Objective: To clone the cDNA of human sPLA2-IIA,construct the engineered Escherischia coli expressing human sPLA2-IIA and identify the expressed human sPLA2-IIA. Methods: Total RNAs were purified from human fetal spleen. The cDNA of human sPLA2-IIA was cloned by RT-PCR and inserted into plasmid pET32a(+) between NcoI and EcoRI sites for expressing the recombinant human sPLA2-IIA in Escherischia coli BL21(DE3). The recombinants were screened by SDS-PAGE. The engineered Escherischia coli expressing trxA-human sPLA2-IIA fusion protein was established. The expressed human sPLA2-IIA exists in the form of inclusion body and accounts for about 25% of the total proteins of Escherischia coli BL21(DE3). Conclusion: the engineered E. coli methods are suitable for preparing plenty of human sPLA2-IIA which has laid base for the large-scale expression,purification and basic studies of human sPLA2-IIA.

In this study, we investigated the effect of Cigu Xiaozhi formula on HSC-T6 activity in hypoxic microenvironment based on network pharmacology and computer-aided drug design, and predicted and verified its possible targets and related signaling pathways. The potential active components and targets of Cigu Xiaozhi formula were screened by searching Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), Encyclopaedia of Traditional Chinese Medicine (ETCM) and Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine (BATMAN-TCM) databases, and the liver fibrosis related targets retrieved from Gene Cards and Pharm GK database were integrated to obtain the potential targets of Cigu Xiaozhi formula in the treatment of liver fibrosis. GO enrichment analysis and KEGG signaling pathway enrichment analysis were performed on Omic Share platform, and Cytoscape software was used to construct the "potential active ingredient-key target-pathway" network. The active components and target proteins were subjected to molecular docking analysis by Auto Dock software. According to the results of molecular dynamics simulation and binding free energy calculation, the top 5 active components with degree were scored. The active components stigmasterol and β-sitosterol were subjected to molecular docking. CoCl₂ was used to induce HSC-T6 cells to construct hypoxia model in vitro. The cell viability was detected by CCK-8 assay, and the optimal time and concentration of hypoxia model of HSC-T6 cells was determined to be 100 µmol·L^-1 CoCl₂ for 24 h. Under hypoxia condition, HSC-T6 cells were activated, the wound healing rate was significantly increased, and the fluorescence signal of activation marker protein α-smooth muscle actin (α-SMA) was significantly enhanced. However, 6% drug-containing serum could inhibit the activation of HSC-T6 cells, and the wound healing rate was significantly decreased, and the fluorescence signal of α-SMA was significantly weakened. Further studies showed that the expressions of hypoxia-inducible factor-1α (HIF-1α), α-SMA and key proteins of Hedgehog (Hh) signaling pathway in HSC-T6 cells were up-regulated under hypoxia, while the expressions of HIF-1α, α-SMA, Patched-1 (Ptch-1) and glioma related oncogene homology-1 (Gli-1) were down-regulated in 6% drug-containing serum group, the YC-1 group and the cyclopamine group. These results indicated that HIF-1α and Hh signaling pathways were involved in the activation of HSC-T6 cells, and the traditional Chinese medicine Cigu Xiaozhi formula could inhibit the activation of HSC-T6 cells, and the mechanism may be related to the inhibition of HIF-1α expression and the blocking of Hh signaling pathway. In conclusion, Cigu Xiaozhi formula can inhibit the activation of HSC-T6 cells by directly acting on HIF-1α and Hh signaling pathway, and exert an anti-hepatic fibrosis effect. The animal experimental protocol has been reviewed and approved by Laboratory Animal Ethics Committee of Gansu University of Chinese Medicine, in compliance with the Institutional Animal Care Guidelines.

This study was to improve the way for selecting ura5 mutants of Cryptoccocus neoformans Cap59 capsule-deficient strains.They were induced by Diethyl Sulfate. Ura5 mutants were screened by 5-fluoroorotic acid counter selection method. Using the new method, we obtained two ura5 mutants of Cryptoccocus neoformans Cap59 capsule-deficient strain.A easy method that was used to screen ura5 mutants of Cryptoccocus neoformans has been established.

