Objective To observe the clinical effect of Salmeterol xinafoate and fluticasone propionate powder for inhalation in patients with chronic obstructive pulmonary disease.Methods 70 patients diagnosed with chronic obstructive pulmonary disease from March 2014 to March 2015, were randomly divided into two groups ( n =35 ) .Control group were given basic treatment, observation group was given Salmeterol xinafoate and fluticasone propionate powder for inhalation on the basis of control group , patients were followed up and changes of related indicators wererecorded. Results After treatment one month, serum airway remodeling index b-FGF, TIMP-1 values were (93.86 ±17.36 μg/L, 38.06 ±4.28ng/mL) respectively, more than control group(135.03 ±16.06μg/L, 53.95 ±4.15ng/mL)(P<0.05).After treatment one month, inflammatory markers IL-8, TNF-αwere(7.26 ±1.57 pg/mL, 4.29 ±1.02 ng/L)respectively, were more than the control group (14.27 ±1.71 pg/mL, 8.90 ±1.21 ng/L) (P<0.05).After treatment, the observation group, the total effective rate was 91.43%, higher than 77.14%(P <0.05).Conclusion Salmeterol xinafoate and fluticasone propionate powder for inhalation has good effect in treatment of chronic obstructive pulmonary disease , better than the use of basic treatment alone.

Objective To observe the meridian tropism of Polygoni Avicularis Herba by tissue distribution of avicularin in rats.Methods Tissue distribution of avicularin in rat following a single iv administration was appointed and observed.HPLC method was established and validated for the determination of avicularin in rat tissues.Results Kidney and bladder were the most important target tissue of avicularin.Conclusion HPLC method is successfully applied to tissues distribution study of avicularin after iv administration to rats,and the results explain Polygoni Avicularis Herba on bladder tropism reasonably.

Objective To investigate quercetin's protective effect against oxidative stress in and impact on the biological activity of mouse B10BR melanocytes. Methods B10BR cells were cultured and treated with different concentrations of quercetin followed by additional culture. Then, cell viability was measured by using MTT assay, hydrogen peroxide-induced cell apoptosis by flow cytometry, and cell morphological changes by microscopy. The tyrosinase activity in and melanin synthesis by B10BR cells were measured by dopa oxidation assay and sodium hydroxide (NaOH)-lysis method, respectively. Results After treatment with quercetin of 33.33 μmol/L for 24 hours, the survival rate of B10BR cells reached (94.22 ± 3.36)%, tyrosinase activity (107.15 ± 10.96)%, and melanin content (111.85 ± 9.49)%. A significant difference was observed in tyrosinase activity and melanin content between hydrogen peroxide-induced and 33.33 μmol/L quercetin-treated B10BR cells and those only induced by hydrogen peroxide (both P < 0.01). Flow cytometry revealed that quercetin inhibited hydrogen peroxide-induced apoptosis in melanocytes. Conclusion The protective effect of quercetin against hydrogen peroxide-induced apoptosis in melanocytes may provide a new idea for the treatment of vitiligo.

Objective To review the current situation and developments of researches into important pathogenic microorganisms domestically and abroad,and to suggest the orientation of research work and development in pathogenic microbiology in PLA.Methods The achievements and advances of research work achieved domestically and abroad in the past five years regarding important viruses(such as hepatitis viruses,human immunodeficiency virus,influenza virus,encephalitis viruses and hantaanvirus)and bacteria (such as Mycobacterium tuberculosis,Streptococcus suis serotype 2,Yersinia pestis,Bacillus anthracis and Helicobacterp ylori)were retrieved and reviewed using intelligence research methods.Results Infectious diseases caused by pathogenic microorganisms were the most severe hazards to health and life of human beings.Especially in the past thirty years,newly emerging infectious diseases and recurrence of previonsly controlled infectious diseases had received wide attention.Infectious diseases control had been greatly improved owing to the increasing discoveries in the knowledge about pathogenic microorganisms.Conclusions During the period of "Twelfth Five-Years Plan" ,a big team of science and technology personnel with strong innovative ability in the domain of medical microbiology should be brought up in PLA;and a number of advanced and consummate research bases and technology platforms should be built up;to apply for and realize a batch of major research projects,strive to make a number of scientific achievements with innovation and important application prospects,improve the transformation efficiency of scientific and technological achievements and contribution of scientific and technological progress,and strive to achieve important progresses and breakthrough in mainstream research.

