Effects of Helicobacter pylori and Moluodan on the Wnt/ß-catenin signaling pathway in mice with precancerous gastric cancer lesions.

BACKGROUND: Helicobacter pylori (H. pylori) is the primary risk factor for gastric cancer (GC), the Wnt/ß-Catenin signaling pathway is closely linked to tumourigenesis. GC has a high mortality rate and treatment cost, and there are no drugs to prevent the progression of gastric precancerous lesions to GC. Therefore, it is necessary to find a novel drug that is inexpensive and preventive to against GC. AIM: To explore the effects of H. pylori and Moluodan on the Wnt/ß-Catenin signaling pathway and precancerous lesions of GC (PLGC). METHODS: Mice were divided into the control, N-methyl-N-nitrosourea (MNU), H. pylori + MNU, and Moluodan groups. We first created an H. pylori infection model in the H. pylori + MNU and Moluodan groups. A PLGC model was created in the remaining three groups except for the control group. Moluodan was fed to mice in the Moloudan group ad libitum. The general condition of mice were observed during the whole experiment period. Gastric tissues of mice were grossly and microscopically examined. Through quantitative real-time PCR (qRT-PCR) and Western blotting analysis, the expression of relevant genes were detected. RESULTS: Mice in the H. pylori + MNU group showed the worst performance in general condition, gastric tissue visual and microscopic observation, followed by the MNU group, Moluodan group and the control group. QRT-PCR and Western blotting analysis were used to detect the expression of relevant genes, the results showed that the H. pylori + MNU group had the highest expression, followed by the MNU group, Moluodan group and the control group. CONCLUSION: H. pylori can activate the Wnt/ß-catenin signaling pathway, thereby facilitating the development and progression of PLGC. Moluodan suppressed the activation of the Wnt/ß-catenin signaling pathway, thereby decreasing the progression of PLGC.

Characterization of a gE/gI/TK gene-deleted pseudorabies virus variant expressing the Cap protein of porcine circovirus type 2d.

Porcine circovirus type 2 (PCV2) plays a key role in the etiology of PCV2-associated disease (PCVAD), and its predominant strain is PCV2d which is not completely controlled by most commercially available vaccines against PCV2a strains. Pseudorabies (PR) caused by pseudorabies virus (PRV) variants re-emerged in Bartha-K61 vaccine-immunized swine herds in late 2011, which brought considerable losses to the global pig husbandry. Therefore, it is significantly important to develop a safe and effective vaccine against both PCV2d and PRV infection. In the present study, the PCV2d ORF2 gene was amplified by PCR, and cloned into the BamHI site of PRV transfer plasmid pG vector to obtain the recombinant transfer plasmid pG-PCV2dCap-EGFP. Subsequently, it was transfected into ST cells infected with the three gene deleted PRV variant strain NY-gE-/gI-/TK- to generate a recombinant virus rPRV NY-gE-/gI-/TK-/PCV2dCap+/EGFP+, and then the EGFP gene was knocked out to harvest the rPRV NY-gE-/gI-/TK-/PCV2dCap+ using gene-editing technology termed CRISPR/Cas9 system. The recombinant virus rPRV NY-gE-/gI-/TK-/PCV2dCap+ had similar genetic stability and proliferation characteristics to the parental PRV as indicated by PCR and one-step growth curve test, and the expression of Cap was validated by Western blot. In animal experiment, higher PCV2-specific ELISA antibodies and detectable PCV2-specific neutralizing antibodies could be elicited in mice immunized with rPRV NY-gE-/gI-/TK-/PCV2dCap+ compared to commercial PCV2 inactivated vaccine. Moreover, the recombinant virus rPRV NY-gE-/gI-/TK-/PCV2dCap+ significantly reduced the viral loads in the hearts, livers, spleens, lungs, and kidneys in mice following a virulent PCV2d challenge. Mice immunized with rPRV NY-gE-/gI-/TK-/PCV2dCap+ developed comparable PRV-specific humoral immune responses and provided complete protection against a lethal PRV challenge. Together, the rPRV NY-gE-/gI-/TK-/PCV2dCap+ recombinant strain has strong immunogenicity.

