糖尿病 z-bo 2020-09-09 396

　　Introduction: Diabetic skin complications lead to delayed wound healing and increase the risk of amputation; foot ulcers are the main reason for hospitalization in developed countries and the main incidence of diabetes, often leading to pain, suffering and poor quality of life. Oxidative stress and lipid content have been recognized as mechanisms that undermine skin integrity. However, there are no reports showing local skin changes in diabetic animal models. Several non-genetic mouse models have been established. The most widely used is based on HF diet, with or without STZ chemical ablation of pancreatic beta cells. Two induced diabetic mouse models were used to study skin conditions.

　　Animals: 18 C57BL/6J male mice, 15-21 weeks old (average weight 23±0.5 grams), were divided into 3 groups. The first group was fed LF feed as a control, and the second group was fed HF feed. The fat content in the diet is 10% and 60% respectively. Five days before starting the HF diet, three consecutive IP injections of STZ (40 mg/kg). This compound was dissolved in 0.05 M citrate buffer at pH 4. During the pretreatment period, the control group and HF group were injected with vehicle, and the mice survived for 5 weeks. Data were collected weekly after 6 hours of fasting. Measure triglyceride and blood sugar levels. Insulin was measured with a hypersensitive mouse ELISA kit. A one-time biopsy punch was used from the back of the animal to obtain a skin sample with a diameter of 8 mm and a thickness of 1 mm for in vitro TEER measurement. Place the sample face up on a 12mm polycarbonate filter (0.4μm hole) suspended in a cell culture well containing 1X PBS (500μL), and immediately measure TEER. TBARS detection kit measures oxidative stress. Histological analysis was performed on skin samples fixed with 10% formalin, embedded in paraffin, and stained with HE and MT. To determine the lipid content, deuterium-labeled methanol is extracted from the skin. Each sample was diluted with water to obtain a final methanol concentration of 20%, and then the column was washed with petroleum ether and water containing 0.1% formic acid. The sample was eluted with 200 μl of methanol containing 0.1% formic acid. LC-MS/MS, electrospray ionization-TSQ quantum super triple quadrupole mass spectrometer to quantify eicosapentaene compounds in the sample. The separation of lipids was achieved by using a gradient of 0.1% formic acid and 0.1% formic acid in acetonitrile in water. The eluate from the LC column is directly introduced into the TSQ quantum superconductor. All compounds are analyzed in negative ion polarity mode.

　　Result: The weight of the mice fed with HF feed began to increase in the second week. Animals pretreated with STZ (T2D mice) showed early weight loss, which subsequently became consistent with the control group. The HF diet induces a basal level of hyperglycemia, and STZ has an enhanced effect from the third week. STZ treatment did not change the basal levels of triglycerides and insulin, and the parameters were affected by HF diet. The TEER of the skin of diabetic mice is lower than that of the control group, and the enhancement of STZ is more obvious. However, the level of TBARs only increased significantly in the skin of HF+STZ animals. Macroscopic observation showed that the animals pretreated with STZ had severe spontaneous skin damage. The tissue sections showed epidermal atrophy, no signs of inflammatory cell infiltration or vasculitis, and hair follicle reduction. Consistent with the observation results, the skin ZO-1, E-Cadherin, Occludin and Claudin-4 levels of HF and STZ mice were lower. In addition, compared with HF and control skin, the lipid mediators DHA, EPA, 12-HHT, LTC4, PD1, TXB2, 6-keto-PGF1α, 5-KETE, 12-KETE and 8, in the skin of STZ-HF mice, The level of 9EET is lower, and the content of PGD2, PGE2, 9-HODE, 13-HODE and 7-HDoHE is higher.

　　Discussion: Skin changes are due to abnormal skin metabolism caused by diabetes or complications of diabetes, such as damage to the skin barrier caused by foot ulcers. Previous results using the HF diet model described mild skin changes associated with decreased TEER, increased epidermal thickness, and inflammatory markers. Using the STZ type 1 diabetes rat model, an increase in inflammatory cell infiltration and oxidative stress has been described in the skin. In addition, an increase in TBARs has been reported in the skin of T2D patients and STZ diabetic rats. Recently, it has been described in a mouse model of type 1 diabetes that the destruction of the skin barrier is accompanied by a disorder of tight junctions, which is characterized by changes in the localization of the ZO-1 protein. Consistent with the aforementioned observations, we found that the levels of ZO-1, E-Cadherin, Occludin and Claudin-4 in the STZ-HF T2D model were significantly reduced, which may be related to the reduction of TEER. Our model will represent the severe and uncontrollable blood glucose status of T2D as the main trigger of the conditions proposed in this study. The severity of this phenotype can be attributed to the synergistic effect of glucose levels. The reduction of skin DHA and EPA is related to the change of the epidermal barrier, the reduction of PD1, PGF1, KETE and EET is related to inflammation, TXB2 indicates pathology, and 12-HHT is described as a lipid mediator that promotes regeneration through the BLT2 receptor. On the other hand, the local increase of PGD2, PGE2 and HODEs is related to hair loss, inflammation and oxidative stress. The data in this study describes a highly used animal model that will provide evidence for future research in the field of skin endocrinology.

　　Conclusion: Compared with the control group, the animals treated with the STZ high-fat diet showed skin damage without significant metabolic changes. The skins of these animals exhibit high levels of oxidative stress, low levels of adhesion proteins and lipid mediator changes, and these effects are not produced by the high-fat diet itself. Based on the results of this study and our previous reports, we believe that in the STZ-high-fat diet model, severe hyperglycemia and lack of insulin will cause the burst of pro-inflammatory cytokines and keratinocyte oxidative stress, which activates LOX/ COX depends on the pathway, causing local lipid mediator changes and loosening of epidermal integrity, which may cause spontaneous skin damage.