【作者】 周晓磊；

【导师】 薛承锐；

【作者基本信息】 天津医科大学 ， 中西医结合临床， 2010， 博士

【摘要】 目的：观察慢性胰腺炎大鼠模型糖代谢、肝脏抑制蛋白激酶-β(inhibit kappa B kinase-β, IKK-β)和丝氨酸307磷酸化胰岛素受体底物-1(insulin receptor substrate-1, IRS-1)表达、核因子-κB(nuclear factor-κB, NF-κB)活性和肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α) mRNA水平变化,以及阿司匹林、罗格列酮和柴胡疏肝散对其干预作用。方法：Wistar大鼠随机分为对照组、模型组、阿司匹林组、罗格列酮组、柴胡疏肝组。制备慢性胰腺炎模型6周后,阿司匹林组、罗格列酮组和柴胡疏肝组分别给予阿司匹林50 mg/kg、罗格列酮0.92 mg/kg和柴胡疏肝散2.8 g/kg灌胃,对照组和模型组予等量生理盐水灌胃,持续4周。然后进行葡萄糖耐量、胰岛素耐量实验,检测血清胰岛素水平和胰腺及肝脏病理改变,分离肝细胞进行葡萄糖释放实验。最后处死大鼠,迅速摘取肝脏标本,RT-PCR和Western blot检测肝脏IKK-βmRNA和蛋白表达水平变化,Western blot检测肝脏丝氨酸307磷酸化IRS-1表达水平变化,免疫组织化学法检测肝脏NF-κB活性变化,RT-PCR检测肝脏TNF-αmRNA水平变化。结果：与对照组比较,模型组的糖耐量和胰岛素耐量明显异常,血清胰岛素水平显著降低(p<0.05),胰腺病理显示胰腺导管和腺泡结构破坏、淋巴细胞浸润和纤维组织增生等形态学改变,胰岛素对肝细胞葡萄糖释放的抑制率显著降低(p<0.05),肝脏IKK-βmRNA和蛋白表达水平以及丝氨酸307磷酸化IRS-1蛋白表达水平显著增高(p<0.05),肝脏NF-κB阳性细胞率和TNF-αmRNA水平显著增高(p<0.05)。阿司匹林、罗格列酮和柴胡疏肝散能不同程度地改善上述异常,其中罗格列酮组的糖耐量改善最为明显,柴胡疏肝组可见被破坏的胰腺结构部分修复。阿司匹林组与模型组比较,肝脏IKK-βmRNA和蛋白表达水平无显著性差异(p>0.05),而罗格列酮组和柴胡疏肝组的IKK-βmRNA水平和蛋白表达较模型组显著降低(p<0.05)。此外,三个治疗组的肝脏丝氨酸307磷酸化IRS-1蛋白表达、NF-κB阳性细胞率和TNF-αmRNA水平较模型组均显著降低(p<0.05)。结论：慢性胰腺炎引起糖代谢障碍。胰岛素分泌减少是慢性胰腺炎引起糖代谢障碍的机制之一,肝脏胰岛素抵抗也是其中一个重要环节。慢性胰腺炎可通过上调肝脏IKK-β表达,使IRS-1的丝氨酸307磷酸化增加,并使NF-κB活性和TNF-α基因转录增加,从而影响了胰岛素信号传导,这可能是慢性胰腺炎引发肝脏胰岛素抵抗的分子机制。阿司匹林、罗格列酮和柴胡疏肝散能改善慢性胰腺炎引起的糖代谢障碍,而且作用机制都与改善肝脏胰岛素抵抗有关。阿司匹林可能通过直接抑制肝脏IKK-β的丝氨酸激酶活性,而罗格列酮和柴胡疏肝散通过抑制肝脏IKK-β表达的机制,减少IRS-1的丝氨酸307磷酸化,并降低NF-κB活性和TNF-α基因转录,从而改善慢性胰腺炎肝脏胰岛素抵抗。本研究阐明了慢性胰腺炎引发肝脏胰岛素抵抗的分子机制,并且探讨了不同药物对其干预作用,有助于为临床治疗慢性胰腺炎引起的糖代谢障碍开拓新思路。更多还原

