OBJECTIVETo study the effect of supernatants from silicon dioxide(SiO2) stimulated pulmonary alveolar macrophages(PAM) on the localization of connexin 43(Cx43) so as to explore the inhibition level of SiO2 on alveolar epithelial cellular gap-junctional communication(GJIC).

METHODSThe supermatants from the primary cultured PAM were prepared, and then added 5% (v/v) SiO2 into 2% (v/v) NBS RPMI 1640 to stimulate the normal mink lung epithelial cell line CCL-64 for 24 hours. The localizations of Cx43 in CCL-64 were analyzed by indirect immunofluorescence histochemistry and laser confocal scanning microscopy(LCSM).

RESULTSThe normal cultured CCL-64 cells displayed bright membrane-associated Cx43 plaques labeling and formed dashes at regions of intercellular junction. Being exposed to supernatants from SiO2-stimulated PAM, the CCL-64 cells retained a relative low degree of Cx43 labeling at the cell periphery, localized in cytoplasm, and the individual spot, rather than plaques, were smaller compared to normal cultured cells. Along with the increase of the concentrations of SiO2, the cells displayed a different staining pattern, with clear cluster labeling aggregating towards the nucleus.

CONCLUSIONThe altered localization of the gap-junctional protein Cx43 in alveolar epithelial cells, mediated by SiO2, indicated that the internalization of Cx43 may contribute to the inhibition on GJIC in silica-induced lung epithelium injury.

Animals ; Cell Communication ; drug effects ; Cell Line ; Connexin 43 ; analysis ; Epithelial Cells ; chemistry ; drug effects ; Fluorescent Antibody Technique, Indirect ; Gap Junctions ; drug effects ; Microscopy, Confocal ; Mink ; Pulmonary Alveoli ; chemistry ; drug effects ; Silicon Dioxide ; toxicity

OBJECTIVETo explore the effect of silica dioxide(SiO2) on proliferation and downregulation of gap junctional intercellular communication (GJIC) in pulmonary alveolar epithelial cells (CCL-64 cells).

METHODSThe pulmonary alveolar macrophages(PAMs) were incubated in the serum-free RPMI 1640 containing the various concentration of SiO2 for 24 hours. The supernatants were prepared and added 5% (V/V) into 2% (V/V) NBS RPMI 1640 to stimulate the proliferation of CCL-64 cells for 24 hours. A set of "blank control", run in parallel, contained RPMI 1640 + 2% (V/V) NBS alone. The proliferation of CCL-64 cells was detected using MTT assay(to show as the absorbency, A570nm). GJIC function was measured using the fluorescence redistribution after photobleaching(FRAP) assay [to express as the transfer rate of the fluorescence, K (x 10(-3)/s)], with a laser scanning confocal microscope(LSCM, Leica TCS SP).

RESULTSThe silica-exposed PAM supernatants could induce both the proliferation(F = 9.679, P < 0.01) and downregulation of GJIC(F = 20.587, P < 0.01) of CCL-64 cells. In the range of 50-500 micrograms/ml SiO2 concentrations, the proliferation (A570nm values) and GJIC(the transfer rate, K) were fitted well in a dose-dependent manner(proliferation: r = 0.891, P < 0.05; GJIC: r = -0.943, P < 0.05).

CONCLUSIONBy way of stimulating the PAM, SiO2 could inhibit GJIC function in lung alveolar epithelial cells, and induce epithelial cell proliferation. In the pathogenesis of silicosis, the downregulation of GJIC of the pulmonary epithelial cells may play an important role in silica-mediated alveolar epithelial cell injury.