Background Recently researches indicated that estrogen plays important role in maintaining the normal metabolism of lens. Objective This study was to investigate the changes of estrogen receptor( ER ) α and β expressions in lens upon estrogen level in castrated female rat. Methods Sixty clean adult female Wistar rats were randomized into castrated group,sham operation group,ovariectomy group,ovariectomy with low-dose estradiol eyedropping group,ovariectomy with high-dose estradiol eyedropping group,ovariectomy with low-dose estradiol injecting group and ovariectomy with high-dose estradiol injecting group,and 10 rats for each.The castrated animal models were established by ovariectomy for 5 months.Then 50％,100％ oestradiol benzoate eyedrops were used 4 times per day respectively and 0.2 or 0.4 mg/kg oestradiol benzoate were intramuscularly injected at two-day interval for 6 weeks in corresponding experimental group.Serum estradiol concentration was detected in the rats of various groups at 5 months after ovariectomy and 6 weeks after administration of estradiol benzoate.The animals were sacrificed using the excessive anesthesia method and the lenses were obtained for the assay of ERα and ERβ expressions.The use of the animals complied with the Statement of ARVO. Results No obvious opacification of lenses and the changes of structure and morphology in lens were seen in the rats of various groups under the slit lamp microscope and light microscope during the observing duration after ovariectomy.The significant differences were found in serum estradiol concentrations among the 6 groups ( F=15490.527,P=0.000) or between before and after usage of estradiol benzoate( F=943.236,P =0.001 ).Six weeks after usage of estradiol benzoate,the expressions of ERα and ERβ in the lenses were lower in the castrated group,ovariectomy with high-dose estradiol eyedropping group and ovariectomy with low-dose estradiol injecting group compared with the the sham operative group (P＜0.05),but those in the ovariectomy with low-dose estradiol eyedropping group and ovariectomy with high-dose estradiol injecting group were elevated in comparison with above groups( P＜0.05 ),and expressions of ERα and ERβ in the lenses were similar to the sham operative group ( ERα:28.04±6.80 vs.31.30±7.11 ;ERβ:27.75±7.13 vs.25.38±5.59).Mean A values of ERα and ERβ in the lenses were lower in the castrated group,ovariectomy with high-dose estradiol eyedropping group and ovariectomy with low-dose estradiol injecting group compared with the sham operative group (P＜0.05),but those in the ovariectomy with low-dose estradiol eyedropping group and ovariectomy with high-dose estradiol injecting group were elevated in comparison with above groups ( P＜0.05 ),and mean 4 values of ERα and ERβ in the lenses were similar to the sham operative group (ERα:0.1833 ±0.0087 vs.0.1859 ±0.0067; ERβ:0.1689±0.0059 vs.0.1686±0.0095). Conclusions The expressions of ERα and ERβ in the LECs are associated with the level of serum estradiol.The effects of estrogen on lens were different by different medication way.Low-dose estradiol eyedropping was a more feasible approach to the prevention of cataract.

Purpose To evaluate the expression of miR-21 in the tissues and cell lines of hepatocellular carcinoma,and to try to find its possible target genes.Methods The expression profile of miR-21 was detected in hepatocellular carcinoma tissues and cell lines.Mter miR-21 inhibitor was used,the alterations in the vitality and invasion of hepatocellular carcinoma cells were observed.The possible target gene of miR-21 was predicted by bioinformatics analysis.The influence of miR-21 inhibitors on the target gene activity was evaluated by dual luciferase reporting gene system.Results The expression level of miR-21 was significantly higher in hepatocellular carcinoma tissues than that in the adjacent ones (P ＜0.05).The expression level of miR-21 in hepatocellular carcinoma cells was significantly higher than that in the hepatic cells (P ＜0.01).After inhibiting miR-21,the viability and invasion ability of hepatocellular carcinoma cells were decreased (P ＜ 0.01).The expression level of programmed cell death 4 (PDCD4) in hepatocellular carcinoma tissues was significantly lower than that in the adjacent tissues (P ＜ 0.01).Its expression level in hepatocellular carcinoma cells was significantly lower than that in the hepatic cells (P ＜ 0.01).After interfering with PDCD4,the vitality and invasion ability of liver cancer cells were increased (P ＜ 0.05).Dual luciferase reporter gene assay indicated that by inhibiting miR-21,the expression level of PDCD4 was up-regulated (P ＜ 0.01).The vitality and invasion ability of liver cancer cells were reduced (P ＜ 0.001).Conclusion MiR-21 can regulate the growth and invasion of liver cancer cells through targeting PDCD4.

