美伐他汀对甲状腺相关眼病眶前脂肪细胞分化及炎症反应的抑制作用

Inhibition of mevastatin on inflammation and differentiation of orbital preadipocytes in thyroid-associated ophthalmopathy

背景脂肪增生和炎症反应是甲状腺相关眼病（TAO）活动期的两大主要病理过程，美伐他汀被证明有抑制前脂肪细胞分化的作用。目的观察美伐他汀对TAO来源眼眶前脂肪细胞分化的作用，及对环氧合酶-2（COX-2）与过氧化物酶体增生物激活受体-γ（PPAR-γ）表达的影响，探讨其对脂多糖（LPS）诱导炎症反应及TAO眼眶前脂肪细胞分化的调控作用。方法从4例TAO患者眼眶减压术中获取眼眶球后脂肪组织，用组织块培养法体外培养TAO眼眶成纤维细胞，为观察美伐他汀对TAO炎症反应过程中COX-2的作用，将培养细胞分为5个组，A组用1000μg／L的LPS，B、C、D组采用1000μg／LLPS分别联合5、10、20μmol／L美伐他汀，E组不加任何干预药物作为对照，均作用8h。观察美伐他汀对眶脂肪细胞分化后的作用，将A组再亚分为A1-A6组，1000μg／LLPS作用8h后，加入诱导液诱导各组细胞向脂肪细胞分化，A，组不加任何干预药物，A2、A3、A4组分别在分化全程中加入5、10、20μmol／L美伐他汀，A5、A6组分别在分化的第4天与第8天加入10μmol／L美伐他汀直至分化结束。采用油红O染色法测定分化后脂肪细胞的相对含量，应用Western blot法和逆转录聚合酶链反应（RT-PCR）法检测成纤维细胞中COX-2和PPAR—v蛋白及其mRNA的表达，用酶联免疫吸附试验（ELISA）检测细胞培养上清液中前列腺素E2（PGE，）的表达。结果B、C、D组眶成纤维细胞中COX-2蛋白及其mRNA的表达和PGE，的分泌水平均较A组明显降低（P〈0．05）。随着美伐他汀浓度的升高，COX-2蛋白及其mRNA的表达和PGE2的分泌水平均逐渐降低，各组间总体差异均有统计学意义（F=228．380、101．745、1586．881，P〈0．05）。E组COX-2蛋白及其mRNA的表达和PGE，的分泌水平较A、B、C组明显减弱，差异均有统计学意义（P〈0．05），但与D组比较差异无统计学意义（P〉0．05）。A1、A2、A3、A4组前脂肪细胞分化后吸光度（A492）值逐渐下降，分化后的细胞PPAR-γ蛋白及其mRNA表达均依次下降，组间两两比较差异均有统计学意义（P〈0．05）；A1与A6组间的A492值、PPAR-1蛋白及其mRNA的表达差异均无统计学意义（P〉0．05）；A1、A5、A3组的A492值及PPAR-γ蛋白及其mRNA表达均依次下降，组间比较差异均有统计学意义（P〈0．05）。结论美伐他汀以剂量依赖的方式抑制LPS激活的TAO眼眶成纤维细胞中COX-2表达、TAO眼眶前脂肪细胞的分化、PPAR-γ的表达和PGE2的分泌，前脂肪细胞分化的早期抑制作用更强。

Background Inflammation and adipogenesis are two parallel processes with increasing aetivity in severe thyroid-associated ophthalmopathy （TAO） , and mevastatin was proved to have the inhibiting effect on the differentiation of adipose. Objective The aim of this work was to investigate the effects of mevastatin on the expression of cyclooxygenase-2 （COX-2） and peroxisome proliferator activated reeeptor-γ（PPAR-γ） and differentiation of TAO-derived orbital preadipoeytes,and explore its modulation effects on lipopolysaccharide （LPS）- induced inflammation and the differentiation of TAO-derived orbital preadipocytes in vitro. Methods The retroorbital adipose tissue was obtained from 4- TAO patients during the surgery. The orbital fibroblasts were cultured from orbital adipose tissues using explant culture method. To study the suppressing effect of mevastatin on inflammatory response, cultured cells were divided into 5 groups. The 1000 μg/L LPS orbital fibroblasts were stimulated for 8 hours in group A,and 1000 Ixg/L LPS combined with 5 μmol/L, 10 μmol/L or 20 μmol/L mevastatin were used respectively for the substitute in the group B, group C and group D. The orbital fibroblasts in group E were cultured routinely without any intervention as control. To observe the inhibiting effect of mevastatin on the differentiation of adipose,the group A were then subdivided into group A1 -A6. After 1000 μg/L LPS was used to treat the cells for 8 hours, the cells were induced to differentiate into adipocytes. All orbital preadipoeytes from A1 to A6 were stimulated to differentiate into mature adipocytes with cocktail differentiation medium for a 10-day duration. During the procedure,group A2 ,A3 and A4 were interfered with 5,10 or 20 μmol/L mevastatin, and in the group A5 and A6, 10 μmol/L mevastatin were added at the fourth day or eighth day. Intracellular fat accumulation in differentiated adipoeytes was determined by oil red O staining. The absorption （A492nm ） was measured in the cells by enzyme-linked immunosorbent assay （ ELISA ）. Expression of COX-2 and PPAR-3, mRNA was detected by reverse transcription polymerase chain reaction （RT-PCR）,and the expression of COX-2 and PPAR-γ protein was detected by Western- blot. The level of PGE2 in the supernatant was detected by ELISA. Results The expression of COX-2 protein and mRNA as well as the PGE2 levels in B, C, D group deereased markedly in comparison with those in A group （P〈 0.05）. With the increase of mevastatin concentration, the expression of COX-2 protein and mRNA as well as the PGEz levels in B ,C ,D groups decreased successively（ F=228. 380,101. 745,1586. 881 ,P〈0.05 ）. The expression of COX-2 protein and mRNA and PGE2 levels in E group were lower significantly than those in A,B and C groups （P〈 0.05） ,hut no significant differences were found between E group and D group （P〉 0.05 ）. The A492 value and the expressions of PPAR-γ protein and mRNA in differentiated cells showed the successively decrease in A1 -A4 group with the elevation of mevastatin concentration （P〈0.05） , and the evidently decreased A492 value and the expressions of PPAR-γ protein and mRNA also were seen in A1 and A5 groups compared with A3 group （P 〈 0.05 ）. Conclusions Mevastatin inhibits LPS-induced COX-2 expression, PPAR-γ expression, PGE2 secretion and differentiation of TAO-derived orbital fibroblasts in vitro in dose-dependent manner. Mevastatin plays these effect more prominently in early stage of adipocytes differentiation.