Objective To investigate the relationship between the efficacy of autologous cultured melanocyte transplantation and serum levels of interleukin-17 (IL-17) and FoxP3 in patients with vitiligo.Methods Forty patients with stable vitiligo vulgaris were included in this study,and received autologous cultured melanocyte transplantation.Six months after the transplantation,treatment efficacy was evaluated,and patients were classified into the successfully treated group (n =25) and unsuccessfully treated group (n =15).Peripheral blood was collected from all the patients before the transplantation,and enzyme-linked immunosorbent assay (ELISA) was performed to measure the serum levels of IL-17 and FoxP3.Statistical analysis was done using two independent samples t-test with the SPSS software (version 17.0).Results The successfully treated patients showed lower serum levels of IL-17 ((15.29 ± 7.86) vs.(43.88 ± 13.02) ng/L,P ＜ 0.05),but higher serum levels of FoxP3 ((6.08 ± 2.03) vs.(3.37 ± 1.81) ng/L,P ＜ 0.05) than the unsuccessfully treated patients.Conclusion The increased serum IL-17 and decreased serum FoxP3 may contribute to the failure of autologous cultured melanocyte transplantation in patients with vitiligo.

Objective To investigate the intracellular signal transduction pathways involved in the protective effect of nicotinic acid against ultraviolet B(UVB)-induced damage in human skin keratinocytes.Methods Cultured human keratinocyte HaCaT cells were divided into several groups to be treated with nicotinic acid,UVB irradiation,LY294002(an inhibitor of Akt),U0126(an inhibitor of extracellular signal-regulated kinase(ERK)1/2),SB203580(an inhibitor of P38)alone or in combination for different durations.Then,Western blot was performed to quantify the phosphorylation levels of the protein kinase B(Akt)/MAPK pathwayassociated proteins including Akt,P38,JNK and ERK1/2,MTT assay to evaluate the activity of HaCaT cells,enzyme-linked immunosorbent assay to determine the levels of endothelin-1(ET-1)and basic fibroblast growth factor(bFGF)in the culture supernatant of HaCaT cells,and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL)to evaluate the apoptosis in HaCaT cells.Results As Western blot showed,phosphorylated Akt,P38,JNK and ERK1/2 were markedly activated within 60 minutes after pretreatment with nicotinic acid and irradiation with UVB(all P ＜ 0.01),and the activation was more significant for phosphorylated Akt,P38,and ERK1/2 within 2 hours(all P ＜ 0.01).Nicotinic acid effectively suppressed the UVB-induced cell death and apoptosis in HaCaT cells.The levels of supernatant ET-1 and bFGF were significantly decreased in HaCaT cells treated with the above 3 inhibitors followed by UVB irradiation than in those treated with the inhibitors alone(all P ＜ 0.05),and nicotinic acid pretreatment only reversed the decrease in supernatant bFGF in HaCaT cells treated with SB203580 followed by UVB irradiation.Conclusion The Akt signaling pathway may play a regulatory role in the protection by nicotinic acid against UVB-induced damage in HaCaT cells.

