CAJ | 학술논문

目的探讨乌索酸能否通过抑制高糖状态下系膜细胞内miRNA-21的过表达,上调其靶基因PTEN的表达,抑制磷脂酰肌醇-3-激酶(PI3K)-Akt-哺乳动物雷帕霉素靶蛋白(mTOR)信号通路的激活,诱导自噬,减少细胞外基质堆积,发挥其肾脏保护作用.方法高糖培养大鼠肾小球系膜细胞,以PI3K抑制剂LY294002以及不同剂量乌索酸进行干预,应用甲基噻唑基四唑(MTT)法观察细胞增殖能力,总蛋白/总细胞数测定细胞肥大,Western印迹和实时定量PCR检测PTEN-PI3K-Akt-mTOR信号通路活性、Ⅰ型胶原及自噬标志物.透射电镜观察自噬体的形成.结果与正常对照组相比,高糖培养的系膜细胞出现显著的肥大、增殖,细胞内miRNA-21表达明显上调,PTEN蛋白及mRNA的表达明显下调,p85PI3K、磷酸化(p)-Akt、p-mTOR、Ⅰ型胶原、p62/SQSTMI表达明显增加,LC3 II表达明显降低,差异均有统计学意义(均P＜ 0.01).与高糖组相比,乌索酸干预组及LY294002组细胞肥大、增殖程度均明显降低,p85PI3K、p-Akt、p-mTOR、Ⅰ型胶原、p62/SQSTMI的表达均明显降低,LC3 II表达明显升高,差异均有统计学意义(均P＜ 0.01);但LY294002组细胞内miRNA-21和PTEN的表达与高糖组差异无统计学意义,而乌索酸干预组细胞内miRNA-21的表达明显下调,PTEN的表达明显上调,差异均有统计学意义(均P＜ 0.01).结论乌索酸可能通过抑制高糖培养系膜细胞内miRNA-21的过表达,上调PTEN表达,抑制PI3K-Akt-mTOR信号通路异常活化,增强自噬从而减少细胞外基质堆积,减轻细胞的肥大、增殖.
목적 탐토오색산능부통과억제고당상태하계막세포내miRNA-21적과표체,상조기파기인PTEN적표체,억제린지선기순-3-격매(PI3K)-Akt-포유동물뢰파매소파단백(mTOR)신호통로적격활,유도자서,감소세포외기질퇴적,발휘기신장보호작용.방법 고당배양대서신소구계막세포,이PI3K억제제LY294002이급불동제량오색산진행간예,응용갑기새서기사서(MTT)법관찰세포증식능력,총단백/총세포수측정세포비대,Western인적화실시정량PCR검측PTEN-PI3K-Akt-mTOR신호통로활성、Ⅰ형효원급자서표지물.투사전경관찰자서체적형성.결과 여정상대조조상비,고당배양적계막세포출현현저적비대、증식,세포내miRNA-21표체명현상조,PTEN단백급mRNA적표체명현하조,p85PI3K、린산화(p)-Akt、p-mTOR、Ⅰ형효원、p62/SQSTMI표체명현증가,LC3 II표체명현강저,차이균유통계학의의(균P＜ 0.01).여고당조상비,오색산간예조급LY294002조세포비대、증식정도균명현강저,p85PI3K、p-Akt、p-mTOR、Ⅰ형효원、p62/SQSTMI적표체균명현강저,LC3 II표체명현승고,차이균유통계학의의(균P＜ 0.01);단LY294002조세포내miRNA-21화PTEN적표체여고당조차이무통계학의의,이오색산간예조세포내miRNA-21적표체명현하조,PTEN적표체명현상조,차이균유통계학의의(균P＜ 0.01).결론 오색산가능통과억제고당배양계막세포내miRNA-21적과표체,상조PTEN표체,억제PI3K-Akt-mTOR신호통로이상활화,증강자서종이감소세포외기질퇴적,감경세포적비대、증식.
Objective To investigate the underlying mechanism of ursolic acid in attenuating diabetic mesangial cells injury induced by high glucose (HG).Methods Rat glomerular mesangial cells were cultured in normal glucose,HG,HG with LY294002 and HG with ursolic acid.The cell proliferation and hypertrophy were detected by MTT and the ratio of total protein content to cell number.miRNA-21 was detected by quantitative real-time PCR.The PI3K-Akt-mTOR pathway,autophagy associated protein and collagen I were detected by Western blotting and quantitative realtime PCR.The autophagosomes were observed by electron microscope.Results Compared with normal control group,the cells exposed to HG showed up-regulating miRNA-21 expression(P ＜ 0.01),down-regulating PTEN protein and mRNA expression(P ＜ 0.01),up-regulating p85PI3K,phospho(p)-Akt,p-mTOR,p62/SQSTMI,collagen I expressions and down-regulating LC3II expression(P ＜ 0.01).Ursolic acid and LY294002 inhibited HG-induced mesangial cell hypertrophy and proliferation(P < 0.01),down -regulated the expressions of p85Pl3K,p-Akt,p-mTOR,p62/SQSTMI and collagen I and up-regulated the expression of LC3II(P ＜ 0.01).But LY294002 had no effect on the expression of miRNA-21 and PTEN.Ursolic acid down-regulated miRNA-21 expression(P ＜ 0.01),up-regulated PTEN protein and mRNA expression(P ＜ 0.01).Conclusion Ursolic acid may inhibit high glucose-induced mesangial cell miRNA-21 overexpression,up-regulate PTEN,inhibit the activation of PI3K-Akt-mTOR signaling pathway and the enhanced autophagy to reduce the accumulation of extracellular matrix and ameliorate cell hypertrophy and proliferation.