats were intratracheally instilled with a single dose of BLM (5.0 mg/kg body mass in 1.0 mL phosphate-buffered saline). At 7 or 28 days after instillation the rats were euthanized. Histopathological and biochemical examinations were performed. The activity and protein level of thioredoxin were assessed. The thioredoxin nitration level was determined using immunoprecipitation and immunoblotting techniques. Our results demonstrated that protein tyrosine nitration increased in the BLM-treated group compared with the control group. Trx activity decreased in the BLM group compared with control group, whereas Trx expression and nitration level increased dramatically in the BLM group compared with the control group. Our results indicated that Trx nitration might be involved in the pathogenesis of IPF.

Chlamydophila (Chlamydia) pneumoniae infection promotes vascular smooth muscle cell adhesion and migration through IQ domain GTPase-activating protein 1.

The mechanisms for Chlamydophila (Chlamydia) pneumoniae (C. pneumoniae) infection-induced atherosclerosis are still unclear. Cell adhesion has important roles in vascular smooth muscle cell (VSMC) migration required in the development of atherosclerosis. However, it is still unknown whether IQ domain GTPase-activating protein 1 (IQGAP1) plays pivotal roles in C. pneumoniae infection-induced the adhesion and migration of rat primary VSMCs. Accordingly, in this study, we demonstrated that rat primary VSMC adhesion (P < 0.001) and migration (P < 0.01) measured by cell adhesion assay and Transwell assay, respectively, were significantly enhanced after C. pneumoniae infection. Reverse transcription-polymerase chain reaction analysis revealed that the mRNA expression levels of IQGAP1 in the infected rat primary VSMCs were found to increase gradually to reach a peak and then decrease gradually to a level similar to the control. We further showed that the increases in rat primary VSMC adhesion to Matrigel (P < 0.001) and migration (P < 0.01) caused by C. pneumoniae infection were markedly inhibited after IQGAP1 knockdown by a pool of four short hairpin RNAs. Taken together, our results suggest that C. pneumoniae infection may promote the adhesion and migration of VSMCs possibly by upregulating the IQGAP1 expression.

Progressive hearing loss in mice lacking the cyclin-dependent kinase inhibitor Ink4d.

Maintenance of the post-mitotic state in the post-natal mammalian brain is an active process that requires the cyclin-dependent kinase inhibitors (CKIs) p19Ink4d (Ink4d) and p27Kip1 (Kip1). In animals with targeted deletions of both Ink4d and Kip1, terminally differentiated, post-mitotic neurons are observed to re-enter the cell cycle, divide and undergo apoptosis. However, when either Ink4d or Kip1 alone are deleted, the post-mitotic state is maintained, suggesting a redundant role for these genes in mature neurons. In the organ of Corti--the auditory sensory epithelium of mammals--sensory hair cells and supporting cells become post-mitotic during embryogenesis and remain quiescent for the life of the animal. When lost as a result of environmental insult or genetic abnormality, hair cells do not regenerate, and this loss is a common cause of deafness in humans. Here, we report that targeted deletion of Ink4d alone is sufficient to disrupt the maintenance of the post-mitotic state of sensory hair cells in post-natal mice. In Ink4d-/- animals, hair cells are observed to aberrantly re-enter the cell cycle and subsequently undergo apoptosis, resulting in progressive hearing loss. Our results identify a novel mechanism underlying a non-syndromic form of progressive hearing loss in mice.

Thymosinß4 alleviates cholestatic liver fibrosis in mice through downregulating PDGF/PDGFR and TGFß/Smad pathways.

