| BackgroundLead (Pb) pollution is one of the most serious environmental pollution problemsworldwide. The detrimental effect of lead on children’s development of central nervoussystem is a subject of environmental science and life science that needs further efforts tobe resolved. Lead can be used in the production of car, battery, printing and dyeing,chemical industry and other fields. With the modern urbanization progress, lead levels inenvironment (smelting exhaust gas, paint, automobile exhaust, toys) are greatly elevated,which significantly increased the population exposure levels. Accumulated effects ofchronic exposure to lead caused irreversible damage of the function of central nervoussystem. The toxicity effects of lead on the central nervous system are prevalence,persistence and irreversible. At present, the average child blood lead level in our country is80.7μg/L and about10%-15%of city children’s blood lead exceeds the current action level.Especially in recent years, anecdotes of industry lead pollution and children lead poisoning incidents happened in Shaanxi Fengxiang County, Guangdong QingyuanCounty, Shanghai Kangqiao County, Sichuan Longchang County, not only making seriousimpact on children’s physical and mental health but also influencing social stability.Therefore, the prevention of childhood lead poisoning is an environmental health andsocial problem to which our government and public should pay attention.Chronic lead exposure can cause irreversible damage of brain function in children.But the visual system is as the important branch of central nervous system, the effects ofchronic lead exposure on the function of developmental visual system have also been thefocus of research. Previous epidemiological survey found that lead poisoning can causeamblyopia, blurred vision, with maximum effects like visual field defects, blindness. Themechanism of vision toxicity may be related to cGMP, dopamine, the neurotransmitterrelease and neuronal apoptosis and other factors. But it is unclear that how lead enters thevisual system and the mechanism of injury. In addition, a large number of experimentalevidence revealed that blood retinal barrier (BRB) make an important role in maintainingrelative stability and independence of the visual system and ensure its physiologicalfunction. The damage of BRB is closely related with the visual system diseases. In thisstudy, the lead exposure model in vivo was set up to study the dose-effect pattern of leadexposure on the growth of rat BRB permeability. The relationship among tight junction(TJ) associated protein and PI3K/Akt signaling pathway, phosphorylation and BRBpermeability changes was explored to elucidate the mechanism of lead induced BRBpermeability changes.ObjectiveA lead-exposed rat model was used to study the effect of lead on BRB. To explore therole of Occludin, Claudin5and PI3K/Akt signaling pathway phosphorylation underpiningin the lead induced disruption of BRB. To provide mechanism basis for the prevention oflead induced visual system injury.Methods1. Relationship between blood lead level and ERGUsing Ganzfeld type vision stimulator (LKC Technologies, Inc.), ERG was recordedin dark room. Flash strength is0.01cd·s/m2,10-μs. 2. The establishment of rat model of lead exposure3weeks male SD rats were allowed to drink water containing0.1%,0.2%,0.3%leadacetate solution, where deionized water was served as a control. After6weeks, rat modelof lead exposure was set up. Tungsten atomic absorption method was used to determinethe blood lead concentration.3. The toxicity of lead exposure on rat retinaRETI-scan system of visual electrophysiological examination instrument for ERGinspection was used to test the effects of lead on retinal visual functions. Hocestimmunofluorescence staining was used to observe lead effects on retinal structure.4. Lead exposure and the changes of BRB permeabilityBRB structural integrity was observed by intravenous injection of Evans blue (EB)under fluorescence microscopy. BRB permeability changes and TJ structural changes wasobserved by lanthanum nitrate tracer method under electron microscope.5. Lead exposure and the changes of BRB TJ associated proteins and PI3K/Aktsignal transduction pathwayTJ associated protein and PI3K/Akt signaling pathway phosphorylation associatedprotein expression changes was determined by Western blot method to clarify whetherlead exposure was related to TJ. PI3K/Akt signaling pathway was manipulated by thespecific inhibitor Wortmannin to observe whether I3K/Akt signaling pathway is involvedin the lead-induced TJ opening and increased BRB permeability.Results1. Relationship between blood lead level and ERGSubjects were divided into low lead group and high lead group according to themedian value of blood lead level (70μg/L). There was no statistical difference inamplitude or latency of ERG a wave between the two groups. The results showed that