The Mechanism Of Environmental Contamination PFOS Increasing The Permeability Of Blood-Brain Barrier

Perfluorooctanesulfonate( PFOS) is a kind of perfluoral organic compound, which has been widely in the production of surfactants, cosmetic, pesticides and drug products. Non-sticking cooking pots, food packing bags, some shampoo, soaps and strippants all contain PFOS. Recent studies suggested that the range and extent of environmental contamination caused by PFOS has gone far beyond our anticipation. In 2001, it was put on the black list of persistent contaminants by Environmental Protection Bureau of U. S and will be strictly managed.Olsen and his colleagues detected that the serum concentration of PFOS of workers from PFOS workshops is 2.91mg/L. The concentration of non-occupational population is 34. 9μg/L,50μg/L and 54μg/L in USA,Japan and China respectively. Staito , Geisy and Kannan et al also detected PFOS from environmental sample, blood serum and tissue of wild animals gathered from all over the world. Widely existing PFOS pollution in ecological environment has become the focus that people pay attention to. In addition, PFOS have characters of no degradation in environment and living bodies and of strong biological accumulation. Therefore, as a new persistent contaminant, PFOS will have great influence on the whole ecological system and health of human body.Some literatures have suggested that PFOS can cause body weight loss, hepatomegaly, dysmetabolism of lipids and energy, embryotoxicity and latent neurotoxicity in experimental animals. At present, no reports about the effect of PFOS to the permeability of BBB has been found. This study approached the effect and molecular mechanisms to provide a interventional target in braindevelopmental disorders caused by this contaminant.Methods1. Detection of permeability of HRP through the BBB and transendothelial electrical resistance (TEER) : Set up BBB model with transwell in vitro. Induce for 30 minutes with PFOS of different concentrations, detect permeability of HRP through the BBB and transendothelial electrical resistance (TEER) with EVOM resistance radiometer;Induce for different lengths of time with PFOS of 50 jxM, detect permeability of HRP through the BBB and transendothelial electrical resistance (TEER) with EVOM resistance radiometer.2. Cellular tight junction protein ZO-1 and cytoskeletal protein F-actin by immunofluorescence staining. : Cultivation of human brain microvascular endothelial cell ( HBMEC ) . Induce for 30 minutes with PFOS of different concentrations, detect the change of cellular tight junction protein ZO-1 and cytoskeletal protein F-actin by immunofluorescence staining;Induce for different lengths of time with PFOS of 50|xM, detect the change of cellular tight junction protein ZO-1 and cytoskeletal protein F-actin by immunofluorescence staining.3. The effects of different inhibitors on permeability of HRP through the BBB and transendothelial electrical resistance (TEER) : 30minutes after the inhibitor Y27632 of Cofilin/P-Cofilin's upstream signaling molecule Rock worked, induce for 30 minutes with PFOS of different concentrations or different lengths of time with PFOS of 50|xM, permeability of HRP and TEER;30minutes after the inhibitor LY294002 of AKT/P-AKT upstream signaling molecule PI3K worked, induce for 30 minutes with PFOS of different concentrations or different lengths of time with PFOS of 50jxM, permeability of HRP and TEER;30minutes after the inhibitor Wortmannin of AKT/P-AKT upstream signaling molecule PI3K worked, induce for 30 minutes with PFOS of different concentrations or different lengths of time with PFOS of 50|xM, permeability of HRP and. TEER;30minutes after the inhibitor GO6976 of PKC worked, induce for 30 minutes with PFOS of different concentrations or induce for different lengths of time with PFOS of 50|jlM, permeability of HRP and TEER;30minutes after the inhibitorG06983 of PKC worked, induce for 30 minutes with PFOS of different concentrations or induce for different lengths of time with PFOS of 50|xM, permeability of HRP and TEER;4. The effects of different inhibitors on the change of cellular tight junction protein ZO-1 and F-actin in cytoskeletal protein by immunofluorescence staining: 30minutes after the inhibitor Y27632 of Cofilin/P-Cofilin's upstream signaling molecule Rock worked, induce for 30 minutes with PFOS of different concentrations and induce for different lengths of time with PFOS of 50jxM, detect the change of cellular tight junction protein ZO-1 and F-actin in cytoskeletal protein by immunofluorescence staining;30minutes after the specific inhibitor LY294002 of AKT/P-AKT upstream signaling molecule PI3K worked, induce for 30 minutes with PFOS of different