Lycopene Protects Endoplasmic Reticulum Stress Aganist Mouse Cardiomyocytes Hypoxia/Reoxgenation Injury And Its Underlying Mechanism

Objective：To investigate whether lycopene protects against endoplasmicreticulum stress induced apoptosis in primary cultured neonatal mousecardiomyocytes exposed to hypoxia/reoxgenation, and explore its underlyingmolecular mechanism.Methods：1. Neonatal C57BL/6mice were used for primary cardiomyocytesculture. To achieve a suitable hypoxia/reoxgenation, cardiomyocytes wereincubated with serum-free and glucose-free medium in hypoxia for4hours,and then were returned to a normoxic environment with the normal culturemedium at37℃for different time.2. After cultured for48-72h, cardiomyocytes were randomly divided tofour groups: Control group, Lycopene group, H/R group, Lycopene+H/Rgroup. A Leica DFIL inverted microscope with phase-contrast optics was usedto evaluate the effects of lycopene on cardiomyocytes’ morphology. Theeffects of lycopene on H/R-induced cell viability loss and apoptosis in cellswere respectively assessed by Cell Counting Kit-8, TUNEL andAnnexinⅤ/PI assay.3. The effects of lycopene on H/R-induced intracellular reactive oxygenspecies generation were measured by DCFH-DA with flow cytometry,Microplate Reader and confocal laser scanning microscope.4. The expression of GRP78which is the marker of endoplasmic reticulum stress was determined by Real-time PCR and Western blot incardiomyocytes with H/R or and lycopene pretreatment, the effects oflycopene on H/R-induced ATF6, eIF2α, uXbp-1and sXbp-1mRNAexpression were assessed by Real-time PCR.5. The effects of lycopene on H/R-induced CHOP/GADD153andcaspase-12mRNA expression were assessed by Real-time PCR. The effectsof lycopene on H/R-induced CHOP/GADD153, Bax, Bcl-2, caspase-12andcaspase-3protein expression were determined by Western blot.6.4-phenylbutyric acid (4-PBA) was used to further investigate therelationship of Endoplasmic Reticulum Stress and ROS generation, whichwas to reveal the underlying mechanism of lycopene’s cardioprotection.Cardiomyocytes were randomly divided to four groups: Control group,4-PBA group, H/R group,4-PBA+H/R group. Cell viability and apoptosiswere respectively detected by CCK-8and TUNEL assay. The proteinexpression of GRP78, CHOP/GADD153, Bax, Bcl-2, caspase-12andcaspase-3were determined by Western blot. The ROS production wasmeasured by DCFH-DA with Microplate Reader.Results：1. Cardiomyocytes were cultured for48-72hours which had the normalmorphological feature. The cell viability was significantly decreased afterhypoxia4h(P＜0.01). The cell viability was further decreased afterreoxgenation, especially, reoxgenation for6h.2. The H/R-induced morphological alterations were prevented bypretreatment with5μM lycopene. Pretreatment with lycopene significantlyreduced H/R-induced loss of cell viability(P＜0.01), however, lycopene hadno significant toxic effects on cultured cardiomyocytes when applied alone. The effects of lycopene on H/R-induced apoptosis in cultured cardiomyocyteswere investigated using the TUNEL and Annexin/PI assay. It is found thatlycopene attenuated H/R-induced apoptosis in cardiomyocytes(P＜0.01).3. Lycopene suppressed intracellular ROS overproduction in H/R-treatedcardiomyocytes. The intracellular ROS level, as measured by DCFfluorescence, increased significantly as compared with control in H/R-treatedcardiomyocytes. Pretreatment with5μM lycopene significantly decreasedH/R-induced ROS generation(P＜0.01).4. Pretreatment with lycopene significantly reduced H/R-induced arobust increase in GRP78mRNA and protein levels(P＜0.01). The resultsobtained from Real-time PCR revealed that H/R caused a significant increasein ATF6, eIF2α, uXbp-1and sXbp-1mRNA levels. However, lycopenepretreatment markerly reversed these trends(P＜0.05).5. Pretreatment with lycopene significantly reduced the H/R-induced aincrease in CHOP/GADD153and caspase-12mRNA(P＜0.01), as measuredby Real-time PCR. The results obtained from Western blot revealed that H/Rcaused a significant increase in CHOP/GADD153and Bax levels(P＜0.01),and a significant decrease in Bcl-2levels(P＜0.01), however, lycopenepretreatment markerly reversed these trends(P＜0.01). H/R significantlydecreased pro-caspase-3expression and increased the expression of cleavedcaspase-12and cleaved caspase-3(P＜0.01). However, lycopene pretreatmentincreased pro-caspase-3expression and decreased the expression of cleavedcaspase-12as well as cleaved caspase-3(P＜0.01).6. Pretreatment with4-PBA markerly reduced the H/R-induced loss ofcell viability(P＜0.01). Data from Western blot confirmed that4-PBA attenuated GRP78expression in H/R-treated cardiomyocytes(P＜0.01). Theresults obtained from TUNEL assays indicate that4-PBA suppressedH/R-induced apoptosis(P＜0.01). Datas from Western blot further confirmedthat4-PBA alleviated H/R-induced a increase in CHOP/GADD153and Baxlevels, and a decrease in Bcl-2levels(P＜0.01). Pretreatment with4-PBAsignificantly inhibited the H/R-induced activation of caspase-12andcaspase-3(P＜0.01). Interestingly, there is no significant effect onH/R-induced intracellular ROS generation when4-PBA pretreatmentcardiomyocytes.(P＞0.05)Conclusions: The present study demonstrated that lycopene rescuedcardiomyocytes injury induced by H/R and attenuated endoplasmic reticulumstress induced apoptosis through inhibiting intracellular ROS generation. Inaddition, endoplasmic reticulum is the downstream of ROS in H/R-treatedcardiomyocytes, and4-PBA could significantly inhibit endoplasmic reticulumstress induced apoptosis, while has no effect on ROS generation.