Pharmacological Mechanism of Wumeiwan in Treating Lung Metastasis of Breast Cancer / 中国实验方剂学杂志

Objective:To explore the potential target and mechanism of Wumeiwan in the treatment of lung metastasis of breast cancer by network pharmacological analysis and experimental verification. Method:The databases of active ingredients and targets of Wumeiwan were established through Traditional Chinese Medicine Systems Pharmacology（TCMSP） Database and Analysis Platform，and the targets of lung metastasis of breast cancer were established through the GeneCards database and Online Mendelian Inheritance in Man（OMIM） database，and the data of Chinese medicine targets and disease targets were matched. Cytoscape 3.6.0 software was used to establish the network analysis of traditional Chinese medicine-active ingredients-therapeutic targets，and the interaction relationship between key target proteins was analyzed by STRING database. Target gene ontology（GO） analysis and Kyoto Encyclopedia of Genes and Genomes（KEGG） signal pathway enrichment analysis were performed by using the Biological Information Annotation Database. Result:A total of 108 possible important targets for Wumeiwan in the treatment of lung metastasis of breast cancer were found，including interleukin 6（IL6），cysteine aspartate-specific protease-3（CASP3），vascular endothelial growth factor A（VEGFA），epidermal growth factor receptor（EGFR），mitogen-activated protein kinase（MAPK8）， and others. GO enrichment analysis yielded 29 cell components（CC），1 218 biological processes（BP） and 125 molecular functions（MF） related to lung metastasis of breast cancer，and KEGG enrichment analysis yielded 118 pathways related to lung metastasis of breast cancer（<italic>P<</italic>0.05），including MAPK signaling pathway and apoptosis pathway. <italic>In vitro</italic> experiments showed that cinnamaldehyde， the active ingredient of Wumeiwan， could induce apoptosis，inhibit proliferation and migration of MCF7 cells，partially validating the predicted results of network pharmacology to a certain extent. Conclusion:The therapeutic effect of Wumeiwan on lung metastasis of breast cancer may be multi-target，multi-pathway and multi-mechanism. The results of this study provide more evidence for the clinical application of Wumeiwan.

Expert consensus on Tongsaimai Tablets/Capsules in treatment of peripheral vascular diseases in clinical practice / 中国中药杂志

Tongsaimai Tablets/Capsules are composed of Lonicerae Japonicae Flos, Angelicae Sinensis Radix, Achyranthis Bidentatae Radix, Codonopsis Radix, Dendrobii Caulis, Astragali Radix, Scrophulariae Radix, and Glycyrrhizae Radix et Rhizoma, and are effective in promoting blood circulation, removing blood stasis, supplementing Qi, and nourishing Yin. It is widely used in the treatment of peripheral vascular diseases. With 40 years of clinical application, it has accumulated substantial research data and application experience. Its good clinical efficacy and pharmacoeconomic benefits in improving the clinical symptoms of peripheral vascular diseases have been confirmed by relevant research. Meanwhile, this drug has also been recommended by many expert consensus, guidelines, and teaching materials, serving as one of the most commonly used Chinese patent medicines in clinical practice. To further improve the understanding of the drug among clinicians and properly guide its clinical medication, the China Association of Chinese Medicine took the lead and organized experts to jointly formulate this expert consensus. Based on the questionnaire survey of clinicians and the systematic review of research literature on Tongsaimai Tablets/Capsules with clinical problems in the PICO framework, the consensus, combined with expert experience, concludes recommendations or consensus suggestions by GRADE system with the optimal evidence available through the nominal group technique. This consensus defines the indications, usage, dosage, course of treatment, medication time, combined medication, and precautions of Tongsaimai Tablets/Capsules in the treatment of peripheral vascular diseases, and explains the safety of its clinical application. It is recommended for clinicians and pharmacists in the peripheral vascular department(vascular surgery), traditional Chinese medicine surgery(general surgery), and endocrinology department of hospitals at all levels in China.

The role of autophagy-related gene mTOR signaling pathway in the pathogenesis of systemic lupus erythematosus / 医学研究生学报

