温莪术对TGF-β_1诱导的肾小管上皮细胞转分化的影响

Effect of Curcuma Aromatica on Epithelial-Myofibroblast Transdifferentiation of NRK-52E Cells Induced by TGF-β_1

目的探讨温莪术对转化生长因子-β1(transforming growth factor-β1,TGF-β1)诱导的肾小管上皮细胞转分化(epithelial-myofibroblast transdifferentiation,EMT)拮抗作用。方法将体外培养的正常肾小管上皮细胞随机分为6组:正常对照组(C组)、TGF-β1诱导模型组(T组)、温莪术低浓度组(E1组)、温莪术中浓度组(E2组)、温莪术高浓度组(E3组)、盐酸贝那普利组(Y组),除C组外,各组TGF-β1诱导24h后,温莪术各剂量组及Y组加入药物作用48h。运用倒置显微镜观察细胞形态的改变,细胞免疫荧光法、RT-PCR法及ELISA法检测各组NRK-52E细胞EMT过程中细胞骨架成分β-肌动蛋白(β-actin)的分布,α-平滑肌肌动蛋白(α-smoothmus-cleactin,α-SMA)mRNA、E钙黏蛋白(E-cadherin,E-cad)mRNA表达,纤维粘连蛋白(fibro nectin,FN)浓度。结果 TGF-β1诱导3天后,T组细胞出现肥大、拉长,呈梭形,细胞骨架结构β-actin表达增多(P<0.05),胞浆内出现束状纤维样结构,细胞内α-SMA mRNA表达显著上调(P<0.05),E-cadmRNA表达降低(P<0.05),细胞上清液中FN浓度升高(P<0.05)。与T组比较,E1-3组细胞仅见部分呈成纤维样改变,伴随胞浆内β-actin表达及FN浓度的降低(P<0.05),E2-3组细胞内α-SMA mRNA表达升高(P<0.05),E-cadmRNA表达减少(P<0.05),但E1组E-cad及α-SMA mRNA表达量与之比较,差异无统计学意义(P>0.05)。与E1组比较,E2-3组胞浆内β-actin蛋白表达及FN浓度降低(P<0.05),E3组细胞内E-cadmRNA表达升高伴α-SMA mRNA表达降低(P<0.05)。与Y组比较,E1组细胞浆内β-actin mRNA及细胞上清液中FN浓度均升高(P<0.05),E3组β-actin表达升高(P<0.05),E-cad mRNA表达降低(P<0.05),E1-3α-SMA mRNA表达量则差异无统计学意义(P>0.05)。结论温莪术可抑制TGF-β1诱导的EMT发生,可作为临床治疗慢性肾功能不全的有效药物之一。

Objective To observe the antagonist effect of Curcuma Aromatica （CA） on renal tubular epithelial-myofibroblast transdifferentiation （EMT） induced by transforming growth factor-β1 （TGF- β1 ）. Methods Normal renal tubular epithelial NRK-52E cells in vitro cultured were randomly divided into 6 groups, i.e., the normal control group （Group C）,the TGF-β1 induced model group （Group T）,the low dose CA treated group （Group El）, the moderate dose CA treated group （Group E2） ,the high dose CA group （Group Es ） ,and the Benazepril Hydrochloride Tablet treated group（ Group Y）. Except Group C ,cor- responding medication （with an action of 48 h） was administered to cells in the rest groups after they were induced by TGF-β1 for 24 h. The morphological changes were observed by inverted phase contrast microscope. The distribution of β-actin protein was detected by immunohistochemical assay. The mRNA expressions of eL-smooth muscle actin （ot-SMA） and E-cadherin （E-cad） were detected by real-time PCR. The concentration of fibronectin （FN） was detected by ELISA. Results After induced by TGF-β1 for three days, hypertrophy and elongated cells in fusiform-shape occurred,with increased expressions of β-actin protein in the cytoskeletal structure （P 〈0.05）, bundle fibrous structure occurred inside cytoplasm withsignificantly up-regulated intracellular （x-SMA mRNA expressions （P 〈0.05）, E-cad mRNA expression decreased （P 〈0.05）, the FN content in the supernate increased （P 〈0.05） in Group T. Compared with Group T, partial cells in Group El, E2, and E3 showed fibrous changes, accompanied with decreased ex- pression of 13-actin protein and FN concentration （P 〈0.05）. The expression of （X-SMA mRNA increased and the expression E-cad mRNA decreased in Group E2 and E3（both P 〈0.05）. But there was no statisti- cal difference in the expression levels of E-cad and （x-SMA mRNA（P 〉0.05）. Compared with Group El, the expression of I^-actin protein and FN concentration decreased in Group E2 and E3 （P 〈0.05）. The ex- pressions of （x-SMA mRNA decreased and E-cad mRNA increased in Group E3（P 〈0.05）. Compared with Group Y, the expression of 13-actin mRNA and FN concentration increased in Group E1 （P 〈0.05） ; the ex pression of β-actin mRNA increased in Group E3（P 〈0.05） ; the expression of E-cad mRNA decreased in Group E3（P 〈0.05）. There was no statistical difference in the expression of （x-SMA mRNA among Group E1, E2, and E3 （P 〉0.05）. Conclusion CA could inhibit the occurrence of TGF-β1 induced EMT,which could be used as an effective drug for treating chronic renal insufficiency.