Enzymolysis peptides from Mauremys mutica plastron improve the disorder of neurotransmitter system and facilitate sleep-promoting in the PCPA-induced insomnia mice.

ETHNOPHARMACOLOGY RELEVANCY: For many centuries, Mauremys mutica is highly valued as a food homologous Chinese herbal medicine. It has been considered useful to sedate, nourish brain and promote sleep. However, the animal experimental evidence of its sleep-promoting activity is missing in literature. AIM OF THE STUDY: In this study, PCPA-induced insomnia model was used to explore the sleep-promoting mechanism of enzymolysis peptides from PMM, and its main composition and chemical structure were analyzed. MATERIALS AND METHODS: Experiments were performed using PCPA-induced insomnia model, all animals were intraperitoneally injected with PCPA (350 mg/kg·d) for two days. The sleep-promoting effect evaluated using measuring content of 5-HT, GABA, DA, IL-1, BDNF and expression of 5-HT1A receptor and GABAA receptor α1-subunit in mice brain. Primary structure of peptides was identified by HPLC-ESI-QqTOF-MS/MS. RESULTS: Compared with the model group, the content of 5-HT, GABA, IL-1, BDNF in mice brain of PMM peptide groups was increased to varying degrees, the content of DA was decreased, and the gene transcription and protein expression of 5-HT1A receptor and GABAA receptor α1-subunit were almost all returned to normal levels. In addition, the primary structures of most abundant nine typical peptides in PMM peptides were identified. CONCLUSIONS: The results showed that PMM peptides could improve the disorder of neurotransmitter system, restore compensatory over-expression 5-HT1A receptor and GABAA receptor α1-subunit, and have a good sleep-promoting effect. The specific amino acid composition, sequence and glycosylation modification of PMM peptides may be the key reason for their activity, which lays a foundation for the subsequent development of sleep-promoting peptide products.

Intervention on immunodeficiency mice and structural identification of enzymatic peptides from Mauremys mutica and Cuora trifasciata.

ETHNOPHARMACOLOGY RELEVANCY: Mauremys mutica (Asian yellow pond turtle, YPT) and Cuora trifasciata (Chinese three-striped box turtle, TBT) are traditional Chinese medicine. They possess many biological characteristics, such as immune-enhancement, anti-inflammatory, anti-cancer effects. They have been used as folk anti-cancer drugs in central and southern China for a long time. However, there was no reports of comparing the immune-enhancement effect of YPT and TBT, nor of identifying the structures of YPT peptides and TBT peptides. AIMS OF THE STUDY: The aim of this study was to evaluate the protective efficacy of YPT and TBT on immunodeficient mice and to compare the primary structures of YPT peptides and TBT peptides. MATERIALS AND METHODS: The protein extracts were extracted using 100 °C water, and peptides were obtained by hydrolyzing protein extracts using alkaline protease. Cyclophosphamide (CTX) was used to induce immunodeficiency in mice. The immune enhancement effect was evaluated by measuring body weight gain curve, thymus index, spleen index, serum SOD activity and GSH-Px activity. Primary structure of peptides was identified by HPLC-ESI-MS/MS. RESULTS: The protein extracts and peptides of the YPT and TBT had certain recovery effects on immunodeficient mice. YPT peptide has the best effect on the recovery of damaged immune organs and the improvement of SOD and GSH-Px activities in mice. In the identification of the primary structure of the polypeptide, we find that YPT and TBT contain some similar peptides as well as different peptides, and the concentration of the peptide segments in HPLC data is very different. The difference of biological activity may be determined by both the difference of specific peptide structure and concentration. CONCLUSIONS: Two kinds of healthy turtle protein extracts and peptides could have immune-enhancement function, and peptides obtained by enzymatic hydrolysis of YPT protein extracts have the best immune-enhancement effect. The identification of the primary structure of the peptide segment preliminarily showed that its biological activity was affected by the amino acid sequence and the concentration of part of the peptide segment. It laid a foundation for the follow-up search of immune-enhancement peptides and the development of high-value YPT products.