Objective　The mechanism of autophagy-related gene mTOR signaling pathway is less studied in its pathogenesis of systemic lupus erythematosus. The aim of this study was to investigate the role of autophagy-related gene mTOR signaling pathway in the pathogenesis of SLE.　Methods　72 patients with systemic lupus erythematosus in the Department of Rheumatism and Immunity in the Second Affiliated Hospital of Hainan Medical College from February 2018 to March 2019 were selected as observation group. 67 healthy subjects in the same period were used as control group. The ENA antibody profiles were determined by immunoblotting, including anti-SSA, anti-SSB, anti-Sm, anti-nRNP antibodies, and ds-DNA antibodies. IgG, IgA, IgM, and complement C3, C4 were determined using an IMMAGE protein analyzer. Fluorescence PCR was used to detect the autophagy-related gene mTOR signaling pathway. SPSS Pearson correlation analysis software was used to analyze the correlation between autophagy-related gene mTOR signaling pathway(pI3K mRNA, Akt mRNA, mTOR mRNA) and humoral immunity level in the two groups.　Results　The level of IgG, IgA, and IgM in the observation group was significantly higher than that of the control group (P<0.05). The levels of complement C3 and C4 in the observation group were lower than those in the control group (P<0.05). The expression levels of PI3K mRNA, Akt mRNA and mTOR mRNA in SLE group (0.52±0.06, 0.61±0.08, 0.48±0.05) were significantly lower than those in control group (1.00±0.09, 0.89± 0.07, 0.95±0.08), with statistically significant differences (P<0.05). The results of SPSS Pearson correlation analysis showed that Akt and mTOR signals expression were negatively correlated with IgG, IgA, IgM level,and ds-DNA, anti-SSA, anti-SSB, anti-Sm, anti-nRNP expression leve(P<0.05). On the contrary, they were positively correlated with complement C3, C 4 levels(P<0.05).　Conclusion　The expression of PI3K mRNA, Akt mRNA and mTOR mRNA in mTOR signaling pathway of autophagy related genes in SLE patients is inhibited, which is closely related to immunoglobulin and complement level. Therefore, detection of the autophagy related gene mTOR signaling pathway will be helpful in assessing the status of SLE patients.

Comparison of clinical features and stent placement outcomes between airway stenosis caused by primary pulmonary malignancies and that caused by primary non-pulmonary malignancies / 中华医学杂志(英文版)

BACKGROUND@#Primary pulmonary malignancies (PPMs) and non-pulmonary malignancies (PNPMs) may result in airway stenosis requiring stenting. This study aimed to compare and evaluate the clinical features and stent placement outcomes of airway stenosis caused by PPMs and PNPMs.@*METHODS@#A total of 141 patients with malignant airway stenosis who underwent Micro-Tech stent placements between January 2004 and October 2017 at Department of Respiratory Medicine, Beijing Tian Tan Hospital, Capital Medical University were divided into PPM (n = 100) and PNPM groups (n = 41). Patients' clinical features and stent placement outcomes were collected and analyzed. Chi-square test was used to compare the categorical variables, while independent- or paired-sample t test was used to compare the continuous variables.@*RESULTS@#There were no significant differences in age, sex, treatment history, respiratory symptoms, and incidence of obstructive pneumonia between groups. Multiple airway involvement (63.0% vs. 31.7%; χ = 11.459, P = 0.001) and atelectasis (17.0% vs. 2.4%; χ = 5.536, P = 0.019) were more common in the PPM group, while extraluminal obstruction (24.4% vs. 6.0%; χ = 8.033, P = 0.005) was more common in the PNPM group. Before stenting, the American Thoracic Society Dyspnea Index (ADI) and Karnofsky Performance Scale (KPS) scores showed no significant differences between groups (all P > 0.05). After stenting, a satisfactory rate of symptom improvement was achieved in both groups (98.0% and 100.0% in the PPM and PNPM groups, respectively; χ = 0.016, P = 0.898); ADI and KPS scores, which showed no significant differences between groups (all P > 0.05), were significantly improved in each group (all P < 0.001). Complications after stenting could be effectively managed using bronchoscopic procedures.@*CONCLUSIONS@#Among cases of malignant airway stenosis requiring stenting, those caused by PPM are more likely to involve multiple airways and are associated with atelectasis, while those caused by PNPM are more likely to cause extraluminal obstruction. Micro-Tech stent placement has the same immediate effect in terms of improvement in respiratory symptoms and performance status for both malignant airway stenosis caused by PPM and that caused by PNPM.

Scarring Airway Stenosis in Chinese Adults: Characteristics and Interventional Bronchoscopy Treatment / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Scarring airway stenosis is commonly seen in China as compared to other developed countries, due to the high prevalence of tuberculosis. Nowadays, interventional bronchoscopy treatment has been widely used to treat this disease in China. This study demonstrated the characteristics of scarring airway stenosis in Chinese adults and retrospectively evaluated the efficacy of interventional bronchoscopy treatment of this disease.</p><p><b>METHODS</b>Patients with scarring airway stenosis from 18 tertiary hospitals were enrolled between January 2013 and June 2016. The causes, site, and length of scarring airway stenosis were analyzed, and the efficacy of the interventional bronchoscopy treatment was evaluated.</p><p><b>RESULTS</b>The final study cohort consisted of 392 patients. Endotracheobronchial tuberculosis (EBTB) was the most common cause of scarring airway stenosis (305/392, 77.8%) in Chinese adults with a high rate of incidence in young women. The left main bronchus was most susceptible to EBTB, and most posttuberculosis airway scarring stenosis length was 1.1-2.0 cm. The average clinical success rate of interventional bronchoscopy treatment for scarring airway stenosis in Chinese patients is 60.5%. The stent was inserted in 8.7% scarring airway stenosis in China.</p><p><b>CONCLUSIONS</b>Scarring airway stenosis exhibits specific characteristics in Chinese patients. Interventional bronchoscopy is a useful and safe treatment method for the disease.</p>

