title: Endotoxin induces luteal cell apoptosis through the mitochondrial pathway.
abstract: The effect of endotoxin (lipopolysacharide, LPS) exposure on luteal cells was studied using an in vitro cell culture system. Buffalo luteal cells were isolated from corpora lutea of the late luteal phase (days 14-16 post estrus) and exposed to various LPS doses (5, 10 and 100 microg/ml) for different time periods (6, 12, 18 or 24 h). The cultured cells were subsequently evaluated for oxidative stress (super oxide, nitric oxide, inducible nitric oxide synthase activity, reduced glutathione depletion and lipid peroxidation) and apoptotic markers (mitochondrial membrane potential, DNA fragmentation, apoptotic cells and cell viability). LPS exposure significantly increased the production of super oxide (P<0.05) and nitric oxide (P<0.01) and increased inducible nitric oxide synthase activity (P<0.01). LPS exposure further depleted reduced glutathione (P<0.05) levels and induced lipid peroxidation (P<0.05). LPS exposure also induced the loss of mitochondrial membrane potential (P<0.05), increased DNA fragmentation (P<0.01) and apoptosis (P<0.01) and decreased cell viability (P<0.01). LPS mediated apoptotic pathway in luteal cells was further characterized using a selected LPS dose (10 microg/ml). It was observed that LPS exposure induced mitochondrial translocation of proapoptotic protein Bax, increased the total Bad expression and down regulated the expression of antiapoptotic proteins Bcl2 and BclXL. LPS exposure further induced cytochrome c release and increased Caspase-9 (P<0.01) and Caspase-3 (P<0.01) activities. LPS exposure also inhibited luteal progesterone secretion (P<0.01). It was evident that the LPS mediated apoptotic effects could be prevented by the coincubation of luteal cells with mitochondrial permeability transition pore blocker Cyclosporine A, inducible nitric oxide synthase inhibitor N-[3-(aminomethyl)benzyl]acetamidine and oxidative stress scavenger N-acetyl cysteine. Our study clearly indicates that LPS induces oxidative stress mediated apoptosis in luteal cells through the mitochondrial pathway.
authors.affiliation: National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.
authors.forename: D P
authors.initials: DP
authors.lastname: Mishra
authors.forename: A
authors.initials: A
authors.lastname: Dhali
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D002417:Cattle; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D016572:Cyclosporine / Q000494:pharmacology; D053938:DNA Fragmentation / Q000187:drug effects; D004731:Endotoxins / Q000494:pharmacology*; D005260:Female; D005978:Glutathione / Q000378:metabolism; D015227:Lipid Peroxidation / Q000187:drug effects; D008070:Lipopolysaccharides / Q000494:pharmacology; D008184:Luteal Cells / Q000166:cytology* / Q000187:drug effects*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects*; D009569:Nitric Oxide / Q000096:biosynthesis; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D011374:Progesterone / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D017392:Thiobarbituric Acid Reactive Substances / Q000378:metabolism; D013997:Time Factors
chemical_list: D051017:Apoptosis Regulatory Proteins; D004731:Endotoxins; D008070:Lipopolysaccharides; D017392:Thiobarbituric Acid Reactive Substances; D013481:Superoxides; D009569:Nitric Oxide; D011374:Progesterone; D016572:Cyclosporine; D052247:Nitric Oxide Synthase Type II; D053148:Caspase 3; D005978:Glutathione