Role Of Tricellulin In Lead-induced Changes In Tightness Of Blood-cerebrospinal Barrier And Possible Mechanism

BackgroundAs a ubiquitous environmental heavy metal element, lead (Pb) has been proved to be atoxicant with no biological function. The widely used application of Pb gives rise to abroad spectrum of toxic effects in adults and children and makes lead a public health issue.In recent years, the toxic effect of Pb on the central nervous system (CNS) has receivedworld-wide attention. Early-life lead exposure can cause learning disabilities,developmental delay, attention-deficit disorders and so on. The detailed mechanism oflead-induced neurotoxicity is not very clear, and recent studies focus on the effect of leadon the permeability of brain barriers.As one of the most important organs, our brains have established many mechanisms to keep their homeostasis. The brain barriers, namely the blood-brain barrier (BBB) and theblood-cerebrospinal fluid barrier (BCB), play important roles in maintenance of normalfunction of brain. The BCB is mainly made up of choroidal epithelial cells. The BCBsecretes cerebrospinal fluid (CSF) and confines the entrance of substances from the bloodto CSF.Tight junctions exist in the BCB and function as a seal that regulates lateral diffusionbetween the apical and basolateral plasma membrane, thus contributing to theestablishment of distinct fluid compartments within the body. TJ proteins include occludin,the claudin protein family and junctional adhesion molecules (JAM), which mainly consistof bicellular TJs (bTJs). Tricellulin (TRIC) is the first tight junction protein found at “threecorner” contact site of endothelial cells in vertebrates. TRIC has been found in epithelialcells of intestine, stomach and so on. However, whether TRIC is expressed in choroidalepithelial cells remains unclear.It is reported that BCB is the aim of Pb toxicity, and lead increases the leakage of BCBby affecting the tight junction protein. Based on the previous research, we tried to find therelationship between TRIC expression in choroidal epithelial cells and lead, which hasnever been reported before.AimsOur research focused on the expression of TRIC in the choroidal epithelial cells in vivoand in vitro, and investigated the role of TRIC on the permeability of BCB. We also triedto find the effect of lead exposure on the expression of TRIC in vitro. By testing thechange of microRNA in the lead induced toxicity in choroidal epithelial cells, we tried tounderstand the function of microRNA in the lead-induced changes in the permeability ofbrain barrier and possible mechanism.Methods1. Samples of the choroid plexus were separated from rats ranging from2w to5w.Immunofluorescence staining was used to investigate the localization of TRIC andwestern bolt and PCR were used to detect the expression of TRIC. 2. Z310cell line was used to establish the in vitro BCB model. Small interfering RNA(siRNA) was used to inhibit the expression of TRIC. TEER and flux of FITC-dextran wereused to value the integrity of barriers after changing the level of TRIC.3. Lead exposure cell model was built up by Z310cells. Western blot,immunofluorescence image and over-expression of TRIC were used to testify the role ofTRIC in lead-induced changes in tightness of BCB.4. Bioinformatics technical analysis was used to find potential miRNA binding motifs onTRIC3’UTR. Real time-PCR was used to detect the expression of microRNA in thePb-induced changes in tightness of BCB. MicroRNA inhibitors were used to find the roleof microRNA in the expression of TRIC and Pb-induced barrier leakage.Results1. TRIC protein and mRNA were detected in choroidal epithelial cells in vivo and invitro, and TRIC was only localized at the tricellular contacts and co-localized withoccludin.2. TRIC influenced the blood-cerebrospinal fluid barrier. Down-regulation of TRICincreased the permeability of BCB.3. Lead exposure reduced the expression and distribution of TRIC in Z310cells, andup-regulation of TRIC partly inhibited the lead-induced barrier leakage.4. Bioinformatics technical analysis showed that miR-203might be potential microRNAregulating the expression of TRIC. Lead exposure increased the expression of miR-203invivo and in vitro. By treatment with miR-203inhibitor, we found that reducing of miR-203could in some degree increase the protein expression of TRIC and inhibit the Pb-inducedbarrier leakage.Conclusion1. Our results showed that TRIC was expressed in choroidal epithelial cells in vivo andin vitro，and played an important role in the permeability of BCB.2. Lead exposure reduced the expression of TRIC in vitro and over-expression of TRICcould inhibit the Pb-induced changes in permeability of BCB in some degree. 3. Bioinformatics technical analysis showed that miR-203may be potential miRNAregulation the function of TRIC. Lead exposure increased the expression of miR-203inchoroidal epithelial cells and down-regulation of miR-203might inhibit the Pb-inducedbarrier leakage by increasing the expression of TRIC.In conclusion, our research focused on the TRIC expression in the choroidal epithelialcells in vivo and in vitro, and its underlying role in the lead-induced BCB leakage. Ourresults may further our understanding on the role of TRIC in lead-induced changes inpermeability of BCB and shed new light on the mechanism of lead-induced toxicity.