Objective:To study the toxicity of dioctyl phthalate (DOP) on adrenal pheochromocytoma (PC12) cells and its effect on processing of amyloid precursor protein (APP) -enzymolysis.Methods:In vitro experiments, PC12 cells were divided into blank control (CT) , low DOP (DOP1) , medium DOP (DOP2) , high DOP (DOP3) , low DOP+Aβ 25-35 (DOP1+Aβ) , medium DOP+Aβ 25-35 (DOP2+Aβ) , high DOP+Aβ 25-35 (DOP3+Aβ) , Aβ 25-35 (Aβ) , a total of 8 groups, each with 4 samples. The cell viability was measured by MTT assay, the contents of lactate dehydrogenase (LDH) , malondialdehyde (MDA) and nitric oxide (NO) were measured, and cysteine protease 3 (Caspase-3) was determined by Western blot. In the transfection experiment, the hamster ovary (CHO) cells were transfected with APP695 and treated with different concentrations of DOP. They were divided into V-Flag control (V-Flag) , APP695-Flag (APP695) , low DOP (DOP1+APP695) , medium DOP (DOP2+APP695) , high DOP (DOP3+APP695) , a total of 5 groups, each with 4 samples. Enzyme-linked immunosorbent assay (ELISA) was used to determine the content of Aβ 1-40 and the activity of γ-secretase. In vivo experiment, 50 male Kunming mice of SPF grade, weighing (20±2) g, were selected and randomly divided into control, lead (Pb) , low DOP (DOP1＇) , medium DOP (DOP2＇) , high DOP (DOP3＇) consisted of 5 groups, each with 10 mice, continuously gavage for 6 weeks. Morris water maze method was used to detect the effect of different concentrations of DOP on learning and memory in mice, and ELISA method was used to detect β-secretase, γ-secretase activity and Aβ 1-40 content in brain tissue. Results:Compared with the CT group, the cell viabilities of the DOP2 and DOP3 groups were decreased, and the contents of LDH, MDA, and NO were increased, and the differences were statistically significant ( P<0.05) . Compared with the CT group, the cell viabilities of DOP1+Aβ, DOP2+Aβ and DOP3+Aβ groups were decreased, the contents of LDH, MDA, NO were increased, the differences were statistically significant ( P<0.05) . Compared with the Aβ group, the cell viability of DOP3+Aβ group was decreased, the contents of LDH, MDA, NO were increased, the differences were statistically significant ( P<0.05) . Compared with the Aβ group, the contents of LDH and NO in the DOP2+Aβ group were increased, and the differences were statistically significant ( P<0.05) . Compared with the CT group, the expression levels of Caspase-3 in the DOP2 and DOP3 groups were increased, and the differences were statistically significant ( P<0.05) . Compared with the Aβ group, the expression levels of Caspase-3 in the DOP2+Aβ and DOP3+Aβ groups were increased, and the differences were statistically significant ( P<0.05) . Compared with the APP695 group, the contents of Aβ 1-40 and the activities of γ-secretase of the DOP2+APP695 and DOP3+APP695 groups were increased ( P<0.05) . Compared with the control group, the activities of β-secretase, γ-secretase and the content of Aβ 1-40 in the brain tissue of DOP3＇group were increased, and the differences were statistically significant ( P<0.05) . Compared with the Pb group, the activities of β-secretase, γ-secretase and the content of Aβ 1-40 of the DOP3＇group were increased, and the differences were statistically significant ( P<0.05) . Compared with the control group, the target quadrant stay time and the number of crossings in the DOP2＇and DOP3＇groups were reduced, and the differences were statistically significant ( P<0.05) . Conclusion:DOP has a certain toxic effect on PC12 cells, causing learning and memory impairment in mice, and may promote the pathological progression of Alzheimer＇s disease.

Objective:To study the toxicity of dioctyl phthalate (DOP) on adrenal pheochromocytoma (PC12) cells and its effect on processing of amyloid precursor protein (APP) -enzymolysis.Methods:In vitro experiments, PC12 cells were divided into blank control (CT) , low DOP (DOP1) , medium DOP (DOP2) , high DOP (DOP3) , low DOP+Aβ 25-35 (DOP1+Aβ) , medium DOP+Aβ 25-35 (DOP2+Aβ) , high DOP+Aβ 25-35 (DOP3+Aβ) , Aβ 25-35 (Aβ) , a total of 8 groups, each with 4 samples. The cell viability was measured by MTT assay, the contents of lactate dehydrogenase (LDH) , malondialdehyde (MDA) and nitric oxide (NO) were measured, and cysteine protease 3 (Caspase-3) was determined by Western blot. In the transfection experiment, the hamster ovary (CHO) cells were transfected with APP695 and treated with different concentrations of DOP. They were divided into V-Flag control (V-Flag) , APP695-Flag (APP695) , low DOP (DOP1+APP695) , medium DOP (DOP2+APP695) , high DOP (DOP3+APP695) , a total of 5 groups, each with 4 samples. Enzyme-linked immunosorbent assay (ELISA) was used to determine the content of Aβ 1-40 and the activity of γ-secretase. In vivo experiment, 50 male Kunming mice of SPF grade, weighing (20±2) g, were selected and randomly divided into control, lead (Pb) , low DOP (DOP1＇) , medium DOP (DOP2＇) , high DOP (DOP3＇) consisted of 5 groups, each with 10 mice, continuously gavage for 6 weeks. Morris water maze method was used to detect the effect of different concentrations of DOP on learning and memory in mice, and ELISA method was used to detect β-secretase, γ-secretase activity and Aβ 1-40 content in brain tissue. Results:Compared with the CT group, the cell