The Role And Regulatory Mechanism Of Tight Junction Proteins In The Pb-induced BBB Damage

Lead is one of most common heavy metal pollution in the environment. Through air,dust, food, drinking water and other contacts, lead accumulates in the body and causesmulti-system and multi-organ injury and dysfunction, especially in the central nervoussystem, even low dose of lead in the early stage will cause irreversible injury in centralnervous system. Due to the expansion of lead-related industry, such as batterymanufacturing, metal smelting, transportation, construction and printing, the lead contentin the living environment and population exposure is sustained increasing.Children are very sensitive to lead contamination. Lead poisoning can causechildren’s functional impairment of the nervous system, including learning and memorydamage, cognitive deficits, psychological and behavioral abnormalities, which is acontinuous dose-response process. As identified in environmental toxicology, the toxiceffects of lead have become the "invisible killer" endangering the health of children and have been a worldwide public health risk factors and important social issues.The blood brain barrier (BBB) is a dynamic regulatory interface between thecapillaries and nerve tissue in the brain and spinal cord. This interface maintains thebalanced and stable environment in the central nervous system. Because the BBB structureof the infant is not yet mature, the outside compound can enter the brain tissue more easilythan adults, and nervous system damage is more serious. Our previous studies showed thatlead exposure could damage the structure of the rat BBB and affect the BBB function. Tofurther elucidate the mechanism during lead-induced damage of the structure and functionof the BBB and to explore preventive measures, using the lead-induced BBB injury modelof rat and cell, we clarified the relationship between lead-induced BBB leakage and TJprotein expression, and the relationship between TJ protein expression and correlatedsignal transduction pathways. We found the protective effects of iron on lead-inducedBBB damages and explored the molecular mechanism of lead-induced BBB damages andcorrelated signal transduction pathways in the Pb-induced enhancement of BBBpermeability.ObjectiveTo clarify the relationship between lead-induced BBB tight junction proteinexpression and corelated signaling pathway changes. To observe the effects of iron onlead-induced BBB tight junction protein expression and corelated signaling pathwaychanges.Method1. The rat model of BBB damage was established by use of lead acetate in the drinkingwater. The blood and brain lead level was determined by atomic absorptionspectrophotometry. The BBB leakage was determined by lanthanum nitrate tracertechnique. Morris water maze and open-field test were used to observe the behaviorchange of rats exposed to lead.2. Brain capillaries of rats exposed to lead was enriched and the expression of tightjunction protein ZO-1, occludin, claudin-5was tested by western blot analysis. The signal transduction pathways correlated with the regulation of tight junction proteins wereobserved using protein chip assay and the chip results were verified by western blotanalysis.3. The ferrous sulfate was supplemented to lead exposure rats. Lanthanum nitrate tracertechnique was used to determine the protective effect of iron on lead-induced BBBdamage. Morris water maze and open-field test were used to determine the protection ofthe iron to rats exposed to lead. Western blot analysis was used to detect the expression ofZO-1, occluding, claudin-5and signaling pathway molecules.Results1. Lead exposure could damage the BBB integrity in developing rats.Lead exposure caused rat blood lead level and brain lead level increased comparedwith control group (p<0.05). Electron microscopy showed that lead induced BBB integritydamage. By disrupting the TJ between endothelial cells lanthanum nitrate penetrated intothe brain tissue. Open-field experiments showed that the spatial cognitive ability in thenew environment, the excitement and exploratory behavior of the lead exposure groupsignificantly decreased (p<0.05). Platform hidden experiment showed that the incubationperiod of lead exposure rats increased compared with control group (p<0.05). Spaceexploration experiment showed that the residence time of lead exposure group in the firstquadrant was significantly shorter compared to the control group (p<0.05).2. Lead-induced changes of tight junction proteins and correlated signaling pathways ofrat brain vascular endothelial cells.The western blot results showed that expression of ZO-1, occludin and claudin-5wassignificantly decreased (p<0.05). Protein microarray showed the signal pathway of MAPKand Akt changed significantly (>1.5time). Western blot results confirmed the signalingpathway molecules of ERK1/2, JNK, p38, Akt473and Akt308phosphorylation increasedsignificantly (p<0.05).3. Iron supplement could antagonize the lead-induced TJ damageIron supplementation by gavage reduced blood lead levels in rats exposed to lead compared with the control group (p<0.05). Iron supplementation could reduce thelead-induced permeability increase. Western blot showed that iron supplementation couldreduce the lead-induced TJ protein downregulation and phosphorylation of ERK1/2, JNK,p38, Akt473and Akt308(p<0.05). These results suggested that iron supplementationcould inhibit the effects of lead on tight junction protein expression through the ERK1/2and PI3K/Akt pathways.ConclusionThis study established the lead-induced BBB injury model of rat and cell, and foundtight junction proteins were important target molecules of lead-induced structural andfunctional damage of BBB. Lead could increase the BBB permeability through activatingthe MAPK and PI3K/Akt signal transduction pathways and decreasing the expression oftight junction proteins. Furthermore, Iron supplementation could antagonize theneurotoxicity caused lead.