Bacillomycin D-like compounds isolated from Bacillus amyloliquefaciens HAB-2 are effective against Burkholderia pseudomallei

Background:Burkholderia pseudomallei (Bp) is a gram-negative environmental bacterium that causes melioidosis. It has high mortality and relapse rates regardless of powerful antibiotic therapy. Bacterial pathogens display versatile gene expression to adapt to changing surroundings, especially when they are infected by drugs. A cyclic lipopeptide was isolated from Bacillus amyloliquefaciens HAB-2, which is a bacillomycin D-like compound, named as bacillomycin DC. It is a potent fungicide against Colletotrichum gloeosporioides Penz.Methods:We used this kind of bacillomycin DC to be inhibitor of Bp and in order to find out how does it infect the bacterial pathogens. We observed the morphological changes under transmission electron microscope (TEM) and scanning electron microscopy (SEM) when BP is in the minimum inhibitory concentration (MIC) of ceftazidime and bacillomycin DC. Then we used quantificationgene Real-Time PCR (qRT-PCR) to measure the expression of three drug-assistant genes including MexB, qnrS and oprD2, respectively.Results:Bacillomycin DC treatment caused changes in the shape and microstructure, and the bacterial outer membrane were damaged, the leakage of the cell were observed. The expression level of mexb gene was not high until 72h after ceftazidime and bacillomycin DC treatment. Both ceftazidime and bacillomycin DC caused high expression of oprD2, but higher expression level proves that the DC works more efficiently and quickly. Bacillomycin DC increased the expresssion level of bacteria qnrS gene in 24 h, which proved this compound injured the DNA helicase and topoisomerase of the bacteria in a short time. The results showed that the bacillomycin DC had better inhibitory effects. We also found out that different mechanism of action between ceftazidime and bacillomycin DC.Conclusion:The bacillomycin DC makes bacterial pathogen display more oprD2 and qnrS, which respectively means bacterial pathogen are sensitive to the bacillomycin DC and its DNA gyrase are injured. In short, our study showed for the first time that bacillomycin DC can inhibit Bp in a short time.

Brucine inhibits bone metastasis of breast cancer cells by suppressing Jagged1/Notch1 signaling pathways / 中国结合医学杂志

<p><b>OBJECTIVE</b>To examine the effects of brucine on the invasion, migration and bone resorption of receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis.</p><p><b>METHODS</b>The osteoclastogenesis model was builded by co-culturing human breast tumor MDA-MB-231 and mouse RAW264.7 macrophages cells. RANKL (50 ng/mL) and macrophage-colony stimulating factor (50 ng/mL) were added to this system, followed by treatment with brucine (0.02, 0.04 and 0.08 mmol/L), or 10 μmol/L zoledronic acid as positive control. The migration and bone resorption were measured by transwell assay and in vitro bone resorption assay. The protein expressions of Jagged1 and Notch1 were investigated by Western blot. The expressions of transforming growth factor-β1 (TGF-β1), nuclear factor-kappa B (NF-κB) and Hes1 were determined by enzyme-linked immunosorbent assay.</p><p><b>RESULTS</b>Compared with the model group, brucine led to a dose-dependent decrease on migration of MDA-MB-231 cells, inhibited RANKL-induced osteoclastogenesis and bone resorption of RAW264.7 cells (P<0.01). Furthermore, brucine decreased the protein levels of Jagged1 and Notch1 in MDA-MB-231 cells and RAW264.7 cells co-cultured system as well as the expressions of TGF-β1, NF-κB and Hes1 (P<0.05 or P<0.01).</p><p><b>CONCLUSION</b>Brucine may inhibit osteoclastogenesis by suppressing Jagged1/Notch1 signaling pathways.</p>

