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摘要:
目的 探讨严重烧伤小鼠肠-胰岛轴功能的变化及其作用。 方法 该研究为实验研究。将90只雄性8~10周龄C57BL/6J小鼠按随机数字表法分为假伤组和烧伤组（每组45只小鼠）,于烧伤组小鼠背部制备30%体表总面积Ⅲ度烫伤（以下称烧伤）创面,假伤组小鼠模拟致假伤。伤后24 h,检测空腹血糖（样本数为12）后,行腹腔糖耐量实验和口服糖耐量实验,并绘制血糖浓度-时间变化曲线,计算曲线下面积（样本数为6）；分别于腹腔注射或灌胃给予葡萄糖溶液前及腹腔注射或灌胃给予葡萄糖溶液后30、60、120 min从心脏取血,采用酶联免疫吸附测定（ELISA）法检测血浆胰岛素和胰高血糖素样肽1（GLP-1）水平（样本数为3）；取每组3只小鼠回肠组织,行免疫荧光染色及原位末端标记染色检测肠道L细胞GLP-1表达及凋亡水平；提取每组6只小鼠胰岛行葡萄糖刺激的胰岛素分泌实验,分别经低糖（2.8 mmol/L葡萄糖）和高糖（16.7 mmol/L葡萄糖）孵育后取上清液,采用ELISA法检测胰岛素水平。取36只雄性8~10周龄C57BL/6J小鼠,按随机数字表法分为假伤组、烧伤组和烧伤+艾塞那肽4（Ex-4）组（每组12只小鼠）,对假伤组和烧伤组小鼠行同前对应处理,将烧伤+Ex-4组小鼠同烧伤组小鼠致伤后给予其GLP-1受体激动剂Ex-4。伤后24 h,提取小鼠胰岛,采用蛋白质印迹法检测重链结合蛋白（BIP）、蛋白激酶R样内质网激酶（PERK）、磷酸化PERK（p-PERK）、真核翻译起始因子2α（eIF2α）、磷酸化eIF2α（p-eIF2α）和CCAAT/增强子结合蛋白同源蛋白（CHOP）的蛋白表达并计算p-PERK/PERK、p-eIF2α/eIF2α比值（样本数为3）,采用流式细胞术检测胰岛细胞凋亡率（样本数为3）,同前行葡萄糖刺激的胰岛素分泌实验以检测上清液中胰岛素水平（样本数为6）。 结果 伤后24 h,烧伤组小鼠空腹血糖为（7.3±1.0）mmol/L,显著高于假伤组的（5.1±0.6）mmol/L（t=6.36,P<0.05）。伤后24 h,在腹腔糖耐量实验和口服糖耐量实验中,烧伤组小鼠血糖浓度-时间变化曲线下面积均显著大于假伤组（t值分别为4.32、6.03,P<0.05）；与假伤组相比,烧伤组小鼠经腹腔注射给予葡萄糖溶液前血浆胰岛素水平及经腹腔注射或灌胃给予葡萄糖溶液前血浆GLP-1水平均显著降低（P<0.05）,经腹腔注射或灌胃给予葡萄糖溶液后30、60、120 min血浆胰岛素水平及经灌胃给予葡萄糖溶液后30、60 min血浆GLP-1水平均显著降低（P<0.05）。伤后24 h,与假伤组相比,烧伤组小鼠肠道L细胞GLP-1表达水平显著降低（t=7.74,P<0.05）,凋亡水平显著升高（t=14.28,P<0.05）。伤后24 h,烧伤组小鼠经高糖孵育的胰岛上清液中胰岛素水平为（8.5±0.4）ng/mg,显著低于假伤组的（15.7±0.3）ng/mg（t=18.68,P<0.05）。伤后24 h,与假伤组相比,烧伤组小鼠胰岛中BIP、p-PERK/PERK、p-eIF2α/eIF2α和CHOP的蛋白表达水平均显著升高（P<0.05）；与烧伤组相比,烧伤+Ex-4组小鼠胰岛中BIP、p-PERK/PERK、p-eIF2α/eIF2α和CHOP的蛋白表达水平均显著降低（P<0.05）。伤后24 h,烧伤组小鼠胰岛细胞凋亡率为（32.0±3.0）%,显著高于假伤组的（10.3±2.5）%（P<0.05）；烧伤+Ex-4组小鼠胰岛细胞凋亡率为（20.0±3.6）%,显著低于烧伤组（P<0.05）。伤后24 h,烧伤组小鼠经高糖孵育的胰岛上清液中胰岛素水平显著低于假伤组（P<0.05）,烧伤+Ex-4组小鼠经高糖孵育的胰岛上清液中胰岛素水平显著高于烧伤组（P<0.05）。 结论 严重烧伤后小鼠肠-胰岛轴功能障碍,肠道L细胞凋亡增多、GLP-1合成及分泌减少,胰岛细胞发生内质网应激、凋亡增多,葡萄糖刺激的胰岛素分泌减少；GLP-1受体激动剂Ex-4能保护严重烧伤小鼠胰岛细胞功能,可能通过减轻内质网应激降低胰岛细胞凋亡水平,促进胰岛素分泌。 Abstract:Objective To explore the changes in entero-insular axis function and its role in mice with severe burns. Methods This study was an experimental study. Ninety C57BL/6J male mice aged 8-10 weeks were divided into sham injury group and burn group (with 45 mice in each group) according to the random number table. A full-thickness scald (hereinafter referred to as burn) wound of 30% of the total body surface area was created on the back of mice in burn group, and the mice in sham injury group were simulated to cause a sham injury. Twenty-four hours after injury, the fasting blood glucose was measured (n=12), followed by intraperitoneal glucose tolerance test and oral glucose tolerance test; the curve of blood glucose concentration changes over time was plotted, and the area under the curve was calculated (n=6); the blood was taken from the heart before intraperitoneal injection or gavage of glucose solution and at 30, 60, and 120 minutes