姜黄素对3T3-L1前脂肪细胞增殖分化的影响

Effect of curcumin on the proliferation and differentiation of 3T3-L1 preadipocytes

目的:脂肪细胞分化障碍是肥胖引发胰岛素抵抗的途径之一,观察中药姜黄提取物姜黄素对3T3-L1前脂肪细胞增殖分化的影响,在细胞水平分析姜黄素防治糖尿病可能的机制。方法:实验于2005-12/2006-03在哈尔滨医科大学营养与食品卫生学教研室完成。以3T3-L1前脂肪细胞为靶细胞,在常规培养时,分别加入0,5,10,15,20,25,30,35,40,50,80和100μmol/L浓度的姜黄素(购自美国Sigma公司),每个剂量设12个平行孔,培养3d,在此期间,分别在24,48,72h时,于姜黄素各剂量组中分别选取4个平行孔,以MTT法测定其增殖情况。在诱导分化时,设一组空白对照,实验组与诱导液同步加入0,2.5,5,10,15和20μmol/L的姜黄素,于诱导分化的第6天,采用油红O染色方法测定3T3-L1前脂肪细胞分化程度。结果:①姜黄素作用3T3-L1前脂肪细胞48h,5,10μmol/L剂量组的细胞吸光度值与空白对照相比显著增加(0.67±0.01,0.69±0.02,0.57±0.01,P<0.01),15μmol/L剂量组细胞吸光度值与空白对照相比差异无显著性意义(0.54±0.01,P>0.05),其余剂量组(20,25,30,35,40,50,80和100μmol/L)细胞吸光度值显著降低(0.53±0.01,0.47±0.01,0.42±0.03,0.45±0.03,0.18±0.01,0.2±0.01,0.16±0.04,0.44±0.05,P<0.01)。②姜黄素作用细胞48h时,促进或抑制细胞增殖的作用最强,40μmol/L以上浓度的姜黄素出现细胞毒性作用,大部分细胞成片脱落死亡。72h时,15～35μmol/L姜黄素组细胞的生长率有所增加,而40μmol/L以下姜黄素处理组细胞生长率仍维持在较低水平。③脂肪细胞油红O染色分光光度法结果显示2.5,5,10,15和20μmol/L姜黄素剂量组的细胞吸光度值与空白对照相比显著增加(0.38±0.05,0.49±0.04,0.56±0.06,0.75±0.06,0.77±0.1,0.83±0.06,0.38±0.05,P<0.05～0.01)。结论:不同浓度姜黄素对3T3-L1前脂肪细胞具有促进增殖和抑制增殖双重作用,低浓度姜黄素促进细胞增殖,高浓度姜黄素抑制细胞增殖。姜黄素通过促进3T3-L1前脂肪细胞的分化,从而增加脂肪细胞对胰岛素的敏感性,这可能是其防治糖尿病的机制之一。

AIM： The dysdifferentiation of adipocyte is one of ways in insulin resistance induced by obesity. To investigate the effect of extracts from curcumin on proliferation and differentiation of 3T3-L1 preadipocyte, and analyze the possible mechanism of curcumin in the prevention of diabetes from cellular level. METHODS： The experiment was conducted in the Department of Nutrition and Food Hygiene, Harbin Medical University between December 2005 and March 2006. 3T3-L1 preadipocytes were taken as the target cells, in which the curcumin （bought from America Sigma Company） were added at different concentrations of 0, 5, 10, 15, 20, 25, 39, 35, 40, 50, 80, 100 μmol/L respectively during routine culture with 12 pores for each concentration for totally 3 days. Four parallel pores were selected from each curcumin group respectively at the 24^th, 48^th and 72^nd hour, and MTT was adopted to detect the proliferation. One group was taken as the blank control group during induced-differentiation. The cells curcumin in the experiment group were cultured with inducer and 0,2.5,5,10,15 and 20 μmol/L curcumin respectively. On the 6^th day of induceddifferentiation, the differentiation of preadipocyte 3T3-L1 was detected by oil red O staining. RESULTS：① Forty-eight hours after the effect of curcumin, the absorbanee of cells was obviously increased in the 5 and 10 μmol/L groups than the blank control group （0.67±0.01,0.69±0.02,0.57±0.01 ,P 〈 0.01）,while there were no significant differences in absorbanee between the 15/μmol/L group and blank control group （0.54±0.01,P 〉 0.05）. The absorbanee in other groups （20,25,30,35,40,50,80 and 100 μmol/L） were obviously decreased （0.53±0.01,0.47±0.01,0.42±0.03,0.45±0.03, 0.18±0.01,0.2±0.01,0.16±0.04,0.44±0.05,P 〈 0.01）. ②The promoting and inhibiting effects of eureumin were the best on the 48^th hour, and there was eytotoxie effect at the concentration above 40 μmol/L with most cells died in great amounts. The growth rate of cells in 15, 20, 25, 30 μmol/L eureumin groups were increased at the 724 hour, while that in eurcumin groups at the concentration below 40 μmol/L were in lower levels. ③ Results of oil red O staining showed that the absorbanee in the 2.5, 5, 10, 15 and 20 μmol/L eureumin groups were obviously increased than the blank control group （0.38 ±0.05,0.49 ±0.04,0.56 ±0.06,0.75 ±0.06, 0.77±0.1,0.83±0.06,0.38±0.05,P 〈 0.05-0.01 ）. CONCLUSION： Cureumin has dual effects of on 3T3-L1 preadipoeyte, which can promote the cell proliferation at low concentration and inhibite the cell proliferation at high concentration. Cureumin may enhance the sensitivity of adipoeyte to insulin by promoting differentiation of 3T3-L1 preadipocyte, which may be one of important mechanisms of anti-diabetes.