Effect Of Organgermanium (Ge-132), Metformin And Their Mixture On The Treatment Activity To2Type Diabetic Rats And Its Mechanism

In recent years, with the improvement of people’s living standards, changes in lifestyle and living environment, the surge in the incidence of diabetes has become a global disease. According to The WHO statistics, the incidence of diabetes in2000was2.8%, about171million patients; in2030is expected to grow to4.4%, the number of patients up to366million.Currently no cure for type2diabetes and mainly symptomatic treatment. Normally takes medication taken for life, but also the adverse reactions of hypoglycemia reaction, gastrointestinal reactions. A heavy burden to the patient’s physical and economic.Recent studies have found that trace elements play an extremely important role in the generation of insulin, role to play as well as people with diabetes the energy metabolism. Has been reported that organic germanium (Ge-132) has obvious role in the prevention of blood sugar in diabetic mice, however, at home and abroad has not been reflected in the experimental study of type2diabetes treatment, this experiment is further validation organic germanium (Ge-132) for the treatment of type2diabetes, and to explore its mechanisms.ObjectiveJoint metformin hydrochloride for treatment of type2diabetic rats by observing organic germanium (Ge-132), the experimental, explore its mechanisms in order to provide new ideas and directions for the prevention and treatment of type2diabetes.Methods1. Preparation of a rat model of type2diabetesIn this study, the high fat diet induced prepared by intraperitoneal injection in combination with low-dose STZ animal model of type2diabetes, the specific method:(A) Animal grouping and treatmentAfter rats adaptability keeping one week, were randomly divided into two groups, the experimental group40,10blank control group. The experimental group fed a special feed; diabetic rats with normal diet of the control group was fed the rats. After four weeks, all rats but water fasting12h, fasting glucose and weighed, and then an intraperitoneal injection to the experimental rats STZ30rag/(kg.Bw), type2diabetic rat model.(B) Glucose tolerance testPrepared to verify the diabetic rat model of type2diabetes model, the oral glucose tolerance test:the experimental group and the control group randomly selected from each of10rats,30,60,120,180min after the glucose load, real-time blood glucose was measured calculate the area under the curve of insulin and serum insulin, homeostasis model assessment of insulin resistance. The standard insulin resistance:HOMA-IR compared with control group difference was statistically significant.2. Organic germanium joint metformin hydrochloride impact on blood sugar levels in type2diabetic rats(A) Animal groupingModeling successful40SD rats were randomly divided into four groups:model control group, metformin group, organic germanium (Ge-132) group, metformin and Organic Germanium (Ge-132) group.(B) The experimental treatmentThe experimental rats blood sugar stable for a week after the experimental rats divided into model group; metformin group; organic germanium (Ge-132) group; the metformin and organic germanium (Ge-132) group. Weekly one, five measuring weight; Tuesday, six measured food intake, water intake; Sundays fasting glucose. Group trial intervention lasted for8weeks. Fasting glucose differences observed in each group.3. The preliminary study of the mechanism of the sensitizing effect of organic germanium (Ge-132) for the treatment of type2diabetes rats(A) A packet of experimental animalsGrouping of the experimental animals with the second portion.(B) The experimental treatment1, Western blotting measurement of type2diabetes in rat skeletal muscle GLUT4protein level:(1) Tissue homogenate preparationThe intervention After8weeks, the rats in each group were sacrificed, take the right amount of the right thigh of the soleus muscle100mg,1:9dilution into1%homogenate analyte.(2) Determination of protein concentration of the determination of the protein content (BCA method) Determination of the content of the target protein in the sample according to the number of samples, the protein concentration by the BCA method, A562absorbance was measured according to the standard curve to calculate the concentration of a protein.(3) Western BlottingThe extracted proteins After protein quantification, SDS-PAGE electrophoresis. The proteins were transferred to PVDF membrane, PVDF membrane to X-ray film put together coincide box came to light, to be color the application image analysis software analysis.3Immunohistochemical staining (SP method) measurement of skeletal muscle GLUT4protein expression:(1) Tissue slice preparationThe soleus muscle of the right amount of the right thigh with4%paraformaldehyde fixed,20%sucrose sedimentation overnight (4℃), the OCT embedded the constant cold box frozen slicer slices made15μm skeletal muscle slice.(2) ABC method for immunohistochemical stainingABC staining skeletal muscle slice conventional production transmission electron microscopy specimens, JEM-1200EX transmission electron microscope observation of the distribution of GLUT4in skeletal muscle cells.4. Statistical MethodsUsing SPSS20.0statistical software for the analysis of experimental data processing, the difference between the two groups into a set of design data, experimental data are presented as mean±standard deviation (χ±s) said, with P<0.05as statistically significant.Results1. Preparation of a rat model of type2diabetes(A) Adaptive feeding period rats generallyRats a good state of mind, a special diet for4weeks in diabetic rats, the experimental group compared with the blank control group had increased body weight (Body weight, BW) and FPG, the difference was statistically significant (P<0.05).