重度烫伤大鼠早期高血糖和急性胰岛素抵抗的实验研究
发布时间：2018-08-01 14:14
【摘要】：研究背景 近年来，急性高血糖引起人们越来越多的关注。此种高血糖往往发生在重度烧伤、感染、创伤、心梗和大手术后等危重症状态下，表现为血糖迅速升高和患者既往无糖尿病病史的特点，而由于在多项临床研究中，血糖升高幅度与机体损伤程度、患者死亡率等不良预后指标高度相关,因而又常被称为“危重症糖尿病”。 在急性高血糖的同时，患者往往出现高胰岛素血症和糖耐量受损，提示其存在胰岛素抵抗现象。而在严重烧伤、体外循环术后、心梗急性期等患者均有用胰岛素控制血糖效果不佳的报道，进一步反映危重症情况下机体对胰岛素敏感性降低。作为慢性病“共同土壤”的胰岛素抵抗往往经年累月才能出现，而危重症患者所出现的急性胰岛素抵抗（AIR）则具有快速发生的特点，仅需要几天，几小时甚至数分钟就可出现，这种在出现时间上的巨大差异是否意味着二者在产生机制和其它表现形式上有所不同，其临床意义何在，目前均不清楚。 另外，从Greet Van den Berghe等人进行的Leuven研究开始，人们就开始关注用胰岛素严格控制血糖水平对ICU病人预后的影响。但随后名为“NICE-SUGAR”的多中心大样本随机对照研究得出了强化胰岛素治疗对患者死亡率没有影响，且会增加低血糖风险的阴性结论。由于褒贬不一，强化胰岛素治疗目前仍难以在临床广泛应用。但是否AIR的出现是导致强化胰岛素治疗效果欠佳的重要原因，成为当前亟待回答的重要问题。 在大面积重度烧伤病人和相应烧、烫伤动物模型中，高血糖的出现均较为典型。因此，本研究拟以重度烫伤大鼠作为实验动物模型，深入研究高血糖和急性胰岛素抵抗出现的特点，以期有助于指导临床危重症患者的诊疗工作。研究目的 1.观察重度烫伤大鼠早期高血糖特点及其与大鼠死亡率之间的关系。 2.探究重度烫伤后大鼠是否存在急性胰岛素抵抗和其临床意义。 实验方法 将80只雄性SD大鼠（200-220g）腹、背部固定区域剃毛后随机分为（1）假伤组(37℃温水处理)；（2）烫伤组（95℃热水处理，背部烫伤15s，腹部8s）；（3）早期胰岛素治疗组（烫伤后立即皮下注射胰岛素2.5IU/kg，目的在于控制烫伤后第1个高血糖峰）；（4）晚期胰岛素治疗组（于烫伤后2.5h皮下注射胰岛素2.5IU/kg，目的是控制烫伤后出现的第2个高血糖峰），每组20只，乙醚麻醉后用自制模具造成40%总体表面积（TBSA）重度烫伤，腹腔注射生理盐水抗休克（40ml/kg）后分组给予不同处理。 1.于大鼠烫伤前和烫伤后30min、1h、3h、6h、12h分别剪尾采血观察急性期各组大鼠血糖变化； 2.于大鼠烫伤前和烫伤后30min、1h、3h、6h、12h颈动脉取血，静置离心后取血清，用放免法测定各时间点胰岛素、胰高血糖素和糖皮质激素的变化； 3.计算烫伤组大鼠HOMA-IR指数［血清胰岛素(μU/ml)×血糖(mM)/22.5］作为胰岛素抵抗程度参考，采用腹腔注射葡萄糖耐量实验（IPGTT）和胰岛素敏感性实验(IST)分别测定烫伤后3h和10h组大鼠的胰岛素敏感性； 4.在烫伤后3h测定各组大鼠血流动力学指标：平均动脉压（MABP）、左室收缩压（LVSP）、心率（HR）、左室收缩/舒张压最大变化速率（±LVdP/dtmax）和皮肤微循环灌注情况； 5.用Western-blot测定假伤组和烫伤组大鼠心肌、骨骼肌组织pAkt/Akt，pGSK3β/GSK3β和peNOS/eNOS在烫伤后30min、1h、3h、6h、12h的表达量与活性变化； 6.分别记录各组大鼠烫伤后12h内生存曲线与14d内烫伤大鼠的体重、糖耐量和存活率变化情况。 实验结果 1.重度烫伤大鼠在伤后12h内出现两个血糖峰值，分别是在烫伤后30min（7.4±0.3mM,较基础血糖升高13%, n=32, P0.05）和烫伤后3h（10.0±1.0mM,较基础血糖升高35%, n=31, P0.01）。进行相关分析发现两血糖峰值之间呈线性相关（r2=0.787, P0.01, n=31）。以烫伤后3h大鼠血糖值中位数（8mM）作为分组依据，可发现烫伤后较低血糖组大鼠生存率（BG8mM,92%）明显高于高血糖组（BG8mM,44%）（n=12-16, P0.05）。 2.