过表达THEM4抑制高糖诱导小鼠系膜细胞细胞外基质分泌

Effects and mechanism of up-regulation of THEM4 on secretion of extracellular matrix in mouse mesangial cells treated with high glucose

目的观察过表达硫酯酶超家族成员4(thioesterase superfamily member 4,THEM4)对高糖诱导的小鼠系膜细胞细胞外基质蛋白分泌的影响。方法体外培养小鼠系膜细胞,分别给予高糖(30 mmol/L)刺激0、6、12、24和48 h后收集细胞。提取RNA,RT-PCR技术检测THEM4 mRNA的表达;提取总蛋白,Western blot法检测THEM4、phospho-Akt(Ser 473)、TGF-β1、α-SMA的表达;ELISA法检测细胞上清液Ⅳ型胶原(collagenⅣ,ColⅣ)、纤维粘连蛋白(fibronectin,FN)的分泌情况。为进一步检测过表达THEM4对高糖诱导的细胞外基质沉积的影响及可能机制,将常规培养的系膜细胞随机分为正常对照组(5.5mmol/L)、高糖组(30 mmol/L)、高糖+p Yr-ads-4-THEM4质粒转染组(高糖+THEM4组)、高糖+p Yr-adshuttle-4空质粒对照组(高糖+V组)。后3组经高糖刺激48 h后终止培养并进行上述检测。结果随着高糖刺激时间的延长,小鼠系膜细胞THEM4表达呈下降趋势,phospho-Akt(Ser 473)、TGF-β1、α-SMA的表达增强,伴随细胞外基质蛋白(包括ColⅣ、FN)的分泌增加;过表达THEM4能逆转高糖刺激所导致小鼠系膜细胞Akt蛋白的活化,下调α-SMA、TGF-β1的表达,抑制细胞外基质蛋白(包括ColⅣ、FN)的分泌。结论过表达THEM4能降低高糖诱导的小鼠系膜细胞细胞外基质蛋白的分泌,可能是通过抑制Akt蛋白的活化,下调α-SMA、TGF-β1表达而实现。

Purpose To investigate the effect and possible mechanism of thioesterase superfamily member 4（THEM4） expression on secretion of extracellular matrix in mouse mesangial cells（MMC） treated with high glucose. Methods In vitro,cultured MMC cells were respectively treated with high glucose（glucose,HG,30 mmol/L） for 0,6,12,24 and 48 h. Then cells were collected for the extractions of RNA and total protein.THEM4 mRNA was detected by real-time PCR. The THEM4,phospho-Akt（Ser 473）,TGF-β1 and α-SMA protein expressions were examined by Western blot. As well,Col Ⅳ and FN were detected using the competitive sandwich ELISA kit. In order to examine the direct effect of THEM4 expression vector on PI3 K/Akt pathway and extracellular matrix secretion, MMC cells were randomly divided into four groups： normal glucose group（Control）,high glucose group（HG）,high glucose plus p Yr-ads-4-THEM4 vector group（HG ＋ THEM4 vector） and high glucose plus p Yr-adshuttle-4 vector group（HG ＋ V vector）. After 48 h with HG stimulation,the cells were collected and relative analyses were performed. Results THEM4 expression decreased and phospho-Akt（Ser 473）,TGF-β1,α-SMA and extracellular matrix increased in MMC cells with the prolonged stimulation of high glucose. Up-regulation of THEM4 reversed high glucose-induced decreased THEM4,increased phospho-Akt（Ser 473）,TGF-β1,α-SMA,secreted extracellular matrix in MMC cells. Conclusion The up-regulation of THEM4 might decrease extracellular matrix secretion by inhibiting the phosphorylation of Akt and down-regulating the expression of TGF-β1 and α-SMA in high glucose-induced MMC cells.