Cell Communication ; drug effects ; Cell Proliferation ; drug effects ; Epithelial Cells ; drug effects ; Gap Junctions ; drug effects ; Pulmonary Alveoli ; drug effects ; Silicon Dioxide ; toxicity ; Silicosis ; etiology

Objective: To investigate the correlation between serum lysosomal-associated membrane protein 2 (LAMP-2), LAMP-2 antibody levels and the progression of renal function in patients with chronic kidney disease (CKD). Methods: A total of 80 patients with CKD 3 to 5 stage (CKD group) and 50 healthy controls (control group) from August 2016 to August 2017 in Affiliated Dongfeng Hospital, Hubei University of Medicine were enrolled. The levels of hemoglobin, creatinine, urea, albumin, LAMP-2 and LAMP-2 antibody in fasting elbow venous blood of 2 groups were detected, and the estimate glomerular filtration rate (eGFR) was calculated. The CKD patients were followed up for 1 year, and renal function deterioration was defined as eGFR declined more than average value; the follow-up was over when the patients started dialysis or died, and those patients also defined as renal function deterioration. The patients were divided into high level group and low level group according to the serum LAMP-2 and LAMP-2 antibody levels. Results: The eGFR, hemoglobin and albumin in CKD group were significantly lower than those in control group: (24.60 ± 5.79) ml/min vs. (119.20 ± 9.52) ml/min, (111.36 ± 24.41) g/L vs. (144.60 ± 17.85) g/L and (36.83 ± 3.84) g/L vs. (45.92 ± 6.37) g/L, the creatinine, urea, LAMP-2 and LAMP-2 antibody were significantly higher than those in control group: (306.17 ± 49.24) μmol/L vs. (83.24 ± 5.55) μmol/L, (15.17 ± 3.39) mmol/L vs. (5.57 ± 1.33) mmol/L, (24.76 ± 5.47) μg/L vs. (12.93 ± 4.43) μg/L and (20.33 ± 4.89) μg/L vs. (9.98 ± 2.20) μg/L, and there were statistical differences (P<0.01). All 80 patients with CKD patients completed follow-up, and the follow-up time was 3 to 12 (8.14 ± 1.95) months. The eGFR at the end of followed-up was significantly lower than that at enrolment: (19.38 ± 7.30) ml/min vs. (24.60 ± 5.79) ml/min, the creatinine and LAMP-2 antibody at the end of followed-up were significantly higher than those at enrolment: (397.56 ± 52.32) μmol/L vs. (306.17 ± 49.24) μmol/L and (22.35 ± 4.74) μg/L vs. (20.33 ± 4.89) μg/L, and there were statistical differences (P<0.01); there were no statistical differences in hemoglobin, albumin, urea and LAMP-2 between the end of follow-up and enrollment (P>0.05). Among the 80 patients with CKD, renal function deterioration was in 42 cases, and renal function stability was in 38 cases. In renal function deterioration patients, the eGFR and urea at the end of followed up were significantly lower than those at enrolment: (18.28 ± 6.92) ml/min vs. (24.46 ± 5.76) ml/min and (13.51 ± 1.92) mmol/L vs. (14.81 ± 3.32) mmol/L, the creatinine, LAMP-2 and LAMP-2 antibody at the end of followed up were significantly higher than those at enrolment: (412.47 ± 53.21) μmol/L vs. (303.16 ± 47.87) μmol/L, (29.07 ± 5.42) μg/L vs. (25.89 ± 5.39) μg/L and (25.03 ± 3.30) μg/L vs. (20.95 ± 4.86) μg/L, and there were statistical differences (P<0.01 or <0.05); there were no statistical differences in hemoglobin and albumin between the end of follow-up and enrolment (P>0.05). In renal function stability patients, the eGFR at the end of follow-up was significantly lower than that at enrolment: (20.38 ± 7.58) ml/min vs. (24.73 ± 5.89) ml/min, the creatinine at the end of follow-up was significantly higher than that at enrolment: (381.07 ± 46.64) μmol/L vs. (309.49 ± 51.15) μmol/L, and there were statistical differences (P<0.01); there were no statistical differences in hemoglobin, albumin urea, LAMP-2 and LAMP-2 antibody between the end of follow-up and enrolment (P>0.05). In CKD patients, 38 cases were in LAMP-2 high level group (≥ 24.75 μg/L), and 42 cases were in LAMP-2 low level group (<24.75 μg/L); 38 cases were in LAMP-2 antibody high level group (≥ 20.33 μg/L), and 42 cases were in LAMP-2 antibody low level group (<20.33 μg/L). Kaplan-Meier curve analysis result showed that the renal function deterioration risk in LAMP-2 high level group was significantly higher than that in LAMP-2 low level group, LAMP-2 antibody high level group was significantly higher than that in LAMP-2 antibody low level group, and there were statistical differences (P<0.01). Conclusions Serum LAMP-2, LAMP-2 antibody levels are increased in patients with CKD, and higher serum LAMP-2 and LAMP-2 antibody levels may be associated with high risk of adverse kidney outcomes and become a promising marker to predict CKD progression.