Liver fibrosis is an important health problem without adequate and effective therapeutics. In this study, effects of thymosinß4 (Tß4) on hepatic fibrogenesis and the underlying molecular mechanisms were explored in bile duct ligation (BDL)-induced mice cholestatic liver fibrosis model. Results showed exogenous Tß4 significantly reduced the mortality and liver/body weight ratio in BDL mice. Histological examinations and biochemical analyses demonstrated that BDL induced evident portal fibrosis and a significant increase in hepatic collagen contents. However, these changes were significantly attenuated by exogenous Tß4. Quantitative real-time PCR assays showed that Tß4 suppressed BDL-induced increases in many fibrotic genes expression including α-smooth muscle actin (α-SMA), collagen I, III and fibronectin, TGFß1, TGFßR II, Smad2, Smad3, and PDGFRß. Results from immunohistochemistry and Western blots also showed that Tß4 reduced TGFß1 and PDGFRß protein levels in the liver tissues of BDL mice. In vitro studies using LX-2 cells demonstrated that Tß4 could decrease PDGFRß and TGFßR II levels in hepatic stellate cells. Taken together, findings in our present studies suggested that exogenous Tß4 alleviated BDL-induced cholestatic liver fibrosis through downregulating PDGF/PDGFR and TGFß/Smad pathways.

3-methyadenine attenuates chloroform-induced hepatotoxicity via autophagy activation.

Chloroform is a common contaminant in the drinking water. Exposure of human to chloroform leads to severe hepatotoxicity. In the present study, chloroform-induced acute liver injury was investigated in mice using 3-methyadenine (3-MA), a common autophagy inhibitor. At 24 h after intraperitoneal injection of 0.5 mL/kg chloroform, serum alanine aminotransferase (ALT) levels were increased significantly; extensive necrosis and inflammation occurred as identified by histological examinations. Moreover, chloroform induced an increase in lipid peroxidation as demonstrated by increased formation of malondialdehyde (MDA) in the liver tissues. Hepatic antioxidants including glutathione (GSH) and superoxide dismutase (SOD) were decreased by chloroform treatment. All these changes were significantly inhibited by 3-MA treatment. Further mechanistic insights demonstrated that chloroform up-regulated pro-inflammatory cytokine, IL-1ß, in the livers and blood, which was suppressed by 3-MA. Surprisingly, Western blots results showed that after 24-hours of chloroform treatment 3-MA activated autophagy as indicated by decreased levels of LC3B II and p62 protein. Co-treatment of chloroquine with 3-MA to inhibit autophagy would abrogate the hepatoprotection of 3-MA in chloroform hepatotoxicity. Taken together, findings in the present study suggested that a widely-used autophagy inhibitor, 3-MA, significantly reduced chloroform hepatotoxicity in mice via autophagy activation. Findings in this study also suggested that caution should be exercised when using 3-MA to modulate autophagy in vivo.

Inhibition of acetaminophen-induced hepatotoxicity in mice by exogenous thymosinß4 treatment.

OBJECTIVE: To study the effects of exogenous thymosinß4 (Tß4) treatment in acetaminophen (APAP)-induced hepatotoxicity. METHODS: Liver injury was induced in mice by a single intraperitoneal injection of APAP (500 mg/kg). Exogenous Tß4 was intraperitoneally administrated at 0 h, 2 h and 4 h after APAP injection. Chloroquine (CQ) (60 mg/kg) was intraperitoneally injected 2 h before APAP administration to inhibit autophagy. Six hours after APAP injection liver injury was evaluated by histological examinations, biochemical measurements and enzyme linked immunosorbent assay (ELISAs). Western blots were performed to detect proteins expression. RESULTS: Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were significantly increased 6 h after APAP administration, but were significantly reduced by co-administration of Tß4. Histological examinations demonstrated that Tß4 reduced necrosis and inflammation induced by APAP. Immunofluorescence showed that Tß4 suppressed APAP-induced translocation of high mobility group box-1 protein (HMGB1) from the nucleus to cytosol and intercellular space. Hepatic glutathione (GSH) depletion, malondialdehyde (MDA) formation and decreased superoxide dismutase (SOD) activities induced by APAP were all attenuated by Tß4. APAP-induced increases in hepatic nuclear factor-κB (NF-κB) p65 protein expression and inflammatory cytokines production including interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) were reduced by Tß4 treatment. Increased LC3 and p62 proteins in the liver tissues of APAP-treated mice were decreased by Tß4 treatment, which indicated the enhancement of autophagy flux by Tß4. Furthermore, inhibiting autophagy by CQ abrogated the protective effects of Tß4 against APAP hepatotoxicity. CONCLUSION: Exogenous Tß4 treatment exerts protective effects against APAP-induced hepatotoxicity in mice. The underneath molecular mechanisms may involve autophagy enhancement and inhibition of oxidative stress by Tß4.

