菩人丹对糖脂毒导致的INS-1细胞损伤的修复作用及机制研究
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摘要
[背景]
2型糖尿病是严重危害人类健康的重大疑难疾病。胰岛p细胞功能的衰竭和胰岛素抵抗是其主要发病机制,其中胰岛p细胞数量的异常减少和胰岛素分泌功能的下降是导致2型糖尿病发生和发展的关键因素。葡萄糖和脂肪虽然是机体必需的营养物质,但是长期高水平的葡萄糖和游离脂肪酸将造成胰岛p细胞损伤,致使细胞发生凋亡并逐步丧失胰岛素分泌的功能,最终导致2型糖尿病的不可逆发生。而胰岛p细胞上的胰岛素信号转导通路,调控着p细胞的增殖、分化和胰岛素的合成、分泌,在机体糖脂代谢中发挥着重要作用。我国传统中医药治疗糖尿病已有上千年的历史,中药复方多靶点、多途径的作用特点,以及较少的毒副作用,在预防糖尿病发生以及并发症出现方面具有显著优势。
[目的]
本实验试图通过体外实验研究,证实高浓度葡萄糖、软脂酸及软脂酸+高糖对胰岛p细胞的损伤作用及其差异。在此基础上考察“益气养阴清热、活血化瘀通络”中药复方菩人丹对胰岛p细胞活力、凋亡以及胰岛素分泌的影响,阐明菩人丹保护胰岛p细胞“质”“量”之功效,并从分子水平研究菩人丹对胰岛p细胞胰岛素信号转导中IRS2/PI3K/Akt通路的影响,揭示菩人丹修复“糖脂毒”导致的胰岛p细胞损伤的作用机制。
[方法]
选取大鼠胰岛素瘤细胞系INS-1细胞为胰岛p细胞的体外研究对象,以33.3mM葡萄糖、0.5mM软脂酸及0.5mM软脂酸合并33.3mM葡萄糖分别构建糖毒性、脂毒性及糖脂毒性p细胞损伤模型。在各损伤模型基础上,分别给予5%菩人丹含药血清、10%菩人丹含药血清干预24小时,并以10%二甲双胍含药血清为阳性对照。采用CCK-8试剂盒检测INS-1细胞活力；Hoechst荧光染色法和流式Annexin V-FITC/PI双染法检测细胞凋亡；caspase-3和caspase-8活性检测试剂盒检测细胞caspase-3/-8活性；葡萄糖刺激胰岛素释放实验检测INS-1细胞的胰岛素分泌；采用Western blot法分析胰岛素信号转导通路相关蛋白IRS2、Akt、BAD、FOXO1、mTOR、P70s6k和PTP1B的蛋白质表达水平,以及IRS2(ser731)、Akt(thr308)、BAD(serl36)、 FOXO1(ser256)、mTOR(ser2448)和P70s6k(thr389)的磷酸化水平。
[结果]
1.三种模型的特点和胰岛素分泌功能变化规律：
(1)33.3mM葡萄糖作用于INS-1细胞,12小时后,与对照组比较细胞活力(P=0.013)和细胞BIS以及GSIS均显著升高(BIS:P<0.001, GSIS: P=0.046);培养24小时后,细胞活力和胰岛素分泌出现下降(P<0.001),其降低呈现时间依赖性；此时出现明显的细胞凋亡(P<0.001)。
(2)0.5mM软脂酸作用于INS-1细胞,6小时后细胞活力即开始下降,但此时细胞BIS有所升高,然而二者变化均无统计学意义(P>0.05)；培养24小时后,细胞活力和GSIS均明显降低(P<0.001),但BIS则明显升高(P=0.001)；同时观察到明显的细胞凋亡(P<0.001),且凋亡率大于高糖损伤细胞。
(3)0.5mM软脂酸和33.3mM葡萄糖同时作用于INS-1细胞,6小时后,细胞活力和GSIS即出现显著降低(细胞活力：P=0.041; GSIS:P<0.001),细胞BIS在12小时后也开始显著下降(P<0.001),其下降呈现时间依赖性；细胞凋亡率则远高于单纯高糖组和软脂酸组。
2.菩人丹对糖脂损伤INS-1细胞的保护作用：
(1)菩人丹对糖脂损伤INS-1细胞的活力影响：高糖、软脂酸、软脂酸+高糖均使INS-1细胞活力显著降低(vs.对照组,P<0.001)。低剂量菩人丹对损伤细胞的活力无明显作用(vs.模型组,P>0.05),而高剂量菩人丹可使细胞活力明显升高(vs.模型组,P=0.003或P<0.001),作用效果与阳性对照药二甲双胍相当。
(2)菩人丹对糖脂损伤INS-1细胞凋亡的影响：高糖、软脂酸、软脂酸合并高糖均明显促进INS-1细胞的凋亡(vs.对照组,P<0.001),升高caspase-3和caspase-8的活性。低剂量菩人丹对细胞凋亡无明显作用(vs.模型组,P>0.05),而高剂量菩人丹可显著降低caspase-3和caspase-8的活性,抑制细胞凋亡(vs.模型组,P<0.001),并与阳性对照药二甲双胍作用效果相当。
