Objective To investigate the roles of SENP1 in regulation of biological characteristics of NK cells.Methods Lentivirus-mediated-Senp1-small-hairpinRNA (shRNA) transduction was applied to NK92 cells.The expression of SENP1 in NK92 cells was determined by real-time PCR and Western blot.The proliferation of NK92 cells was detected by CCK-8 assay.The apoptosis of NK92 cells was determined by Annexin Ⅴ and PI labeling.The cytotoxicity of NK92 cells against K562 cells was evaluated by luciferase reporter assay.Results Treatment of NK92 cells with IL-21 resulted in SENP1 upregulation.Lentivirus mediated SENP1 knockdown reduced proliferation and increased apoptosis in NK-92 cells,but SENP1 inhibition had slight impact on the cytotoxic ability of NK92 cells to kill K562 cells.Conclusion SENP1 mediates the regulatory effect of IL-21 on the proliferation and survival of NK92 cells.

OBJECTIVETo investigate the effect of Trx-LHRH, a new GnRH crasis protein, on antibody production and male reproductive function.

METHODSTrx-LHRH produced in vitro with a new crasis gene which crasised Trx gene and GnRH gene together, was used as vaccine, and hydroalaminum base as adjuvant, in adult SD rats. After 5 weeks of the first treatment, the same dosage was used again to enhance the effect of vaccine. Antibody level was measured by ELISA, and androgen level by RIA.

RESULTSTrx-LHRH induced successfully the polycolonal antibody at the level of 1 :1 280 approximately 2 560 after 4 weeks of the first treatment, and 1 : 2 000 after 6 weeks of the enhanced treatment. Testosterone level was reduced significantly (P < 0.01) by ELISA, but there was reasonable variation among individuals. Sperm count was also reduced by Trx-LHRH treatment.

CONCLUSIONTrx-LHRH can be used as effective vaccine to induce antibody production, and at the same time, restrain the function of hypothatamas-pituitary-testis axis in vivo.

Animals ; Antibodies ; blood ; Gonadotropin-Releasing Hormone ; immunology ; Male ; Rats ; Rats, Sprague-Dawley ; Recombinant Fusion Proteins ; immunology ; Sperm Count ; Testosterone ; blood ; Thioredoxins ; immunology ; Vaccines, Synthetic ; immunology

Objective: To investigate the effects of antimicrobial peptides merecidin on the biological functions of human lung adenocarcinomaA549 cells and the potential signaling pathways and targets that involved in promoting apoptosis, by studying the changes of phosphorylation levels of proteins in A549 cells after merecidin treatment. Methods: The antibacterial peptide mericidin (9 μmol/L) was applied to treat A549 cells for 6 h, and the total protein was collected and extracted. The peptide was digested by trypsin and labeled with TMT, and then fractionated by HPLC. The phosphorylated peptides were enriched with IMAC-Fe, and finally subjected to mass spectrometry analysis. Library identification and quantification of phosphorylated peptides obtained by mass spectrometry were processed using MaxQuant software, to further analyze the functions and pathways of differentially expressed phosphorylated proteins by combining with bioinformatic analysis. Results: Through IPA analysis of phosphorylated proteins in the normal control group and the antibacterial peptide merecidin treatment group, 753 differentially phosphorylated proteins in mericidin treatment group were screened out under the conditions of |Fold Change|≥2 and P<0.05, including 229 significantly up-regulated genes and 417 down-regulated genes. Among them, the differentially expressed proteins related to apoptosis included RB1, MAPK1, ARAF, PTK2, FOXO, MARCKSandsoon.Theresultsofbiologicalprocessanalysisshowedthatdifferentiallyexpressed phosphorylated proteins were mainly concentrated in cell signal transduction, degradation and transport of nucleic acid, and cellular energy metabolism, protein translation and synthesis, and cytoskeleton formation etc. The enrichment results showed that the differentially expressed phosphorylated proteins were mainly involved in apoptosis-related MAPK, ErbB, PI3K-Akt, and Ras signaling pathways. Protein-protein interaction analysis indicated the associations among apoptosis-related proteins PTK2, PRKCA, MA2PK2, MAPK1, and LMNA. Conclusion: The antibacterial peptide merecidin may induce apoptosis and alteration of other cell functions by affecting a variety of genes and signaling pathways such as RB1, MAPK1,ARAF, PTK2, FOXO and MARCKS etc.

Objective:To explore the interventional effect of β-sitosterol on ovalbumin(OVA)-induced allergic asthma rats and its potential mechanism.Methods:SD male rats were randomly divided into normal group(CON),model group(M),positive drug dexamethasone group(DEX,0.075 mg/kg)and β-sitosterol group(Sit,50 mg/kg).A rat model of allergic asthma was estab-lished by intraperitoneal injection of OVA with aluminum hydrogen solution,and nebulized inhalation of OVA to stimulate.Rats were given intragastric administration 30 min before aerosol challenge,and after continuous administration for 7 days,the indicators of cough and asthma and tracheal phenol red excretion were detected.HE staining was used to observe pathological changes of lung tis-sue.Flow cytometry was used to detect reactive oxygen species(ROS)generation,apoptosis level and ratios of Th17 and Treg cells in peripheral blood.Biochemical method was used to detect contents of MDA,and activities of T-SOD and GSH-Px in rat lung tissues.ELISA was used to detect levels of Th17 and Treg-related cytokines(TNF-α,IL-4,IL-6,IL-17A,and IL-35).Results:Compared with model group,β-sitosterol significantly prolonged the incubation period of cough and gasp in rats with allergic asthma,reduced the frequency of cough and gasping,and promoted the excretion of phenol red in trachea;significantly reduced inflammatory infiltration in lung tissue of asthmatic rats;observably reduced MDA content in lung tissue,ROS of primary lung cell and apoptosis levels of asthmatic rats,increased the activities of T-SOD and GSH-Px;markedly reduced proportion of Th17 cells and levels of pro-inflammatory cyto-kines TNF-α,IL-4,IL-6 and IL-17A,increased proportion of Treg cells and levels of anti-inflammatory cytokine IL-35.Conclusion:β-sitosterol can ameliorate airway inflammation and oxidative damage in OVA-induced allergic asthmatic rats,and its mecha-nism may be related to the regulation of β-sitosterol on Th17/Treg immune imbalance and oxidative stress response.

