镰形棘豆提取物对人肾小管上皮细胞转分化的影响

Study the effect of extract of Oxytropis falcate Bunge on trans-differentiation of human renal tubular epithelial cell HK-2 induced by TGF-β1

目的:本实验通过观察镰形棘豆提取物含药血清对转化生长因子-β1(TGF-β1)诱导人肾小管上皮细胞(HK-2)转分化的作用,探讨其在肾间质纤维化方面的作用及机制。方法:采用镰形棘豆提取物(300mg/kg)给大鼠灌胃,每天2次,连续3天。3天后大鼠股动脉采血,分离血清,即得镰形棘豆提取物含药血清。体外培养HK-2细胞,培养基为含10%胎牛血清的DMEM/F12(1:1);细胞分为4组:空白对照组、空白血清对照组(TGF-β1 10 ng/ml+10%空白血清)、镰形棘豆提取物组(TGF-β1 10 ng/ml+10%镰形棘豆提取物含药血清)、转化生长因子-β1诱导组(TGF-β1 10 ng/ml)。药物干预细胞后,采用免疫组化技术测α-平滑肌肌动蛋白(α-SMA)和E-钙粘蛋白(E-cadherin)的表达,ELISA技术测Ⅰ型胶原(ColⅠ)、Ⅲ型胶原(ColⅢ)和纤连蛋白(FN)的含量,荧光定量PCR检测转化生长因子-β受体I(TβRI)、受体II(TβRII)的mRNA表达,Western blot检测Smad 2、smad 3的蛋白表达。结果:正常的HK-2细胞只表达E-cadherin,不表达α-SMA。但经TGF-β1诱导后细胞转分化,E-cadherin的表达开始减弱,α-SMA表达逐渐增强,药物干预后,α-SMA表达明显减弱,E-cadherin表达增强。与空白对照组相比,TGF-β1诱导组ColⅠ、ColⅢ、FN的含量显著增加,在24h时分别为(197.4±0.7)、(32.7±0.2)、(754.5±4.1),72h时分别为(216.4±0.8)、(38.3±0.1)(995.4±1.5),P﹤0.05;与TGF-β1诱导组相比,镰形棘豆提取物组ColⅠ、ColⅢ、FN的分泌显著下降,72h时作用更为显著,含量分别为(205.5±0.7)、(35.0±0.3)、(892.4±0.9),P﹤0.05。与空白对照组相比,TGF-β1诱导组TβRI(11.08±0.10)、TβRII(11.57±0.12)的mRNA表达和Smad 2(0.971±0.017)、smad 3(0.972±0.011)的蛋白表达显著上升,P﹤0.05;与TGF-β1诱导组相比,镰形棘豆提取物组TβRI(9.788±0.379)、TβRII(8.452±0.250)mRNA表达和Smad 2(0.536±0.013)、Smad3(0.530±0.035)蛋白表达也减少,P﹤0.05。空白血清则无此作用。结论:镰形棘豆提取物能够减少细胞外基质成分分泌,调控Smads信号通路,抑制肾小管上皮细胞转分化,在一定程度上具有防治肾间质纤维化的作用。

Objective： Through studying the effect of extract of Oxytropis falcate Bunge on transdifferentiation of human renal tubular epithelial cells HK-2 induced with transforming growth factor-βl （ TGF-β1 ）, to explore the mechanism of it on renal fibrosis. Methods： Rats were given extract of Oxytropis falcate Bu.nge（300mg/kg） orally, 2 times a day for three days. Three days later, rat femoral artery blood serum was sepa- rated, that was the extract of Oxytropis falcate Bunge containing serum. The HK-2 cells were cultured in DMEM / F12（ 1：1 ）with 10% fetal bovine serum. Cells were divided into control group, rat serum control group（ TGF-β110 ng/ml ＋ 10% rat serum）, therapy group （TGF- β110 ng/ml ＋ 10% extract of Oxytropis falcate Bunge-containing rat serum）, TGF-β1 group （TGF-IM 10 ng/ml）. After 24h, α-SMA and E-cadherin expression were observed by immunohistochemistry assay, the mRNA expression of TgRI and TβRII were tested by fluorescence quantitatiye PCR, and the protein expression of Smad 2 and smad 3 by Western-blot. And after 24h, 48h and 72h, the concentration of type [ collagen, type llI collagen and FN in culture medium superuatant were detected by ELISA assay. Results： After TGF-β1 induction the ex- pression of α-SMA increased and E-cadherln was markedly decreased, but through treating with extract of Oxytropis falcate Bunge, the α- SMA began decrease and E-cadherin increase. Compared with control group, the secretion of type I collagen, type lI collagen and FN were markedly increased in TGF-β1 group, at 24h they wre respectively （ 197.4± 0.7）, （32.7 ± 0.2） and （754.5 ± 4.1 ）, at 72h they wre re- spectively （216.4 ± 0. 8）, （ 38.3 ±0.1 ） and （995.4 ± 1.5 ）, P 〈 0.05. Compared with TGF-IM group, the secretion of type I collagen, type m collagen and FN were markedly inhibited in therapy group, at 72 h the effect was the most significantly, they wre respectively（205. 5 ±0.7） ,（35.0 ±0.3） and（892.4 ±0.9） ,P 〈 0.05. The expression of TβRI （11.08 ±0.10） ,TβRII （ 11.57 ±0.12） mRNA and Smad 2 （ 0.971 ± 0.017 ） , smad 3 （ 0. 972 ± 0. 011 ） protein in TGF-β1 group were increased compared with control group （ P 〈 0. 05 ） , and signif- icantly decreased in therapy group TβRI （9.788 ±0.379） ,TβRII（8. 452 ±0.250） mRNA,Smad2（0.536 ±0.013） and smad3（0.530 ±0. 035 ） , compared with TGF-β1 group （ P 〈 0.05 ）. Rat serum had no such effect. Conclution ： The results showed that extract of Oxytropis faleate Bunge could prevent the development of renal fibrosis to a certain extent.