after intraperitoneal injection or gavage of glucose solution for measuring the plasma insulin and glucagon like peptide-1 (GLP-1) levels using enzyme-linked immunosorbent assay (ELISA), with a sample number of 3; the ileal tissue was taken from 3 mice in each group for detecting the GLP-1 expression and apoptosis levels of intestinal L cells by immunofluorescence staining and TdT-mediated dUTP nick-end labeling staining; the pancreatic islets were collected from 6 mice in each group for glucose-stimulated insulin secretion experiments. After incubation with low glucose (2.8 mmol/L glucose) and high glucose (16.7 mmol/L glucose), the supernatant was taken and the insulin level was detected using ELISA. Thirty-six C57BL/6J male mice aged 8-10 weeks were divided into sham injury group, burn group, and burn+exendin-4 (Ex-4) group (with 12 mice in each group) according to the random number table. The mice in sham injury group and burn group were subjected to the same corresponding treatment as before. The mice in burn+Ex-4 group were injured in the same way as the burn group mice followed by treatment with GLP-1 receptor agonist Ex-4. Twenty-four hours after injury, mouse pancreatic islets were collected, the protein expressions of heavy-chain binding protein (BIP), protein kinase R-like endoplasmic reticulum kinase (PERK), phosphorylated PERK (p-PERK), eukaryotic translation initiation factor 2α (eIF2α), phosphorylated eIF2α (p-eIF2α), and CCAAT/enhancer-binding protein homologous protein (CHOP) were detected using Western blotting, and the p-PERK/PERK and p-eIF2α/eIF2α ratios were calculated (n=3), the apoptosis rate of pancreatic islet cells was detected using flow cytometry (n=3), the glucose stimulated insulin secretion experiment was conducted as before to detect insulin levels in the supernatant (n=6). Results Twenty-four hours after injury, the fasting blood glucose of mice in burn group was (7.3±1.0) mmol/L, which was significantly higher than (5.1±0.6) mmol/L in sham injury group (t=6.36, P<0.05). Twenty-four hours after injury, in the intraperitoneal glucose tolerance test and oral glucose tolerance test, the areas under the curve of blood glucose concentration changes over time of mice in burn group were significantly larger than those in sham injury group (with t values of 4.32 and 6.03, respectively, P<0.05); compared with those in sham injury group, the plasma insulin levels of mice before intraperitoneal injection of glucose solution and the plasma GLP-1 levels of mice before intraperitoneal injection or gavage of glucose solution in burn group were significantly decreased (P<0.05), and the plasma levels of insulin of mice at 30, 60, and 120 minutes after intraperitoneal injection or