Interventional Bronchoscopic Therapy in Adult Patients with Tracheobronchial Mucoepidermoid Carcinoma / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Tracheobronchial mucoepidermoid carcinoma (MEC) is a rare airway tumor in adults for which surgery is considered a first-line treatment. However, some patients already lost the best opportunity of a surgical intervention when diagnoses are confirmed, and surgery causes considerable trauma resulting in partial loss of pulmonary function. Moreover, the tumor is resistant to radiotherapy and chemotherapy. These factors make the treatment of tracheobronchial MEC challenging. This study aimed to evaluate the safety and efficacy of interventional bronchoscopic therapy in adult patients with tracheobronchial MEC.</p><p><b>METHODS</b>We retrospectively analyzed the clinical manifestations, bronchoscopic interventions, complications, and outcomes of 11 adult patients with tracheobronchial MEC. Paired t-test was used to analyze the parameters of the American Thoracic Society Dyspnea Index and the Karnofsky Score before and after the first interventional bronchoscopic therapy.</p><p><b>RESULTS</b>All tumors occurred in the main bronchus and were easily visualized by bronchoscopy. After interventional bronchoscopic therapy, the symptoms of all patients showed significant improvement. The American Thoracic Society Dyspnea Index decreased from 1.91 ± 1.22 to 0.27 ± 0.47 (t = 6.708, P < 0.001) and the Karnofsky Score increased from 78.18 ± 16.62 to 95.46 ± 8.20 (t = -5.190, P < 0.001). Bronchoscopic intervention did not result in serious complications or mortality. During the follow-up period between 3 and 96 months after the first therapy, the following results were noted: (1) among the eight patients with low-grade tracheobronchial MEC, only one patient had a relapse and agreed to surgical treatment; (2) among the three patients with high-grade tracheobronchial MEC, one patient required repeated bronchoscopic interventions, one patient died of pulmonary infection, and one patient died of systemic failure owing to tumor metastasis.</p><p><b>CONCLUSIONS</b>Interventional bronchoscopic therapy, as an alternative treatment, shows promise in some adult patients with low-grade tracheobronchial MEC confined to the bronchus. However, for adult patients with high-grade tracheobronchial MEC, early diagnosis and surgical treatment are still strongly recommended.</p>

Current status of medical instrument cleaning in 127 hospitals in five coastal provinces or cities in China / 中华护理杂志

Objective To investigate the current situation of medical instrument cleaning in Central Sterile Supply Departments(CSSD),in order to identify problems and provide references for further improvements of equipment cleaning.Methods Questionnaire survey was adopted,data of 127 CSSDs in five coastal provinces or cities in China in 2017 were collected,and status of each link of medical instrument cleaning was analyzed.Results The allocation rate of ultrasonic cleaner and spray cleaner were 96.6％ and 78.7％,respectively;62.2％ of hospitals used tap water to pre-rinse,and 10.2％ used normal saline or sterile water to pre-rinse,11.0％ used tap water for finial rinsing;78.7％ of hospitals did not perform continuous humidification in transit,and 18.1％ of hospitals did not perform continuous humidification and their time of transit was more than 2 hours;66.9％ of CSSDs only used multi-enzyme detergent.More than half of the hospitals did not record time for manual rinsing/manual final rinsing.Conclusion The configuration of medical instrument cleaning equipment of 127 CSSDs is good.However,it is necessary to pay more attention to cleaning details.Specifications for instrument cleaning water should be established;continuous humidification during transportation of medical instruments should be promoted;cleaning methods and cleaning detergents should be selected reasonably;standardized rinsing should be enhanced to ensure quality of medical instrument cleaning.

Effects of dengue type 2 virus on the apoptosis and autophagy of primary HHSECs and the expres-sion of related genes / 中华微生物学和免疫学杂志

Objective To investigate the effects of dengue type 2 virus(DENV-2)on the apopto-sis and autophagy of primary human hepatic sinusoidal endothelial cells(HHSECs)and the expression of ICAM-1 and Beclin-1 at mRNA level and to analyze the possible pathogenic mechanism of DENV-2. Meth-ods Immunohistochemistry(IHC)and flow cytometry analysis(FCM)were performed to identify HHSECs by detecting factor Ⅷ and CD31. The DENV-2 strain was identified by using PCR and HindⅢ. The 50%tissue culture infective dose(TCID50 )of DENV-2 was calculated after infecting C6 / 36 cells with DENV-2. Dynamic changes of DENV-2 NS1 were measured by real-time PCR after infecting HHSECs with DENV-2. CCK-8 was used to dynamically detect the cytotoxicity of DENV-2 to HHSECs. The transcriptional levers of Beclin-1 and ICAM-1 in DENV-2-infected HHSECs were detected by real-time PCR. FCM was performed to analyze the apoptosis of HHSECs and the expression of LC3B and ICAM-1. Results The cells in the exper-imental group were stained brown by DAB and the positive expression rate of CD31 reached 87. 1% . The TICD50 of DENV-2 to C6 / 36 cells was 10-6. 845 / 0. 1 ml. Compared with the uninfected cells,partial se-quences of NS1 gene were expressed in DENV-2-infected HHSECs. DENV-2 suppressed the cell activities of HHSECs. The suppression rates of DENV-2 to HHSECs at 12 h,24 h,36 h and 48 h were respectively (10. 90±1. 24)% ,(16. 40±0. 42)% ,(17. 00±0. 46)% and(29. 60±0. 26)%(P﹤0. 05). The tran-scriptional levels of Beclin-1 and ICAM-1 in HHSECs were significantly increased at the time point of 24 h after DENV-2 infection,the 2-△△Ct values of which were 46. 77±2. 55 and 40. 97±4. 91,respectively. The expression of LC3B and ICAM-1 in DENV-2-infected HHSECs were increased,the peaks of which were reached at 24 h(14. 7% )and 36 h(35. 5% ),respectively. The apoptosis of DENV-2-infected HHSECs was remarkably enhanced at 12 h with an apoptosis rate of 13. 17% . Conclusion HHSECs was susceptible to DENV-2. DENV-2 induced the upregulation of ICAM-1 and the activation of HHSECs. Moreover,autoph-agy and apoptosis of HHSECs could also be induced by DENV-2.

