汪秋艳，孙晓辉

白藜芦醇对间歇低氧大鼠胰岛素抵抗的影响

汪秋艳，孙晓辉*

目的 观察白藜芦醇对间歇低氧大鼠胰岛素抵抗的影响,探讨其可能的作用机制。方法 8周龄SD雄性大鼠40只，随机分为5组，对照组(A组)、间歇低氧组(B组)、白藜芦醇低剂量组[C组,6 mg/(kg·d)]、白藜芦醇中剂量组[D组,30 mg/(kg·d)]、白藜芦醇高剂量组[E组,60 mg/(kg·d)]。C组、D组、E组大鼠间歇低氧4周，同时按不同剂量白藜芦醇灌胃。实验结束后，测大鼠空腹胰岛素和血糖水平，取肝脏，采用RT-PCR和Western blot法测定肝脏SIRT1。结果 白藜芦醇干预后间歇低氧大鼠的胰岛素抵抗降低，呈剂量依赖性，伴随SIRT1的表达增高，各组间差异有统计学意义(P<0.01)。结论 白藜芦醇能减轻间歇低氧大鼠的胰岛素抵抗，呈剂量依赖性，其可能是通过提高SIRT1的表达起作用。

白藜芦醇；间歇低氧；胰岛素抵抗

0 引言

阻塞性睡眠呼吸暂停综合征是目前常见的一种睡眠呼吸紊乱性疾病，在人群中的发病率高达2%～4%，在肥胖人群中达到25%～35%[1-2]，老年人患病率更高。研究表明,OSA(Obstructive sleep apnea)是胰岛素抵抗的独立危险因素[3-4]，鼠类模型提示，间歇性低氧(Intermittent hypoxia，IH)是一个重要因素。IH可以导致健康瘦鼠急性胰岛素抵抗[5]。白藜芦醇(Resveratrol，RSV)是沉默信息调节因子1(Silent information regulator 1，SIRT1)的强有力的激动剂[6-8]，研究发现，其具有改善胰岛素敏感性的作用[9-10]，可以减轻缺氧组织的胰岛素抵抗[11-12]，但机制尚未明确。

本研究旨在应用不同剂量的白藜芦醇对间歇低氧胰岛素抵抗大鼠进行干预，探讨白藜芦醇减轻间歇低氧大鼠胰岛素抵抗的可能机制。

1 材料和方法

1.1 实验动物和分组处理

1.1.1 实验动物 8周龄SD雄性大鼠，适应性喂养1周后开始实验。常规饲料喂养，温度(22±2)℃，湿度50%左右，明/暗周期12 h，自由进食和饮水。

1.1.2 动物分组及处理 40只大鼠随机分为5组，对照组(A组)、间歇低氧组(B组)、白藜芦醇低剂量组(C组)、白藜芦醇中剂量组(D组)、白藜芦醇高剂量组(E组)。A组为对照组，B组间歇低氧4周，4周后大鼠禁食12 h，测空腹胰岛素和血糖水平，计算稳态模型胰岛素抵抗指数(HOMA-IR),确定大鼠产生明确的胰岛素抵抗，建模成功。按建模方式处理C组、D组、E组大鼠，同时分别按6、30、60 mg/(kg·d)白藜芦醇灌胃4周。

1.2 实验方法

1.2.1 间歇低氧方式 大鼠每日间歇低氧8 h(09∶00～17∶00)，将其置入自制间歇低氧设备，入气口经气体交换控制系统控制，分别给予8%氧气(氮气平衡)和室内空气(21%氧气)，每间隔90 s给低氧1次。

1.2.2 稳态胰岛素评估模型胰岛素抵抗指数 HOMA-IR指数=空腹胰岛素浓度(μU/mL)×空腹血糖浓度(mmol/L)/22.5。大鼠胰岛素测定应用大鼠胰岛素检测试剂盒(millipore公司)。

1.2.3 RT-PCR方法测定肝脏SIRT1 SIRT1上游引物：ACCCTCAATTTCTGTTCTGC；下游引物：TTGGACATTACCACGTCTGC。Western blot方法测SIRT1。

2 结果

2.1 间歇低氧4周，大鼠产生明显的胰岛素抵抗,SIRT1的转录和表达明显下降。白藜芦醇干预后，大鼠胰岛素抵抗减轻，呈剂量依赖性。C组、D组、E组胰岛素抵抗指数与B组之间差异均有统计学意义(P<0.01)。见表1。

