The Protective Effects Of Melatonin Against Diabetic Retinopathy And The Underlying Mechanisms

Diabetic retinopathy (DR) is a common and potentially devastating microvascular complication of diabetes; it is a leading cause of acquired blindness among people at occupational age . It is now well accepted that oxidative stress and inflammation play important roles in the pathogenesis of DR. Any shift in the delicate balance between the oxygen radical or inflammatory cytokines production and elimination could result in cellular damage. Thus, any beneficial treatment that limits oxidative and inflammatory effects of diabetes could greatly reduce its consequences at retinal level. Melatonin, synthesized mainly by the pineal gland, is considered as effective antioxidant and anti-inflammatory agent. In addition to the pineal gland, the retina is another site of melatonin synthesis. Thus, melatonin is a potent compound with potential for therapeutic applications in diabetic retinopathy.The goals of our current study were to investigate the protective effects of melatonin against hyperglycemia-induced injury in primary rat Muller cells and in the retinas of diabetic rats. Furthermore, the underlying mechanisms were also investigated.Part I:The protective effects of melatonin against hyperglycemia-induced injury in primary rat Muller cellsPurpose:To investigate the protective effects of melatonin against hyperglycemia-induced injury in primary rat Muller cells and to explore the underlying mechanisms.Methods:Cultured Muller cells were divided into several groups, treated with either high glucose medium (containing 30mM glucose) alone or combined with different concentrations of melatonin (lOnM to 0.1mM) for various time. In the osmotic control group, cells were treated with medium containing 5.5mM glucose and 24.5mM mannitol. In some experiments, cells were pretreated with either a PI3K inhibitor, LY294002 (10μM), or a melatonin receptor antagonist, luzindole (50μ M). Melatonin receptors MT1 or MT2 were examined with immunocytochemistry. VEGF protein concentrations were analyzed with Enzyme-linked immunoassay. Expression levels of the mRNA and protein of MT1, MT2, catalytic and modulatory subunits of glutamate-cysteine ligase (GCLc, GCLm), heme oxygenase 1 (H0-1), Nrf2, phosphorylated Akt, IL-1β,TNF-α and NF-κB, were detected with Real time RT-PCR and Western Blot.Results:Immunocytochemical studies demonstrated that GS (glutamine synthetase) is expressed in almost all Muller cells. High glucose induced upregulation of MT1/MT2 in Muller cells. Hyperglycemia stimulated VEGF production and co-treatment with melatonin significantly reduced the amount of released VEGF. Melatonin could upregulated the expressions of phosphorylated Akt, HO-1, Nrf2, GSH, GCLc and GCLm compared to the high-glucose counterparts. Whereas the effects of melatonin could be partly inhibited by LY294002 or Luzindole. Inflammatory factors IL-1 β,TNF-α and NF-κB elevated under high glucose treatment, while melatonin treatment significantly decreased the expressions. Conclusions:The data suggest that melatonin can exert protective effects in Muller cells against high glucose-induced oxidative stress, inflammation and VEGF overproduction. PI3K/Akt-Nrf2 signaling pathway and NF-κ B play an important role in its effects.Part Ⅱ:Inhibitory effect of melatonin on the retinal inflammation and oxidative stress in experimental diabetic retinopathyPurpose:To determine whether melatonin could ameliorate retinal injury in streptozotocin (STZ)-induced diabetic rats.Methods:Animals were divided into three groups:non-diabetic, diabetic, and melatonin (MT)-treated diabetic group based on the baseline blood glucose and body weight. Diabetes was induced by a single intraperitoneal injection of STZ (60 mg/kg body weight).3 days later, the development of diabetes was confirmed by measuring glucose levels in blood samples taken from the tail vein. Animals with glycemia level of 16.7 mmol/L or above were considered diabetic and included in the experimental group. Non-diabetic rats were injected with vehicle in the same volume. In the animals of MT-treated diabetic group, melatonin (10 mg/kg/daily) was administered. Body weight and glycemia were measured twice a week throughout the entire course of the experiments. After diabetes induction for 4,8,12 weeks, the rats were sacrificed, their retinas were removed for histological and biochemical examination. The distribution of melatonin membrane receptors MT1 and MT2 were detected with Immunofluorescence. Real time RT-PCR and Western Blot were used to detect the expressions of VEGF, H0-1, iNOS, IL1 β, TNF-α, Nrf2, phosphorylated I K B, phosphorylation of Akt and nuclear NF-κB levels. ERG was used to evaluate the functional status of the retina.Results:The STZ-induced diabetic rats showed significant rise in plasma glucose and significant decline in body weight as compared to age matched normal control rats. Treatment with melatonin did not produce any effect on blood glucose level or body weight of the STZ-treated rats. A strong immunoreactivity of MT1 and MT2 were identified in the ganglion cell layer and in the inner plexiform layer of the retina. Increased expressions of MT1 and MT2 were observed in retinas of diabetic rats compared with that in the non-diabetic group. Melatonin could reduce the VEGF overproduction and the upregulated expressions of TNF-α, IL-1β, phosphorylated I K B and NF-K B in retinas of STZ-induced diabetic rats. The levels of phosphorylation of Akt, Nrf2 and H0-1 were significantly increased in response to melatonin treatment. The results of ERG shown decreased amplitude of a wave and b wave in diabetic rats compared with the age-matched controls, whereas it did not decrease in diabetic rats with melatonin treatment.Conclusions:Melatonin plays a potential protective role in diabetic retinopathy, partially mediated through the attenuation of inflammation by NF-K B inhibition and the prevention of the decreased activity of antioxidant enzymes via the Akt-induced Nrf2 pathway. It also reduced the elevated VEGF expression in diabetic retinas. Thus, melatonin is a potent compound with potential for therapeutic applications in diabetic retinopathy.