gavage of glucose solution, as well as the plasma levels of GLP-1 of mice at 30 and 60 minutes after gavage of glucose solution were significantly decreased in burn group (P<0.05). Twenty-four hours after injury, compared with those in sham injury group, the GLP-1 expression level of intestinal L cells of mice in burn group was significantly decreased (t=7.74, P<0.05), and the apoptosis level was significantly increased (t=14.28, P<0.05). Twenty-four hours after injury, the insulin level in the supernatant of mice pancreatic islet incubated with high glucose in burn group was (8.5±0.4) ng/mg, which was significantly lower than (15.7±0.3) ng/mg in sham injury group (t=18.68, P<0.05). Twenty-four hours after injury, compared with those in sham injury group, the protein expression levels of BIP, p-PERK/PERK, p-eIF2α/eIF2α, and CHOP in the pancreatic islets of mice in burn group were significantly increased (P<0.05); compared with those in burn group, the protein expression levels of BIP, p-PERK/PERK, p-eIF2α/eIF2α, and CHOP in the pancreatic islets of mice in burn+Ex-4 group were significantly decreased (P<0.05). Twenty-four hours after injury, the apoptosis rate of pancreatic islet cells of mice in burn group was (32.0±3.0)%, which was significantly higher than (10.3±2.5)% in sham injury group (P<0.05); the apoptosis rate of pancreatic islet cells of mice in burn+Ex-4 group was (20.0±3.6)%, which was significantly lower than that in burn group (P<0.05). Twenty-four hours after injury, the insulin level in the supernatant of mice pancreatic islet incubated with high glucose in burn group was significantly lower than that in sham injury group (P<0.05), while the insulin level in the supernatant of mice pancreatic islet incubated with high glucose in burn+Ex-4 group was significantly higher than that in burn group (P<0.05). Conclusions After severe burns, the mice display dysfunction of the entero-insular axis, increased apoptosis of intestinal L cells, decreased synthesis and secretion of GLP-1, endoplasmic reticulum stress and increased apoptosis in pancreatic islet cells and a decrease in glucose-stimulated insulin secretion. The GLP-1 receptor agonist Ex-4 can protect the function of pancreatic islet cells of mice with severe burns, reducing the apoptosis level of pancreatic islet cells and promoting insulin secretion possibly via the alleviation of endoplasmic reticulum stress. -
Key words:
- Burns /
- Insulin /
- Glucagon-like peptide 1 /
- Endoplasmic reticulum stress /
- Hyperglycemia /
- Entero-insular axis
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参考文献
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图 1 2组小鼠伤后24 h行腹腔糖耐量实验和口服糖耐量实验后的血糖浓度-时间变化曲线。1A.腹腔糖耐量实验；1B.口服糖耐量实验