Objective To investigate the effect of nuclear translocation of E2p45 related factor 2 (Nrf2)on the biological activity of melanocytes.Methods Plasmid vectors containing wild-type nrf2 gene (pcDNA-nrf2) and nls-deleted nrf2 gene (pcDNA-nrf2△nls) were constructed.B10BR normal murine melanocytes were classified into three groups,i.e.,untransfected group,wild-type nrf2 group transfected with pcDNA-nrf2,and mutated nrf2 group transfected with pcDNA-nrf2△nls.Each of the above groups were further divided into three subgroups:control subgroup receiving no treatment,hydrogen peroxide (H2O2) subgroup treated with H2O2 of 200 μmol/L for 24 hours,and combined subgroup pretreated with tert-butyl hydroquinone (TBHQ) followed by treatment with H2O2 of 200 μmol/L for 24 hours.Subsequently,methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate the proliferative activity of cells,dopa oxidation assay to determine tyrosinase activity,Transwell assay to estimate cell migration ability,Western blot to quantify the expressions of Nrf2 and his tag fusion protein.Results TBHQ significantly enhanced the nuclear expression of Nrf2 in B10BR cells transfected with pcDNA-nrf2 or pcDNA-nrf2△nls (both P ＜ 0.01).No significant difference was observed in tyrosinase activity between untreated wild-type nrf2 group,mutated nrf2 group,and untransfected group (P ＞ 0.05).There was a statistical decrease in tyrosinase activity in the two H2O2-treated transfected groups compared with the untreated transfected groups (both P ＜ 0.05),and the decrease was reversed by TBHQ pretreatment in the wildtype nrf2 group (P ＜ 0.05),but not in the mutated nrf2 group (P ＞ 0.05).Further more,the proliferative activity of B10BR cells experienced no obvious changes in the wild-type nrf2 group (P ＞ 0.05),but was significantly reduced in the untransfected group (P ＜ 0.05) and mutated nrf2 group (P ＜ 0.01) after the H2O2 treatment compared with the corresponding untreated groups.TBHQ could protect the pcDNA-nrf2-transfected B10BR cells,but not pcDNA-nrf2△nls-transfected B10BR cells,from H2O2-induced oxidative damage.Transwell assay showed no significant difference in migration ability among these nine groups (P ＞ 0.05).Conclusions Abnormal nuclear translocation of Nrf2 could affect antioxidant activity of,proliferative activity of and tyrosinase activity in melanocytes.TBHQ may enhance the tyrosinase activity in,proliferative activity and antioxidant activity of melanocytes via activating the nuclear expression of wild type Nrf2.

Objective To express and purify the epitope peptide of human melanin-concentrating hormone receptor 1, and to evaluate its performance in the detection of autoantibodies in vitiligo patients. Methods The target gene encoding the epitope peptide of human melanin-concentrating hormone receptor 1 was synthesized, cloned to prokaryotic expression vector pGEX-4T-2 which was then transferred to E. coli BL21. The protein expression was induced by isopropy-β-D-thiogalactoside (IPTG) and identified with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Blocking ELISA was carried out with membrane proteins extracted from melanocytes as the blocking antigen. The antigenicity of the peptide was detected in sera from 100 patients with progressive vitiligo and 30 healthy human controls. Results The recombinant expression vector was successfully constructed, and the target protein was successfully expressed in E.coli, which was evidenced by SDS-PAGE and Western blot. With the glutathione S-transferase (GST) purification kit, the purity of the recombinant protein reached 100% when the sampling weight was less than 0.625 μg.The binding of the target protein with serum IgG antibodies from vitiligo patients could be blocked by natural membrane antigen of melanocytes. Of the 100 sera from patients with progressive vitiligo, 36 were reactive with the target protein. Conclusions The epitope peptide of human melanin-concentrating hormone receptor 1 has been successfully expressed and purified. The purified protein can bind with serum IgG antibodies from vitiligo patients, and may be applied to the detection of autoantibodies against human melanin-concentrating hormone receptor 1.