Effects of exogenous thymosin ß4 on carbon tetrachloride-induced liver injury and fibrosis.

The present study investigated the effects of exogenous thymosin ß4 (TB4) on carbon tetrachloride (CCl4)-induced acute liver injury and fibrosis in rodent animals. Results showed that both in mice and rats CCl4 rendered significant increases in serum alanine aminotransferase and aspartate aminotransferase, hepatic malondialdehyde formation, decreases in antioxidants including superoxide dismutase and glutathione, and up-regulated expressions of transforming growth factor-ß1, α-smooth muscle actin, tumor necrosis factor-α and interleukin-1ß in the liver tissues. Hydroxyproline contents in the rat livers were increased by CCl4. Histopathological examinations indicated that CCl4 induced extensive necrosis in mice livers and pseudo-lobule formations, collagen deposition in rats livers. However, all these changes in mice and rats were significantly attenuated by exogenous TB4 treatment. Furthermore, up-regulations of nuclear factor-κB p65 protein expression by CCl4 treatment in mice and rats livers were also remarkably reduced by exogenous TB4 administration. Taken together, findings in this study suggested that exogenous TB4 might prevent CCl4-induced acute liver injury and subsequent fibrosis through alleviating oxidative stress and inflammation.

Ginsenoside Rg3 sensitizes human non-small cell lung cancer cells to γ-radiation by targeting the nuclear factor-κB pathway.

At present, it is elusive how non-small cell lung cancer (NSCLC) develops resistance to γ-radiation; however, the transcription factor nuclear factor-κB (NF-κB) and NF-κB-regulated gene products have been proposed as mediators. Ginsenoside Rg3 is a steroidal saponin, which was isolated from Panax ginseng. Ginsenoside Rg3 possesses high pharmacological activity and has previously been shown to suppress NF-κB activation in various types of tumor cell. Therefore, the present study aimed to determine whether Rg3 could suppress NF-κB activation in NSCLC cells and sensitize NSCLC to γ-radiation, using an NSCLC cell line and NSCLC xenograft. A clone formation assay and lung tumor xenograft experiment were used to assess the radiosensitizing effects of ginsenoside Rg3. NF-κB/inhibitor of NF-κB (IκB) modulation was ascertained using an electrophoretic mobility shift assay and western blot analysis. NF-κB-regulated gene products were monitored by western blot analysis. The present study demonstrated that ginsenoside Rg3 was able to sensitize A549 and H1299 lung carcinoma cells to γ-radiation and significantly enhance the efficacy of radiation therapy in C57BL/6 mice bearing a Lewis lung carcinoma cell xenograft tumor. Furthermore, ginsenoside Rg3 suppressed NF-κB activation, phosphorylation of IκB protein and expression of NF-κB-regulated gene products (cyclin D1, c-myc, B-cell lymphoma 2, cyclooxygenase-2, matrix metalloproteinase-9 and vascular endothelial growth factor), a number of which were induced by radiation therapy and mediate radioresistance. In conclusion, the results of the present study suggested that ginsenoside Rg3 may potentiate the antitumor effects of radiation therapy in NSCLC by suppressing NF-κB activity and NF-κB-regulated gene products, leading to the inhibition of tumor progression.

Establishment and characterization of a schwannoma cell line from a patient with neurofibromatosis 2.

By using retroviral mediated gene transfer technique, a primary schwannoma culture from a 56-year-old Neurofibromatosis type 2 (NF2) patient was immortalized with HPV E6-E7 genes. This cell line, HEI193, has a unique splice site mutation of the NF2 gene. Both immunocytochemistry and molecular biology techniques were used to demonstrate that this cell line is of Schwann cell origin. Comparison of the primary tumor with HEI193 revealed the same NF2 mutation and an identical pattern of allele loss at multiple loci, indicating that the established cell line had maintained many of the properties of the original tumor. The immortalized cell line was non-tumorigenic in both severe combined immunodeficient (SCID) mice and nude mice, but has altered growth properties such as higher proliferation rate and independence of Schwann cell growth factors. To our knowledge, this is the first attempt to establish permanent cell lines from human NF2 patients. This Schwann tumor-derived cell line may provide a useful model system for the study of familial NF2 tumor pathogenesis, for elucidating NF2 functions and for testing new gene-based therapeutic approaches.

