Objective:To observe the effect of Gongyanping Capsule on genital tract infected by ureaplasma urealyticum and fertility of mice, and to explore its mechanism.Methods:One hundred female ICR mice were divided into normal control group, model control group, azithromycin group, the low and high-dose group of Gongyanping Capsule according to random number table method. Except for the normal control group, the other groups were infected with ureaplasma urealyticum to establish the reproductive tract inflammation model. The azithromycin group was given 40 mg/kg of azithromycin, the low and high-dose groups were given 50 and 100 mg/kg of Gongyanping Capsule respectively, the normal control group and model control group were given equal volume of normal saline, once a day, for 4 consecutive weeks. The fertility status of each group was recorded. HE staining was used to observe the pathological changes of the vaginal tissues of the mice in each group, and the content of monocyte chemoattractant protein 1 (MCP-1), IL-4, IL-12, TNF-α myeloperoxidase (MPO) and GSH-Px of the mice in each group were determined; RT-PCR and Western blot were used to determine the vagina level of mRNA and protein expressions of TLR4 and NF-κB.Results:Compared with the model control group, the birth time and the number of dead mice in the azithromycin group and the low and high dose groups of Gongyanping Capsule decreased ( P<0.05), and the number of born mice increased ( P<0.05). The level of MCP-1, MPO, IL-4, IL-12, TNF-α decreased ( P<0.05), the level of GSH-Px increased ( P<0.05), the expression of TLR4 mRNA (1.25±0.33, 2.97±0.92, 2.32±0.72 vs. 3.69±1.32), NF-κB mRNA (1.48±0.42, 2.91±0.99, 2.13±0.70 vs. 3.83±1.41) decreased ( P<0.05), the expression of TLR4 (0.63±0.13, 1.32 ± 0.34, 1.04 ± 0.33 vs. 1.63 ± 0.41), NF-κB (0.63 ± 0.14, 1.36 ± 0.32, 1.03 ± 0.30 vs. 1.94 ± 0.58) decreased ( P<0.05), and had a certain dose-dependence. Conclusion:Gongyanping Capsule has obvious therapeutic effect on genital tract mice infected by ureaplasma urealyticum, and can significantly improve the fertility of mice; the mechanism may be related to that Gongyanping Capsule could inhibit the vaginal TLR4/NF-κB pathway in mice.

Enterokinase is a class of serine proteases that specifically recognize the cleavage DDDDK sequences. Therefore, enterokinase has been widely used as a tool enzyme in the field of biomedicine. Currently, the expression level of enterokinase in Pichia pastoris is low, which hinders related practical applications. In this study, the effects of six different signal peptides SP1, SP2, SP3, SP4, SP7 and SP8 on the secretory expression of enterokinase in Pichia pastoris were studied. Compared with α-factor, SP1 significantly increased the secretory expression of enterokinase (from 6.8 mg/L to 14.3 mg/L), and the enterokinase activity increased from (2 390±212) U/mL to (4 995±378) U/mL in shaking flask cultures. On this basis, the enterokinase activity was further enhanced to (7 219±489) U/mL by co-expressing the endogenous protein Kex2. Moreover, the activity that the mutant strain with N-terminal fusion of three amino acids of WLR was increased to (15 145±920) U/mL with a high specific activity of (1 174 600±53 100) U/mg. The efficient secretory expression of enterokinase laid a foundation for its applications in near future.

Heparin and heparan sulfate are a class of glycosaminoglycans for clinical anticoagulation. Heparosan N-sulfate-glucuronate 5-epimerase (C5, EC 5.1.3.17) is a critical modifying enzyme in the synthesis of heparin and heparan sulfate, and catalyzes the inversion of carboxyl group at position 5 on D-glucuronic acid (D-GlcA) of N-sulfoheparosan to form L-iduronic acid (L-IdoA). In this study, the heparin C5 epimerase gene Glce from zebrafish was expressed and molecularly modified in Escherichia coli. After comparing three expression vectors of pET-20b (+), pET-28a (+) and pCold Ⅲ, C5 activity reached the highest ((1 873.61±5.42) U/L) with the vector pCold Ⅲ. Then we fused the solution-promoting label SET2 at the N-terminal for increasing the soluble expression of C5. As a result, the soluble protein expression was increased by 50% compared with the control, and the enzyme activity reached (2 409±6.43) U/L. Based on this, site-directed mutations near the substrate binding pocket were performed through rational design, the optimal mutant (V153R) enzyme activity and specific enzyme activity were (5 804±5.63) U/L and (145.1±2.33) U/mg, respectively 2.41-fold and 2.28-fold of the original enzyme. Modification and expression optimization of heparin C5 epimerase has laid the foundation for heparin enzymatic catalytic biosynthesis.
Animals ; Carbohydrate Epimerases ; biosynthesis ; chemistry ; genetics ; Escherichia coli ; Gene Expression ; Heparin ; metabolism ; Heparitin Sulfate ; metabolism ; Iduronic Acid ; metabolism ; Zebrafish Proteins ; biosynthesis ; chemistry ; genetics

