Exogenous ghrelin ameliorates acute lung injury by modulating the nuclear factor κB inhibitor kinase/nuclear factor κB inhibitor/nuclear factor κB pathway after hemorrhagic shock.

Previous studies have shown that ghrelin, a peptide produced in the stomach, attenuates acute lung injury (ALI) in various animal models, and that some of these effects are associated with inhibition of the nuclear factor κB signaling pathway. This study investigated whether ghrelin exerts beneficial effects on hemorrhagic shock (HS)-induced ALI by modulating nuclear factor κB inhibitor kinase/nuclear factor κB inhibitor/nuclear factor κB (IKK/IκBα/NF-κB) pathway activity. HS was induced in male SD rats by withdrawing blood to a mean arterial pressure (MAP) of 40 mm Hg for 1 h; rats then received ghrelin (10 nmol/kg) or vehicle intravenously and were resuscitated with the shed blood and an equal volume of Ringer lactate solution followed by observation for 2 h. After resuscitation, samples were collected and analyzed for lung histopathology, wet to dry weight ratio (W/D), bronchoalveolar lavage fluid (BALF) protein, neutrophil infiltration, plasma inflammatory cytokines (TNF-α and IL-6), and cytoplasmic phosphorylated IKKß, IκBα, phosphorylated IκBα and nuclear NF-κB expression. Compared to those in the two sham groups, lung injury, W/D, BALF protein, neutrophil infiltration, plasma TNF-α and IL-6 levels, and IKK/IκBα/NF-κB pathway activation were significantly increased in HS rats. After ghrelin administration, all parameters analyzed were decreased compared to those without ghrelin in HS rats. Moreover, ghrelin alleviated the decreased MAP after resuscitation compared to that in HS rats. Exogenous ghrelin attenuates the inflammatory response and acute lung injury after HS. These beneficial effects appear to be mediated through inhibition of IKK/IκBα/NF-κB signaling.

N-myc downstream-regulated gene 4, up-regulated by tumor necrosis factor-α and nuclear factor kappa B, aggravates cardiac ischemia/reperfusion injury by inhibiting reperfusion injury salvage kinase pathway.

N-myc downstream-regulated gene 4 (NDRG4) is expressed weakly in heart and has been reported to modulate cardiac development and QT interval duration, but the role of NDRG4 in myocardial ischemia/reperfusion (I/R) injury remains unknown. In the present study, we analyzed the expression as well as potential function of cardiac NDRG4 and investigated how NDRG4 expression is regulated by inflammation. We found that NDRG4 was weakly expressed in cardiomyocytes and that its expression increased significantly both in I/R injured heart and in hypoxia-reoxygenation (H/R) injured neonatal rat ventricular myocytes (NRVMs). The increased NDRG4 expression aggravated myocardial I/R injury by inhibiting the activation of the reperfusion injury salvage kinase (RISK) pathway. Forced over-expression of NDRG4 inhibited RISK activation and exacerbated injury not only in I/R injured heart, but also in H/R treated NRVMs, whereas short hairpin RNA (shRNA)-mediated knock-down of NDRG4 enhanced RISK activation and attenuated injury. Upon injury, myocardial NDRG4 expression was induced by tumor necrosis factor-α (TNF-α) through nuclear factor kappa B (NF-κB), and we found that pre-treatment with inhibitors of either TNF-α or NF-κB blocked NDRG4 expression as well as I/R injury in vivo and H/R injury in vitro. Our study indicates that up-regulation of NDRG4 aggravates myocardial I/R injury by inhibiting activation of the RISK pathway, thereby identifying NDRG4 as a potential therapeutic target in I/R injury.

Abnormal Accumulation of Collagen Type I Due to the Loss of Discoidin Domain Receptor 2 (Ddr2) Promotes Testicular Interstitial Dysfunction.