(3)菩人丹对糖脂损伤INS-1细胞胰岛素分泌的影响：高糖、软脂酸合并高糖使INS-1细胞的BIS和GSIS降低(vs.对照组,P<0.001),而软脂酸使INS-1细胞GSIS降低的同时(vs.对照组,P<0.001),使BIS升高(vs.对照组,P=0.012)。低剂量菩人丹对细胞胰岛素分泌无显著影响(vs.模型组,P>0.05),而高剂量菩人丹可明显增加糖脂损伤INS-1细胞的BIS及GSIS(vs.模型组,P<0.001),对软脂酸导致的细胞BIS上升虽有所抑制,但差异无统计学意义(vs.模型组,P=0.099)。菩人丹对糖脂损伤INS-1细胞胰岛素分泌的作用效果与二甲双胍相当。
3.菩人丹对INS-1细胞胰岛素信号转导通路的影响：
高糖、软脂酸、软脂酸合并高糖均可显著下调IRS2蛋白质表达水平(P<0.001),降低Akt(thr308)、B AD(ser136)、FOXO1(ser256)、mTOR(ser2448)和P70s6k(thr389)的磷酸化水平(P<0.001),上调FOXO1和PTP1B的蛋白质表达,升高IRS2(ser731)磷酸化水平(P<0.001)。菩人丹干预24小时后,促进了各损伤模型细胞IRS2的蛋白表达(P<0.001)以及Akt(thr308)、 BAD(ser136)、FOXO1(ser256)、mTOR(ser2448)和P70s6k(thr389)的磷酸化(P<0.001),同时抑制了FOXO1和PTP1B的蛋白质表达(P<0.001)以及IRS2(ser731)的磷酸化(P<0.001),并且与阳性对照药二甲双胍作用效果相当。
[结论]
1.高浓度葡萄糖在短时间内可以促进INS-1细胞的增殖和胰岛素分泌,而持续高糖则表现出促进细胞凋亡、抑制细胞活力和胰岛素分泌的作用,并且对GSIS的影响较BIS更为显著；软脂酸可抑制INS-1细胞活力,促进细胞凋亡,并且在短时间内可轻微增加胰岛素分泌,但长时间作用表现为增加细胞BIS而抑制GSIS;软脂酸合并高糖可抑制INS-1细胞活力、促进细胞凋亡、降低胰岛素分泌,且作用强于单纯高糖和软脂酸,对INS-1细胞的损伤具有协同性和叠加性。
2.菩人丹可以显著抑制糖毒性、脂毒性和糖脂毒性导致的胰岛p细胞凋亡、提高细胞活力、降低caspase-3和caspase-8活性、促进p细胞葡萄糖刺激的胰岛素分泌,保护p细胞的“质”和“量”,从而发挥菩人丹“益气养阴清热、活性化瘀通络”治疗2型糖尿病之功效。
3.菩人丹可以减少“糖脂毒”损伤INS-1细胞IRS2蛋白的异常降解和丝氨酸(ser731)磷酸化,保证足量的IRS2可以被胰岛素受体的酪氨酸激酶活化,继而减轻糖脂毒对胰岛p细胞Akt活性的抑制作用,使Akt苏氨酸(thr308)磷酸化增强,激活胰岛素信号转导系统中的PI3K/Akt通路,将胰岛素信号继续向下游传递,使下游效应因子BAD、FOXO1、mTOR和P70s6k被活化,得以发挥其各自的生物学功能,从而抑制“糖脂毒”对胰岛p细胞凋亡的促进作用、修复“糖脂毒”导致的胰岛素分泌损伤,改善“糖脂毒”损伤p细胞的“质”“量”。
Background:Type2diabetes mellitus (T2DM) is a chronic disease that could result disability and shortened life expectancy. Abnormal reduced cell mass and dysfunction of insulin secretion of pancreatic islet β cell are the key causes of type2diabetes mellitus. Although glucose and fat are essential nutritional elements of organisms, high level of them for long-term might cause apoptosis of β cells and decline of insulin secretion, and will ultimately induce type2diabetes mellitus. Insulin signaling pathways in islet β cell have regulation effects on β cell mass and insulin secretion, which play an important role in glucolipid metabolism.
