Lycopene Antagonizes Oxidative Stress Induced Neuronal Damage And Its Underlying Molecular Mechanism

Background:Oxidative stress is the imbalance of ROS (reactive oxygen species) production andscavenging which leading to the tissue and cellular damage. Neurodegenerative diseases areone of the diseases that threaten human health. Nowadays, it is generally accepted that theoccurrence of oxidative stress played an important role in the occurrence and developmentin neurodegenerative diseases. Therefore, antioxidants are considered as potential drugcandidates for neurodegenerative diseases therapy. Lycopene, one of the members ofcarotenoids family, has received greater attention as a result of studies indicating that it is ahighly efficient antioxidant and free radical scavenger. Among naturally occurringcarotenoids, lycopene has the strongest ability to scavenge free radicals; being10-fold,47-fold and100-fold more effective in quenching singlet oxygen than α-tocopherol,β-carotene and vitamin E respectively. Lycopene has high liposolubility and so can passthrough the blood-brain barrier, suggesting it may be a potentially useful treatment for braindiseases associated with oxidative stress.Trimethyltin (TMT) is a short-chain trialkyltin used as a stabilizer of plastics。 It isalso a known neurotoxin, producing significant and selective neurodegeneration in thelimbic system of both human and animals, and is particularly toxic to the hippocampalformation. TMT intoxication in both humans and experimental animals caused profoundbehavioral alterations (hyperactivity and aggression) and cognitive impairments (memoryloss and learning impairment) that were attributed to severe hippocampal damage. Althoughthe molecular mechanisms underlying TMT-induced neurotoxicity are not fully understood,it is widely accepted that oxidative stress initiates TMT-induced apoptotic cell death.Neurodegeneration evoked by TMT shares crucial pathogenic mechanisms with manyneurodegenerative diseases, including oxidative stress and selective neuronal apoptosis, so TMT is regarded as a useful tool to model neuropathology. Based on these resultsmentioned above, we hypothesized that lycopene can inhibit TMT-induced oxidativedamage may offer protection against many neurological diseases. In the first part of thisstudy, we investigated the effects of lycopene on TMT-induced neurotoxicity in primarycultured rat hippocampal neurons, and demonstrated that lycopene protects against TMTtoxicity by suppressing oxidative stress and blocking activation of the mitochondrialapoptosis pathway.Alzheimer's disease (AD) is a common neurodegenerative disorder. Several studieshave demonstrated the toxic properties of Aβ to neurons, suggesting a causative role of thispeptide in the pathogenesis of AD. Thus, identifying a way to reduce the Aβ-inducedneurotoxicity would be beneficial for AD treatment. Therefore, in the second part, toexamine whether lycopene has the neuroprotective effects against Aβ-induced neurotoxicityin cultured rat cortical neurons. In addition, we explored possible mechanisms for theobserved effect.PartⅠ Lycopene Antagonizes Trimethyltin-Induced Neuronal Damage And theRelated Molecular MechanismMethods:1. Primary cultures of hippocampal neurons. Newborn Sprague–Dawley (SD) ratswere used for primary hippocampal neuronal cultures. Cultured neurons were used for invitro studies at day8(DIV8). To evaluate neuronal morphology, cultures were observedusing a Leica DFIL inverted microscope with phase-contrast optics. Cultured neurons weredetermined by immunofluorescence with β-Tublin III. Neurons were exposed to severalconcentration of TMT to determine a working time for subsequent assays. Cell viabilitywas then assessed by CCK-8.2. Hippocampal neurons were used for in vitro studies at day8which divided to fourgroup: Control, Lycopene, TMT, Lycopene+TMT. A Leica DFIL inverted microscopewith phase-contrast optics was used to evaluate the effects of TMT on neuronal morphologyand the influence of lycopene. The effects of lycopene on TMT-induced cell viability lossand apoptosis in hippocampal neurons were determined by Cell Counting Kit-8andTUNEL assay.3. The effects of lycopene on TMT-induced intracellular and mitochondrial reactive oxygen species overproduction were measured by DCFH-DA and Mito-SOX.4. The Colorimetry mPTP Detection Kit which is based on quenching of calceinfluorescence by cobalt The opening of the mPTP and JC-1were used for Measurement ofthe effects on mitochondrial permeability transition pore (mPTP) opening and membranepotential (Ψm).5. Cytochrome c release was determined by immunofluorescence and Caspase-3activity was determined using the Caspase-3Activity Kit.Results:1. Hippocampal neurons dissected from SD rat were cultured for8days which had thenormal neuronal morphological feature. Neurons were immunostained using a β-Tublin IIIantibody indicating that the degree of pollution by non-neuronal cells is low, and neuronsare with high purity. Treatment with5μM TMT for24h was selected for experimentstesting lycopene-mediated