Primary mechanism of changing permeability in DENV-2 infected primary human dermal micro-vascular endothelial cells / 中国免疫学杂志

Objective:To reveal the primary mechanism of changing permeability in DENV-2 infected pHDMECs. Methods:pHDMECs was incubated by DENV-2 on the concentration of 103 TCID50 ,and the penetrability of the cell was detected by Transwell at 4,8,12,24,48 h,respectively. Then,the partial sequence of DENV-2 NS1 was analyzed by Real time-PCR,and NS1 protein was detected by immunofluorescence and flow cytometer (FCM). The apoptosis rate of pHDMECs was assayed by FCM. Finally,IL-6 and IL-8 secreted by pHDMECs were analyzed by Real time-PCR and double antibody sandwich ELISA. Results:The relative expression of NS1 gene was elevated but NS1 protein was not detected;the permeability of DENV-2 infected pHDMECs had dramatically increased both at 24,48 h,but the apoptosis rate has little changed even been influenced by DENV-2 at 72 h. However,the relative expression of IL-6/IL-8 mRNA was boosted at 8,24 h[(2. 49±0. 50) and (6. 82±1. 69) fold,respectively,P<0. 05]. In protein level,compared with control(869. 6±50. 70)pg/ml,IL-6 secreted by DENV-2 infected pHDMECs could reach by(1 248. 8±86. 9)pg/ml(P<0. 05),and IL-8 was(1 331. 0±86. 3)pg/ml(P<0. 05) while the control was (967. 6±156. 6)pg/ml. Conclusion:Indeed,pHDMECs can be infected by DENV-2;the increasing permeability of DENV-2 infected pHDMECs may not be caused by the pHDMECs′ apoptosis but the enhancing of pro-inflammatory cytokine IL-6 /IL-8.

Paclitaxel Drug-eluting Tracheal Stent Could Reduce Granulation Tissue Formation in a Canine Model / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Currently available silicone and metallic stents for tracheal stenosis are associated with many problems. Granulation proliferation is one of the main complications. The present study aimed to evaluate the efficacy of paclitaxel drug-eluting tracheal stent in reducing granulation tissue formation in a canine model, as well as the pharmacokinetic features and safety profiles of the coated drug.</p><p><b>METHODS</b>Eight beagles were randomly divided into a control group (bare-metal stent group, n = 4) and an experimental group (paclitaxel-eluting stent group, n = 4). The observation period was 5 months. One beagle in both groups was sacrificed at the end of the 1st and 3rd months, respectively. The last two beagles in both groups were sacrificed at the end of 5th month. The proliferation of granulation tissue and changes in tracheal mucosa were compared between the two groups. Blood routine and liver and kidney function were monitored to evaluate the safety of the paclitaxel-eluting stent. The elution method and high-performance liquid chromatography were used to characterize the rate of in vivo release of paclitaxel from the stent.</p><p><b>RESULTS</b>Compared with the control group, the proliferation of granulation tissue in the experimental group was significantly reduced. The drug release of paclitaxel-eluting stent was the fastest in the 1st month after implantation (up to 70.9%). Then, the release slowed down gradually. By the 5th month, the release reached up to 98.5%. During the observation period, a high concentration of the drug in the trachea (in the stented and adjacent unstented areas) and lung tissue was not noted, and the blood test showed no side effect.</p><p><b>CONCLUSIONS</b>The paclitaxel-eluting stent could safely reduce the granulation tissue formation after stent implantation in vivo, suggesting that the paclitaxel-eluting tracheal stent might be considered for potential use in humans in the future.</p>