BACKGROUND: Loss of functional allele for discoidin domain receptor 2 (Ddr2) results in impaired Leydig cell response to luteinizing hormone (LH), low testosterone production and arrested spermatogenesis in older male Ddr2slie/slie mice. However, the underlying mechanism responsible for this phenotype remains unknown. Herein, we reported for the first time that the deregulated expression of Ddr2 cognate ligand, namely collagen type I (COL1), may account for the disruption of the testicular steroidogenesis in Ddr2slie/slie mutant testes. METHODOLOGY/PRINCIPAL FINDINGS: Expression of Ddr2 increased gradually along postnatal development, whereas COL1 expression became negligible from adulthood onwards. In Ddr2slie/slie mutant testis, however, in contrast to the undetectable staining of Ddr2, COL1 expression was constantly detected, with the highest values detected during adulthood. In the experimental vasectomy model, Ddr2slie/slie mutant mice exhibited an early androgen deficiency than wild-type mice, along with the accumulation of fibrotic tissue in the interstitium. Functionally, ablation of endogenous Ddr2 resulted in a significant decrease of testosterone (T) level in TM3 cells in the presence of higher concentration of COL1 treatment. Conversely, overexpression of Ddr2 could help TM3 cells to maintain a normal testicular steroidogenesis even in the presence of high concentration of COL1. Additionally, attenuated expression of Ddr2 correlates to the deregulated level of serum T levels in human pathological testes. CONCLUSIONS: Abnormal accumulation of interstitial COL1 may be responsible for the steroidogenic dysfunction in Ddr2slie/slie mutant testes.

Percutaneous transhepatic embolization of gastroesophageal varices combined with partial splenic embolization for the treatment of variceal bleeding and hypersplenism.

This study aims to evaluate the therapeutic results of percutaneous transhepatic embolization of gastroesophageal varices combined with partial splenic embolization in patients with liver cirrhosis, and to explore the role of this minimally invasive treatment as an alternative to surgery. 25 patients with liver cirrhosis were received percutaneous transhepatic embolization of gastroesophageal varices combined with partial splenic embolization. Another 25 patients with liver cirrhosis underwent Hassab's operation. They were followed up, and received endoscopy, B ultrasound, liver function and hematologic examination at 24 months after the therapy. In minimal invasive group, before treatment and after 24 month following up after treatment, improved varices, improved portal hypertension and improved hypersplenism were showed comparing with the surgery group, and that they were measured by endoscopic visualization, ultrasound and blood counts. the white blood cell and platelet count were 2.33±0.65 (10(9)/L) and 3.63±1.05 (10(10)/L), 7.98±3.0 (10(9)/L) and 16.3±9.10 (10(10)/L) (P<0.05); the diameter of the portal vein were 1.47±0.25 cm, 1.31±0.23 cm (P<0.05). Esophageal varices passed from grade III to lower grade II in 11 patients, and from grade II to lower grade I in 6 patients at 24 month following up. In surgical group, the white blood cell and platelet count were 2.2±0.60 (10(9)/L), 4.1±1.25 (10(10)/L) before treatment; 9.3±2.56 (10(9)/L), 32.1±12.47 (10(10)/L) after the treatment at 24 month following up (P<0.05). The diameter of the portal vein were 1.43±0.22 cm before the treatment and 1.28±0.18 cm after the treatment (P<0.05). Esophageal varices passed from grade III to lower grade II in 13 patients, and from grade II to lower grade I in 7 patients. The combination of PGEV and PSE can be considered as an option for the treatment of variceal bleeding with hypersplenism.

[The influences of Tet system on activity and tissue specificity of HBV Cp.].

AIM: Analyze the effect of Tetracycline-controlled system on Hepatitis B virus core promoter (Cp) activity and tissue specificity. METHODS: The 7 Teto sequence was amplified from plasmid pTL-8 using polymerase chain reaction (PCR) and cloned into T vector pMD19-T Simple. After sequencing, the 7 teto sequence was subcloned into upstream of Cp in pGL3-Basic/Cp. In order to observe the effect of Tetracycline-controlled system on Cp activity and tissue specificity, the plasmid pTL-8 which luciferase encoding sequence was excised and pGL3-Basic/7t/Cp were cotransfected into different tissue-derived cell lines including HepG2, Hela, COS-7, MDA-MB-231 and HT-29. The dual luciferase activities were determined by Dual Luciferase Report (DLR) assay system. RESULTS: Have successfully constructed plasmid pGL3-Basic/7t/Cp. The transcriptional activity of Cp was increased significantly after the teto sequence was cloned upstream of Cp. CONCLUSION: The transcriptional activity of Cp is augmented significantly by Tetracycline-controlled system. But the tissue specificity of Cp lost at the same time.

[Adenovirus-mediated delivery of targeted ribonuclease against HBV replication in vitro].

