PI3K/AKt/mTOR信号通路参与缺氧诱导因子-1α在急性胰腺炎大鼠的表达调节
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摘要
目的:探讨PI3K/AKt/mTOR信号通路对急性胰腺炎(AP)大鼠HIF-1α表达的调节。
方法: 56只雄性SD大鼠随机分为空白对照组(n=8)、假手术组(n=8)、AP模型组(n=8)、wortmannin预处理组(n=8)、rapamycin预处理组(n=8)、wortmannin+rapamycin预处理组(n=8)、溶剂对照组(n=8)。采用逆行胰胆管注射5%牛磺胆酸钠方法分别制备大鼠AP模型, wortmannin预处理组、rapamycin预处理组、wortmannin+rapamycin预处理组分别于AP模型制备前30分钟腹腔注射wortmannin、rapamycin、wortmannin+rapamycin DMSO/PBS溶液,溶剂对照组仅注射DMSO/PBS溶液。模型复制成功后6小时处死大鼠,留取胰头部组织标本,应用RT-PCR检测HIF-1αmRNA的变化情况;采用Western blot检测HIF-1α、Akt、p-Akt、p70s6k、p-p70s6k蛋白表达的变化情况。
结果:HIF-1αmRNA在空白对照组和假手术组大鼠胰头组织中仅有极少量表达,两组间比较无统计学差异(P>0.05);在AP模型组、Wortmannin预处理组、Rapamycin预处理组、Wortmannin+Rapamycin预处理组、溶剂对照组均较空白对照组明显升高(P<0.01),各预处理组与AP模型组相比差异无统计学意义(P>0.05)。Akt、p70s6k蛋白在胰腺组织中表达稳定,各组间无统计学差异(P>0.05),HIF-1α、p-Akt、p-p70s6k蛋白在空白对照组和假手术组中均有极少量表达,两组间无统计学差异(P>0.05)。AP模型组HIF-1α、p-Akt、p-p70s6k蛋白含量与空白对照组相比明显升高(P<0.01);Wortmannin预处理组、rapamycin预处理组、wortmannin+rapamycin预处理组中HIF-1α、p-p70s6k蛋白表达明显高于空白对照组(P<0.01),但较AP模型组明显降低(P<0.01),其中HIF-1α蛋白表达在wortmannin+rapamycin预处理组比wortmannin预处理组、rapamycin预处理组降低更明显(P<0.05)。HIF-1α、p-Akt、p-p70s6k蛋白含量在溶剂对照组和AP模型组之间无明显差异(P>0.05)。
结论:HIF-1αmRNA和蛋白质在急性胰腺炎大鼠胰头部组织中表达明显增强,PI3K/AKt/mTOR信号通路阻断剂wortmannin、rapamycin能够显著降低急性胰腺炎大鼠HIF-1α的表达,表明PI3K/AKt/mTOR信号通路参与了HIF-1α的表达调控,提示PI3K/AKt/mTOR信号通路可作为急性胰腺炎防治中的新靶点。
Objective : To observe PI3K/AKt/mTOR signaling pathwany regulate the expression of hypoxia inducible factor-1αin rats with acute pancreatitis. Methods: Fifty-six male SD rats were randomly divided into seven groups as blank control group(n=8), sham operation group(n=8), acute pancreatitis(AP) group (n=8), wortmannin pretreatment group (n=8), rapamycin pretreatment group (n=8) , wortmannin+rapamycin pretreatment group(n=8)and the solvent of DMSO/PBS control group(n=8). The rat model with AP was made by injection with 5% sodium deoxycholate through retrogradely bilio pancreatic ducts. For the pretreatment groups of wortmannin pretreatment, rapamiycin pretreatment and wortmannin+rapamycin were given intraperitoneal injection with the reagents of wortmannin, rapamiycin and wortmannin+rapamycin respectively prior to the AP model made, the solvent control group only given the DMSO/PBS solution,and rats in the sham group just only given operation procedure. The head of pancrease in rats were harvested at 6 hours after the model set up, the expression of HIF-1αmRNA were detected by RT-PCR, the expression of HIF-1α, Akt, p-Akt, p70S6K and p-p70S6K proteins were detected by western blot respectively in pancreatic tissues. Results: The expression of HIF-1αmRNA in the head of pancrease in rats were seldom both in the blank control group and the sham operation control group, and there was no significant difference between these two groups(P>0.05). The expression of HIF-lαmRNA were significantly higher in the AP group, wortmannin group, rapamycin group, wortmannin+rapamycin group and solvent control group than that in the blank control group(P<0.01), and there