与假伤组相比，烫伤组大鼠血清胰高血糖素在伤后30min显著升高（n=6, P0.05），之后在6h逐渐回落（n=6, P0.05），，而烫伤组糖皮质激素和胰岛素在烫伤后12h内较假伤组始终处于显著升高水平(n=6,P0.05)。 3. HOMA-IR值提示重度烫伤后大鼠出现胰岛素抵抗。在烫伤后3h和10h行腹腔糖耐量实验和胰岛素敏感性实验，结果表明烫伤后存在急性胰岛素抵抗（AIR）现象，且在烫伤后3h机体胰岛素敏感性最低(n=6,P0.05)。 4. Western-blot结果显示，烫伤组大鼠心肌和骨骼肌中pAkt/Akt,pGSK3β/GSK3β和peNOS/eNOS比值均随时间发生变化，表现为烫伤后30min起开始升高，至1h达到峰值(n=4, P0.05)，3h后逐渐下降，至12h趋于稳定。表明在重度烫伤所致的应激条件下，尽管机体胰岛素水平升高，但其信号通路下游分子在伤后3h出现激活障碍。 5.与假伤组相比，大鼠在烫伤后3h MABP（97.6±5.7vs.126.7±6.7mmHg, n=6, P0.05），LVSP（97.6±6.7vs.146.7±7.7mmHg, n=6,P0.01），HR（348.8±17.4vs.450±17.1bpm, n=6, P0.01），±LVdP/dtmax（2746.8±432.4vs.5581±384.7mmHg/s, n=6, P0.01；1955±244.7vs.4532±439.0mmHg/s, n=6, P0.01）和体表微循环灌注均显著降低（n=6, P0.05），表明重度烫伤后心血管功能明显受损。 6.与烫伤组相比，在烫伤后立即给予胰岛素治疗可以抑制随后出现的30min与3h血糖峰值，显著提高烫伤后3h与10h的胰岛素敏感性（n=6,P0.05），增强3h心脏血流动力学和体表微循环灌注（n=6, P0.05），最终使早期胰岛素治疗组大鼠烫伤后12h内存活率较烫伤组显著提高（93%vs.71%, n=34-43, P0.05），14d内存活状态（体重，糖耐量和生存率）也较烫伤组明显改善（n=6-43, P0.05）。 7.在烫伤后2.5h予以同等剂量胰岛素治疗后，与烫伤组相比，虽然能暂时降低烫伤后3h血糖峰值，但随后另一血糖峰会在伤后8h出现，12h内大鼠存活率较烫伤组有下降趋势但未见显著差异(55.6%vs.71%, n=18-34, P=0.17)，且14d内存活状态（体重，糖耐量和生存率）与烫伤组相比无改善（n=6-34, P=0.21）。 结论 1．重度烫伤大鼠在急性期会出现两个高血糖峰，其中第2个血糖峰的出现往往与预后不良密切相关。 2．从整体胰岛素敏感性和分子信号两方面证明重度烫伤大鼠出现急性胰岛素抵抗现象（AIR）,且在烫伤后3h最为严重。 3．分别干预两个血糖峰，发现针对第一个血糖峰（H1）的胰岛素治疗不仅能有效降低血糖，还可减轻AIR，改善心功能和体表微循环灌注，最终提高大鼠存活率;而用同等剂量胰岛素控制第2个血糖峰（H2）则难以发挥相应保护作用，推测其机制可能与AIR的出现有关。
[Abstract]:Research background
In recent years, acute hyperglycemia has attracted more and more attention. This hyperglycemia often occurs in severe burns, infections, trauma, myocardial infarction, and major postoperative critical conditions, characterized by rapid rise in blood sugar and no history of diabetes in the patient, but the increase in blood sugar and the body's injury process in many clinical studies It is highly correlated with adverse prognostic indicators such as mortality, and is often referred to as "critical diabetes".