Objective To explore the possible mechanism of Jianyi Recipe for improving the function of islet cells from the aspects of synthesis,secretion and inactivation of glucagon-like peptide-1 (GLP-1).Methods The diabetic rat model was established by feeding with high-lipid food combined with injection of streptozotocin (STZ).The rats were randomly divided into model group,Jianyi Recipe group,and normal group.The treatment for the rats lasted for 4 weeks.The blood glucose level was detected by the rapid blood glucose meter.The plasma levels of GLP-1 and insulin were detected by Luminex liquid phase protein chip technology.Pancreatic duodenal homeobox-1 (PDX-1) mRNA expression level was detected by quantitative real-time fluorescence polymerase chain reaction (PCR).The level of GLP-1 in ileum L cells was detected by immunohistochemistry,and dipeptidyl peptidase Ⅳ (DPP-Ⅳ)level was detected by enzyme-linked immunosorbent assay.Results Jianyi Recipe could decrease the levels of fasting blood glucose and postprandial glucose (P ＜ 0.05),promote the secretion of insulin (P ＜ 0.05),and increase PDX-1 mRNA expression level in the pancreas of the diabetic rats.Compared with the model group,plasma GLP-1 level,and ileal GLP-1 positive expression area and integrated optical density were increased (P ＜ 0.05) in Jianyi Recipe group,while the differences of serum DPP-Ⅳ levels were insignificant between the two groups (P＞ 0.05).Conclusion Jianyi Recipe maybe regulate the synthesis and secretion of GLP-1 to promote PDX-1 gene expression and insulin secretion,so as to reduce blood glucose in diabetic rats.

This study aimed at analyzing the medication regularity based on differentiation in traditional Chinese medicine (TCM) for losing weight using text mining technique.All the references over losing weight were retrieved in CNKI,Wangfang Database,VIP Database and Pubmed.The drugs from the references were classified in accordance with drug property,drug flavor,channel tropism and drug efficacy.Frequency and constituent ratio of a single drug in TCM prescriptions for losing weight were put into analysis using chi square test and factor analysis to find out the medication regularity.It was found that the properties of TCM drugs in the prescriptions contained both cold and warm,while the flavors of the drugs involved pungent,sweet and light.The channel tropism of the drugs mainly belonged to spleen meridian,liver meridian,stomach meridian and lung meridian.They were mostly tonic,relieving,blood-activating,qi regulating,inhibiting-damp and antipyretic drugs.Through factor analysis we found that the common formula compatibilities were concluded as:cassia seed,lotus leaf,hawthorn,salvia miltiorrhiza,polygonum cuspidatum and radix polygonum multiflorum;capillary artemisia,epimedium herb,stephania tetrandra and ligusticum wallichii;dried tangerine peel,pinellia ternata and poria cocos;plantain seed,pericarpium arecae and selfheal;paeonia lactiflora,angelica sinensis,scutellaria baicalensis and ligusticum wallichii;poria cocos,cassia twig,atractylodes and glycyrrhiza;immature bitter orange and bark of magnolia;radix bupleuri,lycium chinensis and jujube;Chinese yam and coix seed;and astragalus,pueraria lobata and polygonatum.In conclusion,formula compatibility mainly combined syndrome differentiation with disease differentiation for the treatment of obesity in clinic,using the drugs belonging to liver meridian,spleen meridian,stomach meridian and lung meridian with the flavors of sweet,bitterness or pungent and the nature of both warm and cold.

