Tsuboi Tomoe, Maeda Morihiko, Hayashi Toshio
Department of Geriatrics, Nagoya University Graduate School of Medicine School of Health Sciences, Daiko-Minami, Higashi-ku, Nagoya, Aichi, Japan.
Chubu University Graduate School of Bioscience and Biotechnology, Matsumoto-cho, Kasugai, Aichi, Japan.
PLoS One. 2018 Feb 7;13(2):e0192252. doi: 10.1371/journal.pone.0192252. eCollection 2018.
L-citrulline and L-arginine supplementation has been shown to have several beneficial effects on the cardiovascular system. Nitric oxide (NO) protects against the progression of atherosclerosis and is synthesized by nitric oxide synthase (NOS), which converts L-arginine (L-Arg) into L-citrulline (L-Cit). Our previous study revealed that chronic administration of a combination of L-Cit and L- Arg has a better therapeutic effect on high cholesterol-induced atherosclerosis in rabbits. We investigated how L-Arg and L-Cit affect endothelial function, aging and atherosclerosis. Following a 3-day stimulation of human umbilical venous endothelial cells (HUVECs) with high glucose (HG: 22 mM) and L-Arg (300 μM), L-Cit (300 μM) or L-Arg plus L-Cit (LALC: each 150 μM) supplementation, endothelial senescence and function were evaluated. These amino acids were also administered to dyslipidemic type 2 diabetic (ZDFM) rats fed a high cholesterol diet. They were fed L-Arg or L-Cit or LALC for four weeks. Aortic senescence was investigated by measuring senescence-associated ß-galactosidase (SA-ß-gal), telomerase activity, DNA damage and p16INK4a protein expression. Only L-Cit and LALC supplementation retarded the HG-induced endothelial senescence, as evaluated by SA-ß-gal activity, a widely used marker of cellular senescence, p16INK4a expression, a senescence-related protein, and DNA damage. Under HG conditions, L-Cit and LCLA restored telomerase activity to levels observed under normal glucose (NG) conditions. Under HG conditions, L-Cit decreased ROS production, as measured by CM-H2DCFDA and the expression of p67phox, a major component of NADPH oxidase. Under HG conditions, L-Cit and LALC increased NO production, as measured by DAF-2AM. Endothelial NO synthase (eNOS) and phosphorylated eNOS were decreased under HG conditions and L-Cit and LALC significantly increased these levels. Arginase 2 protein expression increased under the HG conditions, and L-Cit and LALC significantly attenuated this effect. In ZDFM rats, SA-ß-gal activity was detected on the aortic endothelial surface; however, L-Cit and LALC reduced these levels. L-Cit and LALC both decreased the proportion of senescent cells. Furthermore, treatment with LALC for 4 weeks increased plasma NO production. Therefore conclusively, L-citrulline supplementation rescued NO levels better than L-arginine supplementation by inhibiting ROS production and arginase 2 protein expression. Consequently, L-Cit and LCLA supplementation retaeded HG-induced endothelial senescence.
补充L-瓜氨酸和L-精氨酸已被证明对心血管系统有多种有益作用。一氧化氮（NO）可预防动脉粥样硬化的进展，它由一氧化氮合酶（NOS）合成，该酶将L-精氨酸（L-Arg）转化为L-瓜氨酸（L-Cit）。我们之前的研究表明，长期联合给予L-Cit和L-Arg对高胆固醇诱导的兔动脉粥样硬化具有更好的治疗效果。我们研究了L-Arg和L-Cit如何影响内皮功能、衰老和动脉粥样硬化。在用高糖（HG：22 mM）和L-Arg（300 μM）、L-Cit（300 μM）或L-Arg加L-Cit（LALC：各150 μM）补充剂对人脐静脉内皮细胞（HUVECs）进行3天刺激后，评估内皮衰老和功能。这些氨基酸也被给予喂食高胆固醇饮食的2型糖尿病血脂异常（ZDFM）大鼠。给它们喂食L-Arg或L-Cit或LALC四周。通过测量衰老相关的β-半乳糖苷酶（SA-β-gal）、端粒酶活性、DNA损伤和p16INK4a蛋白表达来研究主动脉衰老。如通过SA-β-gal活性（一种广泛使用的细胞衰老标志物）、衰老相关蛋白p16INK4a表达和DNA损伤所评估的，只有补充L-Cit和LALC能延缓HG诱导的内皮衰老。在HG条件下，L-Cit和LCLA将端粒酶活性恢复到正常葡萄糖（NG）条件下观察到的水平。在HG条件下，通过CM-H2DCFDA测量，L-Cit减少了活性氧（ROS）的产生以及NADPH氧化酶的主要成分p67phox的表达。在HG条件下，通过DAF-2AM测量，L-Cit和LALC增加了NO的产生。在HG条件下内皮型一氧化氮合酶（eNOS）和磷酸化eNOS减少，而L-Cit和LALC显著提高了这些水平。在HG条件下精氨酸酶2蛋白表达增加，而L-Cit和LALC显著减弱了这种作用。在ZDFM大鼠中，在主动脉内皮表面检测到SA-β-gal活性；然而，L-Cit和LALC降低了这些水平。L-Cit和LALC都降低了衰老细胞的比例。此外，用LALC治疗4周增加了血浆NO的产生。因此可以确定，补充L-瓜氨酸通过抑制ROS产生和精氨酸酶2蛋白表达比补充L-精氨酸能更好地挽救NO水平。因此，补充L-Cit和LCLA可延缓HG诱导的内皮衰老。