At the same time of acute hyperglycemia, patients often have hyperinsulinemia and impaired glucose tolerance, suggesting the presence of insulin resistance. In severe burns, patients with acute myocardial infarction, after cardiopulmonary bypass, are used to report the poor effect of insulin on blood glucose control, further reflecting the sensitivity of the body to insulin sensitivity in critical conditions. Low. Insulin resistance, as a chronic disease "common soil", often occurs over the years, and the acute insulin resistance (AIR) of critically ill patients has a rapid occurrence, only a few days, a few hours or even a few minutes. Whether the huge difference in time means that the two are producing the machine. It is unclear what the clinical significance of the system differs from other forms.
In addition, from the Leuven study conducted by Greet Van den Berghe and others, people began to pay attention to the effect of insulin on the prognosis of ICU patients strictly controlled by insulin. However, the subsequent multicenter, randomized controlled study called "NICE-SUGAR" has concluded that intensive insulin therapy does not affect the mortality of patients and increases the low blood pressure. The negative conclusion of sugar risk is still difficult to be widely used in clinical practice because of mixed treatment. But whether the emergence of AIR is an important cause of the poor effect of intensive insulin therapy and is an important question to be answered urgently.
In the large area of severe burn patients and the corresponding burn and scald animal models, the appearance of hyperglycemia is more typical. Therefore, this study intends to use severe scald rats as an experimental animal model to further study the characteristics of hyperglycemia and acute insulin resistance in order to help guide the diagnosis and treatment of clinical critical patients.
1. to observe the relationship between hyperglycemia and mortality in severely scalded rats.
2. to investigate whether acute insulin resistance and its clinical significance exist after severe scald in rats.
Experimental method
After shaving 80 male SD rats (200-220g), the back fixed area was randomly divided into (1) false injury group (37 C warm water treatment); (2) scald group (95 C hot water treatment, back scald 15s, abdominal 8s); (3) early insulin treatment group (immediately after scald, subcutaneous injection of insulin 2.5IU/kg, aimed at controlling first hyperglycemia peaks after scald); (4) late late. The insulin treatment group (2.5h subcutaneous injection of insulin 2.5IU/kg after scald was designed to control second hyperglycemia peaks in each group), 20 in each group. After eether anesthesia, the total surface area (TBSA) of the total surface area (TBSA) was severely scalded, and the intraperitoneal injection of saline against Hugh (40ml/kg) was given to different treatments.
1. blood samples were taken from rats before scalding and scalding for 30min, 1H, 3h, 6h and 12h respectively to observe the blood glucose changes in each group during acute phase.
2. the blood of 30min, 1H, 3h, 6h, 12h was taken before and after scald in rats. The serum was taken after static centrifugation. The changes of insulin, glucagon and glucocorticoid were measured by radioimmunoassay at all time points.
3. the HOMA-IR index [serum insulin (micron) (mM) /22.5] of rats in the scald group was calculated as a reference for insulin resistance. The insulin sensitivity was measured by intraperitoneal injection of glucose tolerance test (IPGTT) and insulin sensitivity test (IST), respectively, in group 3H and 10h group after scald.
4. after the scald, the hemodynamic indexes of the rats were measured by 3H: mean arterial pressure (MABP), left ventricular systolic pressure (LVSP), heart rate (HR), the maximum change rate of left ventricular systolic / diastolic pressure (+ LVdP/dtmax) and skin microcirculation perfusion.
5. the myocardium of rats in the injured group and the scald group was measured by Western-blot. The expression and activity of 30min, 1H, 3h, 6h, 12h in the skeletal muscle tissue pAkt/Akt, pGSK3 beta /GSK3 beta and peNOS/eNOS were changed after the scald.
