Metalloproteinases MMP2/9 Lead-induced Brain Barrier Destruction Mechanism

Background Lead(Pb) is a well-known neurotoxicant and a risk factor forneurologic disorders. Pb accumulation in human body results in lead poisoning, especially in children. Lead poisoning in children was found to cause irreversible impairment in the central nervous system(CNS), as shown by includingdecreased intelligence quotient(IQ) and cognitive deficits. Therefore, children lead poison is one of the major security issues of public health in the world. Brain barrier system composed of three parts: blood-brain barrier(BBB), blood-CSF barrier(BCB) and cerebrospinal fluid-brain barrier(CBB). Brain barriers play important roles in blocking the transport of harmful meterials between blood and brain tissue, regulating the transport of essentialmetal ions in the brain, and removing metabolites orunwanted mate-rials from brain extracellular fluid to the blood. Previous studies in this lab has demonstrated that developmental Pb exposure in rats resulted in BBB impairment, accompanying with decreased levels of tight junction proteins; However, the regulatory mechanisms are not clear.Matrix metalloproteinases(MMPs) are important components of extracellular matrix proteasome and affect the remodeling and degradation of extracellular matrix greatly. Some study showed that MMP-2 and MMP-9 cause leakage of brain barrier by degradation of tight junction proteins claudin-5 and occludin. Whether MMP-2 and MMP-9 have influence on the lead-induced brain barrier loss is never reported. Our studies mainly aim to investigate the roles of MMP-2 and MMP-9 in lead-induced brain barrier system disorder. The results will provide research basis for the study of Pb-induced neurotoxicity, and the protective measures.Objective To establish in vitro BBB/BCB model and investigate the impact of Pb exposure on c tight junction proteins and MMP-2/9, and further study the role of MMP-2/9 in lead-induced BBB/BCB injuries model and underlying mechanism.Methods 1. The in vitro BBB model was established by C6 cell line co-cultured with HBMEC cell line and in vitro BCB model was set up by Z310 cell line. MTT assay was used to select the proper concentration of lead. 2. TEER and flux of FITC-dextran were performed to determine the permeability of in vitro BBB/BCB model. 3. Western blot, immunocytochemical methods and PCR were used to observe the expression of TJ proteins and MMP-2/9. 4. Gelatin zymography was used to measure the activity of MMP-2/9. 5. MMP-2 selected inhibitors were used to inhibit the activity of MMP-2, and then TEER and flux of FITC-dextran were determined to study the permeability of BBB modeland expression of TJ Prs.Results 1. Mechanism of MMP2/9 in BBB model 1) The impact of C6 cells in BBB : a) Without added lead treatment C6 conditioned medium, after 10μM lead exposed on BBB, it will not cause any major changes in barrier permeability(p> 0.05), ZO-1 and Occludin change is not significant(p> 0.05). b) After adding lead treatment C6 conditioned medium, after 10μM lead exposed, BBB permeability significantly increased(p <0.05), and the expression of ZO-1 and Occludin significantly decreased(p <0.01), and a dose-dependent manner; 2) The impact of MMP2 / 9 of the BBB after lead exposure Lead cause MMP2 and MMP9 in astrocytes m RNA expression levels increased(p <0.05), the expression increased(p <0.05), and the activity of MMP2 significantly improved(p <0.05), having a certain dose-dependent effects. 3) MMP2 inhibitor added impact BBB barrier function After adding APR100(MMP2 tissue-specific inhibitor), lead exposure increases Zo-1 and Occludin protein expression(p <0.05), the permeability of the barrier has been partially reversed(p <0.05). 2. Mechanism of MMP2/9 in BCB model 1) The impact of lead exposure on the barrier function of BCB Lead increased the leakage of permeability in BCB model(p <0.05), and induced decrease on the protein expression of ZO-1 and Occludin(p <0.01). 2) The impact of MMP2 / 9 of the BCB after lead exposure After 10μM Pb exposure in BCB model, the MMP2 / 9 m RNA levels and protein expression levels were not significant changed(p> 0.05).Conclusions 1. A low dose of lead exposure causes the breakdown of BBB barriers by reducing theexpression of ZO-1 and Occludin. 2. Astrocytes play an important role in the BBB model MMP2 level in astrocytes under lead exposure significantly increased(including protein levels, m RNA levels and activity), and in a dose-dependent manner; MMP-2 inhibitor treatment partially reversed the protein expression of TJPrs after lead treatment, and prevent partly lead induced barrier loss in BBB model. 3. In the BCB model, barrier function and change TJPrs, but no change in the expression of MMPs, means description MMPs no obvious role in the Lead-lead damage in the BBB model. This study can be used as the theoretical basis of the study of childhood lead poisoning, provide some basis for the prevention of childhood lead poisoning.