Mishra D P, Dhali A
National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.
Prostaglandins Other Lipid Mediat. 2007 Feb;83(1-2):75-88. doi: 10.1016/j.prostaglandins.2006.10.002. Epub 2006 Nov 22.
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.
采用体外细胞培养系统研究了内毒素（脂多糖，LPS）暴露对黄体细胞的影响。从黄体后期（发情后14 - 16天）的黄体中分离出水牛黄体细胞，并将其暴露于不同剂量的LPS（5、10和100微克/毫升）中不同时间段（6、12、18或24小时）。随后对培养的细胞进行氧化应激（超氧化物、一氧化氮、诱导型一氧化氮合酶活性、还原型谷胱甘肽消耗和脂质过氧化）和凋亡标志物（线粒体膜电位、DNA片段化、凋亡细胞和细胞活力）的评估。LPS暴露显著增加了超氧化物的产生（P<0.05）和一氧化氮的产生（P<0.01），并增加了诱导型一氧化氮合酶活性（P<0.01）。LPS暴露进一步消耗了还原型谷胱甘肽水平（P<0.05）并诱导了脂质过氧化（P<0.05）。LPS暴露还诱导了线粒体膜电位的丧失（P<0.05），增加了DNA片段化（P<0.01）和凋亡（P<0.01），并降低了细胞活力（P<0.01）。使用选定的LPS剂量（10微克/毫升）进一步表征了LPS介导的黄体细胞凋亡途径。观察到LPS暴露诱导促凋亡蛋白Bax的线粒体易位，增加了总Bad表达，并下调了抗凋亡蛋白Bcl2和BclXL的表达。LPS暴露进一步诱导了细胞色素c的释放，并增加了Caspase-9（P<0.01）和Caspase-3（P<0.01）的活性。LPS暴露还抑制了黄体孕酮的分泌（P<0.01）。很明显，通过将黄体细胞与线粒体通透性转换孔阻滞剂环孢素A、诱导型一氧化氮合酶抑制剂N-[3-（氨基甲基）苄基]乙脒和氧化应激清除剂N-乙酰半胱氨酸共同孵育，可以预防LPS介导的凋亡作用。我们的研究清楚地表明，LPS通过线粒体途径诱导黄体细胞中氧化应激介导的凋亡。
