Ávila-Mendoza José, Mora Janeth, Carranza Martha, Luna Maricela, Arámburo Carlos
Departamento de Neurobiología Celular y Molecular, Instituto de Neurobiología, Campus Juriquilla, Universidad Nacional Autónoma de México, Querétaro, Qro. 76230, Mexico.
Departamento de Neurobiología Celular y Molecular, Instituto de Neurobiología, Campus Juriquilla, Universidad Nacional Autónoma de México, Querétaro, Qro. 76230, Mexico.
Gen Comp Endocrinol. 2016 Aug 1;234:57-67. doi: 10.1016/j.ygcen.2016.04.004. Epub 2016 Apr 7.
It is known that growth hormone (GH) is expressed in extrapituitary tissues, including the nervous system and ocular tissues, where it is involved in autocrine/paracrine actions related to cell survival and anti-apoptosis in several vertebrates. Little is known, however, in reptiles, so we analyzed the expression and distribution of GH in the eye of green iguana and its potential neuroprotective role in retinas that were damaged by the intraocular administration of kainic acid (KA). It was found, by Western blotting, that GH-immunoreactivity (GH-IR) was expressed as two isoforms (15 and 26kDa, under reducing conditions) in cornea, vitreous, retina, crystalline, iris and sclera, in varying proportions. Also, two bands for the growth hormone receptor (GHR)-IR were observed (70 and 44kDa, respectively) in the same tissues. By immunofluorescence, GH-IR was found in neurons present in several layers of the neuroretina (inner nuclear [INL], outer nuclear [ONL] and ganglion cell [GCL] layers) as determined by its co-existence with NeuN, but not in glial cells. In addition, GH and GHR co-expression was found in the same cells, suggesting paracrine/autocrine interactions. KA administration induced retinal excitotoxic damage, as determined by a significant reduction of the cell density and an increase in the appearance of apoptotic cells in the INL and GCL. In response to KA injury, both endogenous GH and Insulin-like Growth Factor I (IGF-I) expression were increased by 70±1.8% and 33.3±16%, respectively. The addition of exogenous GH significantly prevented the retinal damage produced by the loss of cytoarchitecture and cell density in the GCL (from 4.9±0.79 in the control, to 1.45±0.2 with KA, to 6.35±0.49cell/mm(2) with KA+GH) and in the INL (19.12±1.6, 10.05±1.9, 21.0±0.8cell/mm(2), respectively) generated by the long-term effect of 1mM KA intraocular administration. The co-incubation with a specific anti-GH antibody, however, blocked the protective effect of GH in GCL (1.4±0.23cell/mm(2)) and INL (11.35±1.06), respectively. Furthermore, added GH induced an increase of 90±14% in the retinal IGF-I concentration and the anti-GH antibody also blocked this effect. These results indicate that GH and GHR are expressed in the iguana eye and may be able to exert, either directly of mediated by IGF-I, a protective mechanism in neuroretinas that suffered damage by the administration of kainic acid.
已知生长激素（GH）在垂体外组织中表达，包括神经系统和眼组织，在几种脊椎动物中，它参与了与细胞存活和抗凋亡相关的自分泌/旁分泌作用。然而，在爬行动物中却知之甚少，因此我们分析了绿鬣蜥眼中GH的表达和分布及其在经眼内注射海藻酸（KA）损伤的视网膜中的潜在神经保护作用。通过蛋白质免疫印迹法发现，在角膜、玻璃体、视网膜、晶状体、虹膜和巩膜中，GH免疫反应性（GH-IR）以两种异构体（还原条件下为15和26kDa）的形式表达，比例各不相同。同样，在相同组织中观察到生长激素受体（GHR）-IR的两条条带（分别为70和44kDa）。通过免疫荧光法发现，神经视网膜的几层（内核层[INL]、外核层[ONL]和神经节细胞层[GCL]）中的神经元存在GH-IR，这是通过其与NeuN共存确定的，但在神经胶质细胞中未发现。此外，在同一细胞中发现了GH和GHR的共表达，表明存在旁分泌/自分泌相互作用。KA给药诱导了视网膜兴奋性毒性损伤，这是通过细胞密度显著降低以及INL和GCL中凋亡细胞出现增加来确定的。针对KA损伤，内源性GH和胰岛素样生长因子I（IGF-I）的表达分别增加了70±1.8%和33.3±16%。添加外源性GH显著预防了GCL（从对照组的4.9±0.79，到KA处理后的1.45±0.2，再到KA+GH处理后的6.35±0.49个细胞/mm²）和INL（分别为19.12±1.6、10.05±1.9、21.0±0.8个细胞/mm²）中因1mM KA眼内长期给药产生的细胞结构和细胞密度丧失所导致的视网膜损伤。然而，与特异性抗GH抗体共同孵育分别阻断了GH对GCL（1.4±0.23个细胞/mm²）和INL（11.35±1.06）的保护作用。此外，添加的GH使视网膜IGF-I浓度增加了90±14%，抗GH抗体也阻断了这种作用。这些结果表明，GH和GHR在鬣蜥眼中表达，并且可能能够直接或通过IGF-I介导，对因海藻酸给药而受损的神经视网膜发挥保护机制。
