Tao-Hong-Si-Wu decoction reduces ischemia reperfusion rat myoblast cells calcium overloading and inflammation through the Wnt/IP3R/CAMKII pathway.

Limb ischemia reperfusion (LIRI) injury is associated with serious local and systemic effects. Reperfusion may augment tissue injury in excess of that produced by ischemia alone. Calcium overloading and inflammation are considered to be two of the pathological mechanisms of limb ischemia/reperfusion (I/R) injury. Tao-Hong-Si-Wu decoction (THSWD) is a traditional Chinese herbal medicine with a powerful anti-inflammatory properties. We studied the probable restorative effect of THSWD on limb I/R-induced calcium overloading and inflammation in myoblast obtained from gastrocnemius muscle tissues of Sprague-Dawley rats (Frizzled Z5,a wnt5a blocker; KN-93, a calmodulin-dependent protein kinase II (CamkII) blocker; XeC, a IP3R blocker as positive controls). The simulated ischemia and reperfusion(I/R) solutions were used to imitate LIRI environment. The results showed that after I/R treatment, the secretion of proinflammatory factors (TNF-α and IL-1ß) and Wnt5a/Ca2+ signal molecules (wnt5a, camkII, and IP3R) upregulated significantly, the Ca2+ concentration enhanced too in myoblast cells. THSWD pretreatment decreased the secretion of TNF-α and IL-1ß, Ca2+ concentration; and abated the Wnt5a/Ca2+ signal molecules of wnt5a, camkII and IP3R expression activated by I/R injury; but could not abated the Wnt11 and protein kinase C (PKC) expression significantly, the results was similar with Frizzled Z5 treatment cells. Our research illustrated that THSWD may have a mitigating effect on LIRI targeting Wnt/IP3R/CAMKII but not Wnt/IP3R/PKC signaling pathway for the first time. This study may encourage the use of THSWD in the critical clinical settings with LIRI.

Bactericidal mechanism of electrolyzed oxidizing water against Pseudomonas aeruginosa / 中国感染控制杂志

Objective To investigate the bactericidal mechanism of electrolyzed oxidizing water (EOW) against Pseudomona aeruginosa (P.aeruginosa).Methods Bactericidal mechanism of EOW against P.aeruginosa was studied through intracellular protein leakage,nucleic acid,and cell membrane calcium ion permeability,2 ％ glutaraldehyde was used as positive control group,and normal saline (NS) was used as negative control group.Results The killing rates of EOW and 2％ glutaraldehyde to P.aeruginosa were both＞99.99％ with 30-second contact time,and 100.00％ with 60-second contact time.After 60-second contact with EOW,NS,and 2％ glutaraldehyde,the protein leakage of P.aeruginosa detected by bicinchoninic acid (BCA) were (96.00 ± 7.42),(94.15 ± 7.49),and (216.97 ± 10.35)μg/mL,respectively,difference was significant(F =613.20,P＜0.01),2％ glutaraldehyde group was higher than EOW group and NS group;protein leakage did not change with the increase of contact time(all P＞0.05).Electrophoretogram of random amplified polymorphic DNA showed high intensity dense band between 500-1000 Kb in EOW group and NS group,while 2％ glutaraldehyde group was without amplified bands.The fluorescence intensity of calcium ion of EOW group and 2％ glutaraldehyde group were both lower than that of NS group.Conclusion Bactericidal mechanism of EOW may be due to the damage of membrane permeability of P.aeruginosa,which causes Ca2+ leakage,but fails to cause protein leakage,the damage to nucleic acid is not obvious,DNA may not be a bactericidal target of EOW.

Research Progress in Airway Epithelial Cell Culture as a Respiratory Disease Model / 病毒学报

Respiratory virus poses a serious threat to human life and health. Airway epithelial cells are the body's first line of defense from a wide variety of foreign pathogens, such as viruses and bacteria. Therefore, successful airway epithelial cell culture can provide a model for investigating the mechanisms underlying respiratory pathogenic diseases following airway virus infection. This respiratory disease model can also be used for the potential development of novel therapeutics. Here we provide a brief review of recent developments on the culture of cells derived from human trachea-bronchial airway epithelium, and the application of this model for studying respiratory virus and disease.

