高糖及糖基化终末产物对肾小管上皮细胞转分化的影响及小檗碱的干预

Berberine Attenuated Epithelial Messenchymal Transition( EMT) Induced by High Glucose and Advanced Glycation End Products

目的:探讨小檗碱对糖基化终末产物(advanced glycation end product,AGE)和高糖诱导下肾小管上皮细胞转分化的影响及机制。方法:人肾小管上皮细胞株(HK-2细胞)于含10%胎牛血清的D-MEM/F12培养基中进行体外培养。细胞增殖达到70%左右,换无血清培养液继续培养12 h后,进行分组处理。分别用高糖(30 mmol)、AGE(100μg/ml)、小檗碱(10μmol)处理48 h后,倒置显微镜下观察细胞形态,原位细胞免疫组化检测AGE受体(RAGE)、α-SMA、Ⅰ型胶原蛋白(Col-Ⅰ)、平滑肌肌动蛋白(α-SMA)表达,Western blot检测RAGE、α-SMA、转化生长因子(TGF-β1)的蛋白翻译水平。结果:经高糖刺激细胞48 h后,细胞体积逐渐变大,形态变为长梭形,排列紊乱,少部分细胞脱落,高糖+AGE组细胞形态与高糖组相似;小檗碱干预后,细胞形态改变较少见,排列紊乱现象较轻微。免疫组化结果显示对照组及高糖组几乎未见RAGE阳性表达,但高糖+AGE组RAGE呈强阳性表达,Col-Ⅰ和α-SMA显著升高。小檗碱干预后可抑制细胞肥大,并使RAGE和α-SMA的水平有一定下降,同时Col-Ⅰ也有所降低;蛋白印迹法分析显示高糖和AGE可使RAGE蛋白表达显著上调,并刺激TGF-β1表达水平升高,小檗碱可抑制RAGE、TGF-β1的蛋白表达。结论:持续的高糖和AGE作用下可引起肾小管上皮细胞肥大,促进细胞外基质合成,并诱导肾小管上皮细胞转分化。小檗碱能改善高糖和AGE引起的细胞肥大和胶原蛋白合成,抑制肾小管上皮细胞转分化,其机制与下调RAGE,抑制TGF-β1表达有关。

Objective： Increased glucose and advanced glycation end products（ AGEs） are proposed to lead to diabetic kidney disease（ DKD）. Since the tubular epithelial to messenchymal transdifferentiation（ EMT） plays an important role in loss of renal function and progressive of DKD,Berberine,an herbal compound extracted from Rhizoma Coptidis,which have been reported lowering blood glucose level and improving proteinuria,we hypothesis that Berberine might attenuate EMT in tubular epithelial cells induced by high concentration of glucose and AGE. Methods： Cultured human renal tubular epithelial cell line（ HK-2 cells）,treated with high concentration of glucose（ 30 mmol）,AGE（ 100 μg / ml）,Berberine（ 10 μmol） respectively for 48 h,observed the cells＇ morphology,detected the positive deposit of AGE receptor（ RAGE）,type I collagen（ Col- Ⅰ）,and α- smooth muscle actin（ α-SMA） through immunohistochemistry. We also measured the protein translation level of RAGE and transforming growth factor- β_1（ TGF- β_1） by Western blotting. Results： After incubated with 30 mmol glucose or AGE,the cells＇ shape changed from round into fusiformis and showed hypertrophy. The positive optical density（ OD） of RAGE increased dramatically,with the Col- Ⅰ and α-SMA increased significantly compared with the normal glucose group. After treated with Berberine,there showed no more changes of morphology in the cells. The OD value of RGAE and α- SMA significantly decreased,and the Col- Ⅰ was lower. The results of West botting showed high glucose and AGE induced the RAGE up- regulting significantly,and increased the TGF- β_1 expression.Berberine inhibited the expression of RAGE and TGF- β_1 expression. Conclusion： A continuously stimulating with high glucose and AGE resulted renal tubular epithelial cells hypertrophy,increased extracellular matrix synthesis,and induced. tubular EMT. Berberine improved tubular epithelial cells hypertrophy and inhibited cell transdifferentiation. The mechanism might be related to down- regulate the expression TGF- β_1.