To investigate the effect of dihydroartemisinin on apoptosis of human pancreatic cancer cell line JF-305 and the role of reactive oxygen species(ROS) in the apoptosis of JF-305 cells induced by dihydroartemisinin. MTT assays were used to detect effect of different concentrations of dihydroartemisinin on cells proliferation of JF-305 lines. Cell cycle was detected by flow cytometry, and the apoptotic morphology was observed by Hoechst 333258 fluorescence staining. Annexin V fluorescence staining was used to detect the apoptosis changes of JF-305 cells, while DCFH-DA was used to detect the changes of ROS during apoptosis process. Western blot was used to detect the protein expression changes of Bax, Bcl-2, Cleaved caspase-3, Cleaved caspase-9 and Cyto C. As compared with the control group, the JF-305 cells proliferation was inhibited significantly(P<0.05) after treatment with different concentrations of dihydroartemisimin for 48 h; cell cycle was blocked in the G2/M phase; apoptotic morphology of nuclear condensation, aggregation, and fragmentation was found, and the apoptosis ratio was increased(P<0.05). DCFH-DA detection showed that the cell ROS was increased significantly after dihydroartemisinin treatment(P<0.05). Western blot results showed that the expression of Bcl-2 protein was down-regulated; the expression of Bax protein was up-regulated; the ration of Bax/Bcl-2 was increased and the protein expression levels of Cleaved caspase-3, Cleaved caspase-9 and Cyto C were increased after dihydroartemisinin treatment. Therefore, dihydroartemisinin could induce apoptosis of JF-305 cells, and the possible mechanism may be related to the formation and increasing of ROS.

OBJECTIVE: To explore the influence of clinical practice on mental health of male nursing college students,and to investigate the effects of psychological intervention. METHODS: Using the cluster sampling method,408 clinical practice male nursing students from 5 medical colleges in Jilin Province were randomly divided into intervention group and control group. The male nursing students were investigated with Chinese Perceived Stress Scale(CPSS),M to R sections of Cornell Medical Index(CMI) and Symptom Checklist 90(SCL-90). After the first questionnaire survey in the 9 weeks of clinical practice(before psychological intervention),male nursing students in intervention group received psychological intervention,while those in control group received no action. The investigation were then performed in the 20th(11 weeks of intervention) and 30 th weeks(21 weeks of intervention) of clinical practice. RESULTS: In the 11 and 21 weeks of intervention,the positive rates of CPSS,M-R and mental symptoms in intervention group were lower than those of timematched control group(P < 0. 01),and were significantly lower than those before intervention(P < 0. 01). Before intervention,the scores of CPSS,M-R and SCL-90 in two groups had no statistical significance(P > 0. 05). After the 11 and 21 weeks of intervention,the scores of CPSS,M-R and SCL-90 in intervention group were lower than those of control group(P < 0. 01). With the increasing time of intervention,the scores of CPSS and M-R in intervention group decreased(P < 0. 01). After the 11 and 21 weeks of intervention,the scores of SCL-90 in intervention group were lower than those before intervention(P < 0. 01). CONCLUSION: The results suggest that clinical practice is a stress for the mental health of male nursing college students. Psychological supporting system can be used to improve students' psychological health.

OBJECTIVE To study the immunomodulatory effect of Guipi pills on D-galactose（D-gal）-induced aging mice. METHODS The immune-related targets and related pathways for Guipi pills to exert immune effects were screened by network pharmacology and verified through pharmacodynamic experiments. Totally 105 male ICR mice were randomly divided into blank control （CON） group， model control （MOD） group， positive control （POS） group， Guipi pills low-dose （GD） group and Guipi pills high-dose （GG） group. Except for the CON group， other groups were subcutaneously injected with 400 mg/kg D-gal to induce the aging model； CON group and MOD group were given distilled water， POS group was given 300 mg/kg pidotimod oral solution intragastrically， GD group and GG group were given Guipi pills 300， 600 mg/kg intragastrically， once a day， for 8 weeks. After medication， the serum and spleen were collected， and the contents of interleukin 2 （IL-2）， IL-4， IL-6 and tumor necrosis factor α （TNF-α）， and the contents of immunoglobulin G （IgG）， IgM and IgA were detected. The spleen index was calculated and the histopathological changes in the spleen were observed. The activities of superoxide dismutase （SOD）， glutathione peroxidase （GSH- Px） and malondialdehyde （MDA）， and the content of 8-hydroxy-2 deoxyguanosine （8-OHdG） in spleen were detected； the expression of TNF/phosphoinositide-3-kinase-threonine protein kinase （PI3k-Akt）-related proteins in spleen was detected except for POS group. RESULTS The results of network pharmacology showed that TNF， IL-6 and Akt1 were core targets. The results of pharmacodynamic study showed that compared with MOD group， the contents of IL-2， IL-4， IgG， IgM and IgA were increased significantly in Guipi pills groups， while the contents of TNF-α and IL-6 were decreased significantly； the spleen index was increased significantly （P＜0.05 or P＜0.01）. The phenomenon of diffuse proliferation of lymphocytes was improved， the spleen cells were closely arranged， and the line between the white pulp and red pulp was clear. The activities of SOD and GSH-Px in spleen were increased significantly， while the activity of MDA， the content of 8-OHdG， and the protein expressions of TNF-α， PI3K and p-Akt were decreased significantly （P＜0.05 or P＜0.01）. CONCLUSIONS Guipi pills can regulate the immune function of D-gal-induced aging mice， which is related to regulating the TNF/PI3k-Akt pathway， thereby reducing oxidative stress damage in spleen tissue of mice， and regulating protein expressions of TNF-α， PI3K and p-Akt.

