翻白草水提液对大鼠胰岛细胞脂毒性损伤的保护作用
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摘要
目的探讨翻白草水提液对大鼠胰岛细胞脂毒性损伤的影响。方法棕榈酸诱导Wistar大鼠原代胰岛细胞凋亡模型后,FDA/PI双染和流式法检测细胞凋亡,ELISA法测定GSIS水平,qPCR检测FAS、CPT-1 mRNA表达,Western blot检测AMPK蛋白表达,硫代巴比妥酸法测定细胞内丙二醛(MDA)活性,分光光度计法测定细胞还原型谷胱甘肽(GSH)活性。结果与对照组比较,棕榈酸组GSH活性、CPT-1 mRNA表达、p-AMPK蛋白表达显著降低,MDA含量、FAS mRNA表达显著增加(P<0.05),而翻白草水提液+棕榈酸能明显逆转棕榈酸组上述作用。与翻白草水提液+棕榈酸组比较,BML-275(AMPK选择性抑制剂)组GSH活性、CPT-1 mRNA表达显著降低,MDA活性、FAS mRNA表达显著增加(P<0.05)。结论翻白草水提液可能通过激活AMPK,抑制脂肪酸合成,促进脂肪酸β氧化,从而抑制氧化应激所致胰岛细胞脂性凋亡,最终改善胰岛素分泌功能。
AIM To explore the effects of aqueous extract of Potentillae discoloris Herba on lipotoxic rat islet cells.METHODS After palmitic acid-induced apoptosis of primary islet cells in Wistar rats, FDA/PI double staining and flow cytometry were used to detect cell apoptosis. GSIS level was determined by ELISA. FAS and CPT-1 mRNA expressions were detected by qPCR. Western blot was adopted in the protein expression detection of AMPK. Intracellular malondialdehyde(MDA) activity was detected by thiobarbituric acid method, and the determination of cellular reduced glutathione(GSH) activity was performed by spectrophotometry.RESULTS Compared with control group, palmitic acid group demonstrated significantly decreased GSH activity, CPT-1 mRNA expression and p-AMPK protein expression, and significantly increased MDA content and FAS mRNA expression(P<0.05), while aqueous extract of Potentillae discoloris Herba+palmitic acid group shared obviously reversed aforementioned indices. Compared with the aqueous extract of Potentillae discoloris Herba+palmitic acid group, BML-275(AMPK selective inhibitor) group displayed significantly reduced GSH activity and CPT-1 mRNA expression, and significantly increased MDA activity and FAS mRNA expression(P<0.05).CONCLUSION Aqueous extract of Potentillae discoloris Herba suggests its activity in inhibition of ischemic stress-induced islet cell lipid apoptosis by activating AMPK to inhibit the synthesis of fatty acids and promote the oxidation of fatty acid β, and thus achieves.improvement in insulin secretion.
AIM To explore the effects of aqueous extract of Potentillae discoloris Herba on lipotoxic rat islet cells.METHODS After palmitic acid-induced apoptosis of primary islet cells in Wistar rats, FDA/PI double staining and flow cytometry were used to detect cell apoptosis. GSIS level was determined by ELISA. FAS and CPT-1 mRNA expressions were detected by qPCR. Western blot was adopted in the protein expression detection of AMPK. Intracellular malondialdehyde(MDA) activity was detected by thiobarbituric acid method, and the determination of cellular reduced glutathione(GSH) activity was performed by spectrophotometry.RESULTS Compared with control group, palmitic acid group demonstrated significantly decreased GSH activity, CPT-1 mRNA expression and p-AMPK protein expression, and significantly increased MDA content and FAS mRNA expression(P<0.05), while aqueous extract of Potentillae discoloris Herba+palmitic acid group shared obviously reversed aforementioned indices. Compared with the aqueous extract of Potentillae discoloris Herba+palmitic acid group, BML-275(AMPK selective inhibitor) group displayed significantly reduced GSH activity and CPT-1 mRNA expression, and significantly increased MDA activity and FAS mRNA expression(P<0.05).CONCLUSION Aqueous extract of Potentillae discoloris Herba suggests its activity in inhibition of ischemic stress-induced islet cell lipid apoptosis by activating AMPK to inhibit the synthesis of fatty acids and promote the oxidation of fatty acid β, and thus achieves.improvement in insulin secretion.