Repeated Endomorphin Analogue MEL-0614 Reduces Tolerance and Improves Chronic Postoperative Pain without Modulating the P2X7R Signaling Pathway.

The clinical treatment of chronic postoperative pain (CPSP) remains challenging. The side effects of chronic morphine treatment limit its clinical application. MEL-0614, a novel endomorphin analogue that is highly selective and agonistic for µ opioid receptor (MOR), produces a more powerful analgesic effect than that of morphine. In this study, we explored the difference in antinociceptive tolerance and related mechanisms between MEL-0614 and morphine in CPSP induced in a skin/muscle incision and retraction (SMIR) mice model. We found that acute administration of MEL-0614 (1, 3, 5, and 10 nmol, i.t.) produced a dose-dependent analgesic effect that was superior to that of morphine in the SMIR mice model. Long-term MEL-0614 treatment (10 nmol, i.t.) did not induce tolerance compared with morphine. Notably, tolerance induced by morphine could be greatly prevented and/or inhibited via cross-administration or coadministration between MEL-0614 and morphine. In addition, MEL-0614 accelerated the recovery of postoperative pain, whereas morphine aggravated postoperative pain and prolonged its recovery time regardless of preoperative or postoperative treatment. In addition, MEL-0614 did not activate microglia and the P2X7R signaling pathway and showed reduced expression iba1 and P2X7R compared with that observed after morphine administration. Release of inflammatory factors was induced by continued administration of morphine during SMIR surgery, but MEL-0614 did not promote the activation of inflammatory factors. Our results showed that MEL-0614 has superior analgesic effects in CPSP and leads to tolerance to a lesser degree than morphine. Further, MEL-0614 may be used as a promising treatment option for the long-term treatment in CPSP.

Construction and immunogenicity of a gE/gI/TK-deleted PRV based on porcine pseudorabies virus variant.

Pseudorabies (PR) caused by re-emerging pseudorabies virus (PRV) variant has outbroken among PRV vaccine-immunized swine herds on many Chinese pig farms, with severe socioeconomic consequences since late 2011. Here, a gE/gI/TK-deleted recombinant virus (rPRV NY-gE-/gI-/TK-) was constructed based on PRV NY strain from 2012 through homologous DNA recombination and gene-editing technology termed clustered regularly interspaced palindromic repeats (CRISPR)/associated (Cas9) system. The rPRV NY-gE-/gI-/TK- strain showed similar growth kinetics to the parental PRV NY strain in vitro, and was safe for mice. Sixty mice were injected subcutaneously (s.c.) twice with 106.0 TCID50 of rPRV NY-gE-/gI-/TK- and DMEM, respectively, with two-week interval. The levels of PRV gB antibodies and neutralizing antibodies against PRV NY in mice immunized with rPRV NY-gE-/gI-/TK- were higher than those in the DMEM control group. The number of T lymphocyte subclasses CD3+, CD4+ and CD8+ in rPRV NY-gE-/gI-/TK--immunized mice was higher than that in DMEM-injected mice. After challenge with 106.0 TCID50 PRV NY at 42 dpi, all rPRV NY-gE-/gI-/TK--immunized mice survived without exhibiting any pathological lesions in different tissues and intranuclear eosinophilic inclusions of the brain, and the viral genomic copy numbers in various organs of mice were obviously lower than DMEM group. These results showed the rPRV NY-gE-/gI-/TK- could be a promising next-generation vaccine to control now epidemic PR in China.

Characterization of a recombinant pseudorabies virus expressing porcine parvovirus VP2 protein and porcine IL-6.

