大鼠心脏功能 z-bo 2020-11-23 364

　　Introduction: Obesity is a global epidemic, and obesity-related complications such as type 2 diabetes (T2D) and cardiovascular disease (CVD) have also increased. Although this increase is multifactorial in nature, well-known risk factors include a sedentary lifestyle and poor eating habits, such as increasing the calorie intake of sugary drinks (SSB). A large number of risk factors and adverse effects are caused by the body's inability to compensate for excessive calorie consumption. Studies have shown that high SSB intake is related to many risk factors for the onset of cardiometabolic diseases, such as increased visceral fat deposition, increased triglycerides and cholesterol metabolites, and weight gain. The consumption of various carbohydrates (such as sugar and high-fructose corn syrup) can lead to high blood glucose load, inflammation and insulin resistance in the diet. In addition, high SSB consumption will lead to increased blood sugar and insulin concentration, which directly hinders hepatic insulin signal transduction, thereby promoting insulin resistance. SSB intake may not trigger a satiety response, which may be an important link between increased fluid calories and increased health risks. Although there is evidence that the intake of SSB is related to the development of cardiometabolic diseases, the underlying mechanism that causes such complications remains unclear. We evaluate the effect of SSB by establishing a unique in vivo experimental model. In the current study, we investigated whether actual consumption of SSB for 3 months and 6 months would trigger a hypothetical link between metabolic disorders and mitochondrial and cardiac function. We paid special attention to the role of the non-oxidized glucose pathway (nogps), namely the role of the polyol pathway, the hexosamine biosynthesis pathway (hbp) and pkc in this process, because these pathways were previously associated with the occurrence of cardiometabolic complications . Because nogps is a branch pathway of glycolysis, it has an up-regulating effect on the increase of glucose flow, which triggers harmful downstream pathways, which leads to cardiac contractile dysfunction.

　　Animals and experimental protocol: Male Wistar rats weighing ~250 grams were divided into two groups: a, SSB was given; b, pure water was given; the time was 3 months and 6 months (n=6 per group). This product contains 4 grams of sucrose per 100 ml, that is, 176.7 kJ per 250 ml. According to group allocation and body weight classification, rats were gavaged with experimental doses every day. Use the surface area to volume ratio to calculate the dose volume and correct for 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 is measured every week, the rats are weighed every week, and the percentage of weight gain is recorded to evaluate the development of obesity. After completion of the experimental procedure, the rats were euthanized, organs were harvested, weighed, and quick-frozen, and stored at -80°C until further analysis.

　　Blood collection: The animal is fasted overnight (at least 12 hours), then sedated with 3% isoflurane, and then 1 ml of blood is drawn from the right jugular vein. Centrifuge the blood, take the supernatant, and evaluate the levels of uric acid, alanine aminotransferase (ALT), hemoglobin A1C (HbA1c), triglycerides, and total cholesterol (mmol/L) using standard procedures. Different methods are used to determine glycosylated hemoglobin. The NGSP system is the most widely used system, and its results are expressed in percent saccharification. The IFCC method uses high performance liquid chromatography to separate glycated and non-glycated peptides. Mass spectrometry or capillary electrophoresis was used for quantitative analysis of each group, and the results were expressed in mmol/L. Use regression equation to calculate EAG, the measurement unit is mmol/L. Use commercially available enzyme-linked immunosorbent assay kit to measure insulin level.

　　Oral glucose tolerance test: Initially assess the baseline fasting blood glucose level, dissolve glucose powder in distilled water (0.86 g/kg body weight), gavage rats and monitor for 120 minutes. Obtain the results at the following time points 5, 10, 15, 30, 45, 60, and 120 (minutes).

