| Summary: | 碩士 === 元培科學技術學院 === 生物技術研究所 === 93 === ABSTRACT
The intracellular free calcium played a vital role in regulating the cellular physiological and biochemical functions. Endoplasmic Reticular (ER) Ca2+-Store is a major intracellular store to maintain the balance of calcium concentration. In this study, we used thapsigargin (TG), an ER-Ca2+ ATPase irreversible inhibitor, to block ER-Ca2+ ATPase for investigating its effects on inducible nitric oxide synthase (iNOS) expression, nitric oxide (NO) production, protein kinase C (PKC) isoforms and nuclear factor-κB (NF-κB) activation under high glucose condition. When RAW 264.7 cells were treated with 5 g/ml lipopolysaccharide (LPS), increased iNOS expression, NO production and cell apoptosis induction under high glucose condition. In addition, cells were pretreated with TG for 1 hrs and then wash out applied with LPS, the iNOS expression, NO production and apoptosis significantly decreased. On the other hand, PKC-α, βⅡ and ε and NF-κB activation were also inhibited in the same treatment. However, usage of Ca2+ ionophores: A23187, ionomycin and a reversible ER Ca2+-ATPase: DBHQ could not mimic TG to inhibit iNOS expression, NO production, apoptosis, PKC and NF-κB activation. DCF-DA dye were used to detect the production of H2O2. The results showed that, the pretreatment with TG could not reduce LPS-induced H2O2 production. The results showed that ER Ca2+-Store regulated LPS-activated iNOS expression and apoptosis through PKC and NF-κB activation under high glucose condition. Therefore, ER-Ca2+ store may play a vital role not only in regulating the intracellular Ca2+ level but also in modulating signal transduction process.
Base on above analytic data indicated that RAW 264.7 cell treated with LPS could increase more NO production under high glucose condition and we explored the mechanisms. When RAW 264.7 macrophages was shifted from normal glucose medium (5.5 mM) to high glucose medium (25 mM) and immediately treated with LPS, iNOS expression and NO production were significantly increased; the cell viability also decreased after LPS exposure for 48h. Cells pretreated with oxidized adenosine triphosphate (o-ATP), a selective P2X7 receptor antagonist, that LPS-induced iNOS expression, NO production and cell death strongly decreased in high glucose medium. Besides, usage of apyrase, an ATP-hydrolyzing enzyme, also attenuated the high glucose-enhanced effects. LPS-induced the release of endogenous ATP was enhanced measuring by the luciferin-luciferase assay under high glucose condition. However, the production of TNF-α and IL-1β induced by LPS were not significant difference between high and normal glucose conditions. Our results showed that the purinergic receptors may play an important role in enhancing the NO production induced by LPS under high glucose condition.
Keywords: Endoplasmic Reticular (ER) Ca2+-Store, thapsigargin, inducible nitric oxide synthase (iNOS), protein kinase C (PKC) isoforms, nuclear factor-κB (NF-κB), purinergic receptors.
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