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 establish the method to determine the microbial limit tests for Shuyu Capsules.METHODS According to the Chinese Pharmacopoeia 2005 edition,two testing methods had different percentage recovery with 5 control trains.RESULTS Shuyu Capsules were Tradional Chinese Medicine(TCM),and that had antimicrobial effects,which could be eliminated by media dilution method.CONCLUSIONS This method can achieve the destination which is feasible and accurate.

Objective To analyze the value of CT and MRI in the diagnosis of intrahepatic peripheral cholangiocarcinoma (IHPCC ). Methods 32 cases of intrahepatic peripheral cholangiocarcinoma were collected and analyzed retrospectively.25 patients underwent plain and enhanced CT scan,while 18 cases underwent plain MRI and MRCP(8 of them underwent MRI dynamic enhancement scanning).6 cases underwent plain and enhanced scan of CT and MRI simultaneously.Results 32 intrahepatic lesions were found,with CT and MRI revealing all lesions.Lesions of IHPCC showed as hypo-or iso-density on the plain CT,slightly hypo-intensity on T1 WI and uneven mild hyper-intensity on T2 WI.During arterial phase of contrast-enhanced scan,18 lesions showed as marginal mild linear enhancement,3 cases with obvious enhancement and 11 cases with no enhancement.During portal phase,venous phase and delayed scan,19 lesions showed as progressive mild-to-mod-erate uneven enhancement,while 6 cases enhanced homogeneously.25 cases showed characteristic significantly delayed enhance-ment.MRCP could display satisfactory of Intra-and-Extra-hepatic bile ducts.Other signs include intrahepatic bile ducts dilation (23 cases),intrahepatic bile duct stones (6 cases),hepatic lobe atrophy (1 5 cases),and depressed liver capsula (8 cases).Conclusion Both CT and MRI are effective methods for diagnosing of intrahepatic peripheral cholangiocarcinoma.Compared with CT,MRI seems more valuable on presentation the tumor size and border,bile duct involvement,the degree of expansion and portal vein inva-sion,etc.It is more valuable to combine CT with MRI for the diagnosis of intrahepatic peripheral cholangiocarcinoma.

Objective:To explore the possibility of repairing partial nasal alar defects with individualized design of localized skin flaps.Methods:The clinical data of 38 patients with nasal alar region tumor from October 2015 to June 2019 in Dalian Municipal Central Hospital were retrospectively analyzed, including 5 cases with intradermal nevus, 8 cases with junction nevus, 21 cases with basal cell carcinoma, 3 cases with trichoepithelioma, and 1 case with nasal alar sulcus fistula combined with infection. Surgical treatment with local anaesthesia was applied, and intraoperative freezing pathology was used to confirm the diagnosis and determine the safe margin. There was no nasal alar cartilage infiltration in all patients. The defect areas after resection of nasal alar lesions ranged from 1.0 cm × 1.0 cm to 3.0 cm × 2.5 cm. Local skin flap was aesthetically designed in accordance with the location and size of the nasal alar defect to primarily repair the defect. Among them, 15 cases were repaired with pedicled nasolabial groove flap, 10 cases with modified rhomboid flap, 6 cases with rotatory nasolabial groove flap, 5 cases with V-Y push flap, and 2 cases with double lobe flap.Results:One case had blood transportation obstacle after operation caused by compression and bandaging, 1 case had postoperative infection. Healing of the two cases delayed after treatment, and other patients healed properly. All the flaps survived without facial deformity, and the cosmetic effect was good.Conclusions:The primary repair of the nasal alar defect needs to follow the aesthetic requirements of the nose and face, which varies with diseases and experience of doctors. Flap selection should be individualized to achieve both the purpose of repairing defects and beauty.

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.