Objective To evaluate the effect of calcipotriol on the proliferation of and cytokine secretion by melanocytes and perilesional CD8+ cytotoxic T lymphocytes (CTLs) from patients with vitiligo.Methods Melanocytes isolated from abdominal skin and CD8+ CTLs from perilesional skin of patients with vitiligo were subjected to successive culture in vitro.After several passages,the melanocytes and CD8+ CTLs were cultured alone or in combination with or without the presence of various concentrations of calcipotriol for 24 to 48 hours.MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium,inner salt) method was used to evaluate the proliferative activity of cells,enzyme-linked immunosorbent assay to determine the levels of interleukin (IL)-6,tumor necrosis factor (TNF)-α and interferon (IFN)-γ in the culture supematant of cells,flow cytometry to detect cell apoptosis.Some co-cultured melanocytes and CTLs were treated with calcipotriol of 10-8 mol/L and anti-IL-6 antibody of various concentrations (0,1,2,2.5,5,10 mg/L) for two days followed by enumeration of cells.The concentrations of 108 and 10-9 mol/L (calcipotriol) were chosen for relevant tests.Results There was a marked apoptosis in MCs after coculture with CD8+ CTLs.The 24-hour treatment with calcipotriol of 104 and 10-9 mol/L had no obvious effect on the proliferation of melanocytes cultured alone (both P ＞ 0.05),but accelerated the proliferation of melanocytes cocultured with CTLs (both P ＜0.05) as well as that of CD8+ CTLs cultured alone or in combination with melanocytes (all P ＜0.05).A statistical decrease was observed in IL-6,TNF-α and IFN-γlevels in the supernatant of cocultured melanocytes and CTLs compared with those in the supernatant of melanocytes and CTLs cultured alone,and calcipotriol of 10-9 mol/L intensified the decrease in supernatant IL-6 level (t =2.89,P ＜0.05),but no statistical changes were noted for the level of TNF-α or IFN-γin the supernatant of the coculture system after treatment with calcipotriol of 104 or 104 mol/L compared with that before treatment (both P ＞ 0.05).In the coculture system pretreated with calcipotriol of 10-8 mol/L,the number of CD8+ CTLs significantly decreased (t =3.15,P ＜0.05),whereas that of melanocytes significantly increased (t =3.53,P ＜0.05) after the treatment with anti-IL-6 antibody of 5 mg/L.Conclusions Perilesional CD8+ CTLs have a specific cytotoxic effect on melanocytes,and calcipotriol may inhibit the cytotoxic effect of CD8+ CTLs by suppressing the secretion of IL-6,which may partly explain the therapeutic mechanism of calcipotriol for vitiligo.

Objective To evaluate the effects and underlying mechanisms of crocin on glutamate-induced apoptosis of retinal ganglion cells (RGCs) by affecting extracellular calcium influx.Methods Primary rat retinal ganglion cells were isolated and stimulated with glutamate at concentrations of 0.1 mmol/L and 1 mmol/L for 24 h or 48 h,respectively,to establish apoptosis model of RGCs.Afterwards,crocin of different doses (0.1,1.0 and 3.0 μmol/L) was used to treat the glutamate-induced RGCs for 12 h;then cell apoptosis was detected by Annexin V-FITC/PI staining.The intracellular calcium concentration was determined by FIuo-3/AM fluorescent labeling.Western blot was used to examine the effect of crocin on Ca2+-mediated apoptotic signal molecules calpain and CaMKII.The mitochondrial membrane potential was detected by JC-1 staining and mitochondrial apoptosis-related signaling molecules Caspase-3,Caspase-9 and Bcl-2/Bax were evaluated by Western blot,respectively.Results In comparison with the untreated controls,the cell apoptosis of RGCs exposed to 0.1 mmol/L of glutamate for 24 h did not significantly change (P＞ 0.05).However,apoptosis rate of the cells reached (43.050 ± 2.616) ％ when the stimulation time lasted for 48 h and showed a significant increase (P＜0.01).Treatment with higher-dose glutamate (1 mmol/L) significantly increased apoptosis of RGCs at 24 h (46.450±1.061)％ and 48 h (45.500±3.253)％ compared with the controls (P＜0.01).RGCs were induced by 1 mmol/L of glutamate for 12 h,followed by the treatment with crocin at concentrations of 0.1,1.0 and 3.0 μmol/L,respectively.Each dose of crocin could significantly inhibit cell apoptosis in the dose-dependent manner (P＜0.01).In addition,crocin at 1.0 μmol/L blocked glutamate-induced extracellular calcium influx,inhibited the expression of calcium-dependent proteins Calpainl and CaMK Ⅱ.Moreover,crocin at the dose of 1.0 μmol/L also increased mitochondrial membrane potential,suppressed the expressions of Caspase-3 and Caspase-9,and elevated Bcl-2/Bax ratio.Cornclusion Crocin inhibits glutamate-induced apoptosis of retinal ganglion cells through suppressing extracellular calcium influx,thereby blocking calcium-dependent and mitochondria-dependent apoptosis signaling pathways.