Objective: To explore the mechanism of HOTTIP in diabetic retinopathy.
Materials and methods: 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.
Results: 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.
Conclusions: HOTTIP improves diabetic retinal microangiopathy through the p38-MAPK pathway. HOTTIP is expected to become a new target for the treatment of diabetic microangiopathy.