BACKGROUND: Porcine parvovirus (PPV) and pseudorabies virus (PRV) are the important etiological agents of swine infectious diseases, resulting in huge economic losses to the Chinese swine industry. Interleukin-6 (IL-6) has the roles to support host immune response to infections as a pleiotropic cytokine. It is essential to construct a live attenuated vaccine-based recombinant PRV that expresses PPV VP2 protein and porcine IL-6 for prevention and control of PRV and PPV. METHODS: The recombinant plasmid, pGVP2-IL6, was constructed by porcine IL-6 gene substituting for EGFP gene of the PRV transfer plasmid pGVP2-EGFP containing VP2 gene of PPV. Plasmid pGVP2-IL6 was transfected into swine testicle cells pre-infected with the virus rPRV-VP2-EGFP strain through homologous recombination and plaque purification to generate a recombinant virus rPRV-VP2-IL6. The recombinant PRV was further identified by PCR and DNA sequencing, and the expression of the VP2 protein and porcine IL-6 was analyzed by reverse transcription-PCR (RT-PCR) and Western blot. The virus titer was calculated according to Reed and Muench method. The immunogenicity of the recombinant virus was preliminarily evaluated in mice by intramuscular administration twice with the rPRV-VP2-IL6 at 4-week intervals. RESULTS: A recombinant virus rPRV-VP2-IL6 was successfully constructed and confirmed in this study. The properties of rPRV-VP2-IL6 were similar to the parental virus HB98 in terms of growth curve, morphogenesis and virus plaque sizes, and rPRV-VP2-IL6 was proliferated in different cell types. It induced specific antibodies against PPV as well as a strong increase of PPV-specific lymphocyte proliferation responses in mice immunized with rPRV-VP2-IL6, and provided partial protection against the virulent PPV challenge. rPRV-VP2-IL6 also induced a high level of neutralizing antibodies against PRV, and significantly reduced the mortality rate of (1 of 10) following virulent PRV challenge compared with the control (10 of 10). CONCLUSIONS: The recombinant rPRV-VP2-IL6 might be a potential candidate vaccine against PRV and PPV infections in pigs.

Regulation of primordial follicle recruitment by cross-talk between the Notch and phosphatase and tensin homologue (PTEN)/AKT pathways.

The growth of oocytes and the development of follicles require certain pathways involved in cell proliferation and survival, such as the phosphatidylinositol 3-kinase (PI3K) pathway and the Notch signalling pathway. The aim of the present study was to investigate the interaction between Notch and the PI3K/AKT signalling pathways and their effects on primordial follicle recruitment. When the Notch pathway was inhibited by L-685,458 or N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycinet-butyl ester (DAPT) in vitro, the expression of genes in the pathway and the percentage of oocytes in growing follicles decreased significantly in mouse ovaries. By 2 days postpartum, ovaries exposed to DAPT, short interference (si) RNA against Notch1 or siRNA against Hairy and enhancer of split-1 (Hes1) had significantly decreased expression of HES1, the target protein of the Notch signalling pathway. In contrast, expression of phosphatase and tensin homologue (Pten), a negative regulator of the AKT signalling pathway, was increased significantly. Co immunoprecipitation (Co-IP) revealed an interaction between HES1 and PTEN. In addition, inhibition of the Notch signalling pathway suppressed AKT phosphorylation and the proliferation of granulosa cells. In conclusion, the recruitment of primordial follicles was affected by the proliferation of granulosa cells and regulation of the interaction between the Notch and PI3K/AKT signalling pathways.

Enhancement of the immunogenicity of a porcine circovirus type 2 DNA vaccine by a recombinant plasmid coexpressing capsid protein and porcine interleukin-6 in mice.

