Effects Of Lanthanum Chloride Exposure On The Paracellular Permeability Of The Blood-brain Barrier In Rats And Related Molecular Mechanism Research

Objective:Rare earth element?REE?,with their unique physical and chemical properties,have been increasingly widely used in agriculture,industry and medicine fields,thus more and more REE enter into the environment,and into the human body through respiratory tract or food chain then accumulate in the tissues and organs.REE are non-essentia l elements of the body,which cause multiple organ and multi-system damages in high doses.As a result,the impacts on environmental and human health induced by REE accumulat io n have become a non-ignorable health problem.The central nervous system?CNS?is exceptionally sensitive to the toxicity of REE.Both epidemiological and anima l experimental studies have shown that REE can affect the development of the CNS,and impair learning and memory ability.Lanthanum,as a representative of light REE,its neurodevelopmental toxicity mechanism is still unclear,which is currently thought to be associated with disruption of trace elements?calcium and zinc?,enzymes and neurotransmitter systems in the brain,as well as oxidative stress.Recent studies have shown that REE can cross the blood-brain barrier?BBB?and accumulate in the brain,with highest in the hippocampus and cerebral cortex.BBB function is not complete during fetal and early after birth,which makes exogenous environmental toxicant easier into the brain,resulting in CNS dysfunction.Here we speculated that the immature BBB provides an opportunity for lanthanum entering;on the other hand,lanthanum may disrupt the tight junctions?TJs?leading to BBB leakage,consequently more lanthanum entering the brain and therefore accumulation,affecting the neuronal proliferation and differentiation and synaptic structure and function,resulting in neurodevelopmental disorders.BBB is a multicellular vascular structure between CNS and the system circulat io n,which maintains the homeostasis within CNS and al ows for properly neuronal funct io n.BBB barrier properties are mainly determined by the junctional complexes between brain microvascular endothelial cel s,including TJs and adherens junctions?AJs?,TJs are responsible for limiting the paracellular passage of hydrophilic molecules,while AJs promote the formation and maintenance.It is recognized that the regulation of paracellula r permeability is mainly through two aspects:changes in junctional complexes?phosphorylation,expression and redistribution?and cytoskeleton rearrangement?actin-myosin contraction?.Matrix metal oproteinase?MMPs?is a group of zinc-dependent endopeptidase that can directly degrade TJ proteins and perivascular basal membrane and increase BBB permeability,mostly related to gelatinase?MMP-2 and-9?.Rho GTPases RhoA and its downstream effector molecule ROCK activation could induce ctin-myos i n contraction and stress fiber formation,which are closely related to the increased paracelular permeability of BBB.The BBB breakdown due to TJs disruption is an important target of neurotoxicity of heavy metals?such as lead,aluminum and cadmium?,but the effect of lanthanum on BBB permeability and TJs has not been reported so far.In this study,subchronic lanthanum chloride?LaCl₃?-exposed animal model was established by exposure to LaCl₃ through drinking water from birth to one month after weaning.Morphology,biochemistry and molecular biology,and immunofluoresc e nce histochemistry laboratory techniques were used to investigate the effect of LaCl₃ exposure during development period on the BBB paracellular permeability in rats,involvi ng junction proteins changes and cytoskeleton rearrangement,and the mediation by MMP-2/-9 and RhoA/ROCK signaling pathways.And to provide new clues and experime nta l evidence for elucidating the neurodevelopmental toxicity mechanism of lanthanum and effectively preventing the cognitive function impairment caused by lanthanum.Methods:60 healthy adult Wistar rats?male:female=2:1?,240±10g,provided by the China Medical University Experimental Animal Center,were kept at a temperature of 22±1°C,with relative humidity of 50±5%,and 12 h day/night environment.The female rats were randomly divided into control group and low,medium and high lanthanum-dosed group.The mating was performed according to the male and female ratio of 2:1.Presence of vaginal plug or sperm from examination of vaginal secretion indicated pregnancy,recorded as gestation day 0.The rats in the control group were drinking distilled water,while in lanthanum-treated group were drinking 0.25%,0.5%and 1.0%LaCl₃ solutio n respectively.The offspring rats were exposed to lanthanum through milk during lactation,then via drinking LaCl₃ solution for another one month.In