【作者】 张俊；

【导师】 刘晓城；

【作者基本信息】 华中科技大学 ， 内科肾脏病， 2006， 博士

【摘要】 早期肾系膜细胞的增殖以及随后的纤维化，是增殖性肾小球肾炎的显著特征；它也将最终导致终末期肾病。磷脂酰肌醇3激酶／蛋白激酶B／哺乳类雷帕霉素靶蛋白(PI3K／Akt／mTOR)信号途径现被公认是细胞生命活动的重要通路，它调控着细胞的生与死。细胞因子、高糖、机械应力等刺激因素均能通过此信号途径，诱导系膜细胞活化和增殖。姜黄素是从植物姜黄根茎中提取的主要活性成分，具有广泛的生物学效应，如抗炎、抗氧化、抗肿瘤、抗纤维化等。然而，姜黄素在肾脏病和纤维化领域的研究尚不太多。有报道姜黄素减轻阿霉素造成的肾损伤，也有体外肾成纤维细胞研究发现姜黄素能抑制转化生长因子β所致的纤维化。本实验探讨姜黄素对大鼠肾系膜细胞增殖和致纤维化的影响以及可能与磷脂酰肌醇3激酶／蛋白激酶B／哺乳类雷帕霉素靶蛋白信号通路的关系。采用四甲基偶氮唑蓝法检测药物对系膜细胞增殖的影响。姜黄素呈浓度依赖的方式抑制血清刺激系膜细胞的增殖，6.25μM即可显现19.5％的细胞生长抑制率，其后浓度越高，抑制效应越强，在100μM时达到89.7％；其半数生长抑制率约为24.92μM。磷脂酰肌醇3激酶特异抑制剂LY294002也呈浓度依赖方式抑制系膜细胞的增殖，50μM抑制约60％系膜细胞生长。哺乳类雷帕霉素靶蛋白抑制剂雷帕霉素亦随浓度增加，抑制细胞增殖效应增强；1nM雷帕霉素抑制效果达到26.7％，而100nM雷帕霉素抑制效果亦只增加到46.3％；其半数生长抑制率为381.14nM。姜黄素联合雷帕霉素对系膜细胞增殖的抑制效果较同浓度单用组强，其协同治疗指数小于1，二者起协同作用。分别用LY294002和雷帕霉素预孵育系膜细胞30min，姜黄素对系膜细胞增殖的抑制作用较未预孵育减弱；提示姜黄素的抑制效应是依赖PI3K／Akt／mTOR信号途径的。流式细胞周期分析显示姜黄素和雷帕霉素均以浓度依赖的方式阻滞系膜细胞周期于G0／G1期，中等剂量联合应用可得到较单用一种药物更强的阻滞效应。细胞同步生长后，用10％血清刺激系膜细胞，在0～120分钟内磷酸化Akt的表达呈现动态变化，15分钟即可出现明显表达，30分钟时达到最强，此后逐渐减弱，而总Akt的表达未受到明显影响。使用PI3K抑制剂LY294002和mTOR抑制剂雷帕霉素预孵育系膜细胞后，p-Akt表达较阳性对照组减弱，尤其是LY294002，这提示血清刺激的系膜细胞生长是通过PI3K／Akt／mTOR信号途径的。姜黄素对血清刺激后p-Akt的表达呈浓度依赖性抑制；而LY预孵育细胞后，其抑制作用较未预孵育减弱，提示姜黄素的抑制作用部分通过PI3K／Akt信号途径。姜黄素对血清刺激后系膜细胞CTGF和Fn mRNA的表达也起抑制作用，浓度越高其抑制作用越强。LY294002预孵育细胞后，可减弱姜黄素对CTGF和Fn mRNA表达的抑制；而雷帕霉素联合姜黄素持续作用，却可增强姜黄素对CTGF和Fn mRNA表达的抑制。姜黄素亦抑制血清刺激后系膜细胞CTGF蛋白的表达，呈浓度依赖性，LY294002的预孵育细胞可削弱其抑制CTGF蛋白表达的作用，但联合应用雷帕霉素可增强其抑制CTGF蛋白的作用。使用ELISA方法检测姜黄素对系膜细胞上清液中分泌的Fn蛋白，也可得到与CTGF蛋白表达一致的结果。上述结果表明，姜黄素以浓度依赖的方式抑制大鼠肾小球系膜细胞的增殖和致纤维化因子CTGF、细胞外基质Fn的表达；其作用的机制可能与抑制PI3K／Akt／mTOR信号途径相关。因此，姜黄素是一个潜在的抗肾纤维化的药物。更多还原