was no significant difference between the pretreatment groups and the AP group(P>0.05). The expression of Akt、p-p70s6k proteins were stable,and there was no significant difference in groups(P>0.05). The expression of HIF-lα, p-Akt, p-p70s6k proteins were seldom both in the blank control group and the sham operation control group, and there was no significant difference between these two groups(P>0.05). The expression of HIF-1α, p-Akt, p-p70s6k proteins were significantly higher in the AP group than that in the blank control group(P<0.01). In the pretreatment groups of wortmannin, rapamycin and wortmannin+ rapamycin,the expression of HIF-1α、p-p70s6k protein were significantly decreased compared to that in the AP group(P<0.01), but they were still significantly higher than that in the blank control group(P<0.01),and there was no difference between the solvent control group and the AP group(P>0.05). Conclusion:The expression of HIF-1αmRNA and protein in head of pancrease in rats with AP were increased significantly, wortmannin and rapamycin, PI3K/Akt/mTOR signaling pathway inhibitors, reduced the expression of HIF-1αmRNA and protein significantly in rats with acute pancreatitis, these results indicated that the PI3K/Akt/mTOR signaling pathway involved the regulation of the expression of HIF-1α,and suggest that PI3K/Akt/mTOR signaling pathway maybe a potential target for prevent and treatment of acute pancreatitis.
方法: 56只雄性SD大鼠随机分为空白对照组(n=8)、假手术组(n=8)、AP模型组(n=8)、wortmannin预处理组(n=8)、rapamycin预处理组(n=8)、wortmannin+rapamycin预处理组(n=8)、溶剂对照组(n=8)。采用逆行胰胆管注射5%牛磺胆酸钠方法分别制备大鼠AP模型, wortmannin预处理组、rapamycin预处理组、wortmannin+rapamycin预处理组分别于AP模型制备前30分钟腹腔注射wortmannin、rapamycin、wortmannin+rapamycin DMSO/PBS溶液,溶剂对照组仅注射DMSO/PBS溶液。模型复制成功后6小时处死大鼠,留取胰头部组织标本,应用RT-PCR检测HIF-1αmRNA的变化情况;采用Western blot检测HIF-1α、Akt、p-Akt、p70s6k、p-p70s6k蛋白表达的变化情况。
结果:HIF-1αmRNA在空白对照组和假手术组大鼠胰头组织中仅有极少量表达,两组间比较无统计学差异(P>0.05);在AP模型组、Wortmannin预处理组、Rapamycin预处理组、Wortmannin+Rapamycin预处理组、溶剂对照组均较空白对照组明显升高(P<0.01),各预处理组与AP模型组相比差异无统计学意义(P>0.05)。Akt、p70s6k蛋白在胰腺组织中表达稳定,各组间无统计学差异(P>0.05),HIF-1α、p-Akt、p-p70s6k蛋白在空白对照组和假手术组中均有极少量表达,两组间无统计学差异(P>0.05)。AP模型组HIF-1α、p-Akt、p-p70s6k蛋白含量与空白对照组相比明显升高(P<0.01);Wortmannin预处理组、rapamycin预处理组、wortmannin+rapamycin预处理组中HIF-1α、p-p70s6k蛋白表达明显高于空白对照组(P<0.01),但较AP模型组明显降低(P<0.01),其中HIF-1α蛋白表达在wortmannin+rapamycin预处理组比wortmannin预处理组、rapamycin预处理组降低更明显(P<0.05)。HIF-1α、p-Akt、p-p70s6k蛋白含量在溶剂对照组和AP模型组之间无明显差异(P>0.05)。
结论:HIF-1αmRNA和蛋白质在急性胰腺炎大鼠胰头部组织中表达明显增强,PI3K/AKt/mTOR信号通路阻断剂wortmannin、rapamycin能够显著降低急性胰腺炎大鼠HIF-1α的表达,表明PI3K/AKt/mTOR信号通路参与了HIF-1α的表达调控,提示PI3K/AKt/mTOR信号通路可作为急性胰腺炎防治中的新靶点。