ObjectiveTo explore the regulation of claudin-4 expression in endometrial adenocarcinoma cell lines by progesterone.Methods Ishikawa cells were treated with various concentrations of megestrol acetate (MA:2,5,10,15,20 mg/L).After cultured for 24,48 and 72 hours,cells growth were measured by methyl thiazolyl tetrazolium (MTT).The group of Ishikawa cells incubated with MA at the 50％ inhibitory concentration ( IC50 ) was selected for cell apoptosis assay by using transmission electron microscopy and flow cytometry method.Real-time PCR and western blot were used for detecting the mRNA and protein expression levels of claudin-4.The localization of claudin-4 was examined by immunofluorescent staining.Results The inhibitory effects of megestrol acetate on the growth of Ishikawa cells were dosedependent and time-dependent.IC50 of MA on Ishikawa cells was 15 mg/L after incubated for 72 hours.After MA treatment,Ishikawa cells showed shrinkage,nuclear chromatin condensation,fractures of nuclear membrane and endoplasmic reticulum expansion,even round apoptotic bodies were found.The apoptosis rate of cells before MA treatment was (0.076 ±0.024)％,and the rate was (3.934 ±0.816)％ by MA treated for 72 hours,in which there were signicant difference( P ＜ 0.05 ).The relative quantification of claudin-4 mRNA and protein of the cells before MA treatment were 0.64 ± 0.20 and 0.94 ± 0.18,while they were 0.47 -0.15 and 0.62 ±0.15 after MA treated.The expression of claudin-4 was significantly decreased after MA treatment ( P ＜ 0.05 ).The localization of claudin-4 transferred from cytomembrane to cytoplasm and nucleus after MA treatment.Conclusions MA could inhibite the growth of Ishikawa cells,in which the mechanism may be decrease the expression of claudin-4 and the apoptosis of cells.The distribution change of claudin-4 may be related to the anti-cancer effect of progesterone.

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 assess the reliability and validity of the Chinese version of Multidimensional fatigue symptom inventory-short form(MFSI-SF).Methods The reliability and validity of the Chinese version MFSI-SF were assessed in a sample of 203 cancer patients.Statistical software was used to perform the analysis.Results The results showed moderate correlation between items and the total scale,the content validity index was 0.82,and exploratory factor analysis indicated five dimensions of the scale,the cumulative variance contribution was 56.65％.Confirmatory factor analysis showed moderate model fitting:x2/df=l.73,GFI=0.83,AGFI=0.79,NNFI=0.94,RMSEA=0.06,criterion validity was 0.585,and the Cronbach α of the total scale was 0.896.Conclusions The results demonstrated good convergent validity,it is suitable to evaluate fatigue status in Chinese cancer patients.

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 investigate the quantification of CD4~+CD25~+ regulatory T cells and distribution of CD4~+CD8~+ T lymphocyte subgroup in peripheral blood of patients in chronic hepatitis B (CHB),and to reveal relationship between CD4~+CD25~+ regulatory T cells,CD4~+CD8~+ T lymphocyte subgroup and HBV infetion as well.Methods:CD4~+CD25~(high),CD4~+CD25~+Foxp3~+Treg and CD3~+CD4~+CD8~+T lymphocyte subgroup in peripheral blood from 50 patients with CHB and 20 healthy controls was analyzed using flow cytometry.HBV DNA was detected by fluorescence quantitative PCR.Results:The number of CD4~+CD25~(high)Tregs in patients with CHB was obviously higher than that in healthy controls(P＜0.01)and increased with copies of HBV DNA.The same with the change of CD4~+CD25~+Foxp3~+Tregs in patients with CHB and there was a positive correlation between CD4~+CD25~(high)Tregs and CD4~+CD25~+Foxp3~+Tregs(r=0.890,P＜0.001).Compared with healthy controls,the frequency of CD4~+T cells and the ratio of CD4~+/CD8~+ in patients with CHB was declined,but there was no significant difference in the frequency of CD3~+T cells and CD8~+T cells between them(P＞0.05).The variation in the number of CD4~+CD25~(high)Tregs was correlated positively with the copies of HBV DNA(r=0.782,P＜0.001)and glutamic-pyruvic transaminase(ALT)(r=0.432,P＜0.005)separately,but negatively with the frequency of CD3~+,CD4~+,CD8~+T cells and the ratio of CD4~+/CD8~+(P＞0.05).The variation in the frequency of CD3~+,CD4~+,CD8~+T cells and the ratio of CD4~+/CD8~+ was also correlated negatively with the copies of HBV DNA(P＞0.05).Conclusion:The number of CD4~+CD25~(high)Tregs increases in patients with CHB and is in accordance with the copies of HBV DNA and increased level of ALT.Further studies should be done to investigate weather CD4~+CD8~+ T lymphocyte subgroup could be used to monitor the state of community.