We designed the specific primers and TaqMan probes targeting cytochrome b genes of mitochondrial DNA from bovine, goat and sheep. We used different fluorescents to label the probes. After optimization of reaction conditions, we set up a multiplex fluorescent real-time PCR method to detect bovine, goat and sheep derived materials, simultaneously. We finished the detection tests of 17 kinds of animal DNA and 200 DNA samples from different sources with the developed method and the National Standard GB/T 20190Y-2006 routine PCR method. The coincidence rate of these two methods was 100%. Without electrophoresis or restriction digestion, the developed method could reduce the test time to one third as routine PCR and identify three kinds of animal derived materials including bovine, goat and sheep in one reaction. The developed method was approximately 10 times more sensitive than routine PCR, and was applicable to identifications of bovine, goat and sheep derived materials in feed stuff, meat, milk, pelt and grease, etc. The study showed that the developed real-time PCR method is a rapid, sensitive and efficacious detection assay for bovine, goat and sheep derived materials in animal products.
Animal Feed ; analysis ; Animals ; Cattle ; Cytochromes b ; genetics ; DNA Primers ; DNA, Mitochondrial ; analysis ; genetics ; Food Contamination ; analysis ; Genes, Mitochondrial ; Goats ; Meat ; analysis ; Mitochondria, Muscle ; enzymology ; genetics ; Polymerase Chain Reaction ; methods ; Sheep

Objective:To compare the effects of different test meals on postprandial triglycerides and to optimize the standard meal composition and the blood sampling protocol for the oral fat tolerance test.Methods:This study is a prospective, open-label, randomized, cross-over trial. In March 2023, 36 volunteers were recruited in Hebei General Hospital. They underwent a health examination and oral glucose tolerance test. Twenty-six healthy volunteers(11 males and 15 females) were included in this study, with an average age of(39.08±4.56) years. Each volunteer received 75 g protein meal, 75 g fat meal, 700 kcal fixed-calorie high-fat mixed meal, and a high-fat mixed meal with energy adjusted based on 10 kcal/kg body weight. A one-week washout period of regular diet was applied before each trial. Blood was collected at fasting status and 1, 2, 3, 4, 5, and 6 hours after a meal to detect serum triglycerides, total cholesterol, low density lipoprotein-cholesterol(LDL-C), high density lipoprotein-cholesterol(HDL-C), glucose, and insulin. The variations of postprandial metabolic indicators over time following the consumption of different test meals were analyzed. The disparities in postprandial metabolic responses between the two types of mixed meals were compared.Results:The protein meal, fat meal, fixed-calorie high-fat mixed meal, and adjusted-calorie high-fat mixed meal resulted in postprandial triglyceride increases of 22.45%, 115.40%, 77.14%, and 63.63%, and insulin increase of 560.43%, 85.69%, 554.18%, and 598.97%, respectively, and with reductions in total cholesterol, LDL-C, and HDL-C ranging from 5.64%-21.81%, respectively. The blood glucose changed slightly. Changes in metabolic indicators mainly occured within 4 hours. The comparison of the characteristics of postprandial triglycerides between the two high-fat mixed meals showed no statistically significant differences( P>0.05). Conclusion:A standardize protocol with a 700 kcal fixed-calorie high-fat mixed meal as test meal, and blood lipid levels measured at fasting and at 1, 2, 3, and 4 hours after consumption, can serve as an optimized approach for oral fat tolerance test.