动物造模,心脏功能 z-bo 2020-09-10 362

　　Introduction: Obesity is a global epidemic, and obesity-related complications such as type 2 diabetes (T2D) and cardiovascular disease (CVD) are increasing. Although this increase is multifactorial in nature, well-known risk factors include a sedentary lifestyle and poor eating habits, such as increased calorie intake of sugary drinks (SSB). .. The human body’s inability to compensate for excessive calorie consumption can lead to many risk factors and adverse effects. Studies have shown that high SSB intake is associated with many risk factors for the development of heart disease, including increased visceral fat deposition, increased triglycerides and cholesterol metabolites, and weight gain. The intake of various carbohydrates (such as sugar and fructose corn syrup) can cause hyperglycemia, inflammation and insulin resistance in the diet. In addition, increasing the consumption of SSB will increase blood sugar and insulin levels, directly destroy hepatic insulin signaling and promote insulin resistance. Similarly, the intake of SSB may not cause satiety. This may be an important link between increased water calories and increased health risks. Although there is evidence that intake of SSB is associated with cardiovascular and metabolic disorders, the underlying mechanism leading to this complication remains unclear. Evaluate the effect of SSB by establishing a unique in vivo experimental model. The current study investigated whether the actual intake of SSB between 3 and 6 months created a fictitious link between metabolic disorders and mitochondria and heart function. Special attention was paid to the role of the non-oxidized glucose pathway (nogps), namely the polyol pathway, the hexosamine biosynthesis pathway (hbp) and the role of pkc in this process. This is because these pathways were previously associated with the occurrence of cardiovascular complications.

　　Since nogps is a branch pathway of glycolysis, it has an up-regulation effect on increased glucose flow, triggering harmful downstream pathways and causing cardiac contractile dysfunction. Animal experiment protocol: Male Wistar rats weighing no more than 250 grams were divided into two groups: a. Give SSB; b Give pure water; the time is 3 months and 6 months respectively (n = 6 per group). This product contains 4 grams of cream per 100 ml and 176.7 kJ per 250 ml. According to group distribution and body weight classification, the experimental dose was given to rats by gavage every day. Use the surface volume ratio to calculate the dose volume and correct the weight. The dosage here simulates the daily intake of 125 ml (54 calories) of SSB for a 60 kg person. The food intake of the animals was measured every week, the rats were weighed every week, and the weight gain percentage was recorded to assess the development of obesity. After the experimental procedure was completed, the rats were euthanized, the organs were harvested, weighed, quickly frozen and stored at -80°C until further analysis. Blood collection: The animals were fasted overnight (at least 12 hours), then sedated with 3% isoflurane, and 1 ml of blood was collected from the right jugular vein. Centrifuge the blood using standard procedures, take the supernatant, and evaluate the levels of uric acid, alanine aminotransferase (ALT), hemoglobin A1C (HbA1c), triglycerides and total cholesterol (mmol/L). I will. Various methods are used to determine glycosylated hemoglobin. The GSP system is the most widely used system, and the results are expressed in terms of saccharification rate. The IFCC method uses fast liquid chromatography to separate glycosylated and non-glycosylated peptides. Use mass analysis or capillary electrophoresis to quantitatively analyze each group, and the results are expressed in mmol/L. Use regression equation to calculate EAG. The unit of measurement is mmol/L. To measure insulin levels, use a commercially available enzyme-binding immunosorbent measurement kit.

　　Oral glucose tolerance test: First, to assess the baseline of fasting blood glucose level, glucose powder is dissolved in distilled water (0.86 g/kg body weight), administered by gavage, and monitored for 120 minutes. Next time you will get results at 5, 10, 15, 30, 45, 60, and 120 (minutes). Research on mitochondrial respiration: As mentioned above, the polarographic oxygen sensor is used to measure mitochondrial respiration in the 2 ml glass chamber of a 2K oxygen analyzer. Use saponin to penetrate into myocardial fibers (approximately 2 mg), place them in two oxygen tracking chambers, and measure endogenous regular (R) breathing when the oxygen flux is stable. Pyruvate (5 mM), glutamate (10 mM), and malic acid (2 mM) are used to induce glucose oxidation, and octyl carnitine (0.2 mM) and malic acid (2 mM) are used to oxidize fatty acids (FA). I will guide you. Phosphorus (oxphos) was measured by adding 2.5 mM ADP and then adding 2.5 μM oligomycin to induce dyspnea by inhibiting ATP synthase. .. Immersion uncoupled CCCP (0.5M) to increase breathing to the maximum level. This is called the electronic transmission system (ETS) capacity. Next, add rotenone (complex I inhibitor) to a final concentration of 0.5 M, while adding 2.5 M antimycin A to induce residual oxygen consumption (complex III inhibition). Finally, titrate 0.5 mM TMPD and 2 mM ascorbic acid to evaluate complex venous respiration as a representative of mitochondrial content. The oxygen flow rate under all breathing conditions is normalized to a compound venous flow rate to compensate for changes in cell content in the oxygen tracking room. Calculate the excess E-R volume to determine the difference between ETS volume and R breath.

