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目的探讨过氧化物酶增殖活化受体-γ(PPAR-γ)激动剂吡格列酮对胰岛β细胞糖脂毒性损伤的保护及受体相互作用蛋白140(RIP140)在其中的介导机制。方法将胰岛β细胞株MIN6细胞分为NC组、高糖高脂组、吡格列酮干预组。稳定过表达RIP140的MIN6细胞(O-RIP140-MIN6)和过表达绿色荧光蛋白(GFP)的MIN6细胞(GFP-MIN6)。分别予高糖高脂(25 rmmol/L葡萄糖+500μmol/L棕榈酸)和/或10/μmol/L吡格列酮干预。利用MTT分别检测各组细胞增殖率、流式细胞仪检测凋亡率、RT-PCR检测RIP140 mRNA、Western blot检测B淋巴细胞瘤-2(Bcl-2)的表达、硫代巴比妥酸法检测丙二醛(MDA)水平及黄嘌呤氧化酶法检测超氧化合物歧化酶(SOD)含量。结果 NC组、高糖高脂组及吡格列酮干预组MTT吸光值分别为:24 h(1.80±0.04)、(0.95±0.04)及(0.97±0.03);48 h(2.70±0.11)、(1.04±0.06)及(1.30±0.03)。NC组与高糖高脂组比较,差异有统计学意义(24 h:t=25.94,P<0.01,48 h:t=24.00,P<0.01)。高糖高脂组与吡格列酮干预组比较,差异有统计学意义(48 h:t=9.37,P<0.01)。各组间的24 h凋亡率分别为(2.93±0.66)%、(48.08±3.95)%(vs NC组,t=19.54,P<0.01)及(31.38±3.92)%(vs高糖高脂组,t=5.20,P<0.01)。Bcl-2相对表达量分别为(1.14±0.06)、(0.42±0.02)(vs NC组,t=20.52,P<0.01)及(0.86±0.04)(vs高糖高脂组,t=17.71,P<0.01)。RIP140表达量分别为(1.13±0.11)、(2.34±0.21)(vs NC组,t=9.69,P<0.01)及(1.63±0.13)(vs高糖高脂组(t=5.03,P<0.01);高糖高脂组与NC组比较,MDA[(10.13±0.47vs(5.00±0.26)nmol/mg,t=16.57,P<0.01]、SOD[(5.15±1.07)协(12.25±1.25)nmol/mg,t=7.51,P<0.01]比较,差异均有统计学意义。高糖高脂组与吡格列酮干预组比较,MDA[(10.13±0.47)vs(7.83±0.36)nmol/mg,t=6.77,P<0.01]、SOD[(5.15±1.07)v5(8.74±0.59)nmol/mg,t=5.16,P<0.01)差异有统计学意义。O-RIP140-MIN6和GFP-MIN6细胞分别给予高糖高脂及吡格列酮处理后,两组MTT吸光值:24 h(1.04±0.07)vs(1.40±0.16)(t=5.01,P<0.01),48 h(1.16±0.13)vs(1.98±0.14)(t=10.73,P<0.01)。凋亡率为(41.95±4.88)%vs(31.26±2.86)%(t=2.97,P<0.05)、Bcl-2相对表达为(0.22±0.04)vs(0.76±0.03)(t=21.54,P<0.01),SOD为(7.53±0.71)vs(9.62±0.43)nmol/mg(t=4.36,P<0.05),MDA为(10.23±0.28)vs(8.15±0.38)nmol/mg(t=7.63,P<0.01)。结论高糖高脂促进胰岛β细胞损伤,吡格列酮通过下调RIP140表达来抑制高糖高脂对胰岛β细胞的损伤。
Objective To investigate the protective effect of pioglitazone, a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, on the glycolipid-induced pancreatic β-cell injury and the mechanism of its receptor-protein interaction 140 (RIP140). Methods The islet β cell line MIN6 cells were divided into NC group, high glucose and high fat group, pioglitazone intervention group. MIN6 cells overexpressing RIP140 (O-RIP140-MIN6) and MIN6 cells (GFP-MIN6) overexpressing green fluorescent protein (GFP). Intervention with high glucose and high fat (25 mmol / L glucose + 500 mol / L palmitic acid) and / or 10 / mol / L pioglitazone respectively. The proliferation rate of each group was detected by MTT, the apoptosis rate was detected by flow cytometry, the mRNA expression of RIP140 was detected by RT-PCR and the expression of Bcl-2 by Western blot. The thiobarbituric acid The levels of malondialdehyde (MDA) and superoxide dismutase (SOD) were detected by xanthine oxidase method. Results The MTT absorbance values in NC group, high glucose and high fat group and pioglitazone group were 24 h (1.80 ± 0.04), (0.95 ± 0.04) and (0.97 ± 0.03), 48 h (2.70 ± 0.11) and (1.04 ± 0.06) and (1.30 ± 0.03). The difference between the NC group and the high glucose and high fat group was statistically significant (24 h: t = 25.94, P <0.01, 48 h: t = 24.00, P <0.01). Compared with pioglitazone intervention group, the difference was statistically significant (48 h: t = 9.37, P <0.01). The 24 h apoptosis rates among the groups were (2.93 ± 0.66)%, (48.08 ± 3.95)% (vs NC group, t = 19.54, P <0.01) and (31.38 ± 3.92)% Group, t = 5.20, P <0.01). The relative expression of Bcl-2 was (1.14 ± 0.06), (0.42 ± 0.02) vs (NC group, t = 20.52, P <0.01) and (0.86 ± 0.04) P <0.01). (1.13 ± 0.11) and (2.34 ± 0.21), respectively (vs NC group, t = 9.69, P <0.01) and (1.63 ± 0.13) ). Compared with NC group, MDA [(10.13 ± 0.47 vs (5.00 ± 0.26) nmol / mg, t = 16.57, P <0.01] and SOD [(5.15 ± 1.07) (10.13 ± 0.47) vs (7.83 ± 0.36) nmol / mg, t (t = 7.51, P <0.01) = 6.77, P <0.01], SOD (5.15 ± 1.07) v5 (8.74 ± 0.59) nmol / mg, t = 5.16, P <0.01) .O-RIP140-MIN6 and GFP- After treatment with high glucose and high fat and pioglitazone, the MTT absorbance values of the two groups were as follows: 24 h (1.04 ± 0.07) vs 1.40 ± 0.16 (t = 5.01, P <0.01) The apoptotic rate was (41.95 ± 4.88)% vs (31.26 ± 2.86)% (t = 2.97, P <0.05) and the relative expression of Bcl-2 was (0.22 ± 0.04) (P <0.05), vs (10.23 ± 0.28) vs (0.76 ± 0.03) (t = 21.54, P <0.01) (8.15 ± 0.38) nmol / mg (t = 7.63, P <0.01) .Conclusion High glucose and high fat can promote pancreaticβcell injury, and pioglitazone inhibits the expression of RIP140 High glucose and lipid islet β cell damage.