The development of effective vaccines against porcine circovirus type 2 (PCV2) has been accepted as an important strategy in the prophylaxis of post-weaning multisystemic wasting syndrome; a DNA vaccine expressing the major immunogenic capsid (Cap) protein of PCV2 is considered to be a promising candidate. However, DNA vaccines usually induce weak immune responses. In this study, it was found that the efficacy of a DNA vaccine expressing Cap protein was improved by simultaneous expression of porcine IL-6. A plasmid (pIRES-ORF2/IL6) separately expressing both Cap protein and porcine IL-6 was constructed and compared with another plasmid (pIRES-ORF2) expressing Cap protein for its potential to induce PCV2-specific immune responses. Mice were vaccinated i.m. twice at 3 week intervals and the induced humoral and cellular responses evaluated. All animals vaccinated with pIRES-ORF2/IL6 and pIRES-ORF2 developed specific anti-PCV2 antibodies (according to enzyme-linked immunosorbent assay) and a T lymphocyte proliferation response. The percentages of CD3(+), CD3(+)CD8(+), and CD3(+)CD4(+) subgroups of peripheral blood T-lymphocytes were significantly higher in mice immunized with pIRES-ORF2/IL6 than in those that had received pIRES-ORF2. After challenge with the virulent PCV2 Wuzhi isolate, mice vaccinated with pIRES-ORF2/IL6 had significantly less viral replication than those vaccinated with pIRES-ORF2, suggesting that the protective immunity induced by pIRES-ORF2/IL6 is superior to that induced by pIRES-ORF2.

Primordial follicle assembly was regulated by Notch signaling pathway in the mice.

Notch signaling pathway, a highly conserved cell signaling system, exists in most multicellular organisms. The objective of this study was to examine Notch signaling pathway in germ cell cyst breakdown and primordial follicle formation. The receptor and ligand genes of Notch pathway (Notch1, Notch2, Jagged1, Jagged2 and Hes1) were extremely down-regulated after newborn mouse ovaries were cultured then exposed to DAPT or L-685,458 in vitro (P < 0.01). Since DAPT or L-685,548 inhibits Notch signaling pathway, the expression of protein LHX8 and NOBOX was significantly reduced during the formation of the primordial follicles. Down-regulated mRNA expression of specific genes including Lhx8, Figla, Sohlh2 and Nobox, were also observed. The percentages of female germ cells in germ cell cysts and primordial follicles were counted after culture of newborn ovaries for 3 days in vitro. The result showed female germ cells in cysts was remarkably up-regulated while as the oocytes in primordial follicles was significantly down-regulated (P < 0.05). In conclusion, Notch signaling pathway may regulate the formation of primordial follicle in mice.

Di-(2-ethylhexyl) phthalate and bisphenol A exposure impairs mouse primordial follicle assembly in vitro.

Bisphenol-A (BPA) and diethylhexyl phthalate (DEHP) are estrogenic compounds widely used in commercial plastic products. Previous studies have shown that exposure to such compounds have adverse effects on various aspects of mammalian reproduction including folliculogenesis. The objective of this study was to examine the effects of BPA and DEHP exposure on primordial follicle formation. We found that germ cell nest breakdown and primordial follicle assembly were significantly reduced when newborn mouse ovaries were exposed to 10 or 100 µM BPA and DEHP in vitro. Moreover, BPA and DEHP exposure increased the number of TUNEL positive oocytes and the mRNA level of the pro-apoptotic gene Bax in oocytes. These effects were associated with decreased expression of oocyte specific genes such as LIM homeobox 8 (Lhx8), factor in the germline alpha (Figla), spermatogenesis and oogenesis helix-loop-helix (Sohlh2), and newborn ovary homeobox (Nobox). Interestingly, BPA and DEHP exposure also prevented DNA demethylation of CpG sites of the Lhx8 gene in oocytes, a process normally associated with folliculogenesis. Finally, folliculogenesis was severely impaired in BPA and DEHP exposed ovaries after transplantation into the kidney capsules of immunodeficient mice. In conclusion, BPA and DEHP exposures impair mouse primordial follicle assembly in vitro.