Objective : To observe PI3K/AKt/mTOR signaling pathwany regulate the expression of hypoxia inducible factor-1αin rats with acute pancreatitis. Methods: Fifty-six male SD rats were randomly divided into seven groups as blank control group(n=8), sham operation group(n=8), acute pancreatitis(AP) group (n=8), wortmannin pretreatment group (n=8), rapamycin pretreatment group (n=8) , wortmannin+rapamycin pretreatment group(n=8)and the solvent of DMSO/PBS control group(n=8). The rat model with AP was made by injection with 5% sodium deoxycholate through retrogradely bilio pancreatic ducts. For the pretreatment groups of wortmannin pretreatment, rapamiycin pretreatment and wortmannin+rapamycin were given intraperitoneal injection with the reagents of wortmannin, rapamiycin and wortmannin+rapamycin respectively prior to the AP model made, the solvent control group only given the DMSO/PBS solution,and rats in the sham group just only given operation procedure. The head of pancrease in rats were harvested at 6 hours after the model set up, the expression of HIF-1αmRNA were detected by RT-PCR, the expression of HIF-1α, Akt, p-Akt, p70S6K and p-p70S6K proteins were detected by western blot respectively in pancreatic tissues. Results: The expression of HIF-1αmRNA in the head of pancrease in rats were seldom both in the blank control group and the sham operation control group, and there was no significant difference between these two groups(P>0.05). The expression of HIF-lαmRNA were significantly higher in the AP group, wortmannin group, rapamycin group, wortmannin+rapamycin group and solvent control group than that in the blank control group(P<0.01), and there was no significant difference between the pretreatment groups and the AP group(P>0.05). The expression of Akt、p-p70s6k proteins were stable,and there was no significant difference in groups(P>0.05). The expression of HIF-lα, p-Akt, p-p70s6k proteins were seldom both in the blank control group and the sham operation control group, and there was no significant difference between these two groups(P>0.05). The expression of HIF-1α, p-Akt, p-p70s6k proteins were significantly higher in the AP group than that in the blank control group(P<0.01). In the pretreatment groups of wortmannin, rapamycin and wortmannin+ rapamycin,the expression of HIF-1α、p-p70s6k protein were significantly decreased compared to that in the AP group(P<0.01), but they were still significantly higher than that in the blank control group(P<0.01),and there was no difference between the solvent control group and the AP group(P>0.05). Conclusion:The expression of HIF-1αmRNA and protein in head of pancrease in rats with AP were increased significantly, wortmannin and rapamycin, PI3K/Akt/mTOR signaling pathway inhibitors, reduced the expression of HIF-1αmRNA and protein significantly in rats with acute pancreatitis, these results indicated that the PI3K/Akt/mTOR signaling pathway involved the regulation of the expression of HIF-1α,and suggest that PI3K/Akt/mTOR signaling pathway maybe a potential target for prevent and treatment of acute pancreatitis.
引文
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[3] Bai xw, Sun B, Pan SH, et al. Down-Regulation of Hypoxia-Inducible Factor-1αby hyperbaric oxygen attenuates the severity of acute pancreatitis in rats[J]. Pancreas, 2009,38(5):515-522.