Objective:To explore the injury effect of high glucose and palmitic acid on islet β cell, and confirm the protect effect of Pu-Ren-Dan formula (PRD) on injured β cell by investigatin cell viability, apoptosis and insulin secretion. And indicate the effect of PRD on insulin signaling pathways in islet β cell from molecule level, to clarify and the potential mechanisms of protective effect of PRD on islet β cells and provide experimental evidences for clinical application of PRD.
Methods:INS-1cell line was used in this in vitro study, and cultured in33.3mM glucose,0.5mM palmitic acid or both33.3mM glucose and0.5mM palmitic acid to establish the gluco-and lipo-toxicity models. INS-1cell of each model group was treated with PRD serum (5%and10%) and10%metformin (MF) serum for24hours, respectively. Then CCK-8assay was used to measure cell viability; Hoechst33342staining, Flow Cytometry, AnnexinV-FITC/PI staning and caspase-3,-8Activity Assay Kit were deployed to detect INS-1cell apoptosis; and insulin secretion test was performed to evaluate INS-1cell function. And western blotting assay was deployed to investigate protein expressions of IRS2, Akt, BAD, FOXO1, mTOR, P70s6k and PTP1B, as well as the phosphorylations of IRS2(ser731), Akt (thr308), BAD (ser136), FOXO1(ser256), mTOR (ser2448) and P70s6k (thr389).
Results:
1. Compare of cell mass and insulin secretion among three injury models:
(1) Exposure of INS-1cell to33.3mM glucose for12hours, compared to control group, cell viability (P=0.013), basal insulin secretion (BIS, P<0.001) and glucose-stimulated insulin secretion (GSIS, P=0.046) were significantly increased; after24hours, cell viability and insulin secretion showed decreasing time-independently; meantime, apoptosis was observed markedly (P<0.001).
(2) Exposure of INS-1cell to0.5mM palmitic acid for6hours, cell viability was decreased, while BIS was lightly increased with no significant difference (P>0.05); after24hours, cell viability (P=0.001) and GSIS (P<0.001) were significantly reduced, but BIS and apoptosis were increased obviously (P<0.001).
(3) Exposure of INS-1cell to0.5mM palmitic acid and33.3mM glucose simultaneously for6hours, cell viability (P=0.041) and GSIS (P<0.001) were significantly and time-independently decreased; after12hours, BIS also showed decreasing markedly (P<0.001), and apoptosis rate was raised, and higher than other two cell models.
2. Protective effect of PRD on injured INS-1cell:
High glucose, palmitic acid and high glucose with palmitic acid reduced cell viability significantly (P<0.001), but high-dose PRD could inhibit this reduction, and increased it markedly (P=0.003or P<0.001) while low-dose PRD had no significant effect on INS-1cell viability, which was similar to positive drug MF. High glucose, palmitic acid and high glucose with palmitic acid promoted cell apoptosis (P<0.001), and raised activities of caspase-3and caspase-8(P<0.001). Low-dose PRD had no significant effect on INS-1cell apoptosis, but high-dose PRD reduced activities of caspase-3and caspase-8(P<0.001), and apoptosis was inhibited (P<0.001), which was similar to the effects of MF. High glucose, high glucose with palmitic acid decreased INS-1cell BIS and GSIS (P<0.001), while palmitic acid reduced GSIS (P<0.001) but increased BIS (P=0.012). Low-dose PRD had no significant effect on INS-1cell insulin secretion, but high-dose PRD significantly increased INS-1cell BIS and GSIS (P<0.001), except for the BIS of palmitic acid group (P=0.099). The effect of PRD on INS-1cell insulin secretion was similar to MF.