Chlamydia pneumoniae infection induces vascular smooth muscle cell migration via Rac1 activation.

Chlamydia pneumoniae infection has been shown to be associated with the development of atherosclerosis by promoting the migration of vascular smooth muscle cells (VSMCs). However, how C. pneumoniae infection induces VSMC migration is not fully understood. A primary role of Ras-related C3 botulinum toxin substrate 1 (Rac1) is to generate a protrusive force at the leading edge that contributes to cell migration. Whether Rac1 activation plays a role in C. pneumoniae infection-induced VSMC migration is not well defined. In the present study, we therefore examined Rac1 activation in C. pneumoniae-infected rat primary VSMCs and the role of Rac1 activation in C. pneumoniae infection-induced VSMC migration. Glutathione S-transferase pull-down assay results showed that Rac1 was activated in C. pneumoniae-infected rat primary VSMCs. A Rac1 inhibitor, NSC23766 (50 µM,) suppressed Rac1 activation stimulated by C. pneumoniae infection, and thereby inhibited C. pneumoniae infection-induced VSMC migration. In addition, C. pneumoniae infection-induced Rac1 activation in the VSMCs was blocked by LY294002 (25 µM), an inhibitor of phosphatidylinositol 3-kinase (PI3K). Taken together, these data suggest that C. pneumoniae infection promotes VSMC migration, possibly through activating Rac1 via PI3K.

Choline autoradiography of human prostate cancer xenograft: effect of castration.

The purpose of this study was to investigate the effects of castration and tracer uptake time interval on the level of radiolabeled choline accumulation in murine-implanted human prostate tumor xenografts using quantitative autoradiography. We implanted androgen-dependent (CWR22) and androgen-independent (PC3) human prostate cancer cells in castrated (n = 9) and noncastrated (n = 9) athymic male mice and allowed tumors to grow to 1 cm3. The mice were euthanized at 5, 10, and 20 minutes after injection of 5 microCi [14C]-choline. Mice were prepared for quantitative autoradiography with density light units of viable tumor sections converted to units of radioactivity (nCi/mm2) using calibration. Two-group comparisons were performed using a two-tailed Student t-test with unequal variance and with a significance probability level of less than .05. Two-group comparisons between the means of the tracer uptake level for each tumor type at each of three time points for each of two host types showed that (1) the level of tracer localization in the two tumor types was affected little in relation to the host type and (2) PC3 tumor uptake level tended to increase slowly with time only in the noncastrated host, whereas this was not observed in the castrated host or with CWR22 tumor in either host type. The uptake time interval and castration do not appear to significantly affect the level of radiolabeled choline uptake by the human prostate cancer xenograft.

Recombinant thymosin beta 4 can promote full-thickness cutaneous wound healing.

Human thymosin beta 4 (TB4) is a small acidic peptide involved in angiogenesis, wound healing, cancer metastasis and cardiac repair. Currently human TB4 is synthesized chemically for research and this is costly. In order to obtain sufficient biologically active human TB4 economically, we cloned and overexpressed this protein in an Escherichia coli system. We also developed a one-step affinity purification method to purify this fusion protein. After the fusion tag was removed from the fusion protein through autohydrolysis by dithiothreitol (DTT), the biological activity and function of this recombinant human TB4 was evaluated by cell proliferation assay using prepared spleen cells and wound assay using a mouse model, respectively. Our data demonstrated that human recombinant TB4 can promote lymphocyte proliferation and differentiation. Further, it can also promote full-thickness cutaneous wound healing in BALB/c mice. To our knowledge, this is the first report of recombinant human TB4 with the ability to promote wound healing.