The Protective Effect And Mechanism Of EGCG On The Lead-induced Impairments Of Spine Formation In Rat Hippocampus

The function of normal brain depends on the balance of excitatory and inhibitory neurotransmission. Lead is both an environmental pollutant and neurotoxin. Lead exposure mainly impairs the central nervous system and its function. In recent years, it has been reported that lead is an antagonist of NMDAR (N-methyl-D-aspartate), but the effect of lead on inhibitory neurotransmitter and receptors remains unknown.Epigallo-catechin-3-gallate (EGCG) is an important polyphenols in green tea and has obvious free radical scavenging effect. Our laboratory previous studies have shown that EGCG can significantly restore the oxidative stress parameters. Also EGCG can partly reverse the impairment of learning and memory function in lead-exposed rats. But whether EGCG can regulate the synapse formation (the basic infrastructure) and through the Wnt7a/β-catenin signaling to achieve its protective effect of lead poisoning is unclear.The aim of this study is to investigate the effects of lead exposure on the balance of excitatory and inhibitory synapses in cultured hippocampal neurons. Furthermore, the effects and mechanisms of EGCG on the impairment of synapse formation in developmental lead-exposed rats were also explored.Primary hippocampal cultures were made from brains of postnatal SD rats (PO). Cultured neurons were treated with 1 μM Pb2+(lead acetate) for 5 days) and 50 μM EGCG for 48 hours, then the neurons were fixed to observe the dendritic spines under the confocal microscope. Electrophysiologies, Immunocytochemistry, Western blot were used to assay the expression of some proteins after lead exposure. Sprague-Dawley (SD) female rats were feed on 300 ppm lead acetate solution, on parturition day, the pups acquired lead through breast milk from parturition to weaning. From post natal day (PND) 14-21, the off springs were injected intraperitoneally (ip) daily with EGCG (10、25 and 50 mg/kg). Then the pups were sacrificed for Golgi-cox staining; the lead concentration and Wnt7a, β-catenin expression were examined. The results are as follows:1. In cultured neurons, lead exposure (1 μM) significantly reduces the spine density of neurons. Patch clamp recordings showed that lead exposure significantly increase the frequency of mIPSC; while the amplitude of mIPSC, the frequency and amplitude of mEPSCs were not significantly changed. Immunocytochemistry experiments showed that the inhibitory neuro-transmitter transporter vGAT was significantly increased; while Gephyrin and the excitatory neurotransmitter transporter vGlut and PSD-95 have not been altered after lead exposure.2. We further examined the synthesis of GABA and the expression of GABAA receptors. These results showed that lead exposure can up-regulate the expression of GAD65 and GABAAβ2/3.3. In chronic lead-exposed rats, lead exposure significantly decreased dendritic spine density and spine head size of pyramidal neurons in hippocampal neurons, and EGCG (10 and 25 mg/kg) reversed the lead-induced spine damage. In addition, EGCG (10 and 25 mg/kg) recovered the expression of Wnt7a and P-catenin phosphorylation after lead-exposure. However,50 mg/kg of EGCG failed to restore the spine morphology and Wnt/β-catenin pathway activity on lead-exposed rats. Furthermore, in cultured hippocampal neurons, EGCG did not exert any protective effect on lead-induced damages when Wnt7a shRNA applied.In summary, lead exposure could increase the expression and maturation of GAD65 protein and the GABAA receptors, and thus damage the balance of hippocampal neurons suppression. EGCG has a protective effect on dendritic spines formation of lead exposed rats by up-regulating the activity of Wnt/β-catenin signaling pathway.