viabilities of the DOP2 and DOP3 groups were decreased, and the contents of LDH, MDA, and NO were increased, and the differences were statistically significant ( P<0.05) . Compared with the CT group, the cell viabilities of DOP1+Aβ, DOP2+Aβ and DOP3+Aβ groups were decreased, the contents of LDH, MDA, NO were increased, the differences were statistically significant ( P<0.05) . Compared with the Aβ group, the cell viability of DOP3+Aβ group was decreased, the contents of LDH, MDA, NO were increased, the differences were statistically significant ( P<0.05) . Compared with the Aβ group, the contents of LDH and NO in the DOP2+Aβ group were increased, and the differences were statistically significant ( P<0.05) . Compared with the CT group, the expression levels of Caspase-3 in the DOP2 and DOP3 groups were increased, and the differences were statistically significant ( P<0.05) . Compared with the Aβ group, the expression levels of Caspase-3 in the DOP2+Aβ and DOP3+Aβ groups were increased, and the differences were statistically significant ( P<0.05) . Compared with the APP695 group, the contents of Aβ 1-40 and the activities of γ-secretase of the DOP2+APP695 and DOP3+APP695 groups were increased ( P<0.05) . Compared with the control group, the activities of β-secretase, γ-secretase and the content of Aβ 1-40 in the brain tissue of DOP3＇group were increased, and the differences were statistically significant ( P<0.05) . Compared with the Pb group, the activities of β-secretase, γ-secretase and the content of Aβ 1-40 of the DOP3＇group were increased, and the differences were statistically significant ( P<0.05) . Compared with the control group, the target quadrant stay time and the number of crossings in the DOP2＇and DOP3＇groups were reduced, and the differences were statistically significant ( P<0.05) . Conclusion:DOP has a certain toxic effect on PC12 cells, causing learning and memory impairment in mice, and may promote the pathological progression of Alzheimer＇s disease.

Objective:To investigate the role and possible pathogenesis of high mobility group protein B1 (HMGB1) in lipopolysaccharide (LPS)-induced acute lung injury/acute respiratory distress syndrome (ALI/ARDS).Methods:① In vivo, 24 SPFC57BL/6 male mice were randomly divided into normal control group, ALI/ARDS model group, ethyl pyruvate (EP) treatment group and EP control group, with 6 mice in each group. The ALI/ARDS model was established by intraperitoneal injection of 20 mg/kg LPS. Mice in normal control group and EP control group were intraperitoneally injected with the same amount of sterile normal saline. Then, mice in the EP treatment group and EP control group were intraperitoneally injected with 40 mg/kg HMGB1 inhibitor EP. After 6 hours, the mice were sacrificed and lung tissues were collected. The expressions of heparan sulfate (HS), syndecans-1 (SDC-1), heparanase (HPA) and matrix metalloproteinases-9 (MMP-9) in lung tissues were detected by immunofluorescence technique. Orbital blood of mice was collected and serum was extracted to detect the content of HMGB1 by enzyme linked immunosorbent assay (ELISA). ② In vitro, human umbilical vein endothelial cells (HUVECs) were randomly divided into 6 groups: normal control group, HUVECs damage group (treated with 1 mg/L LPS for 6 hours), HMGB1 group (treated with 1 μmol/L recombinant HMGB1 for 6 hours), HMGB1+EP group (treated with recombinant HMGB1 for 1 hour and then added 1 μmol/L EP for 6 hours), LPS+EP group (treated with LPS for 1 hour and then added 1 μmol/L EP for 6 hours), EP group (treated with 1 μmol/L EP for 6 hours). The expressions of HS, SDC-1, HPA and MMP-9 in endothelial cells were detected by immunofluorescence technique. Results:① In vivo, light microscopy showed that the alveolar space was thickened after LPS stimulation, and there were a large number of inflammatory cells infiltrating in the alveolar space. Compared with ALI/ARDS model group, the expressions of HS and SDC-1 in lung tissue of EP treatment group were significantly increased [HS (fluorescence intensity): 0.80±0.20 vs. 0.53±0.02, SDC-1 (fluorescence intensity): 0.72±0.02 vs. 0.51±0.01, both P < 0.05], and the expressions of HPA and MMP-9 were significantly decreased [HPA (fluorescence intensity): 2.36±0.05 vs. 3.00±0.04, MMP-9 (fluorescence intensity): 2.55±0.13 vs. 3.26±0.05, both P < 0.05]; there were no significant changes of the above indexes in EP control group. Compared with ALI/ARDS model group, the content of serum HMGB1 in EP treatment group decreased significantly (μg/L: 131.88±16.67 vs. 341.13±22.47, P < 0.05); there was no significant change in the EP control group. ② In vitro, compared with HMGB1 group, the expressions of HS and SDC-1 in HMGB1+EP group were significantly higher [HS (fluorescence intensity): 0.83±0.07 vs. 0.56±0.03, SDC-1 (fluorescence intensity): 0.80±0.01 vs. 0.61±0.01, both P < 0.05], and the expressions of HPA and MMP-9 were significantly lower [HPA (fluorescence intensity): 1.30±0.02 vs. 2.29±0.05, MMP-9 (fluorescence intensity): 1.55±0.04 vs. 2.50±0.06, both P < 0.05]; the expression of HS, SDC-1, HPA and MMP-9 had no significant changes in EP group. Conclusion:HMGB1 participates in LPS-induced injury of endothelial cell glycocalyx, leading to increased lung permeability, and inhibition of HMGB1 can alleviate lung injury.