OBJECTIVE: To observe the inhibitory effect of targeted ribonuclease delivered via adenovirus against HBV replication in vitro. METHODS: The shuttle plasmids pDC316 were constructed on the basis of the previous plasmids pcDNA3.1(-)/TRL, pcDNA3.1(-)/TR, pcDNA3.1(-)/TRmut, pcDNA3.1(-)/HBVc, and pcDNA3.1(-)/hEDN by subcloning the target gene sequences of TRL, TR, HBVc, and hEDN, respectively. HEK 293 cells were cotransfected with the pDC316 plasmids respectively in the presence of the rescue plasmid pBHGlox(delta)E1,3Cre to yield the recombinant adenoviral vectors which comprised the above genes. After transfection of HepG2.2.15 cells with the vectors, RAd/TRL expression was detected by indirect immunofluorescence staining and RT-PCR. Radioimmunoassay was used to analyse anti-HBV activity of RAd/TRL. RESULTS: Recombinant RAd vectors were prepared successfully. Effective expression of RAd/TRL in HepG2.2.15 cells resulted in a significant decrease of HBsAg and HBeAg concentration in comparison with the controls. CONCLUSION: Adenoviral vector-mediated targeted ribonuclease can effectively inhibit HBV replication.

Anti-HBV activity of TRL mediated by recombinant adenovirus.

AIM: To investigate the inhibitive effect of hepatitis B virus (HBV)-TRL on HBV replication. METHODS: Based on previously constructed pcDNA3.1(-)/TRL, TR, TRmut, HBV core protein (HBVc) and hEDN, interest gene sequences TRL, TR, HBVc and hEDN were inserted into adenovirus shuttle plasmid pDC316 respectively and co-transfected HEK293 cells with rescue plasmid pBHGloxdeltaE1,3Cre to acquire RAd/TRL, TR, HBVc and hEDN. And then RAds were identified, amplified and the titers in HEK293 cells were determined. RAd/TRL and TR were named as the experimental groups, and others were control ones. After HepG2.2.15 cells were infected, RAd/TRL expression was identified by indirect immunofluorescence staining. Supernatant HBV-DNA content was determined by fluorescent quantification PCR. Meanwhile, metabolism of HepG2.2.15 cells was evaluated by MTT colorimetry. RESULTS: RAd vectors with distinct interest gene sequence were successfully constructed. Effective expression of RAd/TRL in HepG2.2.15 cells resulted in a significant decrease of supernatant HBV-DNA content compared to RAd/TR (0.63+/-0.14 vs 1.60+/-0.47, P = 0.0266, <0.05) and other control groups (0.63+/-0.14 vs 8.50+/-2.78, 8.25+/-2.26, 8.25+/-2.29, 8.50+/-1.51, 8.57+/-1.63, P<0.01). MTT assay suggested that there were no significant differences in cell metabolic activity between groups (P>0.05). CONCLUSION: The construction and expression of RAd/TRL has been achieved and it could inhibit HBV replication successfully, which has laid the foundation for further research on anti-HBV activity in vivo.

VP22 fusion protein-based dominant negative mutant can inhibit hepatitis B virus replication.

AIM: To investigate the inhibitory effect of VP22 fusion protein-based dominant negative (DN) mutant on Hepatitis Bvrus (HBV) replication. METHODS: Full-length or truncated fragment of VP22 was fused to C terminal of HBV core protein (HBc), and subcloned into pcDNA3.1 (-) vector, yielding eukaryotic expression plasmids of DN mutant. After transfection into HepG2.2.15 cells, the expression of DN mutant was identified by immunofluorescence staining. The inhibitory effect of DN mutant on HBV replication was indexed as the supernatant HBsAg concentration determined by RIA and HBV-DNA content by fluorescent quantification-PCR (FQ-PCR).Meanwhile, metabolism of HepG2.2.15 cells was evaluated by MTT colorimetry. RESULTS: VP22-based DN mutants and its truncated fragment were expressed in HepG2.2.15 cells, and had no toxic effect on host cells. DN mutants could inhibit HBV replication and the transduction ability of mutant-bearing protein had a stronger inhibitory effect on HBV replication. DN mutants with full length of VP22 had the strongest inhibitory effect on HBV replication, reducing the HBsAg concentration by 81.94%, and the HBV-DNA content by 72.30%. MTT assay suggested that there were no significant differences in cell metabolic activity between the groups. CONCLUSION: VP22-based DN mutant can inhibit HBV replication effectively.

Quantifying anti-HBV effect of targeted ribonuclease by real-time fluorescent PCR.