6. the survival curves of 12h after scald and the changes of body weight, glucose tolerance and survival rate of scalded rats in each 14d group were recorded respectively.
experimental result
In 1. severe scald rats, there were two peak blood glucose peaks in 12h after injury, which were 30min (7.4 + 0.3mM, 13%, n=32, P0.05) after scald and 3H (10 + 1.0mM, 35%, n=31, P0.01) after scald. The correlation analysis showed that the peak of two blood sugar showed linear correlation (r2=0.787, P0.01, n=31). 3 after scald. The median blood glucose value (8mM) of H rats was used as a basis for grouping, and the survival rate of rats in the lower blood glucose group after scald (BG8mM, 92%) was significantly higher than that in the hyperglycemia group (BG8mM, 44%) (n=12-16, P0.05).
2. compared with the false injury group, the serum glucagon in the scald group increased significantly after the injury (n=6, P0.05), and then decreased gradually in the 6h (n=6, P0.05), while the glucocorticoid and insulin in the scald group remained significantly higher than the false group in the scald group (n=6, P0.05) in the scald group after the burn.
3. HOMA-IR values showed insulin resistance in rats after severe scald. In 3H and 10h after scald, intraperitoneal glucose tolerance test and insulin sensitivity test showed that there was acute insulin resistance (AIR) after scald, and the insulin sensitivity was the lowest in 3h after scald (n=6, P0.05).
4. Western-blot results showed that the ratio of pAkt/Akt, pGSK3 beta, /GSK3 beta and peNOS/eNOS in the myocardium and skeletal muscle of the scalded rats varied with time, which showed that 30min began to rise after scald, to the peak of 1H (n=4, P0.05), and the 3H decreased gradually to 12h, indicating that the pancreas in severe scald caused by the stress conditions, despite the body pancreas. The level of islet is increased, but the downstream molecules of signaling pathway are activated after 3H.
5. 3H MABP (97.6 + 5.7vs.126.7 + 6.7mmHg, n=6, P0.05), LVSP (97.6 + 6.7vs.146.7 + 7.7mmHg, n=6, P0.01) after scald, LVSP (97.6 + 6.7vs.146.7 + 7.7mmHg, n=6, P0.01). Perfusion significantly decreased (n=6, P0.05), indicating severe cardiovascular injury after severe scald.
6. compared with the scald group, insulin treatment immediately after scald could inhibit the subsequent peak of 30min and 3H blood glucose, significantly increase the insulin sensitivity of 3H and 10h after scald (n=6, P0.05), enhance 3H cardiac hemodynamics and body surface microcirculation perfusion (n=6, P0.05), and eventually lead to the survival of the early insulin treatment group after the scald in the rat 12h. Compared with the scald group, the survival rate was significantly increased (93% vs. 71%, n = 34-43, P 0.05). The living state (body weight, glucose tolerance and survival rate) in 14 days was also significantly improved (n = 6-43, P 0.05).
7. after the same dose of insulin after the scald, 2.5h was compared with the scald group, although it could temporarily reduce the peak of 3H blood sugar after scald, but then the other blood sugar summit appeared at 8h after injury. The survival rate in 12h rats was lower than that in the scald group, but there was no significant difference (55.6%vs.71%, n=18-34, P=0.17), and the memory of 14d (weight, glucose tolerance) There was no improvement in the volume and survival rate compared with the scald group (n=6-34, P=0.21).
conclusion
1. Two hyperglycemic peaks appear in the acute phase of Severe Scalded rats, and the second one is often closely related to poor prognosis.
2. Acute insulin resistance (AIR) was found in severely scalded rats in terms of overall insulin sensitivity and molecular signaling, and it was most severe at 3 hours after scald.
3. intervention of two blood glucose peaks, and it is found that insulin therapy against the first glucose peak (H1) can not only effectively reduce blood sugar, but also reduce AIR, improve cardiac function and body surface microcirculation perfusion, and ultimately improve the survival rate of rats, while the same dose of insulin control second blood glucose peaks (H2) is difficult to play the corresponding protective effect, speculate the mechanism It may be related to the emergence of AIR.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363
本文编号：2157839
[Abstract]:Research background
In recent years, acute hyperglycemia has attracted more and more attention. This hyperglycemia often occurs in severe burns, infections, trauma, myocardial infarction, and major postoperative critical conditions, characterized by rapid rise in blood sugar and no history of diabetes in the patient, but the increase in blood sugar and the body's injury process in many clinical studies It is highly correlated with adverse prognostic indicators such as mortality, and is often referred to as "critical diabetes".
