模拟失重大鼠胸主动脉和腹主动脉溶酶体相关分子表达改变

谷政慧; 李方方; 谢小萍; 暴军香

doi:10.12125/j.chj.202010018

模拟失重大鼠胸主动脉和腹主动脉溶酶体相关分子表达改变

Remodeling of lysosomal expression and regulation in thoracic and abdominal aorta of simulated weightless rats

摘要

摘要:
目的研究四周尾部悬吊模拟失重大鼠胸主动脉（thoracic aorta, TA）和腹主动脉（abdominal aorta, AA）溶酶体相关分子的基因和蛋白表达改变。
方法采用4周尾部悬吊大鼠模拟失重对心血管系统的影响，通过Western blot技术、免疫组织化学染色和实时定量聚合酶链反应（quantitative real-time polymerase chain reaction，qRT-PCR）检测溶酶体相关膜蛋白1（lysosomal associated membrane protein 1, LAMP1），调控溶酶体合成的转录因子EB（transcription factor EB，TFEB）、转录因子E3（transcription factor E3, TFE3）以及溶酶体相关基因包括LAMP1、酸性神经酰胺酶（N-acylsphingosine amidohydrolase 1，ASAH1)、液泡型ATP酶转运协助蛋白1（adenosine triphosphatase (ATPase) H+ transporting accessory protein 1，ATP6AP1）、液泡型ATP酶H亚基（ATPase H+ transporting V1 subunit H，ATP6V1H）以及粘脂蛋白1（mucolipin 1，MCOLN1）的蛋白表达、分布和mRNA水平变化。
结果 Western blot显示，与对照（control, CTR）组相比，悬吊（hindlimb unloading tail suspension, HU）组大鼠TA和AA的LAMP1蛋白表达均显著降低（P<0.05）。免疫组化则显示LAMP1主要分布于TA和AA的内膜和中膜；CTR大鼠TA内膜、中膜的LAMP1蛋白表达均显著高于AA（P<0.05）；与CTR组相比，HU组大鼠TA、AA内膜LAMP1表达无明显变化，但在TA中膜呈现均匀的表达降低, 而在AA中膜则呈现不均匀的表达降低（P<0.05）；qRT-PCR显示，与CTR组比较，溶酶体相关基因表达在HU组大鼠TA显著增加（P<0.05），但在AA则显著降低（P<0.05）。转录因子TFEB和TFE3呈现与溶酶体相关基因相同变化，蛋白表达在HU组大鼠TA和AA中均显著降低（P<0.05），但基因表达在TA增加，在AA降低（P<0.05）。
结论 HU组大鼠TA和AA溶酶体相关蛋白表达均降低，但基因转录及转录因子表达呈现部位特异性改变，可能参与动脉重塑过程。

Abstract:
AIM To investigate the remodeling of lysosomal expression and regulation in thoracic and abdominal aorta (TA and AA) in simulated weightlessness rats by hindlimb unloading tail suspension (HU) for 4 weeks.
METHODS Four weeks’ HU rat model was used to simulate the effect of weightlessness on the cardiovascular system. Western blotting, immunohistochemical staining and the quantitative real-time polymerase chain reaction (qRT-PCR) were conducted to examine the protein levels of lysosomal associated membrane protein 1 (LAMP1), transcription factor (TFEB), transcription factor E3 (TFE3) and the mRNA abundance of TFEB, TFE3 and lysosome related genes, including LAMP1, N-acylsphingosine amidohydrolase 1 (ASAH1), adenosine triphosphatase (ATPase) H+ transporting accessory protein 1 (ATP6AP1), ATPase H+ transporting V1 subunit H (ATP6V1H) and mucolipin 1 (MCOLN1) in TA and AA.
RESULTS Western blot analysis showed that compared with those in control (CTR) group, the protein levels of LAMP1 of both TA and AA were decreased in HU group (P<0.05). Immunohistochemical staining indicated that LAMP1 was mainly distributed in the intima and the media of TA and AA. Notably, after 4 weeks’ tail suspension, the protein level of LAMP1 in the intima of TA showed no significant difference, while it decreased evenly in the media (P<0.05). However, though the protein level of LAMP1 in the intima of AA showed no significant difference, it decreased unevenly in the media in HU rats (P<0.05). The protein expression of LAMP1 was higher in TA than that in AA either in the intima or the media in CTR group (P<0.05). qRT-PCR results showed that compared with that in CTR group, the mRNA abundance of lysosome related genes was increased in TA, but decreased in AA. The expressions of TFEB and TFE3 showed similar trend to those of lysosome related genes. Compared with those in CTR, the protein levels of TFEB and TFE3 were reduced both in TA and AA in HU rats, while the transcription levels of TFEB and TFE3 were increased in TA, but decreased in AA (P<0.05).
CONCLUSION The amount of lysosomal related proteins of both TA and AA is reduced in HU group, but the transcription levels of TFEB, TFE3 and lysosome related genes display region-specific remodeling, which may contribute to the remodeling of vascular wall.

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