Luo P, Zhang B H, Sun J C, Li Z S, Liu X Z, Yang J Q, Shen Z A
Department of Burns and Plastic Surgery, the Fourth Medical Center of PLA General Hospital, Beijing 100048, China.
Zhonghua Shao Shang Yu Chuang Mian Xiu Fu Za Zhi. 2023 May 20;39(5):424-433. doi: 10.3760/cma.j.cn501225-20230216-00051.
To explore the mechanism of early pancreatic exocrine function changes in severely scalded rats. The experimental research methods was used. Eighty male Sprague-Dawley rats aged 7-8 weeks were divided into simple sham injury group (=8), sham injury+cholecystokinin octapeptide (CCK8) group (=8), severe scald+CCK8 group (=32), and extremely severe scald+CCK8 group (=32) by the random number table, which were treated accordingly. Immediately after injury of rats in the 2 sham injury groups and 1, 2, 3, and 7 days after injury of rats in the 2 scald groups, the improved methods including pancreatic duct puncture and catheterization were used to dynamically collect the pancreatic-bile juice (PBJ) of rats. The PBJ secretory volume within 1 h was recorded, and the content of pancreatic lipase, α-amylase, and trypsin in PBJ was detected by enzyme-linked immunosorbent assay (ELISA), and the number of samples was 8. The femoral venous blood was collected, and the concentrations of pancreatic lipase and α-amylase in serum were detected by standard colorimetry to reflect their activity (=8). The pancreatic tissue was extracted, and the levels of interleukin-1β (IL-1β) and IL-6 in pancreatic tissue were detected by ELISA (=8), the expression of hypoxia-inducible factor 1α (HIF-1α) in pancreatic tissue was detected by immunofluorescence method, and the histopathological changes in pancreatic tissue were observed by hematoxylin-eosin staining, the severity of pancreatic tissue injury in the 2 scald groups was evaluated by modified Schmidt method (=6), and the ultrastructure of acinar cells in pancreatic tissue was observed by transmission electron microscopy. Data were statistically analyzed with analysis of variance for factorial design, Tukey test, independent sample test, and least significant difference test. Compared with the PBJ secretory volume (0.740±0.030) mL in the pancreatic tissue of rats in simple sham injury group within 1 h immediately after injury, the (0.823±0.033) mL in sham injury+CCK8 group was significantly increased (=4.92, <0.05). Compared with that of rats in sham injury+CCK8 group immediately after injury, the PBJ secretory volume of rats within 1 h in severe scald+CCK8 group ((0.681±0.024), (0.608±0.056), (0.525±0.025), and (0.720±0.044) mL) and extremely severe scald+CCK8 group ((0.540±0.025), (0.406±0.021), (0.475±0.036), and (0.690±0.018) mL) was significantly decreased on 1, 2, 3, and 7 days after injury (<0.05). Compared with that in severe scald+CCK8 group, the PBJ secretory volume of rats within 1 h in extremely severe scald+CCK8 group was significantly decreased on 1 and 2 days after injury (<0.05). Compared with that of rats in simple sham injury group immediately after injury, the content of pancreatic lipase, α-amylase, and trypsin in PBJ of rats in sham injury+CCK8 group immediately after injury was significantly increased (with values of 4.56, 3.30, and 4.99, respectively, <0.05). Compared with that of rats in sham injury+CCK8 group immediately after injury, the content of pancreatic lipase and α-amylase in PBJ of rats in severe scald+CCK8 group and extremely severe scald+CCK8 group was significantly decreased on 1, 2, 3, and 7 days after injury (<0.05), the trypsin content in PBJ of rats in extremely severe scald+CCK8 group was significantly decreased on 2 days after injury (<0.05). Compared with that in severe scald+CCK8 group, the content of pancreatic lipase in PBJ of rats in extremely severe scald+CCK8 group was significantly decreased on 1, 2, and 3 days after injury (<0.05), and the content of α-amylase and trypsin in PBJ was significantly decreased on 1 and 2 days after injury (<0.05). There were no statistically significant differences in the activities of pancreatic lipase and α-amylase in serum of rats among the 4 groups at various time points after injury (>0.05). Compared with that of rats in sham injury+CCK8 group immediately after injury, the levels of IL-1β in pancreatic tissue of rats in severe scald+CCK8 group on 1, 2, and 3 days after injury and in extremely severe scald+CCK8 group on 1, 2, 3, and 7 days after injury were significantly increased (<0.05), and the levels of IL-6 in pancreatic tissue of rats in severe scald+CCK8 group and extremely severe scald+CCK8 group were significantly increased on 1, 2, 3, and 7 days after injury (<0.05). Compared with that in severe scald+CCK8 group, the IL-1β level in pancreatic tissue of rats in extremely severe scald+CCK8 group was significantly increased on 2 and 3 days after injury (<0.05), and IL-6 level in pancreatic tissue was significantly increased on 2 days after injury (<0.05). The expression levels of HIF-1α in pancreatic tissue of rats in simple sham injury group and sham injury+CCK8 group immediately after injury were lower; and compared with that in sham injury+CCK8 group immediately after injury, the expression levels of HIF-1α in pancreatic tissue of rats in the 2 scald groups increased to a certain extent at different time points after injury, and the expression position was transited from the edge of the pancreatic tissue to the whole pancreas, the expression levels of HIF-1α in pancreatic tissue of rats in the 2 scald groups tended to be normal on 7 days after injury. Compared with that in simple sham injury group immediately after injury, the proportion of acinar cell cytoplasm in pancreatic tissue of rats in sham injury+CCK8 group was increased; and with the increase of time after injury, edema, hemorrhage, necrosis, and inflammatory infiltration appeared in pancreatic tissue of rats in the 2 scald groups. Compared with that in severe scald+CCK8 group, the scores of edema, inflammatory cell infiltration, bleeding, and necrosis in pancreatic tissue of rats in extremely severe scald+CCK8 group were increased to varying degrees at various time points after injury, and the scores of pancreatic tissue of rats in the 2 scald groups basically recovered to normal on 7 days after injury. Compared with that in simple sham injury group immediately after injury, the number of enzyme granules in acinar cells of pancreatic tissue of rats in sham injury+CCK8 group was increased, and with the increase of time after injury, the enzyme granules in acinar cells of rats in the 2 scald groups were gradually reduced basically. The exocrine functions of pancreas, such as synthesis and secretion of pancreatic enzymes, are decreased in the early stage in severely scalded rats. And the greater the scalded area, the more significant the decline of pancreatic exocrine function. This change may be related to hypoxic injury and inflammation in pancreatic tissue after severe scald.