At the same time of acute hyperglycemia, patients often have hyperinsulinemia and impaired glucose tolerance, suggesting the presence of insulin resistance. In severe burns, patients with acute myocardial infarction, after cardiopulmonary bypass, are used to report the poor effect of insulin on blood glucose control, further reflecting the sensitivity of the body to insulin sensitivity in critical conditions. Low. Insulin resistance, as a chronic disease "common soil", often occurs over the years, and the acute insulin resistance (AIR) of critically ill patients has a rapid occurrence, only a few days, a few hours or even a few minutes. Whether the huge difference in time means that the two are producing the machine. It is unclear what the clinical significance of the system differs from other forms.
In addition, from the Leuven study conducted by Greet Van den Berghe and others, people began to pay attention to the effect of insulin on the prognosis of ICU patients strictly controlled by insulin. However, the subsequent multicenter, randomized controlled study called "NICE-SUGAR" has concluded that intensive insulin therapy does not affect the mortality of patients and increases the low blood pressure. The negative conclusion of sugar risk is still difficult to be widely used in clinical practice because of mixed treatment. But whether the emergence of AIR is an important cause of the poor effect of intensive insulin therapy and is an important question to be answered urgently.
In the large area of severe burn patients and the corresponding burn and scald animal models, the appearance of hyperglycemia is more typical. Therefore, this study intends to use severe scald rats as an experimental animal model to further study the characteristics of hyperglycemia and acute insulin resistance in order to help guide the diagnosis and treatment of clinical critical patients.
1. to observe the relationship between hyperglycemia and mortality in severely scalded rats.
2. to investigate whether acute insulin resistance and its clinical significance exist after severe scald in rats.
Experimental method
After shaving 80 male SD rats (200-220g), the back fixed area was randomly divided into (1) false injury group (37 C warm water treatment); (2) scald group (95 C hot water treatment, back scald 15s, abdominal 8s); (3) early insulin treatment group (immediately after scald, subcutaneous injection of insulin 2.5IU/kg, aimed at controlling first hyperglycemia peaks after scald); (4) late late. The insulin treatment group (2.5h subcutaneous injection of insulin 2.5IU/kg after scald was designed to control second hyperglycemia peaks in each group), 20 in each group. After eether anesthesia, the total surface area (TBSA) of the total surface area (TBSA) was severely scalded, and the intraperitoneal injection of saline against Hugh (40ml/kg) was given to different treatments.
1. blood samples were taken from rats before scalding and scalding for 30min, 1H, 3h, 6h and 12h respectively to observe the blood glucose changes in each group during acute phase.
2. the blood of 30min, 1H, 3h, 6h, 12h was taken before and after scald in rats. The serum was taken after static centrifugation. The changes of insulin, glucagon and glucocorticoid were measured by radioimmunoassay at all time points.
3. the HOMA-IR index [serum insulin (micron) (mM) /22.5] of rats in the scald group was calculated as a reference for insulin resistance. The insulin sensitivity was measured by intraperitoneal injection of glucose tolerance test (IPGTT) and insulin sensitivity test (IST), respectively, in group 3H and 10h group after scald.
4. after the scald, the hemodynamic indexes of the rats were measured by 3H: mean arterial pressure (MABP), left ventricular systolic pressure (LVSP), heart rate (HR), the maximum change rate of left ventricular systolic / diastolic pressure (+ LVdP/dtmax) and skin microcirculation perfusion.
5. the myocardium of rats in the injured group and the scald group was measured by Western-blot. The expression and activity of 30min, 1H, 3h, 6h, 12h in the skeletal muscle tissue pAkt/Akt, pGSK3 beta /GSK3 beta and peNOS/eNOS were changed after the scald.