The effect of centromere protein U silencing by lentiviral mediated RNA interference on the proliferation and apoptosis of breast cancer.

Centromere protein U (CENPU) is a novel transcriptional repressor that is associated with different types of cancer. However, its function in breast cancer is poorly understood. In the present study, it was identified that CENPU was highly expressed in breast cancer tissues compared with expression in normal breast tissues (P=0.001). Furthermore, the CENPU mRNA level in tumors was often elevated, compared with the matched adjacent normal breast cancer tissue specimens in the dataset from The Cancer Genome Atlas database (n=106; P<0.001). To understand the function of CENPU in human breast carcinogenesis, its effects on the proliferation, apoptosis and cell cycle progression of MDA-MB-231 cells were examined using the lentiviral-mediated CENPU knockdown approach. The RNA and protein expression levels in the transfected cells were monitored using reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. The mRNA and protein expression levels of the CENPU gene were significantly lower in the CENPU-shRNA transfected cells than in the control (P<0.01), indicating successful gene expression knockdown. Post-transfection, cell counting and MTT analysis revealed that the proliferation activity was significantly suppressed in CENPU knockdown cells relative to the control (P<0.01). Additionally, fluorescence activated cell sorting analysis revealed that the (G2+S) phase fraction was significantly declined in CENPU knockdown cells relative to the control; while the G1 phase fraction was significantly increased (P<0.01) and the percentage of the apoptotic cells was significantly increased (P<0.01). In conclusion, downregulation of CENPU gene expression may inhibit cell proliferation and cell cycle progression, and increase the apoptosis of the breast cancer cells. These results suggested a possible function of this protein in breast cancer pathogenesis and prognosis.

Association Study of MMP-9 -1562C/T Gene Polymorphism with Susceptibility to Multiple Autoimmune Diseases: A Meta-analysis.

BACKGROUND AND AIMS: Matrix metalloproteinase-9 (MMP-9) -1562 C/T gene polymorphism has been identified as a susceptible gene for multiple autoimmune diseases (ADs), but studies are inconsistent. The aim of this study was to assess the overall association between MMP-9 gene polymorphism and multiple ADs using a meta-analysis. METHODS: Databases of Pubmed, Embase and Web of Science updated to March 1, 2016 were retrieved. Odds ratios (ORs) and corresponding 95% confidence intervals (95% CIs) as effect size were calculated by fixed-effect or random-effect model on the basis of heterogeneity. RESULTS: A total of 12 relevant studies containing 2,034 cases and 1,861 controls were included in this meta-analysis. A significant association between MMP-9 -1562 T allele and AD susceptibility was found in the overall population (OR = 1.269, 95% CI = 1.114-1.444, p <0.001) and the Caucasian populations (OR = 1.222, 95% CI = 1.051-1.422, p = 0.009), but not in the Asian populations (OR = 1.337, 95% CI = 0.989-0.808, p = 0.059). Stratified by disease type, we detected a significant association in other ADs (OR = 1.501, 95% CI = 1.212-1.859, p <0.001), but not in patients with multiple sclerosis (OR = 1.150, 95% CI = 0.977-1.354, p = 0.092). No publication bias was detected in the current meta-analysis. CONCLUSIONS: Data from the present study suggest that the MMP-9 -1562 C/T polymorphism may be associated with multiple AD susceptibility, especially in the Caucasian populations and other ADs. Further epidemiological studies with a larger sample size are needed to confirm these findings.

Complement component 3 deficiency prolongs MHC-II disparate skin allograft survival by increasing the CD4(+) CD25(+) regulatory T cells population.