Establishment and Application of a Model of Well-differentiated Porcine Airway Epithelial Cells in Vitro / 病毒学报

Pigs are increasingly recognized as "natural" hosts of infection by human respiratory viruses because of their similarities to humans in terms of lung physiology, airway morphology, cell types, and distribution of cell receptors in the respiratory tract. We wished to explore the mechanisms of infection by respiratory viruses and screening of drug that could be used to treat respiratory-system diseases. Hence, we developed a model of well-differentiated porcine airway epithelial cells (PAECs) derived from pig-lung tissue and cultured them with serum-free medium under an air-liquid interface condition in vitro. We identified the PAEC model using scanning electron microscopy, electrophysiology, and immunohistology. To evaluate application of gene therapy of adeno-associated virus (AAV)6 on the PAEC model, we generated recombinant adeno-associated virus 6-green fluorescent protein (rAAV6-GFP) using the three-plasmid transfection method and infected PAECs from the apical surface with rAAV6-GFP. Results demonstrated that the PAEC model comprised a multilayer epithelial structure containing ciliated mucous secretory cells, with basal cells located directly beneath the multilayer. rAAV6-GFP could infect PAECs from the apical surface and efficiently transduce PAECs to mediate the long-term expression of the exogenous gene. Establishment of a model of well-differentiated PAECs in vitro could lay a solid foundation for the study of infection by respiratory pathogens, as well as the screening and gene therapy of agents used to treat diseases of the respiratory system.

Investigation on drug resistance status quo of imipenem-resistant Pseudomonas aeruginosa in Xi′an area and carbapenemase genes / 国际检验医学杂志

Objective To investigate the drug resistance status quo of imipenem-resistant Pseudomonas aeruginosa and carbap-enemase gene carrying in Xi′an area for guiding the clinical rational use of antibacterial drugs.Methods 151 strains of imipenem-re-sistant Pseudomonas aeruginosa isolated from clinical samples in 4 hospitals from August 2012 to July 2013 were continuously col-lected.Then the drug resistance characteristics of imipenem-resistant Pseudomonas aeruginosa were investigated by the antimicrobi-al drug sensitivity test.The PCR technique was adopted to detect the carrying situation of carbapenemase drug-resistance genes in imipenem-resistant Pseudomonas aeruginosa.Results Totally isolated 151 strains of imipenem-resistant Pseudomonas aeruginosa were mainly distributed in the neurosurgery ICU (37.1%),neurology ICU (27.1%)and the burn department (19.9%);the detec-ted strains were sensitive to polymyxin B and resistant to other 9 kinds of antibacterial drugs in different degrees;94 strains carried VIM gene,32 strains carried IMP gene,5 strains carried SPM gene and 3 strains carried SIM gene.Conclusion The multidrug re-sistance and pan-drug resistance phenomenon of imipenem-resistant Pseudomonas aeruginosa is serious,its cause might be related with the carbapenemases-producing drug resistant gene expression,the drug resistance genes are dominated by VIM and IMP.Clinic should strengthen the bacterial drug resistance monitoring and use antibacterial drugs rationally and effectively for preventing the spreading of imipenem-resistant Pseudomonas aeruginosa.

Quantitative evaluation of plasma after methylene blue and white light treatment in four Chinese blood centers.

BACKGROUND: Pathogen reduction technology is an important process in the blood safety system, including solvent/detergent treatment, filtration and methylene blue-photochemical technology (MB-PCT). To investigate the quality of MB-PCT-treated plasma, plasma samples from four Chinese blood centers were analyzed over 12 months of storage to determine the recovery of activities and levels of various plasma proteins. MATERIALS AND METHODS: Ten plasma units each from the Suzhou, Yancheng, Chongqing and Shandong blood centers were divided into two aliquots. One was subjected to treatment with one of two methylene blue-photochemical technology instruments and the other was used as control. The treated and untreated sample pairs were stored at -30°C. The recovery rates of coagulation factors, inhibitor proteins, total protein, immunoglobulins, and complement proteins were measured at different time points after storage. RESULTS: The mean recovery of most proteins exceeded 80% after MB treatment. The exceptions were factor XI and fibrinogen, of which only 71.3-74% and 69.0-92.3% were retained during storage. The two equipment types differed in terms of residual MB concentration in the plasma samples (0.18 µM and 0.29 µM, respectively). They had similar protein recovery rates at 0.5 month but differed at later time points. The four blood centers differed significantly with regard to factor II, V, VIII and fibrinogen activities. Only the samples from the Shandong blood center met the methylene blue treated fresh frozen plasma requirement. The major factor that influenced the quality of the MB-FFP samples was the time taken between blood collection and storage. DISCUSSION: Methylene blue treated plasma showed reduced coagulation factor (CF) activity and protein levels. The MB treatment-induced damage to the proteins was acceptable at the four Chinese blood centers, but the quality of the MB-treated plasma in general was not satisfactory. The main factor affecting plasma quality may be the duration of the collection and processing.

