Objective To investigate the killing effect of ethacrynic acid (EA) on lung cancer A549 cells derived spheres and explore the underlying mechanism.Methods A549 spheres were cultured in serum-free medium,and the protein expression of CD133,SOX2,EpCAM and ABCG2 was detected by Western blotting.MTT assay was used to evaluate the cell viability of A549 spheres and A549 cells after treated by 1,2,5,10 and 20 mg/mL cisplatin (DDP) for 48 h.The activity of glutathione S-transferase (GST) was measured by colorimetric method after A549 spheres were treated with 10,50,100 and 200 μmol/L EA,respectively.Flow cytometry,Western blotting,real-time PCR and luciferase assay were used to analyze the levels of cellular reactive oxygen species (ROS),formation of A549 spheres,mRNA and protein expression levels of β-catenin,Sox2 and ABCG2,and promoter activity of β-catenin upon 200 μmol/L EA treated cells for 48 h.A549 sphere was infected with β-catenin adenovirus for 24 h,followed by 200 μmol/L EA treatment (in presence or absence of 5 mg/mL DDP) for 24 h.The expression of β-catenin,Sox2 and ABCG2 at mRNA and protein levels was detected by real-time PCR and Western blotting,and cell growth of A549 spheres was evaluated by MTT assay.Results The A549 spheres,with high expression of tumor stem cells markers CD133,SOX2,EpCAM and drug resistance related molecule ABCG2,and resistance to DDP at different doses,were successfully derived.After 200 μmol/L EA had treated A549 sphere for 48 h,the levels of ROS were significantly increased (P ＜ 0.05),and the mRNA and protein levels of β-catenin,Sox2 and ABCG2,and promoter activity of β-catenin were notably decreased (P ＜ 0.05).The treatment of 200 μmol/L EA enhanced the inhibitory effect on proliferation and the promoting effect on apoptosis in A549 spheres induced by 5 mg/mL DDP (P ＜ 0.05).Up-regulation of β-catenin by adenoviral infection partly reversed the effects of 200 μmol/L EA on suppressing the expression levels of β-catenin,Sox2 and ABCG2,compared to the spheres infected with blank adenovirus.Additionally,β-catenin over-expression significantly remitted the inhibitory effect of 200 μmol/L EA and 5 mg/mL DDP on the proliferation in A549 spheres.Conclusion EA exerts inhibitory effect on the proliferation and stemness of A549 spheres through suppressing GST activity and β-catenin expression,and then promotes cell apoptosis.EA might be a novel drug in treatment of lung cancer and cancer stem cells.

Objective: To investigate the effects of glutathione peroxidase 1 (GPx1) on proliferation and apoptosis of lung adenocarcinoma A549 pheres in vitro, and to explore its possible mechanism. Methods: A549 spheres were cultured in serum-free medium. The proportion of CD133+CD44+ cells in A549 spheres was detected by FCM, and the expression levels of GPx1 protein and stem cell markers Sox2 and Nanog were detected by Western blotting. The A549 spheres and their parental A549 cells were treated with 5 mg/mL cisplatin (DDP) for 48 h, then the cell survival rate was detected by CCK-8 method, the glutathione (GSH) concentrations and GPx1 activities in A549 spheres and parental A549 cells were measured by colorimetric method, while the expression level of GPx1 protein and the level of reactive oxygen species (ROS) were detected by Western blotting and FCM, respectively. When GPx1-siRNA was transfected into A549 spheres, the expression levels of GPx1 mRNA and protein were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. After silencing GPx 1 gene expression in A549 spheres, the level of ROS in A549 spheres was detected by FCM, the expression levels of Sox2 and Nanog proteins were analyzed by Western blotting, and the sphere formation ability was detected by sphere-forming experiment. When A549 spheres were transfected with GPx1-siRNA and treated with DDP (5 mg/mL), the survival rate and the apoptosis rate of A549 sphere cells were measured by CCK-8 and FCM, respectively. Additional, the expression levels of phospho-p38 (p-p38), phospho-activating transcription factor 2 (p-ATF2), p53 and Bax proteins were detected by Western blotting. Results: A549 spheres were obtained successfully. The proportion of CD133+CD44+ cells was (11.7±0.6) % in all A549 spheres, which was higher than (2.2±0.3)% of parental A549 cells. The protein levels of Sox2 and Nanog in A549 spheres were higher than those in parental A549 cells (both P < 0.05). After treatment with DDP (5 mg/mL) for 48 h, the survival rate of A549 spheres was not significantly changed (P > 0.05). The GSH concentration, GPx1 activity and GPx1 protein expression level in A549 spheres were higher than those in parental A549 cells (all P < 0.01), but the ROS level in A549 spheres was lower than that in parental A549 cells (P < 0.05). After GPx1- siRNAs were transfected into A549 spheres, the expression levels of GPx1 and Sox2 were downregulated, and the sphere formation was suppressed (all P < 0.05). After GPx 1 gene-silencing and DDP (5 mg/mL) treatment, the survival rate of A549 spheres was significantly decreased, and the apoptosis rate was elevated (both P < 0.05), while the protein expression levels of p-p38, p-ATF2, p53 and Bax were significantly up-regulated (all P < 0.05). Conclusion: Down-regulation of GPx1 expression may suppress the expression of Sox2 and increase the level of ROS, so as to inhibit the proliferation and induce the apoptosis of A549 spheres via p38-p53 signal pathway. Thus GPx1 may be a novel potential target for lung cancer treatment.