the first part,the permeabilit y of BBB was quantitatively evaluated by Evans blue extravasation assay,the ultrastruct ura l changes of BBB was observed by transmission electron microscopy,the protein and mRNA expression of junction proteins was detected by Western blot and Real-Time PCR,respectively,to illustrate effects of LaCl₃ on the BBB permeability and the expression of BBB junction proteins in rats.In the second part,the activity of MMP-2/-9 was detected by In situ zymography,the expression of MMP-2/-9 proteins was detect using Western blot,the mRNA expression of MMP-2/-9 and its endogenous inhibitor TIMP-2/-9 was examined by Real-Time PCR,to investigate the important role of MMP-2/-9 in LaCl₃-induced increased BBB permeability and down-regulation of tight and adherens junction proteins.In the third part,cerebral vessels was isolated using dextran density gradient centrifugat io n,the activity of RhoA in rats'cerebral vessels was analyzed using Pull-down assay,and proteins expression of ROCK1/2,p-MYPT1,p-MLC2 and MLC2,and MLCK were examined by western blot,to explore the role of myosin phosphorylation and cytoskeleton rearrangement in LaCl₃-induced increased BBB paracel ular permeability.Results:1.Effects of lanthanum chloride exposure on the permeability and ultrastruct ure of BBB in offspring rats.The extravasation of Evans blue in the cerebral hemispher es,cortex and hippocampus in LaCl₃-treated rats all increased significantly,suggesting that LaCl₃-exposure increased the BBB permeability of the offspring rats.LaCl₃-exposure caused ultrastructural changes of the BBB in the offspring rats,with partial dissolved basement membrane and decerased density of the tight junction in the middle-dosed group,while unevenly thickness of the basement membrane,unclear tight junctions,and mild swel ing in astrocyte end-foot were observed in high-dose group.2.Effects of lanthanum chloride exposure on protein expression and mRNA transcription of BBB junction proteins in offspring rats.There was a trend consistency between the effects of LaCl3 on the expression of tight and adherens junction proteins in the cerebral cortex and hippocamp us of offspring rats,demonstrated by significant down-regulation of claudin-5,occludin,and VE-cadherin,with no obvious changes in ZO-1 protein expression.However,the effects on mRNA levels were different,such as occludin and ZO-1 in the low-dose group were higher than the control,but with no significance,whereas claudin-5 and VE-cadherin mRNA were significantly decreased in the cerebral cortex and the hippocampus of high-dose rats.3.Effects of lanthanum chloride exposure on activity of MMP-2/-9 in Rats.The activity of MMP-2/-9 in the brain of LaCl₃-treated rats was significantly higher than the control group.The results of Western blot showed that MMP-9 protein expression was significantly up-regulated in the cerebral cortex and hippocampus of offspring rats.The expression of Mmp9 mRNA in the cerebral cortex and hippocampus was also significa nt l y higher than the control,while Timp1 mRNA was significantly decreased,with no obvious changes in Mmp2 or Timp2 mRNA level.4.Effects of lanthanum chloride exposure on RhoA/ROCK signaling pathway and cytoskeleton rearrangement in brain microvessels of offspring rats.The results of pull-down analysis showed that the ratio of GTP-RhoA/Total-RhoA in the brain microvessels of LaCl₃-treated group was significantly increased,indicating the activity of RhoA was increased.The results of Western blot showed that ROCK1 and ROCK2 protein expression was significantly upregulated,and the relative level of ROCK2 in each group was higher than ROCK1,suggesting that LaCl₃ exposure could activate RhoA/ROCK signaling pathway.In addition,the phosphorylation levels of downstream MYPT1 and myosin MLC2 were significantly increased,and MLCK protein expression was also significantly up-regulated,indicating that ROCK and MLCK co-mediated MLC2 phosphorylation,resulting in cytoskeleton rearrangement.Conclusion:1.Developmental LaCl₃-exposure could impair the ultrastructure of blood-brain barrier,down-regulate protein expression of occludin,claudin-5 and VE-cadherin,and reduce mRNA transcription of claudin-5 and VE-cadherin,consequently caused increased permeability of the blood-brain barrier in offspring rats.2.Developme nta l LaCl₃-exposure could enhance the activity of MMP-2/-9,up-regulate protein expression of MMP-9,and disrupt the balance between MMPs and TIMPs,with increasing mRN A level of MMP-9 simultaneously reducing TIMP-1.3.Developmental LaCl₃-exposure could stimulate the activation of RhoA/ROCK signaling pathway,and up-regulate Ca²⁺/CaM-dependent MLCK protein expression,resulting in increased MLC2phosphorylation,which promoted actin cytoskeleton rearrangement.