This study was aimed to evaluate effect of Qinghua Granules (QHG) on glycometabolism, pancreatic islet function and oxidative stress in type-2 diabetics with heat syndrome. A total of 60 cases of type-2 diabetics with heat syndrome (according to the Syndrome Element Syndrome Differentiation) were enrolled in the clinic of the Department of Endocrinology and Metabolism, Shuguang Hospital Affiliated to Shanghai University of Tradi-tional Chinese Medicine. The average age of enrolled cases was (57.9 ± 6.9) years. Enrolled cases were randomly divided into the treatment group and the control group. The original hypoglycemic plan was continued to use. In the treatment group, QHG was administrated. And in the control group, placebo was given. The administration dosage in both groups was one package per day. The treatment course was 12 weeks. The fasting and postpran-dial (120 min after standard meal) blood samples before and after medication were collected. The main evalua-tion indexes were fasting plasma glucose (FPG), postprandial plasma glucose (PPG) and hemoglobin A1c (HbA1c). The secondary evaluation indexes were homeostasis model assessment (HOMA2-%B, HOMA2-%S, HOMA2%-IR), superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), maleic dialdehyde (MDA), total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL) and low-density lipoprotein (LDL). The anal-ysis of variance was used in the comparison of efficacy between two groups . The results showed that HbA1c in the treatment group was obviously reduced, and HOMA2-%B was obviously increased. There was no significant changes in the control group ( P = 0 . 044 , P = 0 . 016 ) . In the treatment group , SOD increased obviously , MDA reduced obviously. There was no significant change in the control group. There was difference b etween two groups (P = 0.011, P = 0.049). There was no change on blood lipids or other evaluation indexes. It was conclud-ed that QHG is effective in the improvement of glycometabolism, islet β-cell functions and oxidative stress in type-2 diabetics with heat syndrome .

Objective To compare curative effect of tibiofibular fractures treated by external fixation device and interlocking intramedullary nail.Methods A total of 121 patients with tibiofibular fractures were included in the study and were divided into external fixator group (56 patients) and interlocking intramedullary nail group (65 patients),according to diverse surgical approaches.The two groups were compared in indices of postoperative good-excellent rate,intraoperative blood loss,operation time,subsided time of postoperative swelling,hospital stay,fracture healing time,time of fixation removal and incidence of postoperative complications.Results The postoperative excellence rate in external fixator group and interlocking intramedullary nail group was 88％ and 89％,respectively (P ＞ 0.05).Compared with interlocking intramedullary nail group,the intraoperative blood loss was reduced more in external fixator group,with more evidently shortened operation time,subsided time of postoperative swelling and hospital stay (P ＜ 0.01).Fracture healing time and time of fixation removal,however,were significantly shorter in the interlocking intramedullary nail group than those in the external fixator group (P ＜ 0.01).Incidence of postoperative complications was 11％ in the external fixator group and 28％ in the interlocking intramedullary nail group (P ＜0.05).Conclusions External fixation device and interlocking intramedullary nail are both effective in treatment of tibial and fibular fractures.External fixation device with less damage to tissue is relatively more helpful to postoperative recovery.On the contrary,the interlocking intramedullary nail is relatively more conducive to fracture healing.

ObjectiveTo investigate the modification method of Cervical Spine Injury Severity Score System and discuss diagnosis and treatment strategy of lower cervical spine injuries.Methods Treatments of lower cervical spine injuries were selected according to the injury severity graded by the modified Moore' s classification system.Conservative therapy could be adopted for the patients with stability quantification rating ＜ 3 points or for the those with stability quantification rating =3 points but without spinal cord or nerve root compression.Surgical treatment was recommendable for the patients with stability quantification rating =3 points and with spinal cord or nerve root compression.Surgical therapy could be required for the patients with stability quantification rating ≥4 points and with risk of lower cervical instability.The higher the stability quantification score implied the stronger the surgical indications.Lower cervical spine injury combined with spine cord or nerve root compression had absolute surgical indications.At the same time,therapies were selected based on patients' other factors.ResultsBased on basic principles of the modified Moore' s classification system together with opinions of the patients and their relatives,14 patients were managed with surgical treatment and 16 with conservative treatment.Among the patients with complete spinal cord injury (Grade A),two patients treated surgically showed no obvious signs of spinal function recovery,but their nerve root irritation symptoms disappeared; the other one patient who needed surgery but received conservative treatment had no change of the spinal cord function and nerve root irritation.The patients with incomplete spinal cord injury (Grades B,C and D) treated surgically obtained certain degree of spine cord function recovery,with their American Spinal Injury Association (ASIA) score raised by 0.5 grade on average.However,the patients who needed surgery but received conservative treatment gained average increase of ASIA score for 0.5 grade.Imaging examination showed that patients without combined spinal injuries obtained interbody fusion after surgery,with normal alignment and height of the cervical vertebra but without presence of vertebral shift or instability. ConclusionsThe modified Moore' s classification system takes patients' spiaal injury condition and other factors into consideration in selection of conservative or surgical treatment,which improves the Cervical Spine Injury Severity Score System to some extent and has prospect of clinical application.

