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目的研究平目汤对Graves眼病眼眶脂肪细胞FAS、FAS L、CHOP、CASPASE-4蛋白表达的影响,探讨眼眶成熟脂肪细胞凋亡的机制。方法体外培养Graves眼病患者眼眶前脂肪细胞,传代并诱导其分化为成熟脂肪细胞。平目汤含药血清,配成终浓度分别为5%、10%、20%的平目汤低、中、高剂量组。采用MTT法检测前脂肪细胞增殖情况。Western blotting法检测平目汤对眼眶成熟脂肪细胞FAS、FAS L、CHOP及CASPASE-4蛋白表达的影响。结果 (1)MTT法检测结果显示,与空白对照组比较,平目汤低、中、高剂量组的生长曲线显著下移,其中平目汤中剂量组活性下降趋势最明显。地塞米松组与空白对照组差异无统计学意义(P>0.05)。相同的时间点与空白对照组比较,平目汤各组OD值均减小,其中以平目汤中剂量组降低最为明显,差异有统计学意义(P<0.05)。(2)Western blotting结果显示:与空白对照组比较,除地塞米松组外,各组FAS、FAS L蛋白表达均上调,差异有统计学意义(P<0.05),其中FAS蛋白表达以平目汤中剂量组最高,其次为低剂量组,FAS L蛋白表达以平目汤高剂量组最高,其次为中剂量组,但二者比较差异无统计学意义(P<0.05)。与空白对照组比较,除地塞米松组外,平目汤各组CHOP、CASPASE-4蛋白表达均上调,差异有统计学意义(P<0.05),其中平目汤各组组间比较CHOP蛋白表达以高剂量组最高,其次为中剂量组;CASPASE-4蛋白表达以平目汤中剂量组最高,其次为低剂量组。结论平目汤通过死亡受体途径和内质网途径诱导FAS、FAS L、CHOP、CASPASE-4活化,促进成熟脂肪细胞凋亡,进而减少眼眶脂肪细胞积聚,发挥治疗效果。
Objective To study the effect of Pingmu Decoction on the expression of FAS, FAS L, CHOP and CASPASE-4 in orbital fat cells of Graves’s eye disease and to explore the mechanism of apoptosis of orbital mature adipocytes. Methods Human orbital preadipocytes were cultured in vitro and passaged and induced to differentiate into mature adipocytes. Pok nok soup containing serum, dubbed the final concentration of 5%, 10%, 20% of Pork noodle low, medium and high dose groups. The proliferation of preadipocytes was detected by MTT assay. The effects of Pingmu Decoction on the expression of FAS, FAS L, CHOP and CASPASE-4 in orbital mature adipocytes were detected by Western blotting. Results (1) The results of MTT assay showed that the growth curve of Pork decreased significantly in low, medium and high dose groups compared with the blank control group. There was no significant difference between dexamethasone group and blank control group (P> 0.05). At the same time point, compared with the blank control group, the OD value of Pokou decoction decreased, especially in the Pok decoction middle dose group, the difference was statistically significant (P <0.05). (2) Compared with the blank control group, the expression of FAS and FAS L protein in each group were up-regulated by Western blotting (P <0.05), and the expression of FAS protein was The middle dose group was the highest, followed by the low dose group. The expression of FAS L protein was the highest in the Pingtang Decoction high dose group, followed by the middle dose group, but the difference was not statistically significant (P <0.05). Compared with the blank control group, except for the dexamethasone group, the levels of CHOP and CASPASE-4 protein in Pokou Decoction group were all significantly increased (P <0.05), among which the levels of CHOP protein in each group were The highest dose group, followed by the middle dose group; CASPASE-4 protein expression Pingtang Tang middle dose group, followed by the low dose group. Conclusion Pingmu Decoction can induce the activation of FAS, FAS L, CHOP and CASPASE-4 through death receptor pathway and endoplasmic reticulum, promote the apoptosis of mature adipocytes, and then reduce the accumulation of orbital fat cells and exert the therapeutic effect.