动物造模,糖尿病,视网膜病变 z-bo 2020-09-02 409

　　Background: Retinopathy remains one of the most destructive complications of diabetes. Although great progress has been made in basic science and clinical research to understand the complex pathophysiological mechanism of this blindness, the exact mechanism is still unknown and effective treatment methods are still unknown. Diabetic retina and capillary cells can increase oxidative stress and cause mitochondrial damage. In addition to capillary cells, other retinal cells, including photoreceptor cells and retinal pigment epithelial cells, have been observed to increase oxidative stress. Antioxidants include lipoic acid, which can supplement antioxidants and 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 for vascular permeability and angiogenesis. It is elevated in diabetes and the retina and vitreous of animals. This increase is related to the symptoms of diabetic retinopathy. Redox sensitive nuclear transcription factor B and NF-κB are activated. This is very important for regulating the expression of growth factors and cytokines, inflammatory mediators (such as interleukin-1β (IL-1β) and cell adhesion), and increased expression levels of Molecular-1 (ICAM-1). Animal models show that antioxidants that inhibit the development of diabetic retinopathy can inhibit the increase of retinal NF-κB and IL-1β. The combination of high levels of polyunsaturated fatty acids with the highest oxygen absorption and glucose oxidation makes the retina more susceptible to oxidative stress than other tissues. It has been proved that the application of zeaxanthin in diabetic rats can prevent retinal oxidative stress and increase pro-inflammatory cytokines VEGF and ICAM-1, antioxidants containing micronutrients such as vitamin C and vitamin E, β-carotene and N- Acetylcysteine can inhibit the development of diabetic retinopathy. This study aims to investigate the effects of nutritional supplementation on diabetic retinopathy. Animal models of diabetic retinopathy are used to study the effects of multi-component nutrients on retinal capillary cell apoptosis, capillary degeneration and cell function. In order to study the improvement effect of supplements on mitochondrial dysfunction, we quantified the gene expression of inflammatory cytokines, the DNA encoding cytochrome b and ND1, and the expression levels of VEGF, IL-1β, and NF-κB. Methods: Wistar rats (male, 200-225g) were treated with streptozotocin to induce diabetes and were divided into two groups. Several dietary supplements in rat group 1 (Purina 5001) contain carotenoids. eyepromiseDVS is specially formulated to improve the structure and function of the retina. It is currently used in clinical trials of visual function in diabetic patients. Purina diet per kilogram contains vitamin C nutrition (ascorbic acid 300 mg), vitamin D3 (cholecalciferol, 10000IU), vitamin E (vitamin E, 300IU), fish oil ee 70% (1.6g), EPA (eicosapentaene) Acid (650 mg), DHA (docosahexaenoic acid, 500 mg), amphetamine (1 g), alpha lipoic acid (750 mg), comcomin (200 mg), zeaxanthin (40 mg), leaf yellow The patented formula (20 mg) contains 300 types of resveratrol, green tea, turmeric, N-acetylcysteine, pine bark, grape seed extract, coenzyme Q10 zinc (2.65 g) and soybean oil. The second group of rats received a non-purine diet (DIAB) and used normal rats of the same age as the control group. During the entire study period (11 months), the average daily food consumption of diabetic rats was 50 grams. After 11 months from the beginning of the experiment, the rats were killed by carbon dioxide asphyxiation. One eye was suspended in 10% formalin, digested with trypsin to prepare retinal capillaries, and the retina of the other eye was removed to quantify conventional biochemical parameters. Take a part of the liver and check the absorption of certain main components by HPLC. Apoptosis of retinal capillary cells and histopathology: Separate the retina from the formalin-fixed eye and rinse with water overnight. To detach the capillary, digest the retina with 3% crude trypsin containing 200 mM sodium fluoride at 37°C for 45-70 minutes. Terminal deoxyribonucleotide transferase (TDT)-mediated dUTP nick end labeling staining (TUNEL method) is used to detect apoptotic vascular cells. Before the TUNEL reaction started, the retinal blood vessels were exposed to DNase as a positive control. The blood vessels and histology of periodate Schiff and hematoxylin staining were evaluated by TUNEL staining. Count the number of acellular capillaries in the central area of the retina and express it as acellular capillaries in the retina. Function analysis: The retinal function of diabetic rats at 4 months was measured by measuring the electroretinogram (ERG). The rats were adapted to the dark night, anesthetized with ketamine and xylazine, and expanded with 1% tropicamide and 2.5% phenylephrine hydrochloride. Place the rat on the heating table and monitor the body temperature with a rectal thermometer. Place the silver eye of the threaded electrode in a thin layer of 1% methylcellulose on the surface of the cornea, and measure the A and b wave amplitudes. Needle electrodes placed on the tail and cheeks act as ground electrodes and reference electrodes, respectively. Detect reactive oxygen species (ROS) by fluorescence spectrometry with 2,7'-dichlorodiacetate. Incubate the protein (5-10μg) in PBS containing 2uMDCHFDA for 10 minutes. Fluorescence is measured at wavelengths of 485nm and 530nm. Determine the antioxidant capacity of the retina. Mitochondrial genome-specific PCR quantitative extension length and 03XL PCR kit determine the degree of mitochondrial DNA damage. ELISA method quantitatively detects the expression of VEGF, NF-κB and IL-1β.

　　Results: Dietary supplements containing carotenoids can prevent diabetic retinopathy and accelerate capillary apoptosis and histopathology: As expected, retinal vascular TUNEL positive cells in 11-month-old diabetic rats showed 3- Increase and degeneration of 4 capillaries. Dietary supplements containing carotenoids can improve capillary apoptosis caused by diabetes. The number of TUNEL-positive capillary cells in normally fed rats is similar to that in diabetic rats. Compared with rats without dietary supplements, in diabetic rats treated with dietary supplements, the number of degenerated capillaries in the retinal blood vessels was significantly reduced. Evaluation of retinal function by ERG measurement. Figure 2 shows that in diabetic rats, the A wave and B wave amplitude are significantly reduced, while the electroretinogram response is delayed. The decrease in the amplitude of the A wave and the B wave is due to people receiving a nutritionally supplemented diet. Increased oxidative stress and mitochondrial damage in the retina of fed diabetic rats: Compared with normal age-matched rats, the significant increase in ROS levels in the retina and total antioxidants of diabetic rats has greatly reduced the energy value. However, compared with diabetic rats without diabetes, diabetic rats receiving supplements had significantly lower ROS levels and enhanced antioxidant capacity. Compared with diabetic rats that did not receive dietary supplements, diabetic rats that received dietary supplements also prevented mitochondrial damage and significantly increased the expression of mitochondrial DNA genes that encode proteins in the electron transport chain. Compared with normal rats, the levels of ND1, ND6 and Cytb in the retina of diabetic rats were reduced by about 40-80%. Diabetic rats treated with dietary supplements prevented this decline. The use of nutritional supplements can protect the retina from increasing inflammatory mediators. Compared with normal rats, the level of retinal vascular endothelial growth factor in diabetic rats increased by 50%, and the increase of retinal vascular endothelial growth factor was improved by nutritional supplementation. Because inflammation is considered to be the main cause of diabetic retinopathy. Supplementation of nutrition cannot improve the severity of hyperglycemia in diabetic rats: Compared with diabetic rats without supplementation, analysis of macronutrients in liver samples of rats receiving supplementation, then, the level of α-tocopherol increased by 2 (36μg/ g?74μg/g), lutein increased 3 times (0.02μg?0.06μg), and zeaxanthin increased 9 times (0.008μg?0.07μg/g). ..

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