　　Heart function: One week before the end of the experiment, the rat was gently anesthetized with 1.5-2% isoflurane, and then placed on a heating pad. Use VEVO2100 ultrasound system and 13-25 MHz linear array sensor for chest cavity closed echocardiography. In order to evaluate the left ventricular diastolic function, in the apical four-chamber view, the mitral valve E and peak velocity are obtained by pulsed Doppler, and then the E/A ratio is calculated. Using the software installed on the ultrasound system, all measurements are taken offline, averaging at least three consecutive cardiac cycles. For the perfusion test, the rats were anesthetized with sodium pentobarbital (160 mg/kg body weight). The heart is quickly removed, placed in ice-cold (4°C) Krebs-Henslate bicarbonate buffer, and connected to the aortic cannula through the aorta. The left atrium is also cannulated through the pulmonary vein. First, the heart was retrogradely perfused for 10 minutes under a constant hydrostatic pressure (100 cmH2O), and then in the cardiac function mode (preloaded with 15 cmH2O, then loaded with 100 cmH2O) for 20 minutes. The heart does not have electrical pacing, and the myocardial temperature is thermostat-controlled and regularly monitored (constant at 37°C during reperfusion and at 36.5°C during ischemia). Then the proximal left anterior descending branch was ligated and reperfused for 2 hours to make the heart ischemic for 35 minutes. Determine the area of myocardial infarction as described above.

　　Myocardial fat and glucose metabolism: Use the pico probe fluorescence measurement in the triglyceride quantitative analysis kit to assess tissue triglyceride levels. The levels of myocardial glycogen and glycogen synthase 1 were measured using commercially available kits.

　　Result: Changes in body weight and organs: The body weight of the SSB group increased compared to the control group. The area under the curve (AUC) analysis showed that the weight of the SSB group increased significantly at 3 and 6 months. There was no difference in feed consumption between the two groups, nor was there a significant difference in tissue weight (expressed as a percentage of the final weight).

　　blood metabolites and OGTTS: serum levels measured after fasting: uric acid, ALT, HbA1c, cholesterol, triglycerides, glucose. After 3 months, the detection rate of HbA1c in the SSB group increased (compared with the control group, P\u003c0.05), and the serum cholesterol level increased. By 6 months, HbA1c (IFCC) in the SSB group was still elevated (compared with the control group, P\u003c0.05), but there was no significant difference in cholesterol levels. After 6 months, the uric acid level of the SSB group was higher than that of the control group. HOMA-IR data showed that there was no significant difference in SSB consumption between the two experimental time points. OGTT data showed that the AUC of the SSB group was lower than that of the control group at 3 and 6 months. Mitochondrial respiration: The results showed that at 6 months, there was no significant difference in the parameters of the glucose oxidation test between the two groups. Pyruvate, glutamic acid and malic acid are used as oxidation substrates. When examining the response of excess E-R to oxon, LEAK and FA oxidation (caprolactone carnitine and malate substrates), similar results were observed. The addition of FA oxide substrate significantly reduced the ETS ratio of the SSB group. Due to technical issues, we were unable to generate respiratory data within 3 months. In vivo and in vitro cardiac function assessment: echocardiographic analysis showed that there was no significant difference between the control group and the SSB group after 3 and 6 months. Cardiac perfusion data showed that there was no difference in parameters between the SSB treatment group and the control group during the stable and recovery phases after simulated ischemia. In the infarct size measurement, there was no difference between the two groups, but after 6 months, the AR in the SSB group increased. Myocardial lipid and glucose metabolism: Several metabolic indicators were evaluated to better understand mitochondrial respiration and cardiac function data. The levels of triglycerides in the heart tissue between the two groups did not change at both time points. At 6 months, the level of myocardial glycogen in the SSB group decreased significantly, but the level of glycogen synthase 1 did not change significantly.

　　Conclusion: In short, the intake of SSB for 3 months and 6 months will not cause cardiac dysfunction or IR/T2DM. However, over time, early changes at the molecular level put organisms at higher risk for a period of time, especially under stressful conditions.