3. High glucose, palmitic acid and high glucose with palmitic acid significantly down-regulated protein expression of IRS2(P<0.001); reduced phosphorylations of Akt (thr308), BAD (ser136), FOXO1(ser256), mTOR (ser2448) and P70s6k (thr389)(P<0.001); up-regulated protein expressions of FOXO1and PTP1B (P<0.001); increased phosphorylations of IRS2(ser731)(P<0.001). After PRD treatment for24hours, protein expression of IRS2, phosphorylations of Akt (thr308), BAD (serl36), FOXO1(ser256), mTOR (ser2448) and P70s6k (thr389) were increased (P<0.001), while protein expressions of FOXO1and PTP1B, and phosphorylations of IRS2(ser731) were reduced significantly (P<0.001).
Conclusion:
1. High glucose promoted (3cell proliferation and insulin secretion in short-term, but increased apoptosis, inhibit cell viability and insulin secretion in long-term of high glucose. The influence of high glucose on GSIS was more significant than BIS; palmitic acid inhibited (3cell viability and promoted apoptosis and BIS, and GSIS was increased by palmitic acid in short-term, but decreased in long-term; palmitic acid with high glucose inhibited (3cell viability, decreased insulin secretion and promoted apoptosis, and the effects of palmitic acid with high glucose were stronger than that of palmitic acid or high glucose.
2. PRD as an effective antidiabetic Chinese medicine formula could protect islet β cell against gluco-and lipo-toxicity induced injury by inhibiting apoptosis, raising cell viability, reducing activities of caspase-3and-8, and promoting insulin secretion.
3. PRD inhibited the abnormal degradation of IRS2and its serine phosphorylation, which ensured the adequate IRS2proteins and its tyrosine phosphorylation, and alleviated the inhibition of Akt activity by glucolipotoxicity, promoted threonine phosphorylation at thr308to activate the PI3K/Akt pathway, then the downstreat effector proteins BAD, FOXO1, mTORand P70s6k were activated to conduct their biological functions to inhibit P cells apoptosis, promote cell viability, and improve insulin secretion.
2型糖尿病是严重危害人类健康的重大疑难疾病。胰岛p细胞功能的衰竭和胰岛素抵抗是其主要发病机制,其中胰岛p细胞数量的异常减少和胰岛素分泌功能的下降是导致2型糖尿病发生和发展的关键因素。葡萄糖和脂肪虽然是机体必需的营养物质,但是长期高水平的葡萄糖和游离脂肪酸将造成胰岛p细胞损伤,致使细胞发生凋亡并逐步丧失胰岛素分泌的功能,最终导致2型糖尿病的不可逆发生。而胰岛p细胞上的胰岛素信号转导通路,调控着p细胞的增殖、分化和胰岛素的合成、分泌,在机体糖脂代谢中发挥着重要作用。我国传统中医药治疗糖尿病已有上千年的历史,中药复方多靶点、多途径的作用特点,以及较少的毒副作用,在预防糖尿病发生以及并发症出现方面具有显著优势。
[目的]
