糖尿病,视网膜病变 z-bo 2020-11-17 487

　　Background: Retinopathy remains one of the most devastating complications of diabetes. Although basic science and clinical research have made significant progress to understand the complex pathophysiological mechanism of this blinding disease, the exact mechanism is still unclear, and effective treatment methods are still unclear. Diabetic retina and capillary cells can increase oxidative stress and cause mitochondrial damage. In addition to capillary cells, other retinal cells, including photoreceptors and retinal pigment epithelial cells, have also been observed to increase oxidative stress. Antioxidant contains lipoic acid to supplement the antioxidant, which can prevent diabetic retinopathy in diabetic rats. In addition to oxidative stress, the retina also exhibits many abnormalities consistent with other inflammatory diseases. Vascular endothelial growth factor (VEGF) is an important angiogenic factor in vascular permeability and neovascularization. It is elevated in the retina and vitreous of diabetic patients and animals. This increase is related to the manifestations of diabetic retinopathy. Redox-sensitive nuclear transcription factor-B and NF-κB are activated, which is very important for the regulation of growth factor and cytokine expression, pro-inflammatory mediators such as interleukin-1β (IL-1β) and cell adhesion The expression level of molecule-1 (ICAM-1) increases. Animal models show that antioxidants that inhibit the development of diabetic retinopathy can inhibit the increase of retinal NF-κB and IL-1β. The high content of polyunsaturated fatty acids, combined with the highest oxygen uptake and glucose oxidation, makes the retina highly susceptible to oxidative stress compared to any other tissue. It has been proved that the application of zeaxanthin in diabetic rats can prevent the increase of retinal oxidative stress and pro-inflammatory cytokines VEGF and ICAM-1. Antioxidants containing vitamin C and vitamin E, β-carotene, N-acetylcysteine and other micronutrients inhibit the occurrence of diabetic retinopathy. This study aims to investigate the effect of nutritional supplementation on diabetic retinopathy. Using an animal model of diabetic retinopathy, the effects of multi-component nutrients on retinal capillary cell apoptosis, capillary degeneration and cell function were studied. In order to explore the improvement effect of supplements on mitochondrial dysfunction, the gene expression of inflammatory cytokines, DNA encoding cytochrome b and ND1, and VEGF, IL-1β and NF-κB expression levels were quantified.

　　Method: Wistar rats (male, 200-225 g) were induced to diabetes with streptozotocin and divided into two groups. Rat group 1 (purina 5001) multiple nutritional supplements contain carotenoids. eyepromise DVS is specially formulated to improve the structure and function of the retina, and is now used in clinical trials of visual function in diabetic patients. Per kg Purina diet contains vitamin C nutrition (ascorbic acid, 300 mg), vitamin D3 (cholecalciferol, 10000 IU), vitamin E (vitamin E, 300 IU), fish oil ee 70% (1.6 g), EPA ( Eicosapentaenoic acid, 650 mg), DHA (docosahexaenoic acid, 500 mg), Benfotiamine (1 g), alpha lipoic acid (750 mg), Tocomin (200 mg), zeaxanthin (40 Mg), lutein (20 mg) and patented formula containing 300 resveratrol, green tea, turmeric, N-acetylcysteine, pine bark, grape seed extract, coenzyme Q10 zinc (2.65 g), soybean oil . The second group of rats received Purina feed without any supplements, (DIAB), and used normal rats of the same age as a control. During the entire study period (11 months), the average daily food consumption of diabetic rats was 50 grams. The rats were killed by carbon dioxide asphyxiation method 11 months after the start of the experiment. One eye was suspended in 10% formalin and digested with trypsin to prepare retinal microvessels, and the retina of the other eye was detached to quantify the routine biochemical parameters. Take out a piece of liver and confirm the absorption of some main components by HPLC.

　　Retinal capillary cell apoptosis and histopathology: Separate the retina from formalin-fixed eyes and rinse with water overnight. Digest the retina with 3% crude trypsin containing 200mM sodium fluoride at 37°C for 45 to 70 minutes to separate the capillaries. A terminal deoxyribonucleotide transferase (TDT)-mediated dUTP nick end labeling staining (TUNEL method) was used to detect apoptotic vascular cells. Before the TUNEL reaction started, the retinal blood vessels were exposed by DNase as a positive control. By TUNEL staining, vascular and histological evaluation of periodic acid Schiff and hematoxylin staining. Calculate the number of acellular capillaries in the middle areas of the retina and express them as the acellular capillaries of the retina.

