Shi Huiying, Zhao Xinyan, Ding Zhen, Han Chaoqun, Jiang Ye, Qian Wei, Lin Rong, Hou Xiaohua
Department of Gastroenterology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Wuhan Third Hospital, Wuhan, China.
Cell Physiol Biochem. 2017;42(6):2418-2429. doi: 10.1159/000480184. Epub 2017 Aug 21.
BACKGROUND/AIMS: Dysfunctional autophagy has been reported to be associated with aberrant intestinal metabolism. Amino acids can regulate autophagic activity in intestinal epithelial cells (IECs). Na+/H+-exchanger 3 (NHE3) has been found to participate in the absorption of amino acids in the intestine, but whether NHE3 is involved in the regulation of autophagy in IECs is unclear.
In the present study, an amino acid starvation-induced autophagic model was established. Then, the effects of alanine and proline with or without the NHE inhibitor 5-(N-ethyl-N-isopropyl) amiloride (EIPA) were evaluated. Autophagy was examined based on the microtubule-associated light chain 3 (LC3) levels, transmission electron microscopy (TEM), tandem GFP-mCherry-LC3 construct, sequestosome-1 (SQSTM1, P62) mRNA and protein levels, and autophagy-related gene (ATG) 5, 7, and 12 expression levels. The autophagic flux was evaluated as the ratio of yellow (autophagosomes) to red (autolysosomes) LC3 puncta.
Following amino acid starvation, we found the LC3-II and ATG expression levels were enhanced in the IEC-18 cells. An increase in the number of autophagic vacuoles was concomitantly observed by TEM and confocal microscopy. Based on the results, supplementation with either alanine or proline depressed autophagy in the IEC-18 cells. Consistent with the elevated LC3-II levels, ATG expression increased upon NHE3 inhibition. Moreover, the mCherry-GFP-LC3 autophagic puncta representing both autophagosomes and autolysosomes per cell increased after EIPA treatment.
These results demonstrate that NHE (most likely NHE3) may participate in the amino acid regulation of autophagy in IECs, which would aid in the design of better treatments for intestinal inflammation.
背景/目的：据报道，自噬功能障碍与肠道代谢异常有关。氨基酸可调节肠上皮细胞（IECs）的自噬活性。已发现钠氢交换体3（NHE3）参与肠道氨基酸的吸收，但NHE3是否参与IECs自噬的调节尚不清楚。
在本研究中，建立了氨基酸饥饿诱导的自噬模型。然后，评估了添加或不添加NHE抑制剂5-（N-乙基-N-异丙基）氨氯吡咪（EIPA）的丙氨酸和脯氨酸的作用。基于微管相关轻链3（LC3）水平、透射电子显微镜（TEM）、串联GFP-mCherry-LC3构建体、聚集体蛋白-1（SQSTM1，P62）mRNA和蛋白水平以及自噬相关基因（ATG）5、7和12的表达水平来检测自噬。自噬流通过黄色（自噬体）与红色（自溶酶体）LC3斑点的比率来评估。
氨基酸饥饿后，我们发现IEC-18细胞中LC3-II和ATG表达水平升高。通过TEM和共聚焦显微镜同时观察到自噬泡数量增加。基于这些结果，添加丙氨酸或脯氨酸均可抑制IEC-18细胞中的自噬。与LC3-II水平升高一致，NHE3抑制后ATG表达增加。此外，EIPA处理后，每个细胞中代表自噬体和自溶酶体的mCherry-GFP-LC3自噬斑点增加。
这些结果表明，NHE（很可能是NHE3）可能参与IECs中氨基酸对自噬的调节，这将有助于设计更好的肠道炎症治疗方法。