本实验试图通过体外实验研究,证实高浓度葡萄糖、软脂酸及软脂酸+高糖对胰岛p细胞的损伤作用及其差异。在此基础上考察“益气养阴清热、活血化瘀通络”中药复方菩人丹对胰岛p细胞活力、凋亡以及胰岛素分泌的影响,阐明菩人丹保护胰岛p细胞“质”“量”之功效,并从分子水平研究菩人丹对胰岛p细胞胰岛素信号转导中IRS2/PI3K/Akt通路的影响,揭示菩人丹修复“糖脂毒”导致的胰岛p细胞损伤的作用机制。
[方法]
选取大鼠胰岛素瘤细胞系INS-1细胞为胰岛p细胞的体外研究对象,以33.3mM葡萄糖、0.5mM软脂酸及0.5mM软脂酸合并33.3mM葡萄糖分别构建糖毒性、脂毒性及糖脂毒性p细胞损伤模型。在各损伤模型基础上,分别给予5%菩人丹含药血清、10%菩人丹含药血清干预24小时,并以10%二甲双胍含药血清为阳性对照。采用CCK-8试剂盒检测INS-1细胞活力；Hoechst荧光染色法和流式Annexin V-FITC/PI双染法检测细胞凋亡；caspase-3和caspase-8活性检测试剂盒检测细胞caspase-3/-8活性；葡萄糖刺激胰岛素释放实验检测INS-1细胞的胰岛素分泌；采用Western blot法分析胰岛素信号转导通路相关蛋白IRS2、Akt、BAD、FOXO1、mTOR、P70s6k和PTP1B的蛋白质表达水平,以及IRS2(ser731)、Akt(thr308)、BAD(serl36)、 FOXO1(ser256)、mTOR(ser2448)和P70s6k(thr389)的磷酸化水平。
[结果]
1.三种模型的特点和胰岛素分泌功能变化规律：
(1)33.3mM葡萄糖作用于INS-1细胞,12小时后,与对照组比较细胞活力(P=0.013)和细胞BIS以及GSIS均显著升高(BIS:P<0.001, GSIS: P=0.046);培养24小时后,细胞活力和胰岛素分泌出现下降(P<0.001),其降低呈现时间依赖性；此时出现明显的细胞凋亡(P<0.001)。
(2)0.5mM软脂酸作用于INS-1细胞,6小时后细胞活力即开始下降,但此时细胞BIS有所升高,然而二者变化均无统计学意义(P>0.05)；培养24小时后,细胞活力和GSIS均明显降低(P<0.001),但BIS则明显升高(P=0.001)；同时观察到明显的细胞凋亡(P<0.001),且凋亡率大于高糖损伤细胞。
(3)0.5mM软脂酸和33.3mM葡萄糖同时作用于INS-1细胞,6小时后,细胞活力和GSIS即出现显著降低(细胞活力：P=0.041; GSIS:P<0.001),细胞BIS在12小时后也开始显著下降(P<0.001),其下降呈现时间依赖性；细胞凋亡率则远高于单纯高糖组和软脂酸组。
2.菩人丹对糖脂损伤INS-1细胞的保护作用：
(1)菩人丹对糖脂损伤INS-1细胞的活力影响：高糖、软脂酸、软脂酸+高糖均使INS-1细胞活力显著降低(vs.对照组,P<0.001)。低剂量菩人丹对损伤细胞的活力无明显作用(vs.模型组,P>0.05),而高剂量菩人丹可使细胞活力明显升高(vs.模型组,P=0.003或P<0.001),作用效果与阳性对照药二甲双胍相当。
(2)菩人丹对糖脂损伤INS-1细胞凋亡的影响：高糖、软脂酸、软脂酸合并高糖均明显促进INS-1细胞的凋亡(vs.对照组,P<0.001),升高caspase-3和caspase-8的活性。低剂量菩人丹对细胞凋亡无明显作用(vs.模型组,P>0.05),而高剂量菩人丹可显著降低caspase-3和caspase-8的活性,抑制细胞凋亡(vs.模型组,P<0.001),并与阳性对照药二甲双胍作用效果相当。
(3)菩人丹对糖脂损伤INS-1细胞胰岛素分泌的影响：高糖、软脂酸合并高糖使INS-1细胞的BIS和GSIS降低(vs.对照组,P<0.001),而软脂酸使INS-1细胞GSIS降低的同时(vs.对照组,P<0.001),使BIS升高(vs.对照组,P=0.012)。低剂量菩人丹对细胞胰岛素分泌无显著影响(vs.模型组,P>0.05),而高剂量菩人丹可明显增加糖脂损伤INS-1细胞的BIS及GSIS(vs.模型组,P<0.001),对软脂酸导致的细胞BIS上升虽有所抑制,但差异无统计学意义(vs.模型组,P=0.099)。菩人丹对糖脂损伤INS-1细胞胰岛素分泌的作用效果与二甲双胍相当。
3.菩人丹对INS-1细胞胰岛素信号转导通路的影响：
高糖、软脂酸、软脂酸合并高糖均可显著下调IRS2蛋白质表达水平(P<0.001),降低Akt(thr308)、B AD(ser136)、FOXO1(ser256)、mTOR(ser2448)和P70s6k(thr389)的磷酸化水平(P<0.001),上调FOXO1和PTP1B的蛋白质表达,升高IRS2(ser731)磷酸化水平(P<0.001)。菩人丹干预24小时后,促进了各损伤模型细胞IRS2的蛋白表达(P<0.001)以及Akt(thr308)、 BAD(ser136)、FOXO1(ser256)、mTOR(ser2448)和P70s6k(thr389)的磷酸化(P<0.001),同时抑制了FOXO1和PTP1B的蛋白质表达(P<0.001)以及IRS2(ser731)的磷酸化(P<0.001),并且与阳性对照药二甲双胍作用效果相当。
[结论]
1.高浓度葡萄糖在短时间内可以促进INS-1细胞的增殖和胰岛素分泌,而持续高糖则表现出促进细胞凋亡、抑制细胞活力和胰岛素分泌的作用,并且对GSIS的影响较BIS更为显著；软脂酸可抑制INS-1细胞活力,促进细胞凋亡,并且在短时间内可轻微增加胰岛素分泌,但长时间作用表现为增加细胞BIS而抑制GSIS;软脂酸合并高糖可抑制INS-1细胞活力、促进细胞凋亡、降低胰岛素分泌,且作用强于单纯高糖和软脂酸,对INS-1细胞的损伤具有协同性和叠加性。