Objective: To explore the effects of Yueju pill on Nitricoxide-cyclic guanosine monophosphate (NO-cGMP) signaling in mouse hippocampus. Methods: Single dose of Yueju pill was administered, and mouse hippocampi were got out after administration for 12 min, 24 min, 30 min, 3 h and 24 h. The concentrations of NO and cGMP were detected by ELISA. Other mice were randomly divided into the following groups: control group (con), L-arg group (L-arg), Yueju pill group (YJ) and L-arg+ Yueju pill group (L-arg+YJ) . Control group was treated with saline, YJ group was treated with Yueju pill (13.5 g·kg-1), and L-arg group was treated with L-arg (750 mg·kg-1) . Tail suspension test (TST) was measured30 min after administration.Results:12 min after YJ administration, the concentration of NO was significantly lower than that in control group (P ＜ 0.05) and the concentration of c GMP was also significantly lower than that in control group (P ＜ 0.05) . In the TST test, the immobility time in YJ group was significantly shorter than that in control group (P ＜ 0.01), and the immobility time in L-arg+YJ group was significantly longer than that in YJ group (P ＜ 0.01) . In open field test (OFT), the spontaneous activity of mice were not affected by administering L-arg or YJ. Conclusion: Yueju pill may rapidly alleviate depression-like behaviors of mice through regulating NO concentration, then influencing downstream cGMP and activating the NO-cGMP signaling in cells.

[摘 要] 目的：以磷酸化蛋白质组学技术分析抗菌肽merecidin处理人肺腺癌A549细胞后细胞内磷酸化蛋白质表达的差异，探究merecidin对肺腺癌A549细胞蛋白质活性、功能的影响以及涉及的信号通路。方法：采用9 μmol/L merecidin处理肺腺癌A549细胞6 h，收集并提取总蛋白，SDS-PAGE检验全蛋白提取效果，加入胰酶来对蛋白质进行酶解。酶解所获肽段用TMT标记、采用HPLC分级分离、经IMAC磷酸化修饰富集以及液相色谱-质谱联用（HPLC-MS/MS）分离肽段。使用localization probability>0.75的标准对鉴定数据进行过滤，利用GO（Gene Ontology）数据库、KEGG（KyotoEncyclopedia of Genes and Genomes）数据库和STRING数据库对磷酸化蛋白组学数据进行分析。结果：SDS-PAGE 结果显示，经9 μmol/L merecidin处理后的A549细胞全蛋白分离效果清晰、无明显降解，且实验组与对照组条带差异明显；质谱共鉴定出位于3 089个蛋白上的10 320个磷酸化修饰位点，以|Fold change|>或<1.5且P<0.05为阈值从中筛选出差异明显的753个蛋白质及其1 172个磷酸化位点。蛋白质功能富集显示，磷酸化水平显著变化的蛋白质功能主要集中在蛋白质分子结合、代谢活性、分子功能调节、细胞进程、生物功能调节等方面；整合通路生物信息学分析结果显示，差异蛋白与Ras、PI3K/AKT、mTOR、AMPK等多条通路相关联；经过COG数据库筛选，发现差异性磷酸化蛋白主要集中在细胞信号转导、RNA转录、翻译后加工和修饰、核糖体合成蛋白质、细胞骨架蛋白形成及细胞内的物质转运和分泌、囊泡运输等多个方面；蛋白质互相作用层面分析结果显示，merecidin处理后的A549细胞中形成以MAPK1、RPL23A、SRSF3H、NCBP1等为关键蛋白的相互作用网，其中ATG2B、ULK1等蛋白显著上调，MAPK1、AKT1等蛋白显著下调。结论：磷酸化蛋白组学分析结果显示，抗菌肽merecidin可能通过MAPK、RPL23A、SRSF3H和AKT1等关键蛋白质在多方面生物功能和多条信号通路中发挥作用，促进肺腺癌A549细胞凋亡和自噬，从而抑制细胞的增殖。

Silver nanoparticles were prepared by chemical reduction. Fusarium graminearum was used as the test strain. To study the inhibition of F. graminearum by silver nanoparticles, we studied the activities of protective enzymes superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), and the contents of osmotic adjustment substances soluble protein, soluble sugar and malonaldehyde (MDA) in F. graminearum. Silver nanoparticles inhibited F. graminearum and the inhibitory effect was increased with the concentration of silver nanoparticles. The inhibition rate of 10 μg/mL silver nanoparticles was more than 90% and EC50 was 0.59 μg/mL. When the treating time prolonged (2, 4, 6, 8 and 10 h), the activity of SOD, CAT and POD increased firstly and then declined. SOD, POD and CAT reached the maximum at 4 hours, and decreased to minimum at 10 hours. Silver nanoparticles also increased the MDA content and reduced the soluble sugar and protein contents in pathogens. These results indicated that cell integrity was destroyed in the presence of silver. This may be one of the inhibiting mechanisms of silver nanoparticles on the growth of F. graminearum.