鸢尾素通过自噬对棕榈酸诱导的INS-1细胞功能和凋亡的影响

Effect of irisin on function and apoptosis of INS-1 cells induced by palmitic acid through autophagy

摘要:

目的： 探讨鸢尾素对棕榈酸（palmitic acid，PA）诱导的胰岛β细胞功能和凋亡的影响。方法： 培养INS-1细胞，用不同浓度梯度的鸢尾素干预INS-1细胞，根据蛋白结果筛选出最佳鸢尾素干预浓度为1 500 ng/mL。然后将INS-1细胞分为正常组、PA组、PA＋1 500 ng/mL鸢尾素组、PA＋1 500 ng/mL鸢尾素＋雷帕霉素组、PA＋1 500 ng/mL鸢尾素＋3-MA组。用3.3 mmol/L和16.7 mmol/L的葡萄糖浓度刺激检测干预后胰岛素的分泌水平，Western blot检测自噬相关蛋白p-mTOR、mTOR、p62、LC3Ⅱ、LC3Ⅰ的蛋白水平，用mGFP-GFP-LC3腺病毒转染观察自噬流水平，流式细胞仪检测细胞凋亡水平。结果采用SPSS 20.0软件进行统计分析，计量资料以均数±标准差（±s）表示，多组间差异比较采用单因素方差分析及Turkey HSD检验。结果： ①PA组LC3Ⅱ/Ⅰ的比值较正常组增高（0.745±0.035 vs.1.015±0.012，P=0.000），随着鸢尾素干预浓度增加，LC3Ⅱ/Ⅰ的水平较PA组逐渐升高且有统计学意义（I1组1.465±0.028，I2组2.269±0.063，I3组1.858±0.074，I4组2.355±0.074，均P=0.000），鸢尾素浓度为1 500 ng/mL时LC3Ⅱ/Ⅰ的水平最高。②PA组INS-1细胞上清液中胰岛素水平在3.3 mmol/L和16.7 mmol/L葡萄糖刺激下较正常组均下降[（0.447±0.067）ng/mL vs.（0.138±0.012）ng/mL，P=0.000；（0.625±0.041）ng/mL vs.（0.245±0.043）ng/mL，P=0.000]，鸢尾素干预后胰岛素水平较PA组均升高[（0.138±0.012）ng/mL vs.（0.322±0.041）ng/mL，P=0.001；（0.245±0.043）ng/mL vs.（0.414±0.036）ng/mL，P=0.005]。③与正常组相比，PA干预细胞后，自噬体数量增加（2.33±1.53 vs.10.33±1.53，P=0.000），而自噬溶酶体数量无明显变化（2.33±1.53 vs.3.33±1.53，P=0.939），鸢尾素干预后，自噬体数量和自噬溶酶体数量较PA组均升高（10.33±1.53 vs.15.67±1.53，P=0.007；3.33±1.53 vs.12.00±2.00，P=0.001）。④与正常组相比，PA干预后细胞凋亡率增加[（11.417±1.297）%vs.（46.507±0.525）%，P=0.000]，鸢尾素干预后凋亡率降低[（46.507±0.525）%vs.（33.767±1.083）%，P=0.000]。结论： 鸢尾素通过自噬改善PA诱导的INS-1细胞分泌功能和凋亡。

Abstract:

Objective: To investigate the effect of irisin on the function and apoptosis of pancreatic β cells induced by palmitic acid (PA). Methods: Firstly, INS-1 cells were cultivated and treated with different concentration gradients of irisin. The optimal concentration of 1 500 ng/mL was selected according to the protein results of Western blot. Secondly, INS-1 cells were divided into the normal group, the PA group, the palmitate acid+1 500 ng/mL irisin group, the palmitate acid+1 500 ng/mL irisin+rapamycin group and the palmitate acid+1 500 ng/mL irisin+3-MA group. The glucose-stimulated insulin secretion (GSIS) test (with glucose levels of 3.3 mmol/L and 16.7 mmol/L) was performed to determine the insulin level in each group. Western blot was used to determine the levels of mamlian target of rapamycin (mTOR), p-mTOR, p62, LC3Ⅱand LC3Ⅰ; mRFP-GFP-LC3 and flow cytometry were used to determine the autophagic flow and cell apoptosis, respectively. SPSS 20.0 was used to conduct data analysis. Results were presented as the mean±SD, one-way analysis of variance followed by Turkey HSD was used to calculate differences among the various study groups. Results: ①Compared with the normal group, the ratio of LC3Ⅱ/Ⅰin the PA group was higher (0.745±0.035 vs.1.015±0.012, P=0.000). With the increase of irisin concentration, the level of LC3Ⅱ/Ⅰwas gradually increased and had statistical significance (I1: 1.465±0.028, I2: 2.269±0.063, I3: 1.858±0.074, I4: 2.355±0.074; P=0.000). When irisin concentration was 1 500 ng/mL, the level of LC3Ⅱ/Ⅰwas the highest.②GSIS levels in the INS-1 cell supernatant in the PA group was significantly reduced for stimulation of 3.3 mmol/L and 16.7 mmol/L of glucose when compared with the normal group [(0.447±0.067) ng/mL vs. (0.138±0.012) ng/mL, P=0.000; (0.625±0.041) ng/mL vs. (0.245±0.043) ng/mL, P=0.000], while in the irisin treated group were increased when compared with the PA group [(0.138±0.012) ng/mL vs. (0.322±0.041) ng/mL, P=0.001; (0.245±0.043) ng/mL vs. (0.414±0.036) ng/mL, P=0.005].③The number of autophagosome in the PA group was significantly higher than that in the normal group (2.33±1.53 vs.10.33±1.53, P=0.000), but there was no obvious change in the number of autophagolysosome (2.33±1.53 vs.3.33±1.53, P=0.939). In the irisin group, the autolysosome number and autolysosome number was increased when compared with PA group (10.33±1.53 vs.15.67±1.53, P=0.007; 3.33±1.53 vs.12.00±2.00, P=0.001). Treated with PA for 24 hours, the apoptosis rate of INS-1 cells was increased when compared with the normal group [(11.417±1.297) %vs. (46.507±0.525) %, P=0.000], while the irisin-treated group had a significantly reduced apoptosis rate when compared with the PA group [(46.507±0.525) %vs. (33.767±1.083) %, P=0.000]. Conclusion: Irisin ameliorates apoptosis and improves secretion function of INS-1 cells induced by PA through autophagy.