AIM: To quantify the inhibition of HBV replication by targeted ribonuclease by using real-time fluorescent PCR. METHODS: Targeted ribonuclease was introduced into 2.2.15 cells by liposome-mediated transfection or HIV-TAT mediated protein transduction. Forty-eight hours after the transfection and 24 h after the transduction, supernatants of 2.2.15 cells were collected and HBV DNA in the supernatants was quantified by real-time fluorescent PCR with a commercial kit. RESULTS: HBV DNA concentrations in the supernatants of 2.2.15 cells transfected or transducted with targeted ribonuclease were 4.9+/-2.4 x 10(8) copies/L and 8.3+/-4.0 x 10(8) copies/L, respectively. Compared with controls, transfection or transduction of targeted ribonuclease reduced HBV DNA concentration in the supernatants of 2.2.15 cells by 90.4% and 90.1%, respectively (P<0.05). CONCLUSION: Targeted ribonuclease can inhibit HBV replication in 2.2.15 cells.

Effect of vector-expressed siRNA on HBV replication in hepatoblastoma cells.

AIM: To study the effect of siRNA expressed from DNA vector on HBV replication. METHODS: Human U6 promoter was amplified from genomic DNA and cloned into plasmid pUC18 to construct a mammalian siRNA expression vector pUC18U6. Then oligonucleotides coding for a short hairpin RNA against HBV were cloned into pUC18U6 to form pUC18U6HBVsir which was introduced into 2.2.15 cells by using liposome-mediated transfection. 2.2.15 cells transfected by pUC18U6 and pUC18U6GFPsir which expressed siRNA against green fluorescent protein and mock-transfected 2.2.15 cells were used as controls. Concentration of HBsAg in the supernatant of the transfected cells was measured by using solid-phase radioimmunoassay. RESULTS: A mammalian siRNA expression vector pUC18U6 was constructed successfully. Compared with controls, pUC18U6HBVsir which expressed siRNA against HBV decreased concentration of HBsAg significantly by 44%(P<0.05). CONCLUSION: HBV replication in 2.2.15 cells is inhibited by siRNA expressed from the DNA vector.

[The mechanism for up-regulation of HLA-I expression on HepG2 cells by HBV].

AIM: To explore the mechanism of up-regulation of HLA-I expression on HepG2 cells by wild type (WT) and nucleocapsid mutants(L97 and V60) of hepatitis B virus (HBV). METHODS: The HBV-stable expression vectors EBO-WT, EBO-L97 and EBO-V60 were transfected into HepG2 cells via the liposome mediation, respectively. The cells were assayed by semi-quantitative RT-PCR for HLA-A gene and antigen presentation-related genes LMP2, TAP1, and tapasin mRNA expression. Western blot was applied for analysis of HLA-I protein expression in the cells. RESULTS: HepG2 cells transfected by the 3 HBV expression vectors expressed HLA-A and TAP1, while there was no expression of HLA-A and only marginal expression of TAP1 in HepG2 cells transfected by control vector EBO. The expression level of HLA-A in the transfected cells decreased successively in the order of EBO-L97, EBO-WT and EBO-V60. There was no significant difference in the expression level of TAP1 between HepG2 cells transfected by the 3 HBV expression vectors. No detectable expression of LMP2 and tapasin was observed for all transfected cells. CONCLUSION: HBV can induce the expression of HLA-I molecule and TAP1 in HepG2 cells.

Inhibition of HBV targeted ribonuclease enhanced by introduction of linker.

AIM: To construct human eosinophil-derived neurotoxin(hEDN) and HBV core protein (HBVc) eukaryotic fusion expression vector with a linker (Gly(4)Ser) (3) between them to optimize the molecule folding, which will be used to inhibit HBV replication in vitro. METHODS: Previously constructed pcDNA3.1(-)/TR was used as a template. Linker sequence was synthesized and annealed to form dslinker, and cloned into pcDNA3.1(-)/TR to produce plasmid pcDNA3.1(-)/HBc-linker. Then the hEDN fragment was PCR amplified and inserted into pcDNA3.1(-)/HBc-linker to form pcDNA3.1(-)/TNL in which the effector molecule and the target molecule were separated by a linker sequence. pcDNA3.1(-)/TNL expression was identified by indirect immunofluorescence staining. Radioimmunoassay was used to analyse anti-HBV activity of pcDNA3.1(-)/TNL. Meanwhile, metabolism of cells was evaluated by MTT colorimetry. RESULTS: hEDN and HBVc eukaryotic fusion expression vector with a linker (Gly(4)Ser)(3) between them was successfully constructed. pcDNA3.1(-)/TNL was expressed in HepG2.2.15 cells efficiently. A significant decrease of HBsAg concentration from pcDNA3.1(-)/TNL transfectant was observed compared to pcDNA3.1(-)/TR (P=0.036, P<0.05). MTT assay suggested that there were no significant differences between groups (P=0.08, P>0.05). CONCLUSION: Linker introduction enhances the inhibitory effect of HBV targeted ribonuclease significantly.