Objective: To investigate the blood lead level of children living around lead-zinc mining areas, in order to analyze its distribution features of the population. Methods: In August 2016, three natural villiages around lead-zinc mines were selected randomly as survey sites, which were grouped A, B and C according to each distances from the center field, totally 1 379 children from the three villages were selected by cluster random sampling method as the investigation subjects, and their elbow peripheral vein blood samples were collected to determine the blood lead level by graphite furnace atomic absorption spectrometry. The distribution features of blood lead in different district, gender and age groups were compared and analyzed. Results: The median and quartile range of blood lead were 0.066 and 0.058 mg/L respectively. The blood lead levels of 341 children were esceeding 100 μg/L and that account for 24.73%. The median blood lead showed statistical significant differrnce among three groups (P<0.01) , and the medians from high to low were: 0.075 mg/L (A) , 0.065 mg/L (B) and 0.059 mg/L (C) respectively. The average blood lead of male was higher than that of female (P<0.01) . There was significant difference in blood lead levels among different aged groups (P<0.01) , and the high blood lead rate dicreased with the increase of age (P<0.01) . Results of multivariate logistic regression analysis showed that there was a high risk of excessive lead in the blood lead of boys aged 1-6 years from the center of the area. Conclusion The blood lead level of children living around lead-zinc mining areas is relatively high. There are difference in district distribution, gender and age.

Objective:To survey the knowledge levels about diagnosis and management of asthma among general practitioners in community health centers in Shanghai and to analyze influencing factors.Methods:A questionnaire survey was conducted from May to June 2022 among general practitioners from 80 community health care centers in Shanghai. The questionnaire contained the basic information of the responders; knowledge about the diagnosis of asthma (symptoms, diagnostic methods, interpretation of bronchodilation and fractional exhaled nitric oxide, FeNO tests); and treatment of asthma (choosing and usage of therapeutic drugs). Logistic regression was used to analyze.Results:A total 324 general practitioners completed the questionnaire survey. Among them 221 (68.21%) had bachelor degree; 200 (61.73%) were attending doctors; the median working duration was 11 years; 174 (53.70%) had training on asthma knowledge in last 3 years. The survey results showed that 55 (16.98%) responders misinterpreted results of bronchodilation test, and 90(5.86%)misinterpreted results of FeNO test for diagnosis of asthma; 244 responders (75.31%) suspected implication of long-term cough for diagnosis of asthma; 277(85.49%)would order bronchodilation text to suspected patients; 273(84.26%)prescribed inhaled corticosteroid plus long-acting beta-2 agonist to asthma patients. For mild asthma patients, 144 responders(44.44%)suggested to use budesonide formoterol as needed; for moderate to severe asthma, 174(53.70%)suggested to use budesonide formoterol regularly and as needed, 100(30.86%)suggested to use salmeterol fluticasone regularly and salbutamol as needed. Logistic analysis showed that longer working duration was correlated with higher misinterpretation rate of bronchodilation and FeNO tests( OR=0.798,95% CI:0.694-0.918, P=0.002; OR=0.859,95% CI:0.739-0.998, P=0.047). The accuracy rate of all the questions was significantly higher in general practitioners who had training on asthma knowledge than those who didn′t receive training in last 3 years(all P<0.05). Conclusions:Most general practitioners in community health service centers in Shanghai have relatively high levels of knowledge about diagnosis and treatment of asthma. Years of work experience and training experience can affect the levels of asthma knowledge among community general practitioners.