To analyze the regularity in combined medication with Xiyanping injection (Xiyanping for short) in the real world by as- sociation rules. Totally 5 822 patients using Xiyanping injection was collected from the 18 Class III Grade I hospitals nationwide to study the combined medication information of the patient with lung infection and make the analysis by using association rules and Apriori. According to the results, major drugs combined with Xiyanping in treatment of lung infection included compound amino acid, inosine, coenzyme A, cytidine triphosphate, vitamin C. Common drugs combined with Xiyanping can be divided into 5 categories: nutrition support therapy (vitamin C, compound amino acid) , coenzymes (coenzyme A, cytidine triphosphate, inosine), expectorants and antiasthmatics (ambroxol, salbutamol, doxofylline), hormones (dexamethasone, budesonide), antibiotics (mainly cefminox). The main combined medicines mostly conformed to the regularity for drugs treating lung infection. In addition, there were two most common medical combination models: the model for Xiyanping combined a single medicine is Xiyanping + nutrition support therapy, while the model for Xiyanping combined two or more than two medicines is Xiyanping + nutrition support therapy + coenzyme. Pharmacologically, Xiyanping is mostly combined with western medicines with similar pharmacological effects to substitute or supplement the antibiotic effect in treating lung infection. However, further studies shall be conducted for the safety and rationality of the combined medication based on clinical practices, in order to provide reference for clinical medication.
Adult ; Anti-Bacterial Agents ; administration & dosage ; Ascorbic Acid ; administration & dosage ; Cephamycins ; administration & dosage ; Drug Therapy, Combination ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Hospital Information Systems ; Humans ; Lung Diseases ; drug therapy ; Male ; Middle Aged ; Young Adult

OBJECTIVETo explore the changes of rat testicular spermatogenic epithelium stimulated by bacterial lipopolysaccharide (LPS) in vivo.

METHODSTwenty Wistar rats were divided into two groups: control group and experimental group. The control group was treated with pyrogen-free saline (1 ml/kg) and the experimental group was injected ip with saline containing LPS (1 mg/kg) once every two days. Two groups were operated after ten days in order to investigate the testicular pathological changes by HE staining and the expression of proliferating cell nuclear antigen( PCNA), alpha-catenin in spermatogenic epithelium by immunohistochemistry assay.

RESULTSThe testes of the experimental group showed inflammatory changes. The positive expression of PCNA in seminiferous epithelium was significantly lower than that of control group. The number of positive cells in every seminiferous, in which only spermatogonia were stained in experimental group were 59 +/- 5 and it showed significant decrease compared with the control (P < 0.01). Furthermore, the percentage of such seminiferous tubules was 0.673 +/- 0.054 and increased apparently (P < 0.01). The expression of alpha-catenin in testicular tissue of the experimental group declined (P < 0.01), and cellular positive granular light density was 0.150 +/- 0.014.

CONCLUSIONThe ability of spermatogonium proliferation and the function of conglutination of cells under inflammatory condition of the testes declined, which may be one of the etiologies of male infertility.

Animals ; Bacterial Toxins ; Disease Models, Animal ; Male ; Orchitis ; metabolism ; pathology ; Proliferating Cell Nuclear Antigen ; metabolism ; Random Allocation ; Rats ; Rats, Wistar ; Seminiferous Epithelium ; metabolism ; alpha Catenin ; metabolism

AIMTo discover new drugs which may be applied to diseases of the immune system, hemogenesis system diseases and tumors, several high-throughput drug screening cell models based on JAK-STAT signal pathway have been established.

METHODSFour repeats of STAT DNA binding conserved sequences were synthesized, subcloned into pGL-Luc reporter vector and stably transfected into cell lines in vitro. Cell clones with high copy numbers of STAT binding sites and reporter genes were chosen as high-throughput drug screening cell models. The cell models were tested with known anti-allergic drugs and anti-tumor drugs by determining luciferase activity. The reaction was performed in 96 well micro-plates with a final volume of 50 microL.

RESULTSThe cell models by performing rapid fluorescence assay were shown to be highly sensitive and stable after testing with cytokine and drugs. The modification of the expression plasmid simplified this method and made it more practical. It also provided good linear correlation, wide range of assay, highly sensitive and good reproducibility.

CONCLUSIONThe method can be performed by high-throughput drug screening for effective extraction of Chinese traditional herbs.

