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Objective: To evaluate the inhibitory role of a novel oncolytic adenovirus (OA), GP73-SphK1sR-Ad5, on the growth of hepatocellular carcinoma (HCC). Methods: GP73-SphK1sR-Ad5 was constructed by integrating Golgi protein 73 (GP73) promoter and sphingosine kinase 1 (SphK1)-short hairpin RNA (shRNA) into adenovirus serotype 5 (Ad5), and transfecting into HCC Huh7 cells and normal human liver HL-7702 cells. The expression of SphK1 and adenovirus early region 1 (E1A) was detected by quantitative real-time PCR (qRT-PCR) and western blot, respectively. Cell viability was detected by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay, and apoptotic rate was determined by flow cytometry. An Huh7 xenograft model was established in mice injected intratumorally with GP73-SphK1sR-Ad5. Twenty days after injection, the tumor volume and weight, and the survival time of the mice were recorded. The histopathological changes in tumor tissues were observed by hematoxylin-eosin (HE) staining and transmission electron microscopy (TEM). Results: Transfection of GP73-SphK1sR-Ad5 significantly upregulated E1A and downregulated SphK1 in Huh7 cells, but not in HL7702 cells. GP73-SphK1sR-Ad5 transfection significantly decreased the viability and increased the apoptotic rate of Huh7 cells, but had no effect on HL7702 cells. Intratumoral injection of GP73-SphK1sR-Ad5 into the Huh7 xenograft mouse model significantly decreased tumor volume and weight, and prolonged survival time. It also significantly decreased the tumor infiltration area and blood vessel density, and increased the percentages of cells with nucleus deformation and cells with condensed chromatin in tumor tissues. Conclusions: GP73-SphK1sR-Ad5 serves as a novel OA and can inhibit HCC progression with high specificity and efficacy.

Objective Inducible nitric oxide synthase (iNOS) aggravates and endothelial nitric oxide synthase (eNOS) ameliorates fibrosis in the lung. Our previous study demonstrated that aminoguanidine (AG), a preferred iNOS inhibitor, prevents bleomycin-induced injury and fibrosis in the lung. The diethylenetriamine nitric oxide adduct (DETA/NO) is a slow-release NO donor. Here, to clarify the exact role of the nitric oxide (NO) molecule in the pathogenesis of pulmonary fibrosis in vivo, we observed the effects of inhalation of aerosolized DETA/NO on fibrosis in the lungs of bleomycin-exposed rats with AG treatment, including the effects on the myofibroblast number, collagen deposition, peroxynitrite anion (ONOO−) formation, and injury in the lung. Design and methods Rats received a single intratracheal instillation of bleomycin or normal saline (NS) on day 0, followed by a daily intraperitoneal injection of AG or NS from day 1 to day 13. Each group was additionally given a daily inhalation of DETA/NO or placebo from day 1 to day 13. On day 14, half of the rats in each group was euthanized, and plasma nitrite and nitrate (NOx), myofibroblasts, type I collagen, ONOO− and injury in the lung were estimated by the Griess reaction, western blotting, immunohistochemical staining, sirius red staining, and hematoxylin and eosin (HE) staining, respectively. On day 28, the other half of the rats in each group was euthanized, and the total collagen of the lung was evaluated by hydroxyproline assay. Results ① At the day 14 time point, AG reduced the plasma NOx level in bleomycin rats, while this drug had no significant effect on sham rats. Inhalation of aerosolized DETA/NO increased the plasma NOx level of bleomycin + AG rats, sham rats and sham + AG rats. However, due to large areas of airspace obliteration in the lungs of bleomycin rats, DETA/NO inhalation had no significant effect on the plasma NOx level in these rats. ② At the day 14 time point, AG reduced ONOO− formation (marked by nitrotyrosine, NT), injury, myofibroblast number, and type I collagen deposition in the lungs of bleomycin rats, while this drug had no significant impact on the above parameters in the lungs of sham rats. Interestingly, DETA/NO inhalation enhanced the preventive effects afforded by AG on myofibroblast number and type I collagen deposition, but had no significant impact on ONOO− and injury in lung. ③ At the day 28 time point, because rats were not exposed to DETA/NO after day 13, there was no significant difference of the plasma NOx level in sham rats, sham + AG rats, bleomycin rats, and bleomycin + AG rats between DETA/NO inhalation and placebo inhalation. Interestingly, rats administered both DETA/NO and AG still showed a reduction in total collagen of the entire lung compared to rats administered AG alone at this time point. Conclusions Exogenous NO enhances the prophylactic effect afforded by AG on the myofibroblast number and collagen deposition in the lungs of bleomycin-treated rats in vivo. These results suggest that NO has a direct antifibrotic effect in lungs, except for the formation of ONOO− in the development of pulmonary fibrosis in vivo.