Nonstent Combination Interventional Therapy for Treatment of Benign Cicatricial Airway Stenosis / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Benign cicatricial airway stenosis (BCAS) is a life-threatening disease. While there are numerous therapies, all have their defects, and stenosis can easily become recurrent. This study aimed to investigate the efficacy and complications of nonstent combination interventional therapy (NSCIT) when used for the treatment of BCAS of different causes and types.</p><p><b>METHODS</b>This study enrolled a cohort of patients with BCAS resulting from tuberculosis, intubation, tracheotomy, and other origins. The patients were assigned to three groups determined by their type of stenosis: Web-like stenosis, granulation stenosis, and complex stenosis, and all patients received NSCIT. The efficacy and complications of treatment in each group of patients were observed. The Chi-square test, one-factor analysis of variance (ANOVA), and the paired t -test were used to analyze different parameters.</p><p><b>RESULTS</b>The 10 patients with web-like stenosis and six patients with granulation stenosis exhibited durable remission rates of 100%. Among 41 patients with complex stenosis, 36 cases (88%) experienced remission and 29 cases (71%) experienced durable remission. When five patients with airway collapse were eliminated from the analysis, the overall remission rate was 97%. The average treatment durations for patients with web-like stenosis, granulation stenosis, and complex stenosis were 101, 21, and 110 days, respectively, and the average number of treatments was five, two, and five, respectively.</p><p><b>CONCLUSIONS</b>NSCIT demonstrated good therapeutic efficacy and was associated with few complications. However, this approach was ineffective for treating patients with airway collapse or malacia.</p>

Effects of substrate stiffness on the proliferation of primary human umbilical vein endothelial cells and the release of NO and ET-1 during dengue virus infection / 中华微生物学和免疫学杂志

Objective To investigate the effects of substrate stiffness on the proliferation of human umbilical vein endothelial cells ( HUVEC) during dengue virus infection.Methods Polyacrylamide gels were prepared for cell culture [(0±4) kPa].The proliferation of HUVEC cultured on substrates with differ-ent stiffness was determined by using 3-(4,5-diethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-phenyl)-2H-etrazolium,inner salt ( MTS) assay.The cycle and apoptosis of HUVEC were determined by flow cytometry analysis.Dengue virus serotype 2 (DENV-2) strains were propagated and identified by con-ventional assays.The HUVEC were infected with DENV-2 strains at a MOI of 10 and cultured on traditional plastic and hydrogel substrates, respectively.The levels of nitric oxide (NO) and endothelin-1 (ET-1) were detected by nitric acid reductase assay and double antibody sandwich ELISA.Results Young′s modulus E value of the hydrogels was (3030 ±0.44) Pa.The proliferation of HUVEC and the expression of NO and ET-1 were enhanced along the increased substrate stiffness.However, no significant differences with the cell cycle and apoptosis were observed between cells cultured on different substrates.Conclusion The stiffness of substrates affected not only the proliferation of HUVEC, but also the release of cytokines during DENV-2 infection.The development of dengue fever was associated with the decreased secretion of vascular active substances as a result of blood vessel injury.The establishment of hydrogel substrates as the model of vascu-lar basement membranes might provide a new way for the in vitro investigation of the pathogenesis of DENV infection.

Effects of DENV-2 infection on the expression of IL-29 in primary HUVECs cultured on hydrogel / 中华微生物学和免疫学杂志

Objective To analyze the effects of dengue virus 2 ( DENV-2 ) infection on the ex-pression of IL-29 in primary human umbilical vein endothelial cells ( HUVECs) cultured on hydrogel sub-strates .Methods Primary HUVECs were isolated and cultured on hydrogel substrates .DENV-2 stains were used to infect the primary HUVECs at a multiplicity of infection( MOI) of 10.Flow cytometry analysis was performed to detect the apoptosis and infection rate of HUVECs after 48 hours of culturing .The gene chip profiling was performed to analyze mRNA expression .The expression of IL-29 at mRNA and protein levels were measured by real-time fluorescent quantitative PCR analysis and double antibody sandwich ELISA as -say, respectively.Results Compared with 96.36%of baby hamster kidney (BHK) cells that were infected with DENV-2 stains, only 4.71%primary HUVECs cultured on hydrogel substrates were infected .The pri-mary HUVECs cultured on hydrogel substrates with or without DENV-2 infection showed no significant differ-ences with the rates of cell apoptosis and infection (P>0.05).A significant difference was observed with the expression of IL-29 at mRNA level between primary HUVECs cultured on hydrogel substrates and the cells cultured in plastic bottles (P<0.05).The results of the real-time quantitative PCR analysis and ELISA as-say showed that IL-29 was highly expressed in DENV-2 infected primary HUVECs cultured on hydrogel sub-strates as compared with those in control groups (P<0.05).Conclusion The expression of IL-29 was de-tected in DENV-2 infected primary HUVECs cultured on hydrogel substrates , which was significantly differ-ent from that in DENV-2 infected primary HUVECs cultured in plastic bottles .The successful establishment of hydrogel substrates as the model of vascular basement membranes might provide a new way for the investi -gation of the pathogenesis of DENV infection .

