Search of the factors leading neuronal necrosis to apoptosis using the retinal ischemic model.

低密度(LD)培养的皮质神经元坏死，高密度(HD)培养的神经元在无血清条件下发生凋亡。用HD培养制备的条件培养液(CM)延缓LD培养中神经元的死亡。CM将细胞死亡模式从坏死转变为凋亡。CM抑制LD培养中细胞ATP水平和[~(3 H)]-2-脱氧葡萄糖(2-DG)摄取的快速下降。磷脂酶C和蛋白激酶C的抑制剂有效地阻断了CM诱导的存活活性、[~(3 H)]-2-DG摄取和ATP水平的升高，以及坏死-凋亡开关。这些结果提示，CM通过磷脂酶C和蛋白激酶C介导的机制，导致细胞死亡，从坏死模式向细胞凋亡模式转变。另一方面，高糖处理也抑制了坏死细胞的死亡和细胞内ATP水平的快速下降，这可能与LD和无血清条件下2-DG摄取减少有关。此外，高糖处理通过增加Bax水平、细胞色素c释放、caspase-3激活和DNA梯带形成来诱导细胞凋亡。这些结果表明，高糖处理通过抑制细胞坏死而引起细胞死亡模式的转换，同时在无血清条件下诱导细胞凋亡。在小鼠视网膜缺血模型上，缺血后注射葡萄糖可明显抑制包括坏死在内的神经元死亡。这些结果提示，葡萄糖诱导的细胞从坏死向凋亡的转变和内源性神经营养因子可能同时减弱了葡萄糖诱导的细胞凋亡。此外，我们还发现，在LD和无血清条件下，海洋因子可以抑制神经元坏死和视网膜缺血损伤。在本研究中，我们从CM中发现了细胞死亡模式开关因子NDI，并从海洋因子库中发现了坏死抑制因子。

Cortical neurons die in necrosis in the low-density (LD) culture, while in apoptosis in the high-density (HD) culture under the serum-free condition without any supplements. The neuronal death in LD culture was delayed by conditioned medium factors (CM) prepared from the HD culture. The CM switched the cell death mode from necrosis to apoptosis. The CM inhibited the rapid decrease in cellular ATP levels and [^3H]-2-deoxy glucose (2-DG) uptake in the LD culture. Inhibitors of phospholipase C and protein kinase C effectively abolished the CM-induced elevation of survival activity, [^3H]-2-DG uptake and ATP levels, and necrosis-apoptosis switch. All these results suggest that CM caused cell death, mode switch from necrosis to apoptosis through phospholipase C and protein kinase C-mediated mechanisms. Moreover, we identified NDI from CM as a key molecule to switch necrosis to apoptosis.On the other hand, high-glucose treatment also inhibited necrotic cell death and rapid decrease in cellular ATP levels, possibly related to decreased 2-DG uptake under the LD and serum-free condition. Moreover, high-glucose treatment induced apoptosis through increase in Bax levels, cytochrome c release, caspase-3 activation and DNA ladder formation. These results suggest that high-glucose treatment causes cell death mode switch by inhibiting necrosis, while inducing apoptosis under serum free condition. In the retinal ischemia model in mice, the post-ischemic injection of glucose markedly inhibited the neuronal death including necrosis. These results suggest that glucose-induced switch from necrosis to apoptosis and endogenous neurotrophic factors may attenuate glucose-induced apoptosis at the same time. Moreover, we found that ocean factors inhibited neuronal necrosis under the LD and serum free condition and retinal ischemic injury.In the present study, we discovered cell death mode switch factor, NDI, from CM and necrosis inhibition factors from ocean factors library.