concentrations and different lengths of time with PFOS of 50jxM, detect the change of cellular tight junction protein ZO-1 and F-actin in cytoskeletal protein by immunofluorescence staining;30minutes after the specific inhibitor Wortmannin of AKT/P-AKT upstream signaling molecule PI3 K worked, induce for 30 minutes with PFOS of different concentrations and different lengths of time with PFOS of 50jxM, detect the change of cellular tight junction protein ZO-1 and F-actin in cytoskeletal protein by immunofluorescence staining;30minutes after the inhibitor GO6976 of PKC worked, induce for 30 minutes with PFOS of different concentrations and induce for different lengths of time with PFOS of 50jxM, detect the change of cellular tight junction protein ZO-1 and F-actin in cytoskeletal protein by immunofluorescence staining;30minutes after the inhibitor GO6983 of PKC worked, induce for 30 minutes with PFOS of different concentrations and induce for different lengths of time with PFOS of 50jjlM, detect the change of cellular tight junction protein ZO-1 and F-actin in cytoskeletal protein by immunofluorescence staining5. Study of Singnal pathway5. 1 Detect the expression of signal protein Cofilin/P-Cofilin and AKT/P-AKT by Western blot: Detect the expression of Cofilin/P-Cofilin under the induction of different doses of PFOS for 30 minutes;Detect the expression of Cofilin/P-Cofilin under the different lengths of induction time with PFOS of50|i,M;Detect the expression of AKT/P-AKT under the induction of different doses of PFOS for 30 minutes;Detect the expression of AKT/P-AKT under the different lengths of induction time with PFOS of 50 jxM;After the different concentrations specific inhibitor LY294002 of AKT/P-AKT upstream signaling molecule PI3K worked, detect the expression of AKT/P-AKT under 30 minutes with PFOS of 50|xM;After the different concentrations specific inhibitor Wortmannin of AKT/P-AKT upstream signaling molecule PI3K worked, worked detect the expression of AKT/P-AKT under 30 minutes with PFOS of 50|xM5. 2 Detect the activity of protien kinase C by non-radioactivity: Detect the expression of protien kinase C under the induction of different doses of PFOS for 30 minutes;Detect the expression of protien kinase C under the different lengths of induction time with PFOS of 50|xM6. Detect the cellular tight junction protein occludin by Western blot: 30 minutes after the induction of the above-mentioned 5 inhibitors and 50|xMPFOS to human brain microvascular endothelial cell (HBMEC) for 30minutes, detect the cellular tight junction protein occludin.7. In vivo— permeability of HRP through rat's BBB: Set up animal model by continuous abdominal injection of different doses of PFOS for 14 days. Take HRP as a tracer agent, observe the permeability of HRP through BBB under the induction of different doses of PFOS by electron microscopy.8. In vivo— proliferation of rats' hippocampus nerval stem cell: Set up animal model by continuous abdominal injection of different doses of PFOS for 14 days. Take Brdu as a marker, detect the proliferation of rats' hippocampus stem cell under the induction of different doses of PFOS by imrnunohistochemistrys.Results1. PFOS induce the permeability of HRP and TEER changed: As the concentration of PFOS increased, the permeability of HRP through BBB increased , TEER decreased gradually;As the time of induction increased, the permeability of HRP through BBB increased and then decreased, TEER decreases and then increases.2. PFOS induce cellular tight junction protein ZO-1 changed and cytoskeletal protein F-actin rearranged;Induce for 30 minutes with PFOS of different concentrations, cellular tight junction protein ZO-1 decreased and cytoskeletal protein F-actin rearranged as the concentration of PFOS increased. As the time of induction increased, cellular tight junction protein ZO-1 decreased and then increased and cytoskeletal protein F-actin rearranged and then revived gradually3. Different effects of different inhibitors on permeability of HRP through the BBB and transendothelial electrical resistance (TEER) : 30minutes after the inhibitor Y27632 of Cofilin/P-Cofilin's upstream signaling molecule Rock worked, induce for 30 minutes with PFOS of different concentrations and induce for different lengths of time with PFOS of 50jjlM, the permeability of HRP and TEER is the same with that of no inhibition;30minutes after the specific inhibitor LY294002 of AKT/P-AKT upstream signaling molecule PI3K worked, induce for 30 minutes with PFOS of different concentrations and induce for different lengths of time with PFOS of 50pJM, the permeability of HRP and TEER had no obvious change. 