In the current study, we investigated the preventive effects of wolfberry on hyperglycemia-induced retinal damage by application of dietary wolfberry at 1 % (kCal) to the db/db mouse, starting at 6 weeks of age while the fasting blood glucose level had been elevated to 189.4± 31.9 mg/dL. The results have demonstrated for the first time that RPE integrity and retinal structure were altered at the early stage of diabetes in the db/db mouse model, and this abnormality could be ameliorated by dietary wolfberry.
The genetically programmed diabetic db/db mouse is an adequate model for studies on the pathogenesis of retinopathy, no retinal disorder occurs in the new born mouse (34
). It has been reported that the db/db mouse starts to develop hyperglycemia at 8 weeks of age (35
), which was very similar to our observation. Breakdown of blood retinal barrier and leakage of retinal microvessels occurs at 18 weeks of age. About 7 weeks later, severe microvessel leakage and retinopathy are observed (36
). In the current study we observed that fasting blood glucose level was elevated to >300 mg/dL at 10 weeks of age in db/db fed CD but not WT mice, indicating that the db/db mouse developed hyperglycemia and/or hyperinsulinemia () at the similar age as previously reported (34
). According to publications retinal blood microvessels may still be intact, no blood leakage occurs at this early stage of diabetes in db/db mice (36
). Thus, the young db/db mouse (<18 weeks of age) would be an excellent type 2 diabetic model for the study in prevention of the development of diabetic retinopathy.
Hyperglycemia induces biochemical abnormality in diabetic cells and tissues, which leads to retinal degeneration. Diabetic retinal degeneration is associated with ganglion cell loss, and reduction of retinal thickness including INL, IPL, and/or ONL (23
). The db/db leptin receptor deficient mouse is a spontaneous type 2 diabetic mouse model. Our finding in loss of ganglion cells was consistent with the previous report (41
). We also found for the first time that INL and photoreceptor layers were abnormal compared to WT mice (C57BLKS/J).
Wolfberry fruits have been most popularly consumed as a part of Chinese cuisine though it is also considered as an herbal medicine. Wolfberries are being sold as herbal supplements in Western countries as well (14
). Consumption of wolfberries is beneficial to general health, including vision (14
). In this study we applied a low dose (1 % (kCal)) wolfberry to both WT and db/db mice to mimic what humans would eat on a practical basis. Apparently both types of animals fed the wolfberry supplemented diets survived as well as those fed the control diet as shown in gain of body mass and overall organ weight, suggestive of no obvious side effects at the dose range. However, wolfberry at 1 % (kCal) did not systematically affect the levels of fasting blood glucose and insulin in the diabetic mouse at 14 weeks of age. This may be due to the retinal specific accumulation of its bioactive components zeaxanthin and lutein in mice as discussed below.
The bioactive constituents of wolfberry have been well characterized. The major groups of substance in the wolfberry are carotenoids, polysaccharides, and flavonoids (14
). Zeaxanthin and lutein dipalmitates are the most dominant and unique constituents of wolfberry carotenoid fractions (14
). Zeaxanthin and lutein are specifically transported to and preferentially taken up, and accumulated in retinal cells by sharing the transportation pathway of HDL cholesterol (32
). It has been suggested that in the retina zeaxanthin and/or lutein bind to antioxidant enzymes which in turn stabilize the proteins under chronic oxidative stress in the case of macular degeneration (32
). Recently Gierhart and colleagues reported that zeaxanthin administration at 0.02 % or 0.1 % (diet by weight) significantly inhibited diabetes-induced retinal oxidative damage, but no effect was achieved on the severity of hyperglycemia in rats at the early stage of diabetes (47
). In the current study we used wolfberry fruits and pure zeaxanthin and/or lutein to treat diabetic mice and human RPE cells, respectively. The results suggested that wolfberry protected retinal cells against a high glucose challenge locally but not systemically, at least partially, if not all, through zeaxanthin and/or lutein, though there may be some potential synergistic interactions of other bioactive components in wolfberry, such as phenolics and polysaccharides.
AMPK is a cellular energy sensor in mammals. It is down-regulated in type 2 diabetic patients (3
). Targeting AMPK has been suggested as a most successful pharmaceutical strategy for type 2 diabetes. For instance, metformin, an oral anti-diabetic drug, at least partially works through activation of AMPK (6
). Wolfberry induced AMPK activation in wild type mice, increased AMPK at protein level in db/db mice (). Retinal AMPK activation may be due to retinal specific accumulation of zeaxanthin and/or lutein. Further, wolfberry restored FOXO3α (). Data suggested that wolfberry protection of retina from caspase-3 dependent apoptosis may be through regulation of protein expression, centered by AMPK activation.
FOXO3α is regulated by AKT/PKB, extracellular signal-regulated kinases, and AMPK. It is essential to cell survival in diabetes (31
). FOXO3α is being clinically shown to achieve the therapeutic effects (30
). Down-regulation of FOXO3α results in disruption of cellular ubiquitination and inhibition of antioxidant activity. AMPK up-regulation of FOXO3α has been suggested to account for increased expression of antioxidant enzymes thioredoxin and Mn SOD in diabetic endothelial cells (50
). Application of wolfberry caused up-regulation of retinal FOXO3α and thioredoxin and Mn SOD (), which may lead to diminishing cellular ROS generation. Increased thioredoxin and Mn SOD by dietary wolfberry may be due to up-regulation of gene expression and/or stabilization of existing proteins by zeaxanthin and/or lutein under hyperglycemia. The cell culture study further suggested that wolfberry restoration of thioredoxin and Mn SOD would help rebalance cellular redox homeostasis through normalization of cellular ROS status.
ER stress has been exclusively documented to be a major driven force to cellular oxidative retinal damage at the late stage of diabetes (10
). We have demonstrated in this study that hyperglycemia induced elevated protein expression of ER stress biomarkers in early diabetic retina in vivo, indicating that hyperglycemia accounts for retinal endoplasmic reticulum (ER) stress, inhibition of AMPKα, thioredoxin, Mn SOD, FOXO3α, and retinal abnormality. This was further confirmed by in vitro studies on human retinal ARPE-19 cells when they were exposed to a high glucose challenge (). This was not due to osmotic pressure as mannitol alone did not differ from phosphate buffered saline (PBS) controls. Prolonged hyperglycemia (or high glucose) distinctly induces generation of cellular ROS (), which was diminished by administration of whole wolfberries and/or the bioactive fractions zeaxanthin and lutein. AMPKα siRNA knockdown results further suggested that zeaxanthin diminished high glucose-induced cellular ROS levels via restoration of AMPKα. AMPKα is the upstream modulator of FOXO3α and thioredoxin and Mn SOD.
Taken together, dietary wolfberry and/or its bioactive constituents zeaxanthin and lutein functioned as modulators of cell survival/death signaling pathways, through targeting pathways in AMPK and FOXO3α signaling, resulting in normalization of cellular ROS and subsequent attenuation of ER stress. This could lead to prevention of retinal apoptosis and restoration of retinal structure in the type 2 diabetic mouse (). Dietary treatments using individual fractions isolated from the wolfberry would be necessary for further mechanistic studies in vivo. To our knowledge, this is the first report that wolfberry bioactive constituents prevented or delay the onset of the disease of diabetic retinopathy in an animal mode.
Schematic diagram showing wolfberry and/or zeaxanthin and lutein modulated retinal protection in type 2 diabetes