Department of Ophthalmology, Affiliated Hospital of Weifang Medical University, Weifang, China.
Eur Rev Med Pharmacol Sci. 2018 May;22(10):2941-2948. doi: 10.26355/eurrev_201805_15048.
To explore the mechanism of HOTTIP in diabetic retinopathy.
The diabetic rat model was established by a single intraperitoneal injection of streptozocin (STZ). The expression of HOTTIP in the retina of diabetic mice and wild-type mice was detected by reverse transcriptase-polymerase chain reaction (RT-PCR). The wild-type and diabetic rats were injected with HOTTIP shRNA or Scr shRNA adenovirus, and the down-regulated expression of HOTTIP was accessed by RT-PCR. Visual electrophysiology (ERG) was performed to detect the effect of HOTTIP on visual function in rats. Western blot was carried out to detect the expressions of ICAM-1 (intercellular cell adhesion molecule-1), VEGF (vascular endothelial growth factor) and TNF-α (tumor necrosis factor-α) in the retina of rats in each group. Small RNA interference decreased the expression of HOTTIP in RF/6A cells, and then, stimulated with high glucose (or H2O2). The viability of RF/6A cells was detected by MTT. Cell apoptosis was determined by flow cytometry. Western blot was carried out to determine the activation of p38, JNK (c-Jun N-terminal kinase) and ERK1/2 (extracellular regulated protein kinases) in RF/6A cells after high glucose and HOTTIP downregulation, and to investigate whether HOTTIP could activate Mitogen-activated protein kinase (MAPK) Signaling thus regulating the function of retinal endothelial cells.
HOTTIP was significantly upregulated in the retina of diabetic rats and mice. RT-PCR showed that the expression of HOTTIP in the retina of diabetic rats injected with HOTTIP shRNA adenovirus was down-regulated. There was no significant change after injection of shRNA NC adenovirus. Down-regulation of HOTTIP can reduce the visual function decline and apoptosis of retinal cells caused by diabetes. It also reduced the expression of ICAM-1 and VEGF inflammatory factors in the retina. After high glucose or H2O2 treatment, the viability of RF/6A cells decreased, and the viability of living cells was further decreased after HOTTIP was reduced. Down-regulation of HOTTIP resulted in decreased phosphorylation of p38, but had no effect on phosphorylation of ERK1/2 or JNK1/2. Upregulated HOTTIP could increase the viability of RF/6A cells, which was reversed by pretreatment of a p38 inhibitor, SB23580. However, ERK inhibitor or JNK inhibitor had no effects on cell viability.
HOTTIP improves diabetic retinal microangiopathy through the p38-MAPK pathway. HOTTIP is expected to become a new target for the treatment of diabetic microangiopathy.
探讨 HOTTIP 在糖尿病视网膜病变中的作用机制。
通过单次腹腔注射链脲佐菌素（STZ）建立糖尿病大鼠模型。采用逆转录-聚合酶链反应（RT-PCR）检测糖尿病小鼠和野生型小鼠视网膜中 HOTTIP 的表达。将野生型和糖尿病大鼠分别注射 HOTTIP shRNA 或 Scr shRNA 腺病毒，通过 RT-PCR 检测 HOTTIP 的下调表达。进行视觉电生理学（ERG）检测 HOTTIP 对大鼠视觉功能的影响。采用 Western blot 检测各组大鼠视网膜中细胞间黏附分子-1（ICAM-1）、血管内皮生长因子（VEGF）和肿瘤坏死因子-α（TNF-α）的表达。小干扰 RNA 降低 RF/6A 细胞中 HOTTIP 的表达，然后用高葡萄糖（或 H2O2）刺激。采用 MTT 检测 RF/6A 细胞活力。采用流式细胞术检测细胞凋亡。Western blot 检测下调 HOTTIP 后高葡萄糖和 RF/6A 细胞中 p38、c-Jun N-末端激酶（JNK）和细胞外调节蛋白激酶 1/2（ERK1/2）的激活情况，探讨 HOTTIP 是否可以激活丝裂原活化蛋白激酶（MAPK）信号通路从而调节视网膜内皮细胞的功能。
糖尿病大鼠和小鼠视网膜中 HOTTIP 表达显著上调。RT-PCR 显示，注射 HOTTIP shRNA 腺病毒的糖尿病大鼠视网膜中 HOTTIP 的表达下调。注射 shRNA NC 腺病毒后无明显变化。下调 HOTTIP 可减轻糖尿病引起的视网膜细胞功能下降和细胞凋亡，还可降低视网膜中 ICAM-1 和 VEGF 炎症因子的表达。高葡萄糖或 H2O2 处理后，RF/6A 细胞活力下降，下调 HOTTIP 后活细胞活力进一步下降。下调 HOTTIP 导致 p38 磷酸化减少，但对 ERK1/2 或 JNK1/2 的磷酸化没有影响。上调 HOTTIP 可增加 RF/6A 细胞的活力，用 p38 抑制剂 SB23580 预处理可逆转该作用。然而，ERK 抑制剂或 JNK 抑制剂对细胞活力没有影响。
HOTTIP 通过 p38-MAPK 通路改善糖尿病视网膜微血管病变。HOTTIP 有望成为治疗糖尿病微血管病变的新靶点。
