Regulation Of Hydrogen Molecule On Microcirculation Dysfunction In Type 2 Diabetic Mice

ObjectDiabetes is a group of chronic metabolic diseases characterized by elevated blood sugar caused by multiple causes.Vascular lesions are one of the major complications of type 2 diabetes.Recent studies have shown that microcirculatory dysfunction may be related to the occurrence and development of diabetes mellitus and its complications.Even when blood glucose is well controlled,microcirculation in diabetic patients may have been dysfunctional.Microcirculatory disorder is an important pathophysiological basis for the occurrence of diabetic vascular complications.Therefore,early reduction of blood glucose and positive improvement of microvascular function are of great significance for preventing and delaying various complications of diabetes.Hydrogen has selective antioxidant effects,and its preventive and therapeutic effects on atherosclerosis,diabetes mellitus,organ and system inflammation,Parkinson’s disease and other diseases have also been found by scholars.The effect of hydrogen on microcirculation function of diabetes mellitus was studied.Materials and MethodsAnimals:C57BL/6J male mice aged 4-5 weeks（n=30）came from the Institute of Atherosclerosis,Taishan Medical College.The mice were divided into normal control group（NC,n=10）,diabetic model group（DM,n=10）and hydrogen intervention group（DM+H₂,n=10）at random.The temperature and humidity in the laboratory animal feeding room can be controlled,and the light and dark cycle lasts for 12 hours/12 hours.All the experiments were approved by the Ethics Committee of Experimental Animals of Taishan Medical College and followed the National Guidelines for Animal Care and Use.Induced T2DM model of mice:The mice were fed with high-sugar and high-fat diet for5 weeks,and were injected with Streptozotocin（STZ）once intraperitoneally at a dose of100 mg/kg.The mice were continually fed with high-sugar and high-fat diet for 4 weeks to induce the model of T2DM.Normal control group（NC）was only given normal diet and the same amount of citric acid buffer by intraperitoneal injection.Success rate of T2DM model:blood glucose was measured after 1 week of STZ injection,once a week,about 5 weeks later,the success rate of T2DM model was calculated.Monitor of general condition,blood glucose,body weight:Weekly observation of mice diet,drinking water,urine volume,hair,activity,mental state and so on,and weekly weighing of body weight and tail vein blood sampling to measure blood glucose and record.Detection of microcirculation function of skin,pancreas and cerebrum:Dehairing the back and abdomen of mice,fully exposing the skin,Perimed Peri Cam PSI was used to measure microcirculation function of skin.Open the abdominal cavity,expose the pancreas and measure the microcirculation function of the pancreas.Cut the head skin,carefully clean the fascia on the surface of the skull,and measure the microcirculation function of the cerebrum.Histopathological examination of pancreas:After the successful establishment of the model of T2DM,the pancreatic tissue of mice was fixed,dehydrated,transparent,immersed in wax and embedded to make paraffin blocks,paraffin sections and hematoxylin-eosin staining.The pathological changes of pancreatic tissue of mice were observed under the microscope.Immunohistochemical staining of pancreatic platelet endothelial cell adhesion molec-ule-1（PECAM-1）:The pancreatic tissue of mice was stained by immunohistochem-ical staining after paraffin section.The staining was observed under a microscope and the images were collected.Image-Pro Plus6.0 software was used for image analysis.ELISA:Insulin in serum of mice was detected by mouse insulin（INS）ELISA kit.The absorbance value was read by multifunctional enzyme label and the concentration was calculated.Statistical analysis:The experiment was repeated at least three times and analyzed by Graph Pad software.The data are usually expressed asx（?）s.Inter-group data were analyzed by using one-way analysis of variance.The difference was statistically significant when P≤0.05.The results1.Modeling rate of mice model of T2DM induced by high sugar,high fat and low dose STZ:After 5 weeks of STZ injection,blood glucose of mice was measured by tail vein blood glucose monitor.The animals with hyperglycemia fasting blood sugar（≥16.7mmol/L）were considered to have diabetes mellitus,that is,the model of T2DM mice was successfully induced.Among them,2 mice died in DM group and 2 mice did not develop diabetes.The success rate of modeling was about 89%.All animals in NC group survived and were in good condition.2.H₂ can improve the general condition,reduce blood sugar and control body weight of T2DM mice:DM+H₂ mice were intraperitoneally injected with hydrogen-rich saline（5 ml/kg/d）,once a day.NC and DM mice were intraperitoneally injected with the same volume of saline.The results showed that compared with the NC group,DM group mice were obese,less active,drank more water,urine more,their hair lacked luster,and their mental status was not good.Compared with DM group,DM+H₂ group mice were lighter.The blood sugar and body weight of mice in DM group were significantly higher than those in DM+H₂group and NC group,and the data of blood sugar and body weight of mice in three groups were statistically significant.3.H₂ can improve the microcirculatory function of skin and pancreas in T2DM mice,but has no significant effect on regulating the microcirculatory function of cerebrum in mice:Perimed Peri Cam PSI was used to measure the microcirculation function of skin,pancreas and brain in three groups of mice.The results showed that compared with NC group,the microcirculation blood flow of skin and pancreas in DM group was significantly reduced and its function was impaired.The microcirculation blood flow of skin and pancreas in DM+H₂ group was decreased and its function was less impaired than that in DM group.There were significant differences in the degree of microcirculation damage of skin and pancreas among the three groups,but there was no difference in the degree of microcirculation damage of cerebrum among the three groups.4.H₂ can alleviate the degree of pancreatic tissue injury in mice with T2DM:HE staining was used to observe and compare the pathological changes of pancreatic tissue in three groups of mice.The results showed that compared with NC group,the size of islets in DM group was significantly reduced,the number of islets was reduced,the normal structure of islets was destroyed,the number of cells in islets was significantly reduced,the structure was loose,and the location of microvessels was abnormal.Some islet cells and peripheral pancreatic cells appeared vacuolar degeneration.After H₂ intervention,the above pathological changes were alleviated.5.The expression level of PECAM-1 in pancreas tissue of T2DM mice decreased,and H₂ could improve the expression level of PECAM-1 in pancreas tissue of T2DM mice:Immunohistochemical staining was used to detect the expression of PECAM-1 in pancreas tissue of mice.The results showed that the expression of PECAM-1 in pancreas tissue of DM group was significantly lower than that of NC group.The expression of PECAM-1 in pancreas tissue of DM+H₂ group was slightly lower than that of NC group.6.The level of insulin in serum of T2DM mice increased,and H₂ could improve the level of insulin in serum of T2DM mice:The serum insulin level in DM group was significantly higher than that in NC group.The serum insulin level in DM+H₂ group was also higher than that in NC group,but lower than that in DM group.Conclusion1.Hydrogen-rich saline can improve the general condition of T2DM mice,reduce blood glucose,control body weight and improve insulin resistance.2.H₂ can improve microcirculation dysfunction of skin and pancreas in T2DM mice.