引文
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[13] Hardie G D.AMPK:positive and negative regulation,and its role in whole-body energy homeostasis[J].Curr Opin Cell Biol,2015,33:1-7.
[14] Lee J W,Choe S S,Jang H,et al.AMPK activation with glabridin ameliorates adiposity and lipid dysregulation in obesity[J].J Lipid Res,2012,53(7):1277-1286.
[15] 梁斌,袁松,刘艳敏,等.二甲双胍激活AMPK对高糖诱发内皮细胞氧化应激的影响[J].中国组织化学与细胞化学杂志,2015,24(4):302-306.
[16] Wu P,Yan Y,Ma L L,et al.Effects of the Nrf2 modulator salvianolic acid A alone or combined with metformin on diabetes-associated macrovascular and renal injury[J].J Biol Chem,2016,291(42):22288-22301.
[17] 李钺,谢炜星,晋大祥,等.激活AMPK保护氧化应激条件下成骨细胞的作用机制[J].中国骨质疏松杂志,2017,23(3):407-410.
[2] Rhodes C J.Type 2 diabetes-a matter of β-cell life and death?[J].Science,2005,307(5708):380-384.
[3] Maedler K,Spinas G A,Dyntar D,et al.Distinct effects of saturated and monounsaturated fatty acids on β-cell turnover and function[J].Diabetes,2001,50(1):69-76.
[4] Oprescu A I,Bikopoulos G,Naassan A,et al.Free fatty acid-induced reduction in glucose stimulated insulin secretion:evidence for a role of oxidative stress in vitro and in vivo[J].Diabetes,2007,56(12):2927-2937.
[5] 严哲琳,董正平,孙文,等.翻白草水提液对2型糖尿病胰岛素抵抗大鼠糖脂代谢的影响[J].中国实验方剂学杂志,2012,18(7):216-219.
[6] Babu P V,Liu D,Gilbert E R.Recent advances in understanding the anti-diabetic actions of dietary flavonoids[J].J Nutr Biochem,2013,24(11):1777-1789.
[7] Zhang L,Yang J,Chen X Q,et al.Antidiabetic and antioxidant effects of extracts from Potentilla discolor Bunge on diabetic rats induced by high fat diet and streptozotocin[J].J Ethnopharmacol,2010,132(2):518-524.
[8] 韩永明,张业辉,熊迎春,等.中药翻白草对糖尿病大鼠血糖的影响[J].湖北中医学院学报,2002,4(1):3,23-24.
[9] 郭新民,崔荣军,申梅淑,等.翻白草对2型糖尿病大鼠血脂代谢紊乱的影响[J].牡丹江医学院学报,2005,26(2):4-6.
[10] 韩永明,段妍君,袁芳,等.翻白草对糖尿病大鼠胰岛形态结构的影响[J].湖北中医学院学报,2005,7(3):28-29.
[11] 邹志坚,王晓敏,冯劲松,等.翻白草黄酮对糖尿病大鼠抗氧化的作用[J].江西中医学院学报,2007,19(3):64-65.
[12] Fariss W M,Chan B C,Patel M,et al.Role of mitochondria in toxic oxidative stress[J].Mol Interv,2005,5(2):94-111.
[13] Hardie G D.AMPK:positive and negative regulation,and its role in whole-body energy homeostasis[J].Curr Opin Cell Biol,2015,33:1-7.
[14] Lee J W,Choe S S,Jang H,et al.AMPK activation with glabridin ameliorates adiposity and lipid dysregulation in obesity[J].J Lipid Res,2012,53(7):1277-1286.
[15] 梁斌,袁松,刘艳敏,等.二甲双胍激活AMPK对高糖诱发内皮细胞氧化应激的影响[J].中国组织化学与细胞化学杂志,2015,24(4):302-306.
[16] Wu P,Yan Y,Ma L L,et al.Effects of the Nrf2 modulator salvianolic acid A alone or combined with metformin on diabetes-associated macrovascular and renal injury[J].J Biol Chem,2016,291(42):22288-22301.
[17] 李钺,谢炜星,晋大祥,等.激活AMPK保护氧化应激条件下成骨细胞的作用机制[J].中国骨质疏松杂志,2017,23(3):407-410.