2.菩人丹可以显著抑制糖毒性、脂毒性和糖脂毒性导致的胰岛p细胞凋亡、提高细胞活力、降低caspase-3和caspase-8活性、促进p细胞葡萄糖刺激的胰岛素分泌,保护p细胞的“质”和“量”,从而发挥菩人丹“益气养阴清热、活性化瘀通络”治疗2型糖尿病之功效。
3.菩人丹可以减少“糖脂毒”损伤INS-1细胞IRS2蛋白的异常降解和丝氨酸(ser731)磷酸化,保证足量的IRS2可以被胰岛素受体的酪氨酸激酶活化,继而减轻糖脂毒对胰岛p细胞Akt活性的抑制作用,使Akt苏氨酸(thr308)磷酸化增强,激活胰岛素信号转导系统中的PI3K/Akt通路,将胰岛素信号继续向下游传递,使下游效应因子BAD、FOXO1、mTOR和P70s6k被活化,得以发挥其各自的生物学功能,从而抑制“糖脂毒”对胰岛p细胞凋亡的促进作用、修复“糖脂毒”导致的胰岛素分泌损伤,改善“糖脂毒”损伤p细胞的“质”“量”。
Background:Type2diabetes mellitus (T2DM) is a chronic disease that could result disability and shortened life expectancy. Abnormal reduced cell mass and dysfunction of insulin secretion of pancreatic islet β cell are the key causes of type2diabetes mellitus. Although glucose and fat are essential nutritional elements of organisms, high level of them for long-term might cause apoptosis of β cells and decline of insulin secretion, and will ultimately induce type2diabetes mellitus. Insulin signaling pathways in islet β cell have regulation effects on β cell mass and insulin secretion, which play an important role in glucolipid metabolism.
Objective:To explore the injury effect of high glucose and palmitic acid on islet β cell, and confirm the protect effect of Pu-Ren-Dan formula (PRD) on injured β cell by investigatin cell viability, apoptosis and insulin secretion. And indicate the effect of PRD on insulin signaling pathways in islet β cell from molecule level, to clarify and the potential mechanisms of protective effect of PRD on islet β cells and provide experimental evidences for clinical application of PRD.
Methods:INS-1cell line was used in this in vitro study, and cultured in33.3mM glucose,0.5mM palmitic acid or both33.3mM glucose and0.5mM palmitic acid to establish the gluco-and lipo-toxicity models. INS-1cell of each model group was treated with PRD serum (5%and10%) and10%metformin (MF) serum for24hours, respectively. Then CCK-8assay was used to measure cell viability; Hoechst33342staining, Flow Cytometry, AnnexinV-FITC/PI staning and caspase-3,-8Activity Assay Kit were deployed to detect INS-1cell apoptosis; and insulin secretion test was performed to evaluate INS-1cell function. And western blotting assay was deployed to investigate protein expressions of IRS2, Akt, BAD, FOXO1, mTOR, P70s6k and PTP1B, as well as the phosphorylations of IRS2(ser731), Akt (thr308), BAD (ser136), FOXO1(ser256), mTOR (ser2448) and P70s6k (thr389).