　　Study on mitochondrial respiration: As mentioned above, the polarographic oxygen sensor was used to measure mitochondrial respiration in a 2 ml glass chamber of an oxygen analyzer 2K. Use saponin to infiltrate myocardial fibers (~2 mg) and place them in two oxygen tracing chambers, and then measure endogenous regular (R) respiration when the oxygen flux is stable. Pyruvate (5mM), glutamate (10mM) and malate (2mM) are used to induce glucose oxidation, and octylcarnitine (0.2 mM) and malic acid (2 mM) are used to induce fatty acid (FA) oxidation. Oxidative phosphorylation (oxphos) was measured by adding 2.5 mM ADP, and then adding 2.5 μM oligomycin to induce leak respiration by inhibiting ATP synthase. .Titrate with the uncoupling agent CCCP (0.5 M) to increase breathing to the maximum level, which is called the electron transfer system (ETS) capacity. Then rotenone (complex I inhibitor) was added to a final concentration of 0.5 M, and at the same time, 2.5 M antimycin A was added to induce residual oxygen consumption (complex III inhibition). Finally, titrate 0.5 mM TMPD and 2 mM ascorbic acid to evaluate the complex venous connection respiration as a representative of mitochondrial content. The oxygen flow in all breathing states is normalized to the compound venous flow to correct the changes in the cell content in the oxygen tracing chamber. 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 rats were lightly anesthetized with 1.5-2% isoflurane and placed in a supine position on a warming pad. The closed chest echocardiography was performed using the VEVO 2100 ultrasound system and 13-25 MHz linear array sensor. In order to evaluate the left ventricular diastolic function, in the apical 4-chamber view, the mitral valve E and peak velocity are obtained by pulse Doppler, and then the E/A ratio is calculated. All measurements are performed offline on the average of at least three consecutive cardiac cycles with software installed on the ultrasound system. For the perfusion study, rats were anesthetized with sodium pentobarbital (160 mg/kg body weight). The heart was quickly removed, retained in ice-cold (4°C) Krebs-Henseleit bicarbonate buffer, and installed through the aorta to the aortic cannula. The left atrium is also cannulated through the pulmonary vein. First, the heart was perfused retrogradely for 10 minutes in a non-circular manner under constant hydrostatic pressure (100 cmH2O), and then in the working heart mode (preloaded with 15 cmH2O, followed by 100 cmH2O) for 20 minutes. The heart does not have electrical pacing, and the myocardial temperature is thermostatically controlled and regularly monitored (constant at 37°C during reperfusion and 36.5°C during ischemia). Then, the proximal left anterior descending artery was ligated and reperfused for 2 hours to perform 35 minutes of ischemia on the heart. Determine the area of myocardial infarction as described above.

　　Myocardial fat and glucose metabolism: Assessment of tissue triglyceride levels uses the Picoprobe fluorescence measurement triglyceride quantitative analysis kit. The levels of myocardial glycogen and glycogen synthase 1 were measured using commercially available kits.

　　Results: Changes in body weight and organs: Compared with the control group, the SSB group gained more weight. The area under the curve (AUC) analysis showed that the SSB group gained significant weight at three and six months. There was no difference in feed consumption between the two groups, and no significant difference in tissue weight (expressed as a percentage of final body weight).

　　blood metabolites and OGTTS: measured serum levels after fasting: uric acid, ALT, HbA1c, cholesterol, triglycerides and glucose. Three months later, the HbA1c detection in the SSB group increased (P<0.05 vs control), and the serum cholesterol level increased. By 6 months, HbA1c (IFCC) in the SSB group was still elevated (P<0.05 compared with the control group), but there was no significant difference in cholesterol levels. After six months, the SSB group showed higher uric acid levels compared to 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 compared with the control group, the SSB group had lower AUC at 3 and 6 months.

　　Mitochondrial respiration: The results showed that at the 6-month time point, there were no significant differences in the various parameters of the glucose oxidation test between the groups. Pyruvate, glutamic acid and malic acid are used as oxidation substrates. Similar results were also observed when examining the response of oxonyl, LEAK, and excess E-R ratios to FA oxidation (caprinol carnitine and malic acid substrate). When FA oxidation substrate was added, the ETS ratio of the SSB group was significantly reduced. Due to technical problems, respiratory data could not be generated at the three-month time point.

　　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 months and 6 months. Cardiac perfusion data showed that there was no difference in any parameters between the SSB treatment group and the control group during the stable period and the recovery period after simulated ischemia. Infarct size measurement showed no difference between the two groups, although AR increased in the SSB group after 6 months.

　　Myocardial Lipid and Glucose Metabolism: We evaluated several metabolic markers to better understand mitochondrial respiratory and cardiac function data. Heart tissue triglyceride levels between the two groups remained unchanged at both time points. At 6 months, the concentration of myocardial glycogen in the SSB group was significantly reduced, but the level of glycogen synthase 1 did not change significantly.

　　Conclusion: In summary, the intake of SSB is 3 months and 6 months respectively, and it will not cause cardiac dysfunction or IR/T2DM. Nonetheless, early changes at the molecular level may put this organism at higher risk in the long term, especially under stressful conditions.