表1 各组HOMA-IR与SIRT1比较

注：**与A组比较，P<0.01；##与B组比较，P<0.01

2.2 SIRT1在间歇低氧后转录减少，白藜芦醇干预后，表达增加，呈剂量依赖性(P<0.01)。见图1～图3。

图1 各组SIRT1基因值

图2 各组SIRT1蛋白值

图3 各组SIRT1的免疫组化图(200×)

3 讨论

OSA是一个复杂的病理生理过程，其特点是反复上气道阻塞，频繁打鼾，睡眠时呼吸暂停，慢性夜间睡眠缺失，白天嗜睡[13]。OSA可以导致睡眠时慢性间歇低氧(CIH)。OSA与胰岛素抵抗(IR)、糖耐量异常和其他代谢综合征相关[14-18]。研究表明，IH可以通过交感兴奋、系统性炎症反应、胰腺损害、调节相关激素和脂肪酸分泌来影响糖代谢，导致糖代谢紊乱。而慢性间歇性低氧可以加剧胰岛素抵抗和糖耐量异常。本实验应用IH研究其与大鼠胰岛素抵抗的关系，应用不同剂量白藜芦醇干预间歇低氧大鼠，探讨白藜芦醇减轻大鼠胰岛素抵抗的机制。

白藜芦醇是一种天然多酚类化合物，具有明显的改善胰岛素敏感性、降低血糖的作用[19]。Chi等[20]使用白藜芦醇分别干预1型糖尿病、2型糖尿病和胰岛素抵抗小鼠，发现白藜芦醇可通过胰岛素依赖和非胰岛素依赖的机制改善小鼠的胰岛素抵抗,但白藜芦醇对IH情况下的IR的研究报道很少[12]。

SIRT1是一种依赖于烟酰胺腺嘌呤二核苷酸的组蛋白去乙酰化酶，可使多种蛋白质的赖氨酸残基发生去乙酰化[21]。SIRT1也参与调节葡萄糖内环境稳定和胰岛素分泌。研究表明，在胰岛素抵抗细胞和组织中SIRT1的表达下调，下调或者抑制SIRT1可以导致胰岛素抵抗[22]。

本研究发现，间歇低氧4周后，伴随SIRT1表达的下降，大鼠出现了显著的胰岛素抵抗。白藜芦醇干预后，各组的胰岛素抵抗指数均出现显著下降，说明白藜芦醇可以减轻间歇低氧情况下的大鼠胰岛素抵抗。随着白藜芦醇剂量的加大，减轻胰岛素抵抗的效果就更明显，推测其作用的机制为通过提高SIRT1的表达而起效，为白藜芦醇应用于临床提供了理论依据。

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Effects of different doses of resveratrol on insulin resistance in rats with intermittent hypoxia

WANG Qiu-yan,SUN Xiao-hui*

(The First Affiliated Hospital of Dalian Medical University,Dalian 116011,China)

Objective To investigate the effects of resveratrol on insulin resistance in rats with intermittent hypoxia and to explore its underlying mechanisms.Methods Forty 8-week-old SD rats were randomly divided into five groups,control group (group A),intermittent hypoxia group (group B),low dose resveratrol group [group C,6 mg/(kg·d)],median dose resveratrol group [group D,30 mg/(kg·d)],high dose resveratrol group [group E,60 mg/(kg·d)].Rats in group C,group D and group E were treated with different doses of resveratrol by gavage and intermittent hypoxia for 4 weeks.In the end,the fasting insulin and glucose levels of the rats were measured.The expression of SIRT1 in liver was detected by RT-PCR and Western blot.Results The insulin resistance of rats with chronic intermittent hypoxia was decreased,which was dose-dependent,and the expression of SIRT1 was increased,the difference being statistically significant(P<0.01).Conclusion Resveratrol can reduce the insulin resistance in rats with intermittent hypoxia,which is dose-dependent;the mechanism may be by increasing the expression of SIRT1.

Resveratrol;Intermittent hypoxia;Insulin resistance

2016-04-28

大连医科大学附属第一医院，大连 116011

*通信作者

10.14053/j.cnki.ppcr.201702007