bwave amplitude in the high lead group was significantly higher than that in the low leadgroup, while the two groups had no significant difference in the latency.2. The effect of Lead exposure on the retinal structure and functionAccording to the reports, the criteria of rat lead poisoning was200μg/L.3-week male SD rats were allowed to drink water containing0.1%,0.2%,0.3%lead acetate solution,while deionized water was served as a control. After6weeks period, a rat model of leadexposure was established. Blood lead levels in the control group and the0.1%,0.2%,0.3%groups were24.9μg/L(5.8μg/L),226.2μg/L (5.8μg/L),284.2μg/L (35.4μg/L),330.3μg/L (52.3μg/L), respectively, which showed a dose-dependent manner. Since0.1%(the lowest dose) group of blood lead exceeding the reported value of200μg/L, at whichthe central nervous system injury would be observed, ERG test was used to test the visualfunctions of the four groups. Rod-ERG b waves were significantly different comparedwith the control. The amplitudes of b wave in the control and the three experimentalgroups were229.81(24.36),357.12(56.58),489.42(73.54),530.13(48.42), respectively.The results showed that the Rod-ERG b wave was increased by44%compared to thecontrol group, although b wave amplitude and latency in Max-ERG, Ops, Cone-ERG andFlick-ERG showed no significant difference. Using the Hoechst33342immunofluorescence staining to detect the structural changes of retinal, the results showedthat retinal thickness was thinner in the experimental groups as to the control groupespecially that in the0.2%group and the0.3%group, of which the thickness of retinalwas reduced by approximately58%compared to the control group. Further analysisshowed that the inner plexiform layer of retina, inner core layer and outer nuclear layerwere most sensitive to the lead toxicity, and it could be observed that the structuralthicknesses of these tissues were significantly reduced. In summary, the results suggestedthat lead exposure caused functional and structural injuries of retinal.3. The effects of lead exposure on BRB permeabilityBRB structural integrity was observed with immunofluorescence microscopy byintravenous injection of EB. EB leakage was not found in each layers of retinal in thecontrol group, but the leakage could be observed in all the groups. The fluorescenceintensity was the highest in the0.2%group and the0.3%group. The leakage of the corelayer, the outer nuclear layer and the retinal ganglion cell layer were of the most obviously.Lanthanum nitrate tracer method was used to observe lanthanum particles which werecontinuous distributed in the normal retinal, choroidal capillary lumen under the electronmicroscope. Lanthanum particles infiltrated into retinal pigment epithelial cell basal folds by Bruch membrane, but could not pass through the retinal pigment epithelial cell.Lanthanum particles that went through the choroid pigment epithelial cell gap could beobserved in the lead-exposed group with dose-dependent relationship, and it could befound in the outer segment in the0.3%group. The above results demonstrated that leadexposure caused damage of BRB integrity, which resulted in retinal injury.4. The relationship between BRB TJ and Akt phosphorylationBRB tight junction proteins Occludin and Claudin-5expression levels decreasedsignificantly after lead exposure in rats. There were significantly differences between0.2%and0.3%groups (p<0.01). There was no significant difference of the degree ofimmune positive at Thr308site. The degree of immune positive at Ser473site increasedwith exposure dose. Compared with the control, there were significant difference among0.1%group(p<0.01),0.2%group(p<0.01) and0.3%group(0.1%), which demonstratedBRB opening was related to the expression of TJ and Akt phosphorylation.5. PI3K/Akt signaling pathways involved in the regulation of BRB open after leadexposureWortmannin (PI3K/Akt pathway inhibitors) and lead were used synergistically (group0.3%) to confirm that PI3K/Akt pathway was involved in lead poisoning on BRB damage.Compared to the lead exposure group, Wortmannin could reduce the Rod-ERG b waveamplitude21%, which indicated that visual function could partial reversed by Wortmannintreatment. Morphological observation showed that EB leakage was alleviated andlanthanum particles that went through pigment epithelial cells reduced at the same time.Wortmannin could reduce the expression levels of Occludin and clauding-5by29%and16%. Akt Ser473sites phosphorylation level was significantly decreased by Wortmannin(P <0.01).ConclusionIn summary, higher level of blood lead was related to the supernormal amplitude of bwave of ERG. Lead exposure may produce detrimental effect on the structure and functionof retina. The toxic effect was mediated by the integration of BRB, which was controlledby PI3K/Akt pathway at molecular level. |
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