【作者】 任文辉；

【导师】 郑天珍； 李伟； 张小郁；

【作者基本信息】 兰州大学 ， 生理学， 2010， 硕士

【摘要】 近年来研究资料显示,肥胖与胰岛素抵抗之间呈高度的相关性,并且是肥胖者发生2型糖尿病、高血压及脂质代谢紊乱等的重要原因。芍药苷(Paeoniflorin)是芍药的主要有效成分之一,现代医学认为芍药苷具有止痛、镇静、解痉、抗炎作用以及抗自由基损伤作用。我室前期所做的实验工作发现：白芍水提取液可以抑制去卵巢大鼠肥胖,改善脂代谢紊乱、清除体内过多自由基；离体实验证明芍药苷促进离体脂肪组织释放游离脂肪酸及降低丙二醛的含量,提高总抗氧化能力水平。而芍药苷是否对胰岛细胞有直接作用,国内外尚未见报道,所以本实验选择不同浓度的芍药苷观察其对胰岛细胞胰岛素释放的影响；同时用链脲佐菌素建立胰岛细胞损伤模型,通过观察芍药苷对损伤胰岛细胞的保护和修复作用,以研究芍药苷的作用机制,并初步探讨胰岛素释放的可能机制。选用大鼠胰岛素瘤细胞INS-1,细胞传代培养后,以1×105个/ml接种于96孔板,培养24h后,分别加入各浓度药物,作用12h。取细胞培养液测定细胞增殖活力和胰岛素、丙二醛、总抗氧化能力的水平。实验分为九组：①正常对照组；②芍药苷组(10-7mol/L、10-6mol/L、10-5mol/L);③STZ组(损伤组)；④芍药苷(10-7mol/L、10-6mol/L、10-5mol/L)+STZ组(芍药苷保护组)；⑤STZ+芍药苷(10-7mol/L、10-6mol/L、10-5mol/L)组(芍药苷修复组)；⑥阿托品组(10-6mol/L);⑦阿托品(10-6mol/L)+芍药苷(10-7mol/L)组；⑧异搏定组(10-7mol/L);⑨异搏定(10-7mol/L)+芍药苷(10-7mol/L)组。实验数据用均数±标准差(χ±S)表示,组间显著性差异采用单因素方差分析法,P<0.05被认为有显著性差异。实验结果如下：1.INS-1细胞对葡萄糖的刺激反应与正常对照组相比,随着葡萄糖浓度的增加(5.6-16.8 mmol/L范围内),INS-1细胞分泌的胰岛素量也逐渐增加；但22.4mmol/L、33.6 mmol/L的葡萄糖则使胰岛素的分泌量随葡萄糖浓度的增加有所减少。2.芍药苷对INS-1细胞增殖活力的影响与正常对照组相比,各浓度芍药苷对INS-1细胞的增殖活力无明显影响。3.芍药苷对INS-1细胞胰岛素释放的影响与正常对照组相比,10-7mol/L, 10-6mol/L的芍药苷可分别不同程度的促进INS-1细胞释放胰岛素,而10-5mol/L的芍药苷对INS-1细胞释放胰岛素无明显的促进作用。4.阻断剂对INS-1细胞胰岛素释放的影响与正常对照组相比,阿托品组对INS-1细胞胰岛素释放没有显著影响,阿托品+芍药苷组INS-1细胞胰岛素释放显著升高；与阿托品组相比,阿托品+芍药苷组对INS-1细胞胰岛素释放没有显著影响。与正常对照组相比,异搏定对INS-1细胞胰岛素释放没有显著影响,异搏定+芍药苷组INS-1细胞胰岛素释放显著升高；与异搏定组相比,异搏定+芍药苷组对INS-1细胞胰岛素释放没有显著影响。5.芍药苷对STZ所致INS-1细胞损伤的保护和修复作用与正常对照组相比,STZ组INS-1细胞增殖活力明显降低,胰岛素释放明显减少,MDA含量升高,T-AOC下降。与STZ组相比,芍药苷保护组的胰岛素分泌量明显升高,MDA含量明显降低,T-AOC显著升高。而芍药苷修复组胰岛素释放没有明显变化,但MDA含量明显降低,T-AOC显著升高。6.芍药苷对INS-1细胞形态学及细胞周期分布和凋亡的影响与正常对照组相比,STZ组细胞形态变圆,细胞悬浮于细胞液中；在STZ损伤INS-1细胞前后加入各浓度芍药苷,INS-1细胞形态与STZ组相比没有明显改变。正常对照组细胞分布为G1期48.930%,G2期3.386%,S期47.684%。STZ作用12h,细胞被阻滞在S期,G1期细胞减少,G2期无细胞分布。芍药苷+STZ共同作用12h,细胞仍然被阻滞在S期,G1期细胞减少,G2期无细胞分布。正常对照组的细胞凋亡率为0.247%。与正常对照组相比,STZ组的细胞凋亡率明显升高为0.743%。芍药苷+STZ共同作用12h,细胞凋亡率较STZ组显著回降,为0.441%。实验结果表明：INS-1细胞对葡萄糖的刺激具有良好的反应性；芍药苷可以促进INS-1细胞释放胰岛素,其作用可能部分经由细胞膜上的胆碱能M受体和L-型的Ca2+通道所介导；芍药苷对STZ损伤的INS-1细胞有一定的保护和修复作用,此作用可能与其提高INS-1细胞的抗氧化能力有关；芍药苷对INS-1细胞具有一定的抗凋亡作用。更多还原

