Study On The Molecular Toxicological Mechanisms Of Domoic Acid And The Neuroprotective Mechanisms Of Purple Sweet Potato Color

The excitatory neurotoxin domoic acid (DA) was traced to a bloom of thepennate diatom genus Pseudo-nitzschia, which persists in the marine food chain. It isresponsible for a human illness known as amnesic shellfish poisoning (ASP). Inorder to reduce the damage of DA to the marine ecosystem and the impact on humanhealth, this paper aimed to establish mouse model exposed to chronic toxicity of DAand study the biological toxicity and excitotoxic mechanism of DA and theprotective mechanisms of purple sweet potato color (PSPC) against the excitotoxiceffect of DA in the mouse hippocampus via its prophylactic administration. This mayprovide an important foundation to develop a promising antidote for DA. The chiefachievements are as follows:1. The study on the behavioral toxicity of algae toxin DA in mice and theintervention study of PSPCThe results showed that DA significantly impaired the learning and memoryability of mice by step-through passive avoidance task and Morris water maze task.Immunofluorescence analysis showed that the OD value of NeuN immunoreactivityin the hippocampus of DA-treated mice significantly reduced, indicating the neuronloss induced by DA treatment. Western blot analysis showed that the expression ofmemory-related proteins such as NR1, NR2A and PSD95, was significantly reduced,which is the molecular basis of DA-induced cognitive deficits. The study of PSPCshowed that it could significantly increase the expression of these memory-relatedproteins and block DA-induced memory loss; therefore the learning and memoryability of DA mice was improved.2. Mechanism study on the mitochondrial damage induced by neurotoxicity inDA-treated mice and the protective effects of PSPCBy oxygen consumption assays and the measurement of ATP content, we foundthat DA resulted in a decline in mitochondrial respiratory chain function and anincrease in ATP consumption. Western blot showed that DA significantly reduced theexpression of PGC-1α, inhibited the mitochondrial biogenesis pathway. Therefore,the expression of the complexe I-V of the electron transport chain (ETC) wassignificantly reduced, indicating the mitochondrial dyfunction. Accompaied by theincreased expression of ERα, PSPC activated the mitochondrial biogenesis signaling,increased the expression of the complexeI-V and ultimately maintained the mitochondrial function. The ERα siRNA mouse model was constructed and theexpression of ERα was knocked down in the hippocampus of ERα siRNA mice usingsiRNA. The results suggested that ERα-induced mitochondrial biogenesis pathwayplays an important role for the neuroprotective effect of PSPC against DA.3. Molecular mechanism study on the oxidative stress induced by neurotoxicityin DA-treated mice and the protective effects of PSPCDomoic acid could induced the onset of oxidative stress by the increased levelof ROS and protein cabonyl. Mitochondria are the source of free radicals. In additionto the overproduction of ROS induced by mitochondrial dyfunction, DA couldmarkedly enhance the expression of nicotinamide adenine dinucleotide phosphate(NADPH) oxidases, such as p47phoxand gp91phoxand exacerbate oxidative damage.PSPC could inhibit DA-induced oxidative stress, decrease the level of ROS andprotein carbonyl, thereby showing its neuroprotective effect. Furthermore, PSPCcould reduce the oxidative stress in the hippocampus of DA mice by the inhibition ofthe expression of p47phoxand gp91phox.4. Mechanism study on the endoplasmic reticulum (ER) damage induced byneurotoxicity in DA-treated mice and the protective effects of PSPCWestern blot and immunoprecipatation assays showed that DA significantlyenhanced the expression of the ER stress marker glucose-regulated protein(Grp)78,as well as the activation of PERK pathway and IRE1pathway, ultimately resulting inthe onset of ER stress. Interestingly, PSPC could block the activation of these twosignaling pathways and improve mitochondrial function to reduce the generation ofROS to some extent by ERα-mediated signaling pathway, which led to the inhibitionof the onset of ER stress.5. Mechanism study on the apoptosis induced by neurotoxicity in DA-treatedmice and the protective effects of PSPCThe analysis of western blotting and TUNEL staining showed that DA couldinduce apoptosis in mouse hippocampus. Our further experiments investigated therelationship of ER stress and the mechanism of DA-induced apoptosis by the gaugeof ER stress inhibitor PBA. DA activated three ER apoptosis signaling which wasmediated by caspase-12, CHOP and JNK, and induced the onset of apoptosis. PSPCconteracted domoic acid-induced apoptosis. ERα knockdown could inhibited theanti-apoptosis effect of PSPC. It can be inferred from our present results that PSPCprevented DA-induced neuronal apoptosis by the activation of ERα-induced mitochondrial biogenesis pathway and the inhibition of ER stress.Above all, this thesis deeply and comprehensively investigated the mechanismsof domoic acid, which is produced by the toxic red-tide algae Pseudo-nitzschia. Itschronic neurotoxicity does harm to the health of human and marine animals. PSPCadministration alleviated the neurotoxicity of DA. This research may provide a newmethod for the prevention and control of domoic acid, as well as a theoretical basisfor its harmfulness on the marine ecological environment.