【Abstract】 Early renal mesangial cellular proliferation followed by subsequent fibrosis is a prominent hallmark of proliferative glomerulonephritis, and it may ultimately lead to end-stage renal disease. It is accepted that Phosphorylationtol 3 kinase/protein kinase B/mammalian target of rapamycin is an important pathway in cell life activity, it switchs cell life and death. Cytokine, high glucose, mechanical strain and so on can induce activation and proliferation of mesangial cells through the signaling pathway. Curcumin, a major compound extracted from the rhizomes of Curcuma species, has presents widely biological effects, such as anti inflammatory, anti oxidative, anti cancer and anti fibrosis. However, the research of curcumin on the kidney diseases and fibrosis is not well characterized. It is reported that curcumin can prevents adriamycin nephrotoxicity in rats. It is also reported that curcumin blocks TGF- β’s profibrotic actions on renal fibroblasts. In this study, we explored the effect of curcumin on proliferation and fibrogenesis in rat mesangial cells, and possible correlation with intracellular PI3K/Akt/mTOR signaling pathway.The viability and proliferation of mesangial cell was determined with MTT method (tetrazolium salt colorimetry assay); the assay is based on the principle that live cells convert the yellow tetrazolium salt into a blue formazan. After quiescent mesangial cell cultured in vitro were stimulated with 10% fetal bovine serum, curcumin reduced cell proliferation in a concentration-dependent manner. Significant inhibition of cell proliferation was observed at curcumin dosages of 6.25 μM (19.5%) and 100 μM (89.7%), the median inhibition concentration (IC50) of curcumin was approximate 24.92 μM. The specific PI3K inhibitior LY294002 also inhibit cell proliferation in a dose-dependent manner, 50 μM LY294002 can inhibit about 60% cell growth. Rapamycin, the mTOR specific inhibitior, also inhibit cell proliferation in adose-dependent manner; the inhibition effect of 1nM rapamycin achieve 26.7%, but the inhibition effect of 100nM only get 46.3% . The IC50 of rapamycin was 381.14nM. The inhibition effect of curcumin combine rapamycin on mesangial cell is stronger than the effect of curcumin or rapamycin alone, the combination index of curcumin and rapamycin is below 1, this indicating they can exert synergic action. Pretreatment with either LY294002 or rapamycin, they all can weaken the inhibition effect of curcumin. This show the inhibition effect of curcumin on mesangial cells is get across PI3K/Akt/mTOR signal pathway. Flow cytometric analysis of propidium iodide stained mesangial cell demonstrated that cell cycle progression was arrested in G0/G1 phase with curcumin or rapamycin. Curcumin combine rapamycin can strongly induced cell growth arrest in G1 phase than curcumin or rapamycin alone.We performed time course of 10% fetal bovine serum-induced Akt phosphorylation from 0 to 120 min, the phosphorylation peaked at 30 min. Total Akt protein has no change. Either LY294002 or rapamycin reduced the Akt phosphorylation, especially treatment with LY294002. These results suggested that PI3K/Akt/mTOR signal pathway is required for mesangial activation. Curcumin inhibited the expression of Akt phosphorylation in a dose dependent manner, while pretreatment with LY294002 can reduced it. This indicates that PI3K/Akt pathway is involved in growth inhibition of curcumin. Curcumin also reduced the CTGF and Fn mRNA levels. Pretreatment with LY294002 can weaken this effect, but curcumin combine with rapamycin can enhance this effect. When the expression of CTGF and Fn pretein in curcumin induced in mesangial cells was checked, similar findings were observed.These findings suggest that curcumin inhibits mesangial cell proliferation and limits the expression of CTGF and Fn, the mechanism may be involved in the inhibiton of PI3K/Akt/mTOR signal pathway. Thus curcumin might be a potential antifibrotic drug in kidney diseases.更多还原