Elucidation for anti-apoptotic action of tetrahydrobiopterin in osteoblastic cells

我们研究了一氧化氮合酶(NO)的辅因子5，6，7，8-四氢生物蝶呤(BH4)对小鼠成骨细胞系MC3T3-E1细胞NO毒性的影响。1)促炎细胞因子混合物(肿瘤坏死因子-α、白介素1-β和干扰素-γ)和NO生成物SNAP降低细胞存活率，而细胞内转化为BH_4的七叶蝶呤则增加细胞存活率。流式细胞仪分析表明，细胞因子对细胞活力的降低可能是基于细胞凋亡引起的细胞死亡，而不是像坏死那样的溶血性死亡。2)细胞因子处理引起BH4的产生。3)在BH4存在的情况下，细胞因子导致DNA片段量显著减少(P&lt；0.05)。这些结果表明，细胞因子诱导的细胞死亡归因于NO，BH4对其有保护作用。BH4在MC3T3-E1细胞中可能起到对抗和抵消无毒作用的作用。4)伴放线放线杆菌(Aa)参与了局限性青少年牙周炎(LJP)的发病过程，其胞膜样多糖抗原(CPA)是骨吸收的重要介导物。CPA含有一种有效的抗增殖多糖，其活性与小鼠MC3T3-E1和人骨肉瘤细胞的凋亡有关，提示成骨细胞的凋亡与LJP的发病有关。细胞凋亡机制的阐明可能为LJP的治疗提供潜在的信息。

We investigated the effects of 5, 6, 7, 8-tetrahydrobiopterin (BH4), a cofactor for nitric oxide (NO) synthase, on NO toxicity in mouse osteoblastic cell line MC3T3-E1 cells. 1) Proinflammatory cytokine mixture (TNF-α, IL-1β, and IFN-γ) and SNAP, an NO generator, decreased cell viability, but sepiapterin, which was converted intracellularly to BH4, increased it. Flow cytometric analysis showed that the reduction of cell viability by cytokines may be based upon cell death by apoptosis, but not lytic death as in necrosis. 2) Cytokine treatment caused the production of BH4. 3) In the presence of BH4, cytokines resulted in a statistically pronounced reduction (*p<0.05) in the amount of DNA fragmentation. These results suggest that the apoptotic cell death in response to cytokines is ascribed to NO and is protective by BH4. BH4 may act as the counterpart and counterbalance to NO-toxicity in MC3T3-E1 cells. 4) Actinobacillus actinomycetemcomitans (AA) has been implicated in the etiology of localized juvenile periodontitis (LJP) and the capsular-like polysaccharide antigen (CPA) from AA, is a potent mediator of bone resorption. CPA contains a potent antiproliferative polysaccharide whose activity is associated with apoptotic cell death in mouse MC3T3-E1 and human osteosarcoma, suggesting that apoptotic cell death in osteoblastic cells is associated with the pathogenesis of LJP. The elucidation of apoptotic mechanism may provide the therapeutic potential information for LJP.