6. the survival curves of 12h after scald and the changes of body weight, glucose tolerance and survival rate of scalded rats in each 14d group were recorded respectively.
experimental result
In 1. severe scald rats, there were two peak blood glucose peaks in 12h after injury, which were 30min (7.4 + 0.3mM, 13%, n=32, P0.05) after scald and 3H (10 + 1.0mM, 35%, n=31, P0.01) after scald. The correlation analysis showed that the peak of two blood sugar showed linear correlation (r2=0.787, P0.01, n=31). 3 after scald. The median blood glucose value (8mM) of H rats was used as a basis for grouping, and the survival rate of rats in the lower blood glucose group after scald (BG8mM, 92%) was significantly higher than that in the hyperglycemia group (BG8mM, 44%) (n=12-16, P0.05).
2. compared with the false injury group, the serum glucagon in the scald group increased significantly after the injury (n=6, P0.05), and then decreased gradually in the 6h (n=6, P0.05), while the glucocorticoid and insulin in the scald group remained significantly higher than the false group in the scald group (n=6, P0.05) in the scald group after the burn.
3. HOMA-IR values showed insulin resistance in rats after severe scald. In 3H and 10h after scald, intraperitoneal glucose tolerance test and insulin sensitivity test showed that there was acute insulin resistance (AIR) after scald, and the insulin sensitivity was the lowest in 3h after scald (n=6, P0.05).
4. Western-blot results showed that the ratio of pAkt/Akt, pGSK3 beta, /GSK3 beta and peNOS/eNOS in the myocardium and skeletal muscle of the scalded rats varied with time, which showed that 30min began to rise after scald, to the peak of 1H (n=4, P0.05), and the 3H decreased gradually to 12h, indicating that the pancreas in severe scald caused by the stress conditions, despite the body pancreas. The level of islet is increased, but the downstream molecules of signaling pathway are activated after 3H.
5. 3H MABP (97.6 + 5.7vs.126.7 + 6.7mmHg, n=6, P0.05), LVSP (97.6 + 6.7vs.146.7 + 7.7mmHg, n=6, P0.01) after scald, LVSP (97.6 + 6.7vs.146.7 + 7.7mmHg, n=6, P0.01). Perfusion significantly decreased (n=6, P0.05), indicating severe cardiovascular injury after severe scald.
6. compared with the scald group, insulin treatment immediately after scald could inhibit the subsequent peak of 30min and 3H blood glucose, significantly increase the insulin sensitivity of 3H and 10h after scald (n=6, P0.05), enhance 3H cardiac hemodynamics and body surface microcirculation perfusion (n=6, P0.05), and eventually lead to the survival of the early insulin treatment group after the scald in the rat 12h. Compared with the scald group, the survival rate was significantly increased (93% vs. 71%, n = 34-43, P 0.05). The living state (body weight, glucose tolerance and survival rate) in 14 days was also significantly improved (n = 6-43, P 0.05).
7. after the same dose of insulin after the scald, 2.5h was compared with the scald group, although it could temporarily reduce the peak of 3H blood sugar after scald, but then the other blood sugar summit appeared at 8h after injury. The survival rate in 12h rats was lower than that in the scald group, but there was no significant difference (55.6%vs.71%, n=18-34, P=0.17), and the memory of 14d (weight, glucose tolerance) There was no improvement in the volume and survival rate compared with the scald group (n=6-34, P=0.21).
conclusion
1. Two hyperglycemic peaks appear in the acute phase of Severe Scalded rats, and the second one is often closely related to poor prognosis.
2. Acute insulin resistance (AIR) was found in severely scalded rats in terms of overall insulin sensitivity and molecular signaling, and it was most severe at 3 hours after scald.
3. intervention of two blood glucose peaks, and it is found that insulin therapy against the first glucose peak (H1) can not only effectively reduce blood sugar, but also reduce AIR, improve cardiac function and body surface microcirculation perfusion, and ultimately improve the survival rate of rats, while the same dose of insulin control second blood glucose peaks (H2) is difficult to play the corresponding protective effect, speculate the mechanism It may be related to the emergence of AIR.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363
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1 付锋;胰岛素对大鼠心肌梗死后心室重塑和功能的影响及机制[D];第四军医大学;2011年
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