neuroprotection.2. The TMT-induced morphological alterations were prevented by pretreatment with1μM lycopene. Pretreatment with lycopene significantly reduced TMT-induced loss of cellviability and this protection was dose-dependent (0.1μM,1.0μM, or10μM) andtime-dependent (12h,24h, or48h). In contrast, lycopene had no significant toxic effectson cultured hippocampal neurons when applied alone. The effect of lycopene onTMT-induced apoptosis in cultured hippocampal neurons was investigated using theTUNEL assay. It is found that lycopene attenuated TMT-induced apoptosis in hippocampalneurons.3. Lycopene suppressed intracellular and mitochondrial ROS production inTMT-treated hippocampal neurons. The intracellular ROS level, as measured by DCFfluorescence, increased significantly as compared with control in TMT-treated hippocampalneurons. Pretreatment with1μM lycopene decreased TMT-induced ROS generation.Pretreatment of hippocampal cultures with TMT significantly increased MitoSOX Redemission, indicating an increase in mitochondrial ROS production, while lycopenepretreatment markedly inhibited TMT-induced mitochondria-derived ROS production.4. Exposure of TMT for24h caused a significant decrease in mitochondrialfluorescence (in normalized relative fluorescence units, NRFU) compared to controlcultures, indicating cobalt quenching of casein in the inner mitochondrial matrix, consistent with mPTP opening. A comparatively higher NRFU was observed in TMT-treated neuronspretreated with1μM lycopene, however, indicating less casein quenching and suggestingthat lycopene markedly reduced mPTP activation. Exposure to TMT for24h evoked asignificantdecrease in Ψm, while lycopene (1μM) pretreatment largely preventedTMT-induced depolarization.5. The release of cytochrome c were measured by simultaneous cytochrome cimmunofluorescence and MitoTracker Red fluorescence. TMT decreased colocalization ofcytochrome c and MitoTracker Red indicating the release of cytochrome c from themitochondrial matrix and into the cytosol. This subcellular shift in cytochrome c wasmarkedly inhibited by1μM lycopene pretreatment. The influence of lycopene onTMT-induced caspase-3activation was examined by a spectrophotometric assay in celllysates from control cultures, TMT-treated cultures, and cultures treated with TMT pluslycopene. Neurons treated with TMT for24h showed a significant increase in caspase-3activity. In contrast, pretreatment with1μM lycopene significantly inhibited theTMT-induced activation of caspase-3.Conclusion:The present study demonstrated that lycopene rescued hippocampal neurons fromapoptotic cell death induced by trimethyltin (TMT), a neurotoxin that partially mimics thepathogenic mechanisms of many neurodegenerative disorders. These findings indicate thatthe antioxidant lycopene protects against TMT-induced neurotoxicity by inhibiting theROS-initiated mitochondrial apoptotic pathway.Part Ⅱ Lycopene Antagonizes Amyloid [beta]-Induced Neuronal damage Andthe Related Molecular MechanismMethods:1. The effects of lycopene on Aβ25-35-induced cell viability loss and apoptosis weredetermined by Cell Counting Kit-8and TUNEL assay.2. The effects of lycopene on Aβ25-35-induced intracellular reactive oxygen speciesoverproduction and mitochondrial membrane potential depolarization were measured byDCFH-DA and Rhodamine123, respectively.3. Western blot analysis was used for determining the expression of Bcl-2, Bax andcaspase-3. Results:1. Pretreatment with lycopene significantly reduced the Aβ25-35-induced loss of cellviability in a dose-dependent manner. Data from TUNEL assays further confirmed thatlycopene attenuated apoptosis in Aβ25-35-treated neurons.2. The intracellular ROS level, as measured by DCF fluorescence, increasedsignificantly as compared with control in Aβ25-35-treated neurons. Pretreatment with2μMlycopene decreased Aβ25-35-induced ROS generation. The influence of Aβ25-35on the innermitochondrial membrane potential Ψm was assayed by Rhodamine123staining.Exposure to Aβ25-35for24h evoked a significant decrease in Ψm, while lycopenepretreatment largely prevented Aβ25-35-induced depolarization.3. The results obtained from Western blotting revealed that incubation with25μMAβ25-35caused a robust increase in Bax levels, and a significant decrease in Bcl-2levels,and there was an approximate two-fold increase in the ratio of Bax/Bcl-2expression.However, lycopene (2μM) pretreatment significantly reversed this trend. Aβ25-35decreasedpro-caspase-3expression and increased the expression of cleaved caspase-3, which indicatethat Aβ25-35can induce caspase-3activation. However, lycopene (2μM) pretreatmentincreased pro-caspase-3expression and decreased the expression of cleaved caspase-3nearly to control level.Conclusion:The current study demonstrated that lycopene exerts protective effects againstAβ25-35-induced neurotoxicity, as evidenced by the improved cell viability and thedecreased apoptosis. The inhibition of mitochondrial dysfunction and oxidative stress maycontribute to the protection against Aβ-induced neurotoxicity.