To investigate whether connective tissue growth factor (CGGF) is expressed in mast cells (MCs) in lung in the development of bleomycin (BLM)-induced pulmonary fibrosis.Thirty-two male SD rats were randomly divided into 2 groups: BLM group and control group (n=16). The rats in BLM group were received single intratracheal instillation of BLM (5 mg/kg), and the rats in control group received equal volume of 0.9% normal saline(NS) to BLM. The rats in each group were sacrificed for lung tissue sampling on day 14 and day 28 after intratracheal instillation respectively. As the index of the severity of pulmonary fibrosis, the content of hydroxyproline in lungs was analyzed by chloramine T method. Mast cells and CTGF expression in lungs were examined by toluidine blue stain and immunohistochemical assay respectively.(1) On day 28 after intratracheal instillation of BLM, the content of hydroxyproline in lungs of rats was higher than that of control rats (P0.01). (2) Compared to control rats, the rats on day 14 and day 28 after instillation of BLM showed increased number of mast cells (Both P0.01) and up-regulated CTGF expression (Both P0.01). (3) No CTGF immuno-positive MCs were seen in the lungs of control rats whereas CTGF immuno-positive MCs were observed in the pathological areas in lungs of rats on day 14 and day 28 after BLM.CTGF is expressed in MCs in lungs in the development of pulmonary fibrosis, which might be one of the mechanisms underling promoting effect of MCs on fibrosis in lung.

To clarify the mechanism underlying the preventive effect of baicalin (Bai) on fibrosis in lung, we investigated the influence of Bai on the up-regulation of connective tissue growth factor (CTGF) in fibrotic lungs. Male Sprague-Dawley (SD) rats were divided into four groups randomly: normal saline (NS)+NS group (a single intratracheal instillation of NS plus i.p. injection of NS), NS+Bai group (intratracheal instillation of NS plus i.p. injection of Bai), bleomycin (BLM)+NS group (intratracheal instillation of BLM plus i.p. injection of NS) and BLM+Bai group (intratracheal instillation of BLM plus i.p. injection of Bai). All the i.p. injections were performed once daily. On day 28 after intratracheal instillation of BLM or NS, the rats were sacrificed for lung tissue sampling. As the index of the severity of pulmonary fibrosis, the content of hydroxyproline in lungs was analyzed by chloramine T method. The expression levels of CTGF mRNA and protein in the lungs were detected by RT-PCR and immunohistochemistry, respectively. The results showed that, compared to the rats in NS+NS group, the rats in BLM+NS group showed increased hydroxyproline content and higher levels of CTGF mRNA and protein expressions (P0.01), suggesting that BLM had induced fibrosis in lung and up-regulated CTGF expression in the fibrotic lungs. Administration of different dosages of Bai (6, 12.5 and 50 mg/kg per d, for 28 days) into the BLM-treated rats reduced the increased content of hydroxyproline, and ameliorated the up-regulation of CTGF mRNA and protein levels, respectively. These results suggest that Bai could prevent the up-regulation of CTGF expression in fibrotic lungs of rats receiving BLM instillation, which might be one of the mechanisms underlying the preventive effect of Bai on pulmonary fibrosis.

To further explore the role of adenosine A1 receptor in the neuroprotective effect of cerebral ischemic preconditioning, the present study was undertaken to observe the effect of inhibiting expression of adenosine Al receptor with adenosine A1 receptor antisense oligodeoxynucleotide (ARA1 As-ODN) on the neuroprotective effect of cerebral ischemic preconditioning against delayed neuronal death (DND) normally induced by lethal brain ischemia.The rat 4-vessel occlusion global cerebral ischemic model was used. Forty-eight male Wistar rats with permanent occlusion of the bilateral vertebral arteries were divided into 8 groups: Sham, CIP, brain ischemic insult, CIP + brain ischemic insult, Distilled water + CIP + brain ischemic insult, ARA1 As-ODN, ARA1 As-ODN +CIP, ARA1 As-ODN+ CIP + brain ischemic insult(two doses of 10 nmol/5 microl and 20 nmol/5 microl were used) groups. ARA1 As-ODN was dissolved in distilled water and injected into the right lateral cerebral ventricle. To illustrate the profile of DND, histological grade (HG) and neuronal density (ND) in the CA1 region of the hippocampus were examined 7 d after the sham operation or the last time of ischemia under thionin staining.The HG and ND in CIP group were similar to those in sham group. Brain ischemic insult induced obvious DND as represented with the increase in HG and decrease in ND significantly (P0.05 vs. sham and CIP groups). In CIP + ischemic insult group,no obvious DND was observed,which indicated that CIP protected pyramidal neurons against the ischemic insult.While the administration of ARA1 As-ODN in ARA1 As-ODN + CIP + brain ischemic insult group caused obvious increase in HG and decrease in ND compared with CIP + brain ischemic insult group (P0.05) in a dose dependent manner,which indicated that the neuroprotective effect of CIP against DND of hippocampal pyramidal neurons normally induced by ischemic insult was inhibited by the administration of ARA1 As-ODN.The results further demonstrate the association of up-regulation of adenosine A1 receptors with the induction of CIP-mediated BIT.