二甲双胍对异氟醚麻醉小鼠血糖的影响

发布时间：2018-04-27 12:09

  本文选题：异氟醚 + 血糖　； 参考：《南京大学》2017年硕士论文

【摘要】：研究背景:围术期高血糖可引起严重的术后预后不良,增加院内并发症如感染、急性肾损伤等的发病率以及术后30天和1年死亡率。通常认为围术期高血糖的发生与患者术前基础疾病、术中手术操作、术前与术中用药等相关,越来越多的基础及临床研究表明麻醉药也参与了围术期高血糖的发生,吸入麻醉药七氟醚和异氟醚可引起机体葡萄糖利用受损、糖耐量下降、胰岛素分泌减少、胰岛素抵抗。下丘脑是机体能量调节中心,参与了体内多种物质如糖、脂的代谢,腺苷酸活化蛋白激酶(adenosine monophosphate activated protein kinase,AMPK)在其调节过程中起主要作用,目前关于下丘脑在吸入麻醉药与血糖的代谢中的作用尚未有研究。二甲双胍是目前一线降糖药,有着良好的降糖效果和安全性,其降糖作用也与AMPK相关。本研究通过对健康小鼠行异氟醚麻醉观察小鼠血糖的变化,使用二甲双胍来研究围术期高血糖的预防,检测下丘脑AMPK mRNA的表达探索相关机制。研究方法:本研究使用的动物类型为C57BL/6的小鼠、雄性、8周龄,体重约22-28 g。采用持续吸入6 h异氟醚麻醉,通过尾静脉取血加血糖仪来检测血糖,使用生理盐水溶解二甲双胍后通过腹腔给药。血糖检测时间点分别为麻醉前0.5 h,麻醉0.5h、1h、2h、3h、4h、6h,麻醉结束后2h、4h。本研究实验过程主要分为以下三部分:实验一研究不同浓度异氟醚麻醉对小鼠血糖的影响将随机分为未禁食组(non-fasted group)和禁食12 h组(fasted group),各组再随机分为两组:对照组(Cn组/Cf组),麻醉组(An组/Af组)。使用浓度为1.2%或1.8%异氟醚分别进行麻醉,检测麻醉下小鼠血糖水平。实验二研究不同剂量二甲双胍对异氟醚麻醉下小鼠血糖的影响根据实验一结果,选择与对照组血糖值差异较大的浓度进行下一步实验。同样设置禁食组和未禁食组,各组再随机分为四组:对照组(Cn组/Cf组),麻醉组(An组/Af组),二甲双胍组(Mn组/Mf组)和二甲双胍+麻醉组(MAn组/MAf组)。二甲双胍组和二甲双胍+麻醉组于麻醉前1 h腹腔注射二甲双胍40 mg/kg,麻醉组和二甲双胍+麻醉组均行异氟醚6 h麻醉。二甲双胍组(Mn组/Mf组)和二甲双胍+麻醉组(MAn组/MAf组)。二甲双胍组和二甲双胍+麻醉组于麻醉前1 h腹腔注射二甲双胍20 mg/kg或40 mg/kg,麻醉组和二甲双胍+麻醉组均行异氟醚6 h麻醉。通过尾静脉取血检测围麻醉期各时间点血糖值。实验三研究AMPKα1和AMPKα2在二甲双胍预处理对异氟醚麻醉后小鼠血糖变化中的机制关联根据实验二结果,选择对血糖影响较大的二甲双胍给药剂量组设立平行取材组,每组4只,于麻醉1 h、4 h、6 h取材下丘脑,通过Q-PCR检测AMPKα1、AMPKα2 mRNA的表达水平。研究结果:实验一:1)基础值比较:本实验结果表明禁食12h后小鼠基础血糖值为6.5±0.7mmol/L,未禁食组为8.3±1.0 mmol/L,二者比较有显著差异(P0.001)。2)麻醉对血糖的影响:异氟醚1.0 MAC(1.2%)组:与Cn组相比,1.2%异氟醚麻醉可在麻醉0.5 h-4 h引起An组小鼠血糖升高;而与Cf组相比,Af组小鼠血糖在麻醉0.5 h,3 h、4 h显著升高(P0.05)。异氟醚1.5 MAC(1.8%)组:与Cn组相比,An组小鼠血糖在麻醉0.5 h、2 h显著升高(P0.05);与Cf组相比,Af组未出现升高,在麻醉4h和6h出现降低(P0.05)。实验二:结果一表明,异氟醚浓度为1.0 MAC时小鼠血糖变化更大,因此本此实验选择1.2%异氟醚浓度进一步探索二甲双胍对异氟醚麻醉下小鼠血糖的影响。本次研究中单纯异氟醚麻醉组血糖变化与结果一一致,单独给予不同剂量二甲双胍组小鼠与各自对照组无明显差异(P0.05)。1)预给药二甲双胍为20 mg/kg的分组:与Cn组相比,An组小鼠在麻醉3 h出现显著升高的血糖水平(P0.05),随后恢复至与Cn组相同的水平(P0.05),而MAn组小鼠血糖在麻醉1-2 h显著升高(P0.05),随后逐渐降低至与Cn组相同的水平;与Cf组相比,Af组小鼠在麻醉0.5-2 h出现显著升高的血糖水平,随后在麻醉结束至麻醉结束2h显著低于Cf组(P0.05),于麻醉结束3 h后回到与Cf组相同的水平(P0.05);MAf组小鼠在麻醉0.5-1 h出现显著升高的血糖水平,随后在麻醉结束至麻醉结束2 h显著低于Cf组(P0.05),于麻醉结束3 h后回到与Cf组相同的水平(P0.05)。2)预给二甲双胍为40 mg/kg的分组:与Cn组和Cf组相比,An组和Af组分别在麻醉0.5h、1h、2h和1h,2h,3h升高(P0.05);随后出现血糖水平下降,An组在麻醉结束至结束后3 h期间血糖水平低于Cn组(P0.05),Af组血糖降低,在麻醉4 h后与Cf组无统计学差异(P0.05)。与对照组相比,单纯二甲双胍处理Mn组和Mf组血糖水平无明显差异(P0.05),合并异氟醚麻醉的MAn组和MAf组均于麻醉2 h开始出现低血糖并持续(P0.05),这种差异分别在麻醉结束4h和1h后消失。实验三:根据实验二结果表明,40 mg/kg二甲双胍对麻醉小鼠血糖影响更为显著,因此,本次实验选择剂量为40 mg/kg二甲双胍设立平行取材组。下丘脑AMPK mRNA表达:1)麻醉1 h取材组:与Cf组相比,MAf组AMPKα1和AMPKα 2 mRNA表达下调(P0.05)。2)麻醉4 h取材组:与Cn组和Cf组比较,An组、MAn组和Af组AMPKα1表达下调(P0.05),AMPKα2mRNA表达在MAf组降低(P0.05)。3)麻醉6 h取材组:与Cn组和Cf组比较,AMPKα1和AMPKα2在An组和MAn组,Af组和MAf组mRNA表达都降低(P0.05)。实验结论:长时程异氟醚麻醉可使小鼠血糖呈"先高后低"改变,二甲双胍预处理后麻醉小鼠可出现持续低血糖,这一过程与下丘脑AMPK转录活性无明显关联。
[Abstract]:Background: perioperative hyperglycemia may cause severe postoperative poor prognosis, increase the incidence of intraoperative complications such as infection, acute renal injury, and the mortality rate of 30 and 1 years after operation. Basic and clinical studies have shown that anesthetics also participate in the occurrence of hyperglycemia in the perioperative period. Inhalation of anesthetic sevoflurane and isoflurane can cause impaired glucose utilization, decrease of glucose tolerance, decrease of insulin secretion, insulin resistance. The hypothalamus is the body's energy regulation center, and participates in various substances in the body such as sugar, lipid metabolism, adenylate activation. Adenosine monophosphate activated protein kinase (AMPK) plays a major role in