The evolution, structure and antigenic epitopes prediction of Rana dybowskii antimicrobial peptide dybowskin-1ST were carried out using bioinformatics software available online. Its antibacterial mechanism and structural properties were analyzed, and its activity was verified by applying wound healing assay in mice and bacteriostatic assay in vitro. This provides the theoretical basis for the improvement of parental peptide and the development of novel derivative peptides. The software MEGA_X were used to conduct homology alignment and to construct a phylogenetic tree. The online software ProtParam, ProtScale, PeptideCutter, signal, TMHMM Server were respectively used to predict the physicochemical parameters, hydrophilia/hydrophobicity, shear sites, signal peptides, and transmembrane domains of dybowskin-1ST. The online software SOPMA, Jpred4, DNAstar Protean were used to predict the secondary structure of dybowskin-1ST, and SWISS-MODEL, I-TASSER were used to predict the tertiary structure. ABCpred and SYFPEITHI were respectively used to predict its B-and T-cell epitopes. The effect of dybowskin-1ST on the wound healing was observed on experimental mice. Kirby-Bauer method and dilution method were used to determine the bacteriostatic activity of dybowskin-1ST. The dybowskin-1ST consists of 59 amino acid residues, of which leucine accounts for 16.9%, with a molecular formula of C₃₁₈H₅₁₀N₈₀O₉₃S₂. Its theoretical isoelectric point is 5.10 and the charge is -2. The dybowskin-1ST and dybowskin-1CDYa are closely related phylogenetically. The secondary structure of dybowskin-1ST predicted by the three methods were similar, which consisted of α-helix (44.07%), extended strand (16.95%), β-turns (3.39%), and random coil (35.39%). The prediction of tertiary structure showed that dybowskin-1ST was mainly composed of α-helix, and it was regarded as a hydrophilic protein with signal peptide sequence. Subcellular localization analysis showed that the probability of secreting the mitochondrial targeted peptides was 0.944. Dybowskin-1ST is an extracellular protein with no transmembrane structure region, but contains seven phosphorylation sites, three T-cell epitopes and eight B-cell epitopes. The dybowskin-1ST promoted wound healing and effectively inhibited the growth of Escherichia coli and Staphylococcus aureus. However, it had limited antibacterial activity against fungi and drug-resistant bacteria. Although the structure of dybowskin-1ST is rich in α-helix, the verification experiments showed that its antibacterial ability needs to be enhanced. The reason may be that it is a negatively charged and hydrophilic protein, and amino acid modification with the aim of increasing the number of positive charges and changing the hydrophobicity may be used to obtain derived peptides with enhanced activity.
Amino Acid Sequence ; Animals ; Mice ; Phylogeny ; Pore Forming Cytotoxic Proteins ; Protein Structure, Secondary ; Ranidae

Rapid reduction of postprandial blood glucose is very beneficial to diabetics. In order to shorten the onset time of recombinant insulin, the cone snail insulin G1 (cI G1) of Conus geographus was studied. First, the nucleotide sequence of recombinant cone snail proinsulin G1 (cPI G1) was designed and synthesized according to the genes of human proinsulin (hPI) and cPI G1. The codon was optimized according to Escherichia coli (E. coli) codon usage frequency. Then, the plasmid pET22b(+)-cPI G1 was constructed and the recombinant cPI G1 was expressed in E. coli BL21(DE3) host strain. The recombinant cPI G1 was then purified and cleaved specially by trypsin to generate the recombinant cI G1, and its potency is 25.9 IU/mg. Fasting blood glucose test (FBGT) and oral glucose tolerance test (OGTT) suggested that the recombinant cI G1 could rapidly reduce blood glucose in normal and streptozotocin (STZ)-induced diabetic mice, but only for a short duration. This study provides a technical reference for the development of recombinant fast-acting insulin.
Animals ; Conus Snail ; Diabetes Mellitus, Experimental ; Escherichia coli ; Humans ; Hypoglycemic Agents ; Insulin ; Mice

