大黄素通过PI3K／Akt／mTOR信号通路改善单侧输尿管梗阻模型小鼠肾脏纤维化

Protective effect of emodin on renal interstitial fibrosis in mice of unilateral ureterai obstruction via PI3K/Akt/mTOR signaling pathway

目的探讨大黄素（EM）在单侧输尿管梗阻（UUO）小鼠肾间质纤维化中的作川及其机制。方法雄性C57BL／6J小鼠按随机数字表被分为似手术（Sham）组（n=8）、UUO组（n=8）、UUO＋氯沙坦（LST）组（n=8）和UUO＋EM组（n=8）。UUO＋LST组和UUO＋EM组白手术日起每天分别给予10mg·kg-1·d-1LST和20mg·kg-1·d-1EM等量灌胃。LST和EM溶解于生理盐水配制的0．5％羧甲基纤维素钠。假手术组和UUO组以等量O．5％羧甲基纤维素钠灌胃。术后第14天处死小鼠取肾组织。HE、Masson及PAS染色观察肾脏组织间质纤维化分布；实时荧光定量PCR法分圳检测自噬蛋白微管相关蛋白1轻链3（LC3）、Beclin-1及哺乳动物雷帕霉素靶蛋白（mTFOR）mRNA的表达；Western印迹法分别检测转化生长因子β1（TGF—β1）、仅平滑肌肌动蛋白（α-SMA）、E钙黏蛋白（E—cadherin）、自噬标志性蛋白LC3、Beclin—1、磷脂酰肌醇-3-激酶（P13K）、p-Akt、mTOR蛋白的表达。透射电镜观察自噬体变化。结果与Sham组比较，UUO组小鼠第14天肾组织纤维化明显，TGF-β1和α—SMA表达升高（均P〈0．01），E—eadherin表达减少（P〈0．01），P13K及其下游p-Akt、mTOR表达升高（均P〈0．01）。与UUO组比较，UUO＋LST组和UUO＋EM组自噬标志性蛋白LC3和Beelin-1的表达增加（均P〈0．01），自噬体数量增加，TGF—B1和Ⅱ-SMA表达减少（均P〈0．01），E—cadherin表达增加（P〈0．05），P13K、P—Akt和mTOR表达降低（均P〈0．05），肾组织纤维化程度明显减轻。结论大黄素干预后促进UUO模型自噬增加，通过P13K／Akt／mTOR信号通路，改善肾纤维化的程度，保护肾组织。

Objective To investigate the effect and mechanism of emodin （EM） in renal interstitial fibrosis of unilateral ureteral obstruction （UUO） mice. Methods Male C57BL/6J mice were randomly divided into 4 groups, including sham operation group （n=8）, UUO operation group （n= 8）, UUO operation＋losartan （LST） group （n=8） and UUO operation＋EM group （n=8）. The mice in each group were ingested the suspensions by garage for 14 days after surgery. Mice in UUO＋LST and UUO＋ EM groups were given 10 mg· kg-1· d - LST and 20 mg· kg-1 · d-1 EM, respectively. LST and EM were mixed with 0.5% sodium carboxymethyl cellulose. Mice in sham group and UUO group were given 0.5% sodium earboxymethyl eellulose. The mice were sacrificed at the 14th day. Interstitial fibrosis was observed by HE, Masson and PAS stain. Real-time PCR was used to detect LC3, Beclin-1 and roTOR mRNA. Protein expressions of TGF-131, c^-SMA, E-cadherin, LC3, Beclin-1, PI3K, p-Akt and roTOR were detected by Western blotting. The autophagy was observed with transmission electron microscopy in the renal tissue. Results Compared with sham mice, UUO mice at the 14th day displayed obvious renal fibrosis. Meanwhile, UUO mice had increased expressions of TGF-β1 and α-SMA （all P 〈 0.01）, and decreased expressions of E-cadherin （P 〈 0.01）. Their renal expressions of PI3K, p-Akt and mTOR were also raised （all P 〈 0.01）. Compared with those in UUO group, in UUO＋LST group and UUO＋EM group, expressions of autophagy protein LC3 and Beclin- 1 were increased （all P 〈 0.01）, and the number of autophagic was increased. Additionally, expressions of TGF - β1 and α - SMA were reduced in UUO＋LST group and UUO＋EM group （all P 〈 0.01）, while the expression of E-cadherin was increased by emodin treatment （P 〈 0.05）. And expressions of PI3K, p- Akt and mTOR were decreased in UUO ＋ LST group and UUO ＋EM group （all P 〈 0.05）, meanwhile renal tissue fibrosis significantly reduced. Conclusions Emodin can promote autophagy, ameliorate renal interstitial fibrosis and protect renal function through PI3K/Akt/mTOR signaling pathway.