(B) Type2diabetic rat model successfully establishedModeled after the success of the experimental rats eating the amount of water, urine output increased significantly, body weight loss, unresponsive, and yellow coat color, softer stool. Modeled after the success of the experimental group and the control group compared to FPG increased insulin secretion peak delay, HOMA-IR and AUCi are significantly higher than the control group, a significant difference, the rats of the experimental group, the presence of insulin resistance line2The incidence of diabetes features.2. Organic germanium joint metformin hydrochloride impact on blood sugar levels in type2diabetic rats(A) Eight weeks after the intervention of the type2diabetic rat model, rats in each group of the FPG and GSPEight weeks after the intervention of the rat model of type2diabetes, the rats FPG is measured, the results are as follows:FPG, GSP metformin and metformin+organic germanium (Ge-132) group were lower than the model control group, organic germanium (Ge-132) group, a statistically significant difference; metformin+organic germanium (Ge-132) group FPG, the GSP is lower than the metformin group, a statistically significant difference (p<0.01), to organic germanium metformin type2diabetic rats better than with metformin treatment effect, but not reduce type2diabetic rats FPG alone organic germanium.(B) Adjustment interventions, metformin and metformin+organic germanium (Ge-132) group FPG changeMetformin gastric feeding solution was added the organic germanium (Ge-132) CMC-Na suspension was20mg/kg, simultaneous removal of the organic germanium (Ge-132) in the metformin+organic germanium (Ge-132), respectively, in intervention8weeks after the last fasting blood glucose values do statistical analysis found that metformin group added organic germanium (Ge-132) with eight weeks after the last blood glucose no significant difference, whereas metformin+Organic Germanium (Ge-132) group have statistically significant, the same as the repeat test results.3. The preliminary study of the mechanism of the sensitizing effect of organic germanium (Ge-132) for the treatment of type2diabetes rats(A) In rat skeletal muscle GLUT4protein expression by Western blot results Using one-way ANOVA, and found that each group of data compared with the blank control group, the model control rats membrane of GLUT4expression decreased approximately53%; metformin+organic germanium (Ge-132) group than in the metformin group and organic Germanium (Ge-132) group increased1.15times and1.4times, respectively, with a statistically significant difference.(B) Each rat skeletal muscle GLUT4protein expression by immunohistochemical method resultsImmunohistochemical staining results in rat skeletal muscle cytoplasmic brown substance was positive, the results show:the blank control group rat skeletal muscle cell membrane GLUT4widely distributed; significantly reduced GLUT4expression in skeletal muscle cell membrane model control group; metformin+Organic Germanium (Ge-132) group GLUT4expression nearly blank control group; metformin group or organic germanium (Ge-132) on the cell membrane GLUT4expression is only part of the increase. The immunohistochemistry results consistent with the Western blot.ConclusionThis experiment induced by high-fat diet combined with low dose intraperitoneal injection of STZ successfully prepared with type2diabetes animal model, after observation of organic germanium (Ge-132) on the treatment of type2diabetic rats and skeletal muscle GLUT4protein immunoblot detection results of the method and immunohistochemical method, and explore the mechanism of sensitizing effect of organic germanium (Ge-132) on the treatment of type2diabetes. The main conclusions are as follows:(A) Organic germanium Joint metformin hydrochloride on blood glucose levels in type2diabetic ratsEight weeks after the intervention of the type1or type2diabetic rat model, measured rats FPG, organic germanium (Ge-132) in combination with metformin treatment of type2diabetes in rats is better than with metformin treatment effect, but not reduce type2diabetic rats FPG alone organic germanium. After adjustment interventions, metformin and metformin+organic germanium (Ge-132) group FPG change also shows that the organic germanium (Ge-132) on the treatment of type2diabetes have a synergistic effect.(B) In rat skeletal muscle GLUT4protein expression resultsImmunohistochemistry and immunoblotting results show that:the blank control group rat skeletal muscle cell membrane GLUT4widely distributed; significantly reduced GLUT4expression in skeletal muscle cell membrane model control group; the metformin+organic germanium (Ge-132) group GLUT4expression nearly blank control group; metformin group or organic germanium (Ge-132) group on GLUT4expression is only part of the increase of cell membrane.In summary, the effect of the type2diabetic rats FPG、GSP and skeletal muscle in the cell membrane on the content of the expression of GLUT4comparison are the organic germanium (Ge-132) in combination with metformin treatment of type2diabetic rats excellent alone, with metformin treatment effect of organic germanium (Ge-132) for the treatment of type2diabetes have a synergistic effect. This synergy might Organic Germanium (Ge-132) by correcting the GLUT4protein expression in diabetic rat skeletal muscle cell membrane, enhanced skeletal muscle glucose uptake.