Three different bacterial detection systems for platelet concentrates under inter-laboratory conditions.

BACKGROUND: A variety of screening methods are currently used worldwide in order to decrease the risk of transfusion-transmitted sepsis and improve the safety of PCs. METHODS/MATERIALS: PCs inoculated with five different transfusion-relevant species of bacteria at concentrations of 1, 10, and 100 colony-forming units (CFU)ml(-1) were stored at 22°C for 7 days. Flow cytometry (FACS), BacT/Alert automated culturing, and a quantitative real-time PCR (Q-PCR) were then used to detect the presence of bacteria in samples prepared from these PCs. RESULTS: At the initial spiking concentrations of 1, 10, and 100 CFU ml(-1), Q-PCR detected all five bacterial species tested. Screening with the BacT/Alert culture-based system allowed bacterial detection (inoculated on day 0) within a mean time of 15.13 h for all three spiking concentrations. Using FACS, positive signals were obtained for all three concentrations of Escherichia coli and Bacillus cereus on day 1 and for initial spiking concentrations of Pseudomonas aeruginosa and Staphylococcus aureus of 1 CFU ml(-1) on day 2. For Staphylococcus epidermidis, detection of an initial inoculum of 1 CFU ml(-1) was possible only beginning on day 6. CONCLUSION: This study shows that under standard laboratory conditions the sensitivity of FACS in the detection of bacterial contamination of PCs was lower than that of either the BacT/Alert automated culturing method or Q-PCR.

The analysis of key indicators what may affect the acticity index in systemic lupus erythematosus / 中华风湿病学杂志

Objective To investigate the distribution of characteristics,clinical manifestations,laboratory parameters and activity index of patients with systemic lupus erythematosus (SLE) and to determine their role in helping to make correct clinical diagnosis and disease the activity evaluation of SLE.Methods Collect the data of 1037 SLE patients of Ningxia Medical University Affiliated Hospital from January 2006 to June 2010.Data were analyzed with t test and Chi-square test.Results Over the past three years,there were more and more patients were admitted year by year.Among the 1037 cases of SLE patients,most of them 20-40 year-old woman,accounting for 67.5％ of the whole patient population,with a male to female ratio was 1:8.26.Joint pain was the most common initial symptom,accounting for 54.3％,followed by skin rash,accounting for 48.2％.Decreased complement C3 level and platelets counts, proteinuria,and positive anti-dsDNA antibody could be used as indicators for early diagnosis of SLE.SLEDAI activity score higher than 9 were presented in 26.0％ of patients.Factors that could impact the final score of SLEADI were fever,arthritis,skin rash,proteinuria,low complement levels,high titers of anti-dsDNA antibody,pleurisy,alopecia,mucosal ulcers,pericarditis,mental illness and decreased platelets count.Patients with active disease had a higher accidence of fever,arthritis,skin rash,lung damage,alopecia,mucosal ulcers,heart damage,mental illness and renal damaged,low complement levels,high level of anti-dsDNA antibody titers and elevated erythrocyte sedimentation rate.Conclusion SLE is a multi-system disease with multiple organ involvement,with characteristic clinical symptoms and immunological abnormalities,thus early diagnosis is very important.Understanding the characteristics of the diseases,correct judgement of the disease activity,reasonable and effective treatment all can delay the development of organ damage and improve the prognosis.