Effect of simvastatin treatment on pulmonary hypertension and endothelial progenitor cells in rat / 重庆医学

Objective The present study was designed to investigate the efficiency of simvastatin therapy for experimental pul‐monary hypertension (PH) in rat ,and the effects on the number and function of circulating endothelial progenitor cells (EPC) . Methods Twenty four Sprague Dawley rats were divided into 3 groups randomly :model group ,treatment group and control group , 8 rats in each group .Rats were treated with a single subcutaneous injection of monocrotaline to induce PH (PBS used as control) . The rats in the experimental group were administrated with simvastatin 3 days following subcutaneous injection of monocrotaline .In the 21st day ,the number of circulating EPC ,the right ventricle systolic pressure of rat ,pulmonary vascular structural changes and the quantity of cultured EPC were measured .At the same time ,EPC in each group were cultured in vitro ,then the ability of prolif‐eration and function were analyzed and compared .Results The number of circulating EPC in model group was decreased signifi‐cantly compared to both control and model groups (P< 0 .01) .Compared with model group ,simvastatin treatment markedly de‐creased the RVSP and the ratio of media thickness to eternal diameter (P<0 .01) ,but the ratio of vessel area to total arterial area (VA/TAA) was definitively increased(P<0 .01) .After 7 days of culture in vitro ,both the output of EPC and the ability of prolif‐eration ,conglutination and migration of EPC in treatment group were up -regulated compared with those in model group (P<0 .01) .Conclusion This study confirmed that simvastatin effectively treat experimental PH through improving quantity and func‐tion of circulating EPC .

Prokaryotic expression, identification and bioinformatics analysis of fbpB-esxA fusing gene from Mycobacterium tuberculosis

OBJECTIVE@#To obtain fbpB-esxA fusing gene of Mycobacterium tuberculosis (MTB), express the encoded fusing protein in Escherichia coli (E. coli), identify protein acquired, and predict the structure and function of the protein utilizing methods of bioinformatics.@*METHODS@#fbpB and esxA gene were amplified from genome of MTB H37Rv by PCR. The fbpB-esxA fusing gene ligated by (Gly(4)Ser)(3) linker was gained by means of Gene Splicing by Overlapping Extension PCR (SOE-PCR), and fusing gene was cloned into expression vector pET-30a. The recombinant plasmid was sequenced and expressed in E. coli BL21(DE3). The protein was identified by Western blot using anti-HIS antibody. Secondary structure and antigenic epitopes of the protein were predicting using tools of bioinformatics.@*RESULTS@#The DNA sequences of fbpB-esxA were identical with that published by GenBank. The Ag85B-ESAT-6 fusion protein about 50 kDa comprised 485 amino acids was efficiently produced from expression system in E. coli BL21(DE3) under the induction of IPTG. Bioinformatics analysis showed the protein contained one transmembrane region and fourteen potential antigenic epitopes.@*CONCLUSIONS@#The Ag85B-ESAT-6 fusion protein is successfully expressed with N-terminal HIS-tag. Gel filtration demonstrated that it exists as insoluble inclusion bodies mainly. The existence of linker doesn't affect immunogenicity of Ag85B and ESAT-6. It will allow for characterization in vitro and establish a foundation of further function research such as vaccine or diagnostic reagent.

Cloning, expression, identification and bioinformatics analysis of Rv3265c gene from Mycobacterium tuberculosis in Escherichia coli

OBJECTIVE@#To clone and express Rv3265c gene of Mycobacterium tuberculosis in Escherichia coli (E. coli) under optimistic conditions, obtain and identify protein expressed, analyze the structure and characteristics of the protein using bioinformatics methods for future applications.@*METHODS@#Rv3265c gene from Mycobacterium tuberculosis H37Rv was amplified by polymerase chain reaction, and was cloned into the pET-30a vector after purification and recovery. The recombinant plasmid was sequenced and expressed in E. coli BL21(DE3), and then purified and identified by western blotting. The essential physical-chemical properties of the protein were predicated by bioinformatics tools, including subcellular location, secondary structure, domains, antigenic epitopes, etc. Tertiary structure of the protein based on homology modeling was established, while multi-sequence homological alignment and phylogenetic analysis were proformed.@*RESULTS@#The recombinant protein was obtained in soluble fraction from expression system in E. coli BL21(DE3) carrying pET30- Rv3265c plasmid, and Rv3265c gene was expressed correctly. Bioinformatics analysis showed the protein contained no signal peptide and transmembrane helices, located outside of membrane. Secondary structure analysis revealed it contained α-helix, extended strand and random coil, 46.8%, 14.6%, 38.6%, respectively. Furthermore, it possessed six potential antigenic epitopes, one glycosyl transferase domain. A simple three-dimensional model of this protein was constructed by Swiss-model sever. Both sequences and structures were conservative and especial either in gene or in protein.@*CONCLUSIONS@#Rv3265c gene might be a desirable molecular target for anti-tuberculosis drug and vaccine. The purified protein from expression will be utilized to study the kinetics of L-rhamnosyltransferase and to develope an enzyme assay for screening vaccine or drug.