Recent reports suggest that complement system contributes to allograft rejection. However, its underlying mechanism is poorly understood. Herein, we investigate the role of complement component 3 (C3) in a single MHC-II molecule mismatched murine model of allograft rejection using C3 deficient mice (C3(-/-)) as skin graft donors or recipients. Compared with C3(+/+) B6 allografts, C3(-/-) B6 grafts dramatically prolonged survival in MHC-II molecule mismatched H-2(bm12) B6 recipients, indicating that C3 plays a critical role in allograft rejection. Compared with C3(+/+) allografts, both Th17 cell infiltration and Th1/Th17 associated cytokine mRNA levels were clearly reduced in C3(-/-) allografts. Moreover, C3(-/-) allografts caused attenuated Th1/Th17 responses, but increased CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cell expression markedly in local intragraft and H-2(bm12) recipients. Depletion of Treg cells by anti-CD25 monoclonal antibody (mAb) negated the survival advantages conferred by C3 deficiency. Our results indicate for the first time that C3 deficiency can prolong MHC-II molecule mismatched skin allograft survival, which is further confirmed to be associated with increased CD4(+) CD25(+) Treg cell population expansion and attenuated Th1/Th17 response.

Intraperitoneal Injection of Multiplacentas Pooled Cells Treatment on a Mouse Model with Aplastic Anemia.

Coinfusion of hematopoietic and mesenchymal stem cells is more effective than hematopoietic stem cell transplantation alone. It is necessary to explore a safe and routine mixed stem cell intraperitoneal transplantation method. Multiplacentas pooled cells were intraperitoneally injected into a radiation- and immunity-induced mouse aplastic anemia model with single time. Then, mouse survival time, peripheral blood hemoglobin count, bone marrow architecture, and donor cell engraftment were assessed. The recipient mouse exhibited donor cell engraftment in both bone marrow and peripheral blood. Survival time and peripheral blood hemoglobin count increased in placenta pooled cells treated mice, compared with model-only controls (P = 0.048 and P = 0.000, resp.). However, placentas pooled cells failed to cause a significant decrease in bone marrow pimelosis area (P = 0.357). Intraperitoneally transplanted multiplacentas pooled cells can survive and engraft into a host body through blood circulation, which can increase the life span of an aplastic anemia model mice, and delay but not abrogate the development of aplastic anemia. Furthermore, they appear to play a role in increasing peripheral blood hemoglobin level response for increasing the life span of aplastic anemia model mice.

Effective induction of antitumor immunity by immunization with plasmid DNA encoding TRP-2 plus neutralization of TGF-beta.

Plasmid DNA vaccine is an appealing cancer immunotherapy. However, it is a weak immunogen and immunization with plasmid DNA encoding self-antigens, such as melanoma-associated antigens, could not induce antitumor immunity because of tolerance. In this study, we investigated the feasibility of using a plasmid DNA encoding Xenopus laevis transforming growth factor-beta 5 (aTGF-beta5) as an immunogen to induce neutralizing antibodies against murine TGF-beta1 (mTGF-beta1) and thus enhance the efficacy of plasmid DNA vaccine encoding murine tyrosinase-related protein 2 (mTRP-2) through neutralization of TGF-beta. The results showed that immunization with aTGF-beta5 resulted in the generation of mTGF-beta1-neutralizing antibodies, and immunization with a combination of aTGF-beta5 and mTRP-2 induced specific cytotoxic T lymphocytes (CTLs). On the contrary, immunization with mTRP-2 alone could not elicit the CTL response. Moreover, immunization of C57BL/6 wild-type mice with a combination of aTGF-beta5 and mTRP-2 induced the protective and therapeutic antitumor immunity to B16F10 melanoma, whereas the antitumor activity was abrogated in both CD4-deficient mice and CD8-deficient mice on the C57BL/6 background. Our results indicate that immunization with aTGF-beta5 is capable of breaking immune tolerance and induces mTGF-beta1-neutralizing antibodies. Neutralization of TGF-beta can enhance the efficacy of DNA vaccine encoding mTRP-2 and the induction of antitumor immunity by this immunization strategy is associated with CD4+ and CD8+ T cells.