【Abstract】 Objective:To observe the changes of glycometabolism, hepatic IKK-βand IRS-1 phosphorylated on serine 307(Ser307p-IRS-1) expression, hepatic NF-κB activity and TNF-αmRNA level in chronic pancreatitis rats, and the intervention of aspirin, rosiglitazone and chaihushugansan on the changes. Methods:Wistar rats were divided into control group, model group, aspirin-treated group, rosiglitazone-treated group and chaihushugansan-treated group randomly. After 6 weeks was chronic pancreatitis model established, aspirin, rosiglitazone and chaihushugansan was administrated at the dose 50 mg/kg,0.92 mg/kg and 2.8 g/kg in the three medicine-treated groups respectively, and distilled water was administrated in control group and model group for 4 weeks. And then OGTT, ITT, serum insulin levels, pancreatic and hepatic pathological changes and glucose release test of isolated hepatic cells were investigated. Rats were killed and samples of hepatic tissue were dissected out. Hepatic IKK-βmRNA and protein expression levels were determined by RT-PCR and Western blot. Meanwhile, determination of hepatic Ser307p-IRS-1 protein expression levels was performed by Western blot. Hepatic NF-κB activities were determined by immunohistochemical method, and hepatic TNF-αmRNA levels were determined by RT-PCR. Results:Compared with control group, glucose tolerance and insulin tolerance was abnormal, serum insulin levels were significantly decreased(p<0.05), destruction of pancreatic small ducts and acini, lymphocytic infiltration and fibroplasias were observed, the inhibition ratios of hepatocellular glucose release by insulin were significantly lower(p<0.05), hepatic IKK-βmRNA and protein expression levels and Ser307p-IRS-1 protein expression levels were significantly higher (p<0.05), the ratios of hepatic NF-κB-positive cell and TNF-αmRNA levels were significantly higher (p< 0.05) in model group. Aspirin, rosiglitazone and chaihushugansan can improve those abnormalities in various degrees. The best therapeutic effect on glucose tolerance abnormal was observed in rosiglitazone-treated group, and reparation of destructive pancreatic structure was observed in chaihushugansan-treated group. Hepatic IKK-βexpression levels in aspirin-treated group showed no significant difference from model group (p> 0.05). But in both rosiglitazone- treated and chaihushugansan-treated groups, hepatic IKK-βexpression levels were decreased significantly (p<0.05 compared with model group). In addition, hepatic Ser307p-IRS-1 protein expression levels, the ratios of hepatic NF-κB-positive cell and TNF-αmRNA levels were significantly lower in all of the three medicine-treated groups than those in model group (p<0.05). Conclusion: Chronic pancreatitis induces abnormal glycometabolism. Apart from reduced insulin secretion, hepatic insulin resistance is an important mechanism. Chronic pancreatitis increases hepatic serine 307 phosphorylation of IRS-1, NF-κB activity and TNF-αgene transcription through up-regulating IKK-βexpression, thereby blocks the insulin signal transduction pathway, which might be a molecular mechanism on leading to hepatic insulin resistance. Aspirin, rosiglitazone and chaihushugansan can treat the abnormal glycometabolism induced by chronic pancreatitis through improving hepatic insulin resistance. Aspirin may exert the therapeutic effects on hepatic insulin resistance through inhibiting the serine kinase activity of IKK-β, but rosiglitazone and chaihushugansan through down-regulating hepatic IKK-βexpression, resulting in the attenuation of NF-κB activity and TNF-αgene transcription. Our research interprets the molecular mechanism of hepatic insulin resistance in chronic pancreatitis, and investigates the intervention of different medicines, which contributes to open new windows for the clinical treatment of abnormal glycometabolism induced by chronic pancreatitis.更多还原