Influence of microtubule depolymerization of myocardial cells on mitochondria distribution and energy metabolism in adult rats / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the influence of microtubule depolymerization of myocardial cells on distribution and activity of mitochondria, and energy metabolism of cells in adult rats.</p><p><b>METHODS</b>Myocardial cells of SD adult rats and SD suckling rats were isolated and cultured. They were divided into adult and suckling rats control groups (AC and SC, normally cultured without any stimulating factor), adult and suckling rats microtubule depolymerization agent groups (AMDA and SMDA, cultured with 8 micromol/L colchicine containing nutrient solution for 30 minutes) according to the random number table. (1) The expression of polymerized beta tubulin in myocardial cells of adult and suckling rats was detected with Western blot. (2) Myocardial cells of rats in AC and AMDA groups were collected. The expression of cytochrome c was detected with Western blot. Distribution of voltage-dependent anion channels (VDAC) and polymerized beta tubulin in myocardial cells were observed with immunofluorescent staining. Mitochondrial inner membrane potential was determined with immunocytochemical method. Activity of myocardial cells was detected with MTT method. Contents of ATP, adenosine diphosphate (ADP), and adenosine monophosphate (AMP) and energy charge of cells were determined with high performance liquid chromatography.</p><p><b>RESULTS</b>(1) The expression of polymerized beta tubulin:in AMDA group it was 0.52 + or - 0.07, which was obviously lower than that (1.25 + or - 0.12) in AC group (F = 31.002, P = 0.000); in SMDA group it was 0.76 + or - 0.12, which was significantly lower than that (1.11 + or - 0.24) in SC group (F = 31.002, P = 0.000), but was obviously higher than that in AMDA group (F = 31.002, P = 0.009). (2) The expression of cytochrome c in AC group was 0.26 + or - 0.03, which was obviously lower than that (1.55 + or - 0.13) in AMDA group (t = -24.056, P = 0.000). (3) Immunofluorescent staining result: in AC group, microtubules of myocardial cells were in linear tubiform, distributed in parallel with myocardial fiber; VDAC staining result showed that mitochondria were in granular form, distributed in the same direction as microtubules. In AMDA group, the normal distribution regularity of microtubules was destroyed, with weakened immune fluorescence intensity, microtubules structure indistinct, continuity lost, rough in appearance, and the distribution of mitochondria became disrupted. (4) Mitochondrial inner membrane potential in AC group fluorescent intensity was 1288 + or - 84, which was obviously higher than that (331 + or - 27) in AMDA group (t = 26.508, P = 0.000). (5) Cellular activity: in AC group absorbance value was 1.75 + or - 0.11, which was obviously lower than that (0.81 + or - 0.07) in AMDA group (t = 17.348, P = 0.000). (6) Energy metabolism: compared with those in AC group, content of ATP decreased, contents of ADP and AMP increased, and ATP/ADP value and energy charge decreased in AMDA group.</p><p><b>CONCLUSIONS</b>Microtubules and mitochondria distribute in the same direction in normal myocardial cells in adult rats. After microtubule depolymerization, mitochondria are arranged in disorder fashion; cytochrome c leaks from mitochondria; mitochondrial membrane potential, energy supply, and cellular activity decrease in the myocardial cells.</p>

The role and significance of Wnt/beta-catenin signaling pathway regulating the signaling molecules in hepatocellular carcinoma / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the role and significance of Wnt/beta-catenin signaling pathway regulating GSK-3beta, STAT3, Smad3 and TERT in hepatocellular carcinoma (HCC).</p><p><b>METHODS</b>The HCC cell line HepG2 was transfected with small interfering RNA (siRNA) directed against beta-catenin. Proteins were extracted and the expressions of beta-catenin, GSK-3beta, p-GSK-3beta, STAT3, Smad3 and TERT were detected by Western blot at 72 h and 96 h respectively after transfection.</p><p><b>RESULTS</b>beta-catenin expression was inhibited at both time points and the expression at 96 h was higher than that at 72 h (t = 4.43, P < 0.05). Interestingly, GSK-3beta and p-GSK-3beta expressions increased gradually at 72 and 96 h (tGSK-3beta= 4.98, tp-GSK-3beta= 29.83, P < 0.05) respectively, and STAT3 expression showed no alteration after transfection (F = 0.49, P > 0.05). Smad3 expression was increased at 72 h (t = 10.67, P < 0.05) and decreased to normal at 96 h (t = 1.26, P < 0.05), while TERT expression decreased at 72 h (t = 4.18, P is less than 0.05) and increased to normal at 96 h (t = 1.26, P > 0.05).</p><p><b>CONCLUSIONS</b>Wnt/beta-catenin signaling pathway is related to the expressions of GSK-3beta, Smad3 and TERT, but perhaps not related to STAT3 protein expression in HCC. It suggested that Wnt/beta-catenin signaling pathway might participate in HCC genesis and development through regulating the above three factors.</p>