[4]虎琼华,赵世桥,张献全,等.缺氧诱导因子-1α在重症急性胰腺炎患者中的表达及临床意义[J].中国急救医学,2009,29,(9):769-771.
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[2] Cuthbertson CM, Christophi C. Potential effects of hyperbaric oxygen therapy in acute pancreatitis[J]. ANZ J Surg, 2006,76(7):625-630.
[3] Bai xw, Sun B, Pan SH, et al. Down-Regulation of Hypoxia-Inducible Factor-1αby hyperbaric oxygen attenuates the severity of acute pancreatitis in rats[J]. Pancreas, 2009,38(5):515-522.
[4]虎琼华,赵世桥,张献全,等.缺氧诱导因子-1α在重症急性胰腺炎患者中的表达及临床意义[J].中国急救医学,2009,29,(9):769-771.
[5] Uhlig U, Fehrenbach H, Lachmann RA. et a1. Phosphoinositide 3-OH kinase inhibition prevents ventilation-induced lung cell activation[J]. Am J Respir Crit Care Med,2004,169(2):201-208.
[6] Lupia E, Goffi A, De Giuli P, Ablation of phosphoinositide 3-kinase-gamma reduces the severity of acute pancreatitis. Am J Pathol. 2004,165(6):2003-2011.
[7] Frossard JL, Pastor CM . Experimental acute pancreatitis: new insights into the pathophysiology[J]. Front Biosci,2002,7(1):257-287.
[8] Semenza GL. Hypoxia-Inducible Factor 1 (HIF-1) Pathway[J]. Sci STKE,2007,2007(407):1-3.
[9] Yoon D, Pastore YD, Divoky V,et al. Hypoxia-inducible factor-1 deficiency results in dysregulated erythropoiesis signaling and iron homeostasis in mouse development[J]. J Biol Chem.2006,281(35):25703-25711.
[10] Singh VK, Wu BU, Bollen TL,et al. Early systemic inflammatory response syndrome is associated with severe acute pancreatitis[J]. Clin Gastroenterol Hepatol,2009,7(11):1247-1251.
[11] Lin MT, Kuo IH, Chang CC, et al. Involvement of hypoxia-inducing factor-1alpha-dependent plasminogen activator inhibitor-1 up-regulation inCyr61/CCN1-induced gastric cancer cell invasion[J].J Biol Chem,2008,283(23):15807-15815.
[12] Pene F, Claessens YE, Muller O. Role of the Dhosphatidylinositol 3-kinase/Akt and mTOR/P70s6-kinase pathways in the proliferation and apoptosis in multiple myeloma [J]. Oncogene,2002,2l(43):6587-6597.
[13] Flügel D, G?rlach A,Michiels C,et al. Glycogen synthase Kinase 3 phosphorylates hypoxia inducible factor 1αand mediates its destabilization in a VHL in dependent manner[J]. Mol Cell Biol,2007,27(9):3253-3265.
[1] Semenza GL. Hypoxia-inducible factor 1 (HIF-1) Pathway.Sci STKE,2007, 2007(407): 1-3.
[2] Kasuno K, Takabuchi S, Fukuda K, et al. Nitric oxide induces hypoxia-inducible factor 1 activation that is dependent on MΑPK and phosphatidylinositol 3-kinase signaling[J]. J Biol Chem,2004,279(4):2550-2558.
[3] Li J, Davidson G, Huang Y, et al. Nickel compounds act through, phosphatidylinositol-3-kinase/Akt-dependent,p70S6k-independent pathway toinduce hypoxia inducible factor transactivation and Cap43 expression in mouse epidermal Cl41 cells[J]. Cancer Res,2004,64(1):94-101.
[4] Semenza GL. Hypoxia-inducible factor 1: master regulator of O2 homeostasis[J]. Curr Opin Genet Dev, 1998,8(5):588-594.
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