Results:
1. Compare of cell mass and insulin secretion among three injury models:
(1) Exposure of INS-1cell to33.3mM glucose for12hours, compared to control group, cell viability (P=0.013), basal insulin secretion (BIS, P<0.001) and glucose-stimulated insulin secretion (GSIS, P=0.046) were significantly increased; after24hours, cell viability and insulin secretion showed decreasing time-independently; meantime, apoptosis was observed markedly (P<0.001).
(2) Exposure of INS-1cell to0.5mM palmitic acid for6hours, cell viability was decreased, while BIS was lightly increased with no significant difference (P>0.05); after24hours, cell viability (P=0.001) and GSIS (P<0.001) were significantly reduced, but BIS and apoptosis were increased obviously (P<0.001).
(3) Exposure of INS-1cell to0.5mM palmitic acid and33.3mM glucose simultaneously for6hours, cell viability (P=0.041) and GSIS (P<0.001) were significantly and time-independently decreased; after12hours, BIS also showed decreasing markedly (P<0.001), and apoptosis rate was raised, and higher than other two cell models.
2. Protective effect of PRD on injured INS-1cell:
High glucose, palmitic acid and high glucose with palmitic acid reduced cell viability significantly (P<0.001), but high-dose PRD could inhibit this reduction, and increased it markedly (P=0.003or P<0.001) while low-dose PRD had no significant effect on INS-1cell viability, which was similar to positive drug MF. High glucose, palmitic acid and high glucose with palmitic acid promoted cell apoptosis (P<0.001), and raised activities of caspase-3and caspase-8(P<0.001). Low-dose PRD had no significant effect on INS-1cell apoptosis, but high-dose PRD reduced activities of caspase-3and caspase-8(P<0.001), and apoptosis was inhibited (P<0.001), which was similar to the effects of MF. High glucose, high glucose with palmitic acid decreased INS-1cell BIS and GSIS (P<0.001), while palmitic acid reduced GSIS (P<0.001) but increased BIS (P=0.012). Low-dose PRD had no significant effect on INS-1cell insulin secretion, but high-dose PRD significantly increased INS-1cell BIS and GSIS (P<0.001), except for the BIS of palmitic acid group (P=0.099). The effect of PRD on INS-1cell insulin secretion was similar to MF.
3. High glucose, palmitic acid and high glucose with palmitic acid significantly down-regulated protein expression of IRS2(P<0.001); reduced phosphorylations of Akt (thr308), BAD (ser136), FOXO1(ser256), mTOR (ser2448) and P70s6k (thr389)(P<0.001); up-regulated protein expressions of FOXO1and PTP1B (P<0.001); increased phosphorylations of IRS2(ser731)(P<0.001). After PRD treatment for24hours, protein expression of IRS2, phosphorylations of Akt (thr308), BAD (serl36), FOXO1(ser256), mTOR (ser2448) and P70s6k (thr389) were increased (P<0.001), while protein expressions of FOXO1and PTP1B, and phosphorylations of IRS2(ser731) were reduced significantly (P<0.001).
Conclusion:
1. High glucose promoted (3cell proliferation and insulin secretion in short-term, but increased apoptosis, inhibit cell viability and insulin secretion in long-term of high glucose. The influence of high glucose on GSIS was more significant than BIS; palmitic acid inhibited (3cell viability and promoted apoptosis and BIS, and GSIS was increased by palmitic acid in short-term, but decreased in long-term; palmitic acid with high glucose inhibited (3cell viability, decreased insulin secretion and promoted apoptosis, and the effects of palmitic acid with high glucose were stronger than that of palmitic acid or high glucose.
2. PRD as an effective antidiabetic Chinese medicine formula could protect islet β cell against gluco-and lipo-toxicity induced injury by inhibiting apoptosis, raising cell viability, reducing activities of caspase-3and-8, and promoting insulin secretion.
3. PRD inhibited the abnormal degradation of IRS2and its serine phosphorylation, which ensured the adequate IRS2proteins and its tyrosine phosphorylation, and alleviated the inhibition of Akt activity by glucolipotoxicity, promoted threonine phosphorylation at thr308to activate the PI3K/Akt pathway, then the downstreat effector proteins BAD, FOXO1, mTORand P70s6k were activated to conduct their biological functions to inhibit P cells apoptosis, promote cell viability, and improve insulin secretion.
引文
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