Establishment of experimental model of doxorubicin extravasation injury in mice / 中国实用护理杂志

Objective To establish animal models in order to provide an experimental study basis for both the pathogenesis study and taking effective prevention scheme for doxorubicin extravasation injury. Methods A total of 20 Kunming mice for experiments on doxorubicin extravasation injury were divided in-to four groups, I.e., high dose group(2 g/L), medium dose group(1 g/L), low dose group(0.5 g/L) and the control group (injection with water). Dosages were administered with subcutaneous injection on both sides of mice abdomen. The adverse reaction of body, damage areas of extravasation injury, recovery period were observed and histopathologic slides of animal models on both 4 days and 11 days after experiment were performed and compared. Results No significant adverse body reaction was observed after injection for all groups. The damage areas due to extravasation injury were dosage and concentration dependent. In addi-tion, significant differences in recovery period were observed for mice in different groups, that is, the higher injection concentration and dose led to the longer recovery period. Results from the histopathologic study in-dicated that the putrescence of damage area was developed in high dose group mice, and the ulcer occurred after 4 d of dosage in medium dose group mice, respectively. In contrast, no ulcer was observed in low dose group mice. Conclusions It would be feasible to establish a prevention model for mice on doxorubicin extravasation injury by subcutaneous injection at a dosage of 0.05ml(1 g/L).

Cloning and expression of human keratinocyte growth factor-2 and the purification and identification of its products / 中国组织工程研究

BACKGROUND: Human keratinocyte growth factor-2 (hKGF-2) has extensive physiological functions, which plays an important role in embryonic development, tissue-repairing, nervous regeneration, vascularization and development of tumor.OBJECTIVE: To clone hKGF-2 gene, obtain the expression of hKGF-2 in Escherichia coli(E.coli) and determine its bioactivity, so as to provide experimental basis for further investigation.DESIGN: Open experiment.SETTING: Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention.MATERIALS: The experiment was conducted in State Key Laboratory of Viral Genetic Engineering, Institute for Viral Disease Control and Prevention of Chinese Center for Disease Control and Prevention. The temperature control expression vector pBV220 was constructed by State Key Laboratory of Viral Genetic Engineering; EcoR Ⅰ , BamH Ⅰ , T4 DNA ligase (Promega Co., Ltd.); The specific polymerase chain reaction (PCR) of hKGF-2 (Manufactured by Shanghai Boya Biotechnology Co., Ltd.); Heparin-Sepharose CL-6B (Pharmacia Company); PCR rapid purification kit,Trizol kits for total RNA extract, Kits for RT-PCR (GIBCO Co., Ltd.); Kits for rapid extraction of plasmid DNA (Boda Company); BL-21-codon plus compent cells (Stratagene Co., Ltd.).METHODS: High-expression strain BL-21 codon plus competent cells was used to express and purify initially recombinant hKGF-2 protein, and its activity was detected. RT-PCR was adopted to obtain hKGF-2 cDNA from lung tissues of naturally aborted fetus and clone it into pBV220 carri er plasmid. The hKGF-2 protein expressed in BL-21 codon plus competent cells of E.coli. Affinity chromatography and ion exchange chromatography were applied in isolation and purification, and the bioactivity of expression protein was determined in cell proliferation test.MAIN OUTCOME MEASURES: The length and sequence of cDNA segment in hKGF-2, the expression of hKGF-2 gene inE.coli and the purification of hKGF-2 activity.RESULTS: The segment of hKGF-2 cNDA was about 500 bp, and hKGF-2 protein highly expressed in BL-21, which had soluble expression in the supernatant. SDS-PAGE showed that the relative molecular mass was about 20000, and hKGF-2 protein could significantly promote the mitotic activity of NIH3T3 cells. The A value (490 nm) of hKGF-2 in the 1 μg/L, 5 μg/L and 10 μg/L groupswere higher than that in the blank control group, and the differences were significant (which were 0.174±0.022,0.220±0.029,0.306±0.050,0.066±0.004 respectively,P ＜ 0.001).CONCLUSION: hKGF-2 gene is successfully cloned, which highly expresses in BL-21 of the E.coli. Purified hKGF-2 protein can stimulate the proliferation of NIH3T3 cells and significantly promote its mitotic activity.