Anti-Allergic Agents ; isolation & purification ; pharmacology ; Antineoplastic Agents ; isolation & purification ; pharmacology ; Carcinoma, Hepatocellular ; pathology ; DNA-Binding Proteins ; genetics ; metabolism ; Drug Evaluation, Preclinical ; methods ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Humans ; Janus Kinase 2 ; Jurkat Cells ; metabolism ; Liver Neoplasms ; pathology ; Luciferases ; metabolism ; Protein-Tyrosine Kinases ; genetics ; metabolism ; Proto-Oncogene Proteins ; STAT3 Transcription Factor ; Signal Transduction ; Trans-Activators ; genetics ; metabolism ; Tumor Cells, Cultured

OBJECTIVETo explore the specific cytotoxic T lymphocyte (CTL) induced by dendritic cells (DC), which were transfected by the plasmid pC53-SN3 encoding p53 gene.

METHODSDC derived from HLA-A2(+) mononuclear cells of the 24-lung cancer patients was transfected with the plasmid pC53-SN3 by lipofectamine and then co-cultured with auto-unpurified T cells to induce potent CTL (T-pC53-SN3). The cytolysis of specific CTL against Calu-6, a HLA-A2(+) human lung cancer cell line, was measured by using lactate dehydrogenase (LDH) releasing assay.

RESULTSThe expression of CD(1a) and CD(83), the correlative markers of DC, increased apparently after transfected with plasmid pC53-SN3, the expression rate was (5.45 +/- 0.89)% and (3.26 +/- 0.47)% versus (52.15 +/- 11.56)% and (25.78 +/- 12.35)%. CD(14) decreased apparently, but other DC correlative markers of CD(1a), CD(40), CD(86), and HLA-DR remained almost the same as that before transfection. Compared with T-IL-2, the CTL derived from PBMNC stimulated by IL-2 (100 U/ml), the cytolytic activity of T-pC53-SN3 against Calu-6 cell line showed a significant increase, but cytolytic activity was 56.79 +/- 15.67 and 39.33 +/- 9.88, respectively, when effect cells: target cells was 10:1. The expression of the CD(8), CD(69), and CD(45)RO/CD(8) of T-pC53-SN3 cells increased significantly, but that of CD(3), CD(4), CD(86), ect, was not significantly different from those of T-pCMV-neo.

CONCLUSIONSIt showed that DC transfected by p53 gene could induce potent HLA-A(2) restrictive CTL to kill tumor cell efficiently.

Antigens, CD ; analysis ; B7-2 Antigen ; CD40 Antigens ; analysis ; Cell Line, Tumor ; immunology ; Coculture Techniques ; Cytotoxicity, Immunologic ; immunology ; Dendritic Cells ; drug effects ; immunology ; metabolism ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Interleukin-4 ; pharmacology ; Membrane Glycoproteins ; analysis ; T-Lymphocytes ; immunology ; Tumor Suppressor Protein p53 ; genetics ; physiology

The feasibility of simultaneously loading both liposoluble and water-soluble components of Salvia miltiorrhiza in emulsion was discussed, in order to provide new ideas in comprehensive application of effective components in S. miltiorrhiza in terms of technology of pharmaceutics. With tanshinone II (A) and salvianolic acid B as raw materials, soybean phospholipid and poloxamer 188 as emulsifiers, and glycerin as isoosmotic regulator, the central composite design-response surface method was employed to optimize the prescription. The coarse emulsion was prepared with the high-speed shearing method and then homogenized in the high pressure homogenizer. The biphasic drug-loading intravenous emulsion was prepared to investigate its pharmaceutical properties and stability. The prepared emulsion is orange-yellow, with the average diameter of 241 nm and Zeta potential of -35.3 mV. Specifically, the drug loading capacity of tanshinone II (A) and salvianolic acid B were 0.5 g x L(-1) and 1 g x L(-1), respectively, with a good stability among long-term retention samples. According to the results, the prepared emulsion could load liposoluble tanshinone II (A) and water-soluble salvianolic acid B simultaneously, which lays a pharmaceutical foundation for giving full play to the efficacy of S. miltiorrhiza.
Chemistry, Pharmaceutical ; instrumentation ; methods ; Drugs, Chinese Herbal ; chemistry ; Emulsions ; chemistry ; Quality Control ; Salvia miltiorrhiza ; chemistry