注：烧伤组小鼠为30%体表总面积Ⅲ度烫伤,假伤组小鼠模拟致假伤；每组6只小鼠,取各时间点均值绘制曲线,曲线与横坐标围成的图形的面积为曲线下面积
图 2 2组小鼠伤后24 h回肠组织中L细胞GLP-1表达及凋亡情况 四甲基异硫氰酸罗丹明-异硫氰酸荧光素-4',6-二脒基-2-苯基吲哚×100。2A.假伤组,GLP-1表达较多,凋亡细胞较少；2B.烧伤组,GLP-1表达少于图2A,凋亡细胞多于图2A
注：烧伤组小鼠为30%体表总面积Ⅲ度烫伤,假伤组小鼠模拟致假伤；胰高血糖素样肽1（GLP-1）阳性染色为红色,凋亡细胞阳性染色为绿色,细胞核阳性染色为蓝色
图 3 蛋白质印迹法检测的3组小鼠伤后24 h胰岛中内质网应激相关蛋白表达情况
注：烧伤组和烧伤+艾塞那肽4（Ex-4）组小鼠为30%体表总面积Ⅲ度烫伤,假伤组小鼠模拟致假伤,于烧伤+Ex-4组小鼠伤后给予其Ex-4；BIP为重链结合蛋白,eIF2α为真核翻译起始因子2α,p-eIF2α为磷酸化eIF2α,PERK为蛋白激酶R样内质网激酶,p-PERK为磷酸化PERK,CHOP为CCAAT/增强子结合蛋白同源蛋白；条带上方1、2、3分别指示假伤组、烧伤组、烧伤+Ex-4组
Table 1. 2组小鼠伤后24 h经腹腔注射或灌胃给予葡萄糖溶液前后血浆胰岛素水平比较（pg/mg,
组别 样本数 腹腔注射 灌胃 前 后30 min 后60 min 后120 min 前 后30 min 后60 min 后120 min 假伤组 3 6.03±1.97 12.06±2.08 11.67±1.58 8.73±2.97 5.63±2.92 31.93±7.99 32.19±5.99 26.13±3.49 烧伤组 3 1.83±0.31 7.60±2.17 5.07±0.21 2.20±1.01 1.85±0.31 12.23±2.73 19.94±4.99 15.25±4.29 P值 0.042 0.029 0.001 0.001 0.809 <0.001 0.020 0.043 注：烧伤组小鼠为30%体表总面积Ⅲ度烫伤,假伤组小鼠模拟致假伤；以血浆中胰岛素浓度与血浆总蛋白浓度的比值表示胰岛素水平；腹腔注射的处理因素主效应,F=57.23,P<0.001；时间因素主效应,F=13.88,P<0.001；两者交互作用,F=0.80,P=0.508；灌胃的处理因素主效应,F=38.04,P<0.001；时间因素主效应,F=27.27,P<0.001；两者交互作用,F=2.98,P=0.063 
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Table 2. 2组小鼠伤后24 h经腹腔注射或灌胃给予葡萄糖溶液前后血浆GLP-1水平比较（pg/mg,
组别 样本数 腹腔注射 灌胃 前 后30 min 后60 min 后120 min 前 后30 min 后60 min 后120 min 假伤组 3 14.23±0.69 15.55±2.42 17.06±2.40 13.99±0.09 14.93±0.06 34.20±4.66 30.95±3.96 18.28±1.05 烧伤组 3 10.11±1.92 13.08±0.89 14.25±0.63 13.41±0.20 9.33±0.76 20.32±1.14 19.72±2.02 12.98±1.71 P值 0.013 0.200 0.118 0.981 0.049 <0.001 <0.001 0.066 注：烧伤组小鼠为30%体表总面积Ⅲ度烫伤,假伤组小鼠模拟致假伤；以血浆中胰高血糖素样肽1（GLP-1）浓度与血浆总蛋白浓度的比值表示GLP-1水平；腹腔注射处理因素主效应,F=17.62,P<0.001；时间因素主效应,F=5.92,P=0.006；两者交互作用,F=1.50,P=0.252；灌胃的处理因素主效应,F=82.05,P<0.001；时间因素主效应,F=54.69,P<0.001；两者交互作用,F=4.55,P=0.017
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Table 3. 3组小鼠伤后24 h胰岛中内质网应激相关蛋白表达水平比较（
组别 样本数 BIP p-PERK/PERK p-eIF2α/eIF2α CHOP 假伤组 3 1.00±0.10 1.01±0.10 1.00±0.11 1.00±0.10 烧伤组 3 2.03±0.16 2.45±0.11 1.87±0.07 2.19±0.24 烧伤+Ex-4组 3 1.31±0.09 1.93±0.08 1.28±0.16 1.65±0.21 F值 59.29 178.50 44.82 28.41 P值 <0.001 <0.001 <0.001 <0.001 P1值 <0.001 <0.001 <0.001 <0.001 P2值 <0.001 <0.001 <0.001 0.025 注：烧伤组和烧伤+艾塞那肽4（Ex-4）组小鼠为30%体表总面积Ⅲ度烫伤,假伤组小鼠模拟致假伤,于烧伤+Ex-4组小鼠伤后给予其Ex-4；P1值为烧伤组与假伤组比较所得,P2值为烧伤+Ex-4组与烧伤组比较所得；BIP为重链结合蛋白,PERK为蛋白激酶R样内质网激酶,p-PERK为磷酸化PERK,eIF2α为真核翻译起始因子2α,p-eIF2α为磷酸化eIF2α,CHOP为CCAAT/增强子结合蛋白同源蛋白
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Table 4. 3组小鼠伤后24 h经低糖和高糖孵育的胰岛上清液中胰岛素水平比较（ng/mg,
组别 样本数 低糖孵育 高糖孵育 假伤组 6 5.0±0.5 16.2±0.6 烧伤组 6 4.9±0.8 8.9±0.8 烧伤+Ex-4组 6 5.0±0.9 12.7±1.3 F值 0.04 89.20 P值 0.970 <0.001 P1值 — <0.001 P2值 — <0.001 注：烧伤组和烧伤+艾塞那肽4（Ex-4）组小鼠为30%体表总面积Ⅲ度烫伤,假伤组小鼠模拟致假伤,于烧伤+Ex-4组小鼠伤后给予其Ex-4；P1值为烧伤组与假伤组比较所得；P2值为烧伤+Ex-4组与烧伤组比较所得；低糖指2.8 mmol/L葡萄糖,高糖指16.7 mmol/L葡萄糖；“—”表示无此项
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