OBJECTIVE：To study the effects of Purple frute scens leaves polysaccharides （PPLPs）on oxidative stress and PI3K/AKT/GLUT4 signaling pathway of pancreatic tissues in diabetes mellitus （DM）model mice. METHODS ：Totally 60 mice were given intraperitoneal injection of STZ （60 mg/kg）to induce DM model. The 40 successful modeling mice were randomly divided into model group ，metformin group （positive control ，200 mg/kg），PPLPs high-dose and low-dose groups （400，200 mg/kg），with 10 mice in each group. Another 10 healthy mice were selected as the normal group （normal saline ）. They were given relevant medicine intragastrically ，once a day ，for consecutive 28 days. During the experiment ，general information and body weight of mice were observed ；oral glucose tolerance （OGTT）test（determining FBG at 0，30，60，120 min after giving 40％ glucose solution ）was conducted. After last medication ，the changes of related blood glucose indexes （FBG，FINS，ISI，IRI）， blood lipid indexes （HDL-C，LDL-C，TC，TG）and oxidant stress indexes （MDA content and the activities of SOD ，CAT， GSH-Px）as well as the protein expressions of PI 3K，p-AKT and GLUT 4 in pancreatic tissue were determined. RESULTS ：During the experiment ，compared with normal group ，the mice were slow in action ，the feed consumption and water consumption increased，and body weight significantly increased in model group （P＜0.05）. 0，30，60，120 min after giving glucose ，the FBG content of mice were all increased significantly （P＜0.05）. After last medication ，the contents of FINS and HDL-C in serum as well as ISI ，the activities of SOD ，CAT and GSH-Px as well as the protein expressions of PI 3K，p-AKT and GLUT 4 in pancreatic tissue were all decreased significantly （P＜0.05）；the contents of FBG and LDL-C ，TC and TG in serum as well as IRI ， 疗。E-mail：sunguangping83@163.com MDA content in pancreatic tissue were all increased significantly（P＜0.05）. Compared with model group ，the general condition and OGTT of mice in each administration group was improved；the contents of FINS and HDL-C in serum as well as ISI ，the activities of SOD ，CAT and GSH-Px as well as the protein expressions of PI 3K，p-AKT and GLUT 4 in pancreatic tissue were all increased significantly （P＜0.05）；the contents of FBG，LDL-C，TC and TG in serum as well as IRI ，MDA content of pancreatic tissue were decreased significantly （P＜0.05）. CONCLUSIONS：PPLPs has anti-diabetic effects ，which are related to reducting oxidative stress level and promoting the activation of PI 3K/AKT/GLUT4 signaling pathway.

Objective To study China's policy of health human resources accommodated with the socio-economic development and medical demands. Methods The literature consultation method was used to analyze the status and experience in health human resources development in China since the reform and opening-up to the outside world over the past 30 years. Results Health human resources are the main motive power and decisive factor in the development of China's health work. Condition It is imperative to establish a mechanism to enhance and bring the initiative of health professionals into fall play, and set up a unified evaluation system consisting of responsibility, right and benefit, and a system of classified management of health human resources.

OBJECTIVE： To establish the method for PCR identification of bullwhip， and to identify the authenticity of bullwhip at the molecular level. METHODS： DNA samples of bullwhip and its counterfeits （donkey whip， pig whip， sheep whip） were extracted and their integrity， purity and concentration were detected. Using GenBank related information， using mitochondrial cytochrome b （Cyt b） gene of bullwhip as target gene， Primer-BLAST online software was used to design specific primer. PCR amplification was performed for whips of different species， and electrophoretic analysis was conducted for the product. PCR products of bullwhip samples were cloned and confirmed by DNA sequencing. The specificity and repeatability of the established PCR method were verified. RESULTS： DNA purity of the bullwhip and its counterfeits was high， and there was no protein or RNA pollution. 1.5％ agarose gel electrophoresis showed that there were obvious target gene bands of bullwhip samples at 200-300 bp， while no corresponding bands appeared in other counterfeit products. The results of DNA sequencing showed that the nucleotide sequence of the gene fragment of bullwhip was 100％ similar to that of the bullwhip in GeneBank. Results of methodological validation showed that established method was specific and reproducible. CONCLUSIONS： The established PCR identification method based on Cyt b gene in the study is simple， rapid， accurate， specific and reproducible， and can meet the requirements of analysis and identification of bullwhip and its counterfeits.

Aged ; Diskectomy, Percutaneous ; Endoscopy ; Humans ; Intervertebral Disc Displacement/surgery* ; Lumbar Vertebrae/surgery* ; Spinal Stenosis/surgery* ; Treatment Outcome

Objetive: To study the result of manipulation therapy(MT) for frozen shoulders. Method: Before and after MT, the parameters of involoved shoulder extensors and flexors , abductors and adductors were isokinetically recorded on 20 patients with frozen shoulder twice. Result: Compared with the parameters measured pre-therapy most of those factors measured increased significantly afterwards (P＜0.01 ). Conclusion:Effect of MT on frozen shoulder are satisfactory and ROM are accurate and reliable.