Anti-HBV effect of TAT- HBV targeted ribonuclease.

AIM: To prepare and purify TAT-HBV targeted ribonuclease fusion protein, evaluate its transduction activity and investigate its effect on HBV replication in 2.2.15 cells. METHODS: The prokaryotic expression vector pTAT containing TR gene was used in transforming E.coli BL21 (DE3) LysS and TR was expressed with the induction of IPTG. The TAT-TR fusion protein was purified using Ni-NTA-agrose and PD-10 desalting columns, and analyzed by SDS-PAGE. Transduction efficiency of TAT-TR was detected with immunofluorescence assay and the concentration of HBeAg in the supernatant of the 2.2.15 cells was determined via solid-phase radioimmunoassay (spRIA). MTT assay was used to detect the cytotoxicity of TAT-TR. RESULTS: The SDS-PAGE showed that the TAT-TR fusion protein was purified successfully, and the purity of TAT-TR was 90 %. The visualization of TAT-TR by immunofluorescence assay indicated its high efficiency in transducing 2.2.15 cells. RIA result suggests that TAT-TR could inhibit the replication of HBV effectively, it didn't affect cell growth and had no cytotoxicity. CONCLUSION: TAT-TR possesses a significant anti-HBV activity and the preparation of TAT-TR fusion protein has laid the foundation for the use of TR in the therapeutic trial of HBV infection.

Targeted ribonuclease can inhibit replication of hepatitis B virus.

AIM: To study the effect of a novel targeted ribonuclease (TN), the fusion protein of HBVc and human eosinophil-derived neurotoxin (hEDN), on the HBV replication in vitro. METHODS: The gene encoding the targeted ribonuclease was cloned into pcDNA3.1 (-) to form recombinant eukaryotic expression vector p/TN. Control plasmids, including p/hEDN, p/HBVc, and p/TNmut in which a Lys113-Arg mutation was introduced by sequential PCR to eliminate the ribonuclease activity of hEDN, were also constructed. Liposome-mediated transfection of 2.2.15 cells by p/TN, p/TNmut, p/hEDN, p/HBVc, and pcDNA3.1 (-), or mock transfection was performed. After that, RT-PCR was used to verify the transgene expression. Morphology of the transfected cells was observed and MTT assay was performed to detect the cytotoxicity of transgene expression. Concentration of HBsAg in the supernatant of the transfected cells was measured using solid-phase radioimmunoassay. RESULTS: Transgenes were successfully expressed in 2.2.15 cells. No obvious cytotoxic effect of transgene expression on 2.2.15 cells was found. The HBsAg concentration in the p/TN transfected cells was reduced by 58 % compared with that of mock transfected cells. No such an effect was found in all other controls. CONCLUSION: The targeted ribonuclease can inhibit HBV replication in vitro while it has no cytotoxicity on host cells. The targeted ribonuclease may be used as a novel antiviral agent for human HBV infection.

[Construction and expression of prokaryotic expression vector for pTAT-HBV targeted ribonuclease fusion protein].

AIM: To construct Tat-HBV targeted ribonuclease fusion protein prokaryotic expression vector and express it in E.coli. METHODS: The cDNAs encoding HBV targeted ribonuclease, human eosinophil-derived neurotoxin and HBV core protein were respectively cloned into prokaryotic expression vector pTAT-HA. Recombinant plasmids were transformed into E.coli BL21(DE3) LysS, then the transformed cells were induced with IPTG. The expression of the fusion proteins were analyzed by SDS-PAGE and Western blot. RESULTS: The three recombinant plasmids were constructed and expressed after IPTG induction successfully. CONCLUSION: The obtained Tat-HBV targeted ribonuclease fusion protein has laid the foundation for using TR in therapy of HBV infection.