its regulation, and the role of the hypothalamus in the metabolism of inhaled anesthetics and blood glucose has not been studied. Metformin is a frontline hypoglycemic agent at present. It has a good effect on reducing sugar and safety. The hypoglycemic effect of metformin is also associated with AMPK. In this study, the changes of blood glucose in mice were observed by isoflurane anesthesia in healthy mice. Metformin was used to study the prevention of hyperglycemia in the perioperative period and to detect the mechanism of the expression of AMPK mRNA in the hypothalamus. The research method: This study was used in mice of C57BL/6, male, 8 weeks of age, and weight about 22-28 g. by continuous inhalation of 6 h The blood glucose was detected by the caudal vein and the blood glucose meter through the caudal vein. The time point of blood glucose detection was 0.5 h before anesthesia, 0.5h, 1H, 2h, 3h, 4h, 6h, 2h in the end of anesthesia, 4h. in the following three parts: the experiment studied the different concentrations of isofluorine in the experiment. The effects of ether anesthesia on blood glucose in mice were randomly divided into unfasting group (non-fasted group) and fasting 12 h group (fasted group). Each group was then randomly divided into two groups: control group (group Cn /Cf) and anesthetic group (group /Af group An). The blood glucose level of mice under anesthesia was detected with concentration of 1.2% or 1.8% isoflurane respectively. The experiment two studied different doses. The effect of metformin on the blood sugar of mice under isoflurane anesthesia was selected according to the results of the experiment, and the concentration of blood sugar in the control group was selected for the next step. The same group was set up in four groups: control group (group /Cf of Cn group), anesthesia group (Group An /Af), metformin group (group /Mf of Mn group) and two a pair Guanidine + anaesthesia group (group MAn /MAf). The metformin group and metformin + anaesthesia group were intraperitoneally injected with metformin 40 mg/kg 1 h before anesthesia. The anesthesia group and metformin + anaesthesia group were treated with isoflurane 6 h anesthesia. Metformin group (group Mn /Mf group) and metformin + anaesthesia group (group /MAf group MAn). Metformin group and metformin + anaesthesia group were 1 before anesthesia. H intraperitoneal injection of metformin 20 mg/kg or 40 mg/kg, anesthesia group and metformin + anaesthesia group were treated with isoflurane 6 h anesthesia. Blood glucose values were measured at various time points in the perioperative period through the tail vein extraction. Experiment three study the mechanism of AMPK alpha 1 and AMPK alpha 2 on the changes of blood sugar in mice after isoflurane anaesthetized by metformin pretreatment, according to experimental two results The group of metformin, which had more influence on blood glucose, set up a parallel group, 4 rats in each group, 1 h, 4 h, 6 h in the hypothalamus, and the expression level of AMPK alpha 1, AMPK alpha 2 mRNA by Q-PCR. The result of the study: experiment one: 1) base value comparison: the results of this experiment showed that the base blood sugar of the mice after 12h was 6.5 + 0.7mmol/L, no prohibition. The food group was 8.3 + 1 mmol/L, and the two were significantly different (P0.001).2) the effect of anaesthesia on blood sugar: Isoflurane 1 MAC (1.2%) group: compared with