C-Jun氨基末端激酶信号通路在慢性间歇低氧致胰岛素抵抗中的作用研究

Obstructive sleep apnea-hypopnea syndrome (OSAHS) is independently associated with the development of type 2 diabetes mellitus(T2DM). However, the pathogenesis by which OSAHS induces glucose metabolic disorders is not clear. It was reported that chronic intermittent hypoxia(IH) that mimics OSAHS could cause insulin resistance(IR), one of the two main features involved in the pathogenesis of T2DM. Research on the molecular mechanisms underlying IH-induced IR may have great theoretical and clinical significance. The c-Jun amino-terminal kinase (JNK) signaling pathway was reported to play important roles in the development of IR by interrupting insulin signaling in insulin sensitive organs as well as by regulating the expression of cytokines that can influence insulin sensitivity. However, whether JNK signaling pathway is activated in insulin sensitive organs upon exposure to IH is unknown, as its contribution to the development of IR. In our preliminary study, we found 7 weeks of IH exposure caused systemic IR in mice and the phosphorylation of Akt in the insulin signaling pathway was reduced in mice liver and adipose tissue. Simultaneously, markers of JNK signaling pathway were increased in mice liver and adipose tissue after IH exposure. Based on the research background and our discovery in the preliminary study, we make our hypothesis here that OSAHS and its characteristic IH could cause the activation of JNK signaling pathway in insulin sensitive organs, interfere with insulin signaling and consequently cause IR. In order to verify this hypothesis, we will initially detect changes of insulin signaling and JNK pathway in insulin sensitive organs under IH exposure in vivo and in vitro. And secondly, relevant inhibitors, small interfering RNA and JNK1 knock-out animals will be used to explore critical roles of this pathway in the development of IR under such circumstances. Detection of changes and critical sites of action in this pathway may help further elucidate molecular mechanisms underlying IH-induced IR and lay foundation for the further exploration of potential therapeutic targets for metabolic damages caused by OSAHS.

阻塞性睡眠呼吸暂停低通气综合征（OSAHS）与T2DM独立相关，OSAHS致糖代谢异常机制未明。胰岛素抵抗（IR）是T2DM的重要发病机制，OSAHS特征性的慢性间歇低氧可导致IR，阐释其分子机制具有重要意义。c-Jun氨基末端激酶（JNK）信号通路是介导IR的重要通路。JNK可直接影响胰岛素信号转导，亦可通过调节炎症因子的表达间接引起IR。然而间歇低氧是否通过JNK通路活化导致IR尚不清楚。我们研究发现：间歇低氧可致小鼠全身IR，并伴有肝脏和脂肪组织JNK的活化和胰岛素信号通路关键激酶的活化水平减低。因此推测，JNK信号通路可能在介导间歇低氧致IR中起重要作用。本研究拟从整体水平和细胞水平，研究间歇低氧下JNK信号通路的活化及对胰岛素信号转导的影响；并应用相关抑制剂，RNA干扰及基因敲除技术进行干预和论证，寻找关键治疗靶点，为阐释OSAHS致糖代谢异常的机制及代谢损害的综合防治提供依据。

目的：OSAHS特征性的慢性间歇低氧(CIH)可导致胰岛素抵抗(IR)，机制未明。c-Jun氨基末端激酶(JNK)信号通路是介导IR的重要通路，然而CIH是否通过JNK通路活化导致IR尚不清楚。本研究旨在从整体和细胞水平，研究CIH下JNK信号通路的活化及对胰岛素信号转导的影响，为阐释OSAHS致糖代谢异常的机制提供依据。.方法：1）选取雄性C57BL/6J小鼠，分为CIH组和对照组各12只。CIH组每日8h置入间歇低氧箱(低氧事件40次/h，最低氧浓度5%)，对照组置入相同装置(维持氧浓度21%)，共49天。造模结束后采用经腹腔葡萄糖耐量试验(IPGTT)评估小鼠的糖耐量，Westernblot法检测肝脏、脂肪组织JNK信号通路活化情况及胰岛素信号通路的分子表达。2）选取HepG2细胞系和C57BL/6J小鼠肝原代细胞，分为间歇低氧(IH)和常氧组。IH组分别给予10、30、120、240、360循环的IH刺激(1%O2 1min／21% O2 1min)。常氧组细胞常氧培养48h。3）将HepG2细胞分为IH、IH＋SP600125、常氧、常氧＋SP600125组，IH刺激或常氧培养共360个循环。刺激结束前30min更换含胰岛素100nM培养基，30min后裂解细胞，提取总蛋白，Westernblot法检测JNK信号通路活化情况及胰岛素信号通路的分子表达。.结果：1）CIH刺激后小鼠IPGTT试验中多时点的血糖值高于对照组，在0min和60min时点差异有统计学意义(P<0.05)；CIH组小鼠肝脏和脂肪组织中p-JNK、p-c-Jun的蛋白表达显著高于对照组，并伴有胰岛素信号通路中p-Akt的蛋白水平显著低于对照组。2）HepG2细胞经IH刺激360循环后，p-JNK及p-c-Jun蛋白表达较常氧组显著升高；IH刺激后HepG2细胞和小鼠肝原代细胞胰岛素刺激下的p-Akt蛋白和p-GSK3β蛋白表达较常氧组减低，并存在时间依赖性。3）IH刺激同时加用JNK抑制剂SP600125后，IH所致的p-Akt及p-GSK3β蛋白表达受抑得到了一定程度的改善。.结论：CIH刺激使小鼠肝脏和脂肪组织JNK信号通路激活，胰岛素信号转导受抑。 IH刺激使肝细胞JNK信号通路活化，胰岛素信号转导受抑，抑制JNK信号通路可部分改善IH所致的肝细胞胰岛素抵抗。