Objective To understand the primary structure and potential antigenic epitopes of antigen Pf332(Ag332) of P.falciparum iso late FCC1/HN.Methods　Based on the published Pf332 gene sequence , nine pairs of primers were designed for the PCR amplification of the Pf332 gen e fragments from genomic DNA of P.falciparum isolate FCC1/HN. The amplified gene fragments were subcloned into pMD-18T vectors and sequenced. The sequences were aligned using DNAstar software to obtain the full-length sequence of the gene Pf332. The primary structure and sequence homology of Ag332 were analyzed by SAPS, Tmpred, SingalP and Blastn programs. Three fragments, R0, R1 and R2, cor responding to nt#9595-10083, nt#10339-10767 and nt#10855-11247 of Pf332 gene were subcloned into the eukaryotic expression vector pcDNA3-S separately. The Balb/c mice were immunized with pcDNA3-S-R0, pcDNA3-S-R1 and pcDNA3- S-R2 separately, and the expressions of the recombinant proteins were detected by immunohistochemistry assay. The protective immune responses elicited by DNA I mmunization were analyzed by ELISA and parasite growth inhibition tests in vitro .Results　Nine Pf332 gene fragments were specifically amplif ied, subcloned into pMD-18T vectors and sequenced. Pf332 gene of the P.falci parum isolate FCC1/HN was 16,377 bp in length, encoding a protein of 5,458 ami no acids, about 615.28kDa. The Ag332 contains 17 regions of highly degenerated Glu-rich repeats, with 30.18% Glu in total amino acids of Ag332. Ag332 of P.falciparum isolate FCC1/HN and 3D7 exhibited 94.55 % homology in amino acid residues. The results of immunohischemistry assay showed that R0, R1 and R2 were expressed in mice muscle tissue. The amount of IgG antibody of the groups immu nized with pcDNA3-S-R0, pcDNA3-S-R1 and pcDNA3-S-R2 were higher than those of blank and pcDNA3 groups (P＜0.05). The result of parasite growth inhibition test showed that the immunized sera at 1∶5 dilution of groups of pcDNA3-S-R0, pcDNA3-S-R1 and pcDNA3-S-R2 had an incomplete inhibitor y effect on P.falciparum growth. Conclusion　The antigen Pf332 is an large protein containing highly degenerated Glu-rich repeats. Pf332 gene fragments, R1 and R2 encoding potent antigenic epitope repeats.

[Objective] To construct a eukaryotic expression plasmid containing a gene encoding a 41-3 kilodalton blood stage antigen (p41-3) of Plasmodiu falciparum isolate FCC1/HN, and to determine the sequence of p41-3 gene and analyze the homology of the sequences of 341-3 gene of different P. falciparum isolates. [ Methods] Two pairs of primers were designed according to the known sequence of p41-3 gene. Using PCR technique, the p41-3 gene was obtained by amplification from genomic DNA of isolate FCC1/HN. By cloning target gene into a eukaryotic expression vector, pcDNA3, a recombinant plasmid pcDNA3-p41-3 was con structed and trarsferred into E. coli DH5α. The positive clones were screened and identified by agarose gel electrophoresis, endonu clease digestion and PCR technique. The correct recombinant plasmid pcDNA3-p41-3 was used as template, and the nucleotide se quence of p41-3 gene was determined by the dideoxy chain termination method. Using softwares to analyze the structure and sequence homology of p41-3 gene between isolate FCC1/HN and FCBR. [Results] The p41-3 gene was specifically amplified from genomic DNA of Plasmodiumm falciparum isolate FCC1/HN, and the correct recombinant plasmid pcDNA3-p41-3 was screened and identi fied. The result of sequence determination showed that the p41-3 gene of isolate FCC1/HN was 2 137 base pairs in full length, encod ing 375 amino acids. Isolate FCC1/HN and isolate FCBR exhibited 98.98 % homology in the nucleiotide sequences and 99.73 % ho mology in the encoded anino acids of p41-3 gene. [Conclusion] The eukaryotic expression plasmid pcDNA3-p41-3 is successfully con structed and nucleotide sequence of p41-3 gene of isolate FCC1/HN is determined. The p41-3 genes of isolate FCC1/HN and isolate FCBR share quite high homology.