30minutes after the specific inhibitor Wortmannin of AKT/P-AKT upstream signaling molecule PI3K worked, after induce for 30 minutes with PFOS of different concentrations and induce for different lengths of time with PFOS of 50pJM, the permeability of HRP and TEER had no obvious change. 30minutes after the inhibitor G06976 of PKC worked, induce for 30 minutes with PFOS of different concentrations and induce for different lengths of time with PFOS of 50jxM, the permeability of HRP and TEER is the same with that of no inhibition. 30minutes after the inhibitor GO6983 of PKC worked, induce for 30 minutes with PFOS of different concentrations and induce for different lengths of time with PFOS of 50|xM, the permeability of HRP and TEER is the same with that of no inhibition.4. Different effects of different inhibitors on the change of cellular tight junction protein ZO-1 and F-actin in cytoskeletal protein;30 minutes after the inhibitor Y27632 of Cofilin/P-Cofilin's upstream signaling molecule Rock worked, induce for 30 minutes with PFOS of different concentrations and induce for different lengths of time with PFOS of 50pJVt, the change of cellular tightjunction protein ZO-1 and cytoskeletal protein F-actin is the same with that of no inhibition. 30 minutes after the specific inhibitor LY294002 of AKT/P-AKT upstream signaling molecule PI3K worked, induce for 30 minutes with PFOS of different concentrations and induce for different lengths of time with PFOS of 50|xM, the expression of cellular tight junction protein ZO-1 and cytoskeletal protein F-actin had no obvious change. 30minutes after the specific inhibitor Wortmannin of AKT/P-AKT upstream signaling molecule PI3K worked, induce for 30 minutes with PFOS of different concentrations and induce for different lengths of time with PFOS of 50|xM, the expression of cellular tight junction protein ZO-1 and cytoskeletal protein F-actin had no obvious change. 30minutes after the inhibitor GO6976 of PKC worked, induce for 30 minutes with PFOS of different concentrations and different lengths of time with PFOS of 50jxM, the change of cellular tight junction protein ZO-1 and cytoskeletal protein F-actin is the same with that of no inhibition. 30minutes after the inhibitor GO6983 of PKC worked, induce for 30 minutes with PFOS of different concentrations and different lengths of time with PFOS of 50|xM, the change of cellular tight junction protein ZO-1 and cytoskeletal protein F-actin is the same with that of no inhibition.5. Study of Singnal pathway:5. 1 Detect Cofilin/P-Cofilin or AKT/P-AKT protein expression by Western blot: Expression of Cofilin/P-Cofilin under the induction of different doses of PFOS had no obvious difference. Expression of Cofilin/P-Cofilin under the different lengths of time induction of 50|xM PFOS had no obvious difference. Expression of AKT/P-AKT under the induction of different doses of PFOS increased as the concentration increased. Expression of AKT/P-AKT under the different lengths of time induction of 50jxM PFOS increased as the time increased, got to the top at 30 minute, and then decreased. After the different concentrations specific inhibitor LY294002 of AKT/P-AKT upstream signaling molecule PI3K worked, the result suggested that 30jjlMLY294002 inhibited intensively the expression of P-AKT. After the different concentrations specific inhibitor Wortmannin of AKT/P-AKT upstream signaling molecule PI3K worked, the result suggested that lOOnM Wortmannin inhibited intensively theexpression of P-AKT.5. 2 Detection of Protien kinase C by non-radioactivity: Protien kinase C under the induction of different doses of PFOS and under the different lengths of time induction of 50fxMPFOS by non-radioactivity had no expression in phosphorylation6. Detect cellular tight junction protein occludin expression by Western blot;30 minutes after the induction of the above-mentioned 5 inhibitors and 50|xMPFOS to human brain microvascular endothelial cell ( HBMEC) for 30minutes, PFOS decreased insoluble tight junction protein occludin expression , soluble tight junction protein occludin expression is contrary. LY294002 and Wortmannin inhibited this expression.7. Permeability of HRP through rat's BBB: Take HRP as a tracer agent, In high dosage rats HRP is seen in basal laminae of endothelial and pericytes and parenchyma among perivascular, not in the low dosage and control group.8. Proliferation of rats' nerval hippocampus stem cell: Take Brdu as a marker, under the induction of high dosage PFOS positive cells were found in rats' hippocampus stem cell, not in the low dosage and control group.Conclusions1. PFOS can increase the permeability of BBB2. PFOS can cause the rearrangement of cytoskeletal protein of HBMEC and cellular tight junction "open".3. PFOS implements is increase in permeability of BBB through the PKB (AKT/P-AKT) pathway...