the Cn group, 1.2% isoflurane anesthesia could increase the blood sugar of the An mice in 0.5 H-4 h; compared with the Cf group, the blood sugar in the Af group was significantly increased in 0.5 h, 3 h, and 4. (1.8%). Group: compared with group Cn, blood glucose of mice in group An was 0.5 h and 2 h increased significantly (P0.05). Compared with group Cf, there was no increase in Af group and decreased in 4H and 6h in anaesthesia (P0.05). Experiment two: the results showed that the blood glucose in mice was higher when the concentration of isoflurane was 1 MAC, because this experiment selected 1.2% isoflurane concentration to further explore metformin. In this study, the blood sugar changes in the isoflurane anaesthetized group were consistent with the results. There was no significant difference between the different doses of metformin group and the control group (P0.05).1). The group of pre administered metformin was 20 mg/kg: compared with the Cn group, the mice in the An group increased significantly in the 3 h anesthesia. The blood glucose level (P0.05) was then restored to the same level as that in the Cn group (P0.05), while the blood glucose in the MAn group increased significantly (P0.05) at 1-2 h (P0.05), and then gradually decreased to the same level as that in the Cn group. Compared with the Cf group, the serum sugar level of the Af group mice was significantly higher in the 0.5-2 h, and the 2H was significantly lower than that of the group of 0.5-2. 0.05) back to the same level as that in the Cf group after the end of the anesthesia (P0.05); in group MAf mice, the level of blood glucose increased significantly in the anesthetic 0.5-1 h, and then at the end of anesthesia to the end of anesthesia 2 h was significantly lower than that of the Cf group (P0.05), and returned to the same level as the Cf group after the end of the anesthesia 3 h (P0.05). Compared with group Cf, group An and Af group were anesthetized 0.5h, 1H, 2h and 1H, 2h, 3h increased (P0.05), and then decreased blood sugar levels. The glucose level in the An group was lower than that in the 3 h group after the end of anesthesia. There was no significant difference in blood glucose level in group Mf (P0.05). The MAn group and MAf group with isoflurane anesthesia began to appear hypoglycemia and continued (P0.05) at 2 h anesthesia. This difference disappeared after the end of anesthesia 4H and 1H. Experiment three: according to experimental two, 40 mg/kg two metformin had a more significant effect on the blood glucose in the anesthetized mice. Therefore, this experiment 40 mg/kg metformin was selected to set up a parallel selection group. The expression of AMPK mRNA in the hypothalamus: 1) anaesthetized 1 h group: compared with the Cf group, the AMPK alpha 1 and AMPK a 2 mRNA expression of 2 mRNA (P0.05).2) were anesthetized 4 h materials. 6 h anesthesia group: compared with group Cn and Cf group, AMPK alpha 1 and AMPK alpha 2 in group An and MAn group, Af and MAf group mRNA expression decreased (P0.05). Experimental conclusion: long term isoflurane anesthesia can make the blood glucose in mice "first high and low" change, metformin pretreated mice can appear continuous hypoglycemia, this process and hypothalamic AMPK transcriptional activity There is no obvious connection.

【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R614
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本文编号：1810678