　　Functional analysis: by measuring the electroretinogram (ERG), the retinal function of diabetic rats at 4 months was measured. The rats were adapted to the dark night, anesthetized with ketamine and xylazine, and dilated their pupils with 1% tropicamide and 2.5% phenylephrine hydrochloride. Place the rat on a heating platform and monitor the body temperature with a rectal thermometer. Measure the amplitude of A and b waves by placing the silver eye of the embedded threaded electrode through a thin layer of 1% methylcellulose on the surface of the cornea. The needle electrodes placed on the tail and cheeks serve as ground electrodes and reference electrodes, respectively. Quantification of reactive oxygen species (ROS) by fluorescence spectroscopy of 2,7'-dichlorodiacetate. The protein (5-10μg) was incubated in PBS containing 2uM DCHFDA for 10 minutes. Fluorescence is measured at wavelengths of 485 nm and 530 nm. Determine the antioxidant capacity of the retina. The mitochondrial genome-specific PCR quantitative extension length and 03 XL PCR kit determine the degree of mitochondrial DNA damage. Quantify the expression of VEGF, NF-κB and IL-1β by ELISA method.

　　Results: Nutritional supplements containing carotenoids prevent diabetic retinopathy from accelerating capillary cell apoptosis and histopathology: As expected, in 11-month-old diabetic rats, retinal vascular TUNEL-positive cells have 3-4 times more Increase and appearance of degenerated capillaries. Nutritional supplements with carotenoids can improve the apoptosis of capillary cells caused by diabetes, and the number of TUNEL-positive capillary cells in rats with normal diet is similar to that in diabetic rats. In diabetic rats treated with nutritional supplements, the number of degenerated capillaries in retinal blood vessels was significantly reduced compared with rats without nutritional supplements. Evaluation of retinal function by ERG measurement. Figure 2 shows that the A wave and B wave amplitudes are significantly reduced in diabetic rats, accompanied by delayed electroretinogram responses. The decrease in the amplitude of the A and B waves is due to the acceptance of a nutrient-added diet.

　　Diabetic rats receiving nutritional supplements increased oxidative stress and mitochondrial damage in the retina: ROS levels in the retina of diabetic rats were significantly increased, and the total antioxidant capacity was significantly reduced compared with age-matched normal rats. However, compared with diabetic rats without diabetes, the diabetic rats receiving the supplement have significantly lower ROS levels and enhanced antioxidant capacity. Compared with diabetic rats that did not receive nutritional supplements, diabetic rats that received nutritional supplements also prevented mitochondrial damage, and the gene expression of the mitochondrial DNA encoding protein of the electron transport chain was significantly increased. The levels of ND1, ND6 and Cytb in the retina of diabetic rats decreased by about 40-80%, compared with normal rats. The diabetic rats with nutrient supplements prevented this decline.

　　The use of nutritional supplements can protect the retina from the increase of inflammatory mediators: the level of retinal vascular endothelial growth factor in diabetic rats is increased by 50% compared with normal rats, and the increase of retinal vascular endothelial growth factor is improved by supplementation of nutrients. Because inflammation is recognized as a key cause of diabetic retinopathy.

　　Supplementing nutrients does not improve the severity of hyperglycemia in diabetic rats: Compared with diabetic rats without any supplements, the analysis of major nutrients in liver samples of rats receiving nutritional supplements showed that the level of α-tocopherol increased by 2 times ( 36μg/g to 74μg/g), lutein increased 3 times (0.02μg to 0.06μg), zeaxanthin increased 9 times (0.008μg to 0.07 μg/g).

　　Conclusion: Our data show that nutritional supplements can maintain the structure and function of the retina of long-term diabetic patients, while also protecting nerve cells and blood vessel cells, and inhibiting the development of retinopathy. It may be achieved by improving the increase of inflammatory mediators and maintaining mitochondrial homeostasis, thereby protecting the retina from the self-spreading vicious circle of mitochondrial damage.