【Abstract】 In recent years, there is amount of evidence that obesity is highly related to insulin resistance (IR), which is one of the most important reasons for the obese to develop type 2 diabetes, hypertension and lipid disorders.Paeoniflorin is one of the major components of peony. Modern medicine thinks that Paeoniflorin had analgesic, sedative, anti-spasmodic, and anti-inflammatory and anti-free radicals effects. Our early experimental data have shown:White peony Alba of decoction could inhibit the body weight in ablating the bilateral ovaries of the obese rats, improve lipid metabolism, and scavenge excess free radicals. Evidence in vitro has demonstrated that Paeoniflorin can promote the release of free fatty acids of fatty tissue, decrease malondialdehyde production and increase the level of total antioxidant capacity. Whether Paeoniflorin has a direct effect on pancreatic islet cells has not yet been reported. Thus, in this experiment, different concentrations of Paeoniflorin were used to observe insulin release of pancreatic islet cells. At the same time, we established STZ-injured islet cells in order to study its mechanism of Paeoniflorin by observing the protective and repairment effects of Paeoniflorin on the islet cells and to preliminarily explore the possible mechanism of insulin release.In this experiment, we used rat insulinoma cells, INS-1. After subculture, cells were seeded into 96-well plates with a density of 1 X 105/ml and cultured for 24h. Then various concentrations of drugs were treated for 12h. Cell proliferation activity, insulin release, malondialdehyde and total antioxidant capacity levels of cell supernatant were determined. The Experiment was divided into nine groups:①the control;②Paeoniflorin group (10-7mol/L,10-6mol/L,10-5mol/L);③STZ group;④Paeoniflorin (10-7mol/L,10-6mol/L,10-5mol/L)+STZ group (the Paeoniflorin protective group);⑤STZ+Paeoniflorin(10-7mol/L,10-6mol/L,10-5mol/L)group (the Paeoniflorin repairment group);⑥Atropine group(10-6mol/L);⑦Atropine (10-6mol/L)+Paeoniflorin(10-7mol/L);⑧Verapamil group(10-7mol/L);⑨Verapamil (10-7mol/L)+Paeoniflorin (10-7mol/L).The experimental data was expressed with mean±standard deviation (x±S) Statistical comparisons were made using analysis of variance (ANOVA) between different groups. Differences with P<0.05 were considered statistically significant. Our results were shown as follow:1. Effect of glucose stimulation on insulin release of INS-1 cellsCompared with control, the insulin release of INS-1 cells gradually increased with the increasing of glucose concentration (within 5.6-16.8mmol/L). However, glucose of 22.4 mmol/L and 33.6 mmol/L decreased insulin release.2. Effect of Paeoniflorin on proliferation of INS-1 cellsCompared with control, Paeoniflorin did not affect proliferation of INS-1 cells.3. Effect of Paeoniflorin on insulin release of INS-1 cellsCompared with control, paeoniflorin of 10-7mol/L,10-6mol/L could promote the insulin release of INS-1 cells in varying degrees, while paeoniflorin of 10-5 mol/L had no significant effect on insulin release.4. Effect of Blockers on insulin release of INS-1 cellsCompared with control, Atropine group had on significantly effect on insulin release of the INS-1 cells, but insulin release of (atropine+paeoniflorin) group was significantly increased. Compared with Atropine group, (atropine+paeoniflorin) Group did not affect insulin release.Same as Atropine, Verapamil had no significant effect on insulin release compared with control. However, (verapamil+paeoniflorin) group of insulin release was significantly increased. Compared with Verapamil group, (Verapamil+paeoniflorin) group had no significantly effect on the insulin release.5. Protective and Repairment effects of Paeoniflorin on STZ-injured INS-1 cellsCompared with control, STZ significantly inhibited INS-1 cell proliferation, decreased insulin release, increased MDA production and reduced T-AOC. Compared with STZ group, Paeoniflorin protection group significantly increased insulin release, reduced MDA production and incresed T-AOC; Paeoniflorin repairment group did not affect insulin release, whereas the MDA production was lowered and T-AOC was significantly increased.6. Effect of Paeoniflorin on INS-1 cell morphological change and cell cycle distribution and apoptosisCompared with control, INS-1 cells in STZ group became round, and cells were suspended in the cell suspension. Compared with STZ group, INS-1 cells morphology did not change after the addition of Paeoniflorin in STZ injured INS-1 cells.Cellular distribution of control was G1 48.930%, G23.386%, and S 47.684%. After treated with STZ forl2h, cells were arrested in S phase, while G1 phase cells were decreased and G2 phase had cell-free distribution. After administration of Paeoniflorin+ STZ for 12h, cellular distribution was the same as STZ group.Apoptosis rate of control was 0.247%. Compared with the control, apoptosis rate of the STZ group was up to 0.743% while Paeoniflorin+STZ group was 0.437%.In conclusion, our results showed that INS-1 cells have better activity on glucose stimulation. Paeoniflorin can promote the insulin release of INS-1 cells, which it may be partly mediated through cholinergic M receptor of the membrane and L-type Ca2+ channel. Paeoniflorin has protective and repairment effects on STZ-injured INS-1 cells, which it may be associated with the increasing antioxidant capacity of INS-1 cells. Paeoniflorin maybe have certain effect on apoptosis of INS-1 cells.更多还原