Relationships between lead-induced learning and memory impairments and gut microbiota disturbance in mice / 中华劳动卫生职业病杂志

Objective: To explore the effect of lead exposure on the neurobehavior and gut microbiota community structure in mice. Methods: In August 2019, 64 C57BL/6 mice were randomly divided into 4 groups: control group (0 ppm) , low lead exposure group (20 mg/l) , medium lead exposure group (100 mg/l) and high lead exposure group (500 mg/l) . During the experiment, they were free to eat and drink. The drinking water of the lead exposure group was mixed with lead acetate, and sodium acetate was added in the control group. After 10 weeks of exposure, the Morris water maze was used to test the learning and memory ability of each group of mice, and then they were sacrificed for sampling. ICP-MS was used to detect lead content in whole blood and brain tissue. ELISA was used to determine the level of IL-1β in mouse serum. 16S rRNA sequencing was used to detect the structural diversity of the intestinal flora in feces, and then the correlation between the flora and behavior indicators was analyzed. Results: In the Morris water maze experiment, compared with the control group, there was no significant difference in the body weight and swimming speed of the mice in the lead exposure groups. The escape latency of the mice in the 100 mg/l and 500 mg/l dose groups was prolonged, and the number of platform crossings decreased (P<0.05) ; meanwhile, the staying time of the mice in the 500 mg/l Pb-treated group in the target quadrant was lower than that of the control group, and the difference was statistically significant (P<0.05) . Compared with the control group, the blood lead content of the mice in each lead exposure group was significantly increased, and the brain lead content of mice in the 500 mg/l dose group was significantly elevated (P<0.05) . The serum IL-1β levels of mice in each lead exposure group were higher than those of the control group (P<0.05) . At the phylum level, the relative abundance of the Proteobacteria phylum in all of Pb-treated groups was significantly increased (P<0.05) ; at the genus level, Allobaculum, Desulfovibrio, Lachnospiraceae_NK4A136_group, Turicibacter and Ureaplasma were significantly increased (P<0.05) . Among them. The relative abundance of Desuffaoibrio, Turici bacter, and Ureaplasma was negatively correlated with the residence time of mice in the quadrant of the platform (r=-0.32, -0.29, -0.44, P<0.05) . Conclusion: Lead exposure induced learning and memory impairments in mice, which may be related to the disturbance of the gut microbiota.

Effect of cladribine on growth inhibition and autocrining cytokines in hu-man umbilical vein endothelial cell line EA.hy926 / 中国病理生理杂志

AIM: To study the effects of cladribine on growth and secretion activity of human umbilical vein endothelial cell line EA.hy926, and to investigate the mechanism of its anti-tumor effect by inhibiting endothelial cells. METHODS:The effects of cladribine at different concentrations on the cell viability were detected by CCK -8 assay.Apop-tosis and cell cycle distribution were examined by flow cytometry.The protein expression levels were determined by Western blot.The levels of tumor necrosis factor-α(TNF-α), transforming growth factor-β1(TGF-β1)and vascular endothelial growth factor(VEGF)secreted by EA.hy926 cells with cladribine treatment for 48 h were analyzed by ELISA.The nitric oxide(NO)production was measured by Gries method.RESULTS:Cladribine at 0.4~1 μmol/L inhibited the viability of EA.hy926 cells in time-and dose-dependent manners.The IC50was about 3.644 μmol/L.The results showed 43.74% cells in S phase when the concentration of cladribine was 0.4 μmol/L,and 77.23 % cells in S phase when the concentra-tion of cladribine was 1 μmol/L.The apoptosis was not induced by cladribine at 0.4~10 μmol/L.The protein expression of Bax and caspase-3 did not change.The expression of p21 increased and the p53 decreased(P<0.05).The levels of TNF-αand TGF-β1 secreted by EA.hy926 cells increased after cladribine treatment for 48 h.The levels of VEGF and NO decreased.CONCLUSION:Cladribine obviously inhibits the viability of EA.hy926 cells.The mechanism is related to the cell cycle arrest.Cladribine promotes the secretion of TNF-αand TGF-β1 by EA.hy926 cells and inhibits the secretion of VEGF and NO.

CCL3 promotes hBMSC proliferation and inhibits exosome secretion / 中国病理生理杂志

AIM:To explore the regulatory effect of chemokine CCL 3 on exosome secretion from human bone marrow mesenchymal stem cells(hBMSCs).METHODS: hBMSCs were stimulated with chemokine CCL 3 at different concentrations in vitro.The proliferation of hBMSCs was measured by CCK-8 assay and viable cell counting.Exosome se-cretion from hBMSCs was qualitatively analyzed by transmission electron microscope(TEM)and flow cytometry, and the quantitative analysis was carried out by flow cytometry and nanoparticle tracking analysis(NTA).RESULTS:Compared with control group,the viability of the hBMSCs detected by CCK-8 assay was increased when hBMSCs were treated with CCL3(P<0.05).The results of viable cell counting demonstrated that the number of hBMSCs was raised in CCL 3 group in a dose-dependent manner(P<0.05).The results of flow cytometry showed that hBMSCs expressed 3 CCL3-related spe-cific receptors,CCR1,CCR5 and CCR9.Compared with control group,the fluorescence intensity of CCR9 in CCL3 group was obviously enhanced.However,no significant difference of fluorescence intensity for CCR 5 and CCR1 was observed be-tween the 2 groups.The results of NTA demonstrated that the secretion capacity of CCL 3-induced hBMSCs was far less than that in control group(P<0.05).However, the microvesicles larger than 100 nm in CCL3 groups were increased(P<0.05).The above results indicated that the higher concentration of CCL 3 induced the lower secretion of exosomes.In addi-tion,the results of flow cytometry demonstrated that CCL 3-induced hBMSCs showed lower quantity of CD 9 +exosomes than those in control group(P<0.01).CONCLUSION:CCL3 promotes the proliferation of hBMSCs but depresses the secre-tion of exosomes in a dose-dependent manner.CCL3 affects the size distribution of exosomes and increases the number of nonfunctional microvesicles of larger than 100 nm in size.CCL3 induces the expression of CCR9 in hBMSCs.