探讨严重烫伤大鼠早期胰腺外分泌功能变化的机制。采用实验研究方法。将80只7 - 8周龄的雄性Sprague-Dawley大鼠按随机数字表法分为单纯假伤组(n = 8)、假伤+八肽胆囊收缩素(CCK8)组(n = 8)、重度烫伤+CCK8组(n = 32)和极重度烫伤+CCK8组(n = 32),并进行相应处理。2个假伤组大鼠伤后即刻,2个烫伤组大鼠伤后1、2、3及7天,采用改良的胰导管穿刺插管法动态收集大鼠胰胆汁(PBJ)。记录1 h内PBJ分泌量,采用酶联免疫吸附测定(ELISA)法检测PBJ中胰脂肪酶、α淀粉酶和胰蛋白酶含量,样本数为8。采集股静脉血,采用标准比色法检测血清中胰脂肪酶和α淀粉酶浓度以反映其活性(n = 8)。提取胰腺组织,采用ELISA法检测胰腺组织中白细胞介素-1β(IL-1β)和IL-6水平(n = 8),采用免疫荧光法检测胰腺组织中缺氧诱导因子1α(HIF-1α)表达,采用苏木精-伊红染色观察胰腺组织病理变化,采用改良Schmidt法评估2个烫伤组胰腺组织损伤严重程度(n = 6),采用透射电镜观察胰腺组织腺泡细胞超微结构。数据采用析因设计方差分析、Tukey检验、独立样本t检验和最小显著差检验进行统计学分析。与单纯假伤组大鼠伤后即刻胰腺组织1 h内PBJ分泌量(0.740±0.030)mL相比,假伤+CCK8组(0.823±0.033)mL显著增加(P = 4.92,P < 0.05)。与假伤+CCK8组大鼠伤后即刻相比,重度烫伤+CCK8组大鼠伤后1、2、3及7天1 h内PBJ分泌量(分别为(0.681±0.024)、(0.608±0.056)、(0.525±0.025)和(0.720±0.044)mL)及极重度烫伤+CCK8组大鼠伤后1、2、3及7天1 h内PBJ分泌量(分别为(0.540±0.025)、(0.406±0.021)、(0.475±0.036)和(0.690±0.018)mL)显著降低(P < 0.05)。与重度烫伤+CCK8组相比,极重度烫伤+CCK8组大鼠伤后1及2天1 h内PBJ分泌量显著降低(P < 0.05)。与单纯假伤组大鼠伤后即刻相比,假伤+CCK8组大鼠伤后即刻PBJ中胰脂肪酶、α淀粉酶和胰蛋白酶含量显著增加(P值分别为4.56、3.30和4.99,P < 0.05)。与假伤+CCK8组大鼠伤后即刻相比,重度烫伤+CCK8组和极重度烫伤+CCK8组大鼠伤后1、2、3及7天PBJ中胰脂肪酶和α淀粉酶含量显著降低(P < 0.05),极重度烫伤+CCK8组大鼠伤后2天PBJ中胰蛋白酶含量显著降低(P < 0.05)。与重度烫伤+CCK8组相比,极重度烫伤+CCK8组大鼠伤后1、2及3天PBJ中胰脂肪酶含量显著降低(P < 0.05),伤后1及2天PBJ中α淀粉酶和胰蛋白酶含量显著降低(P < 0.05)。伤后各时间点4组大鼠血清中胰脂肪酶和α淀粉酶活性差异无统计学意义(P > 0.05)。与假伤+CCK8组大鼠伤后即刻相比,重度烫伤+CCK8组大鼠伤后1、2及3天和极重度烫伤+CCK8组大鼠伤后1、2、3及7天胰腺组织中IL-1β水平显著升高(P < 0.05),重度烫伤+CCK8组和极重度烫伤+CCK8组大鼠伤后1、2、3及7天胰腺组织中IL-6水平显著升高(P < 0.05)。与重度烫伤+CCK8组相比,极重度烫伤+CCK8组大鼠伤后2及3天胰腺组织中IL-1β水平显著升高(P < 0.05),伤后2天胰腺组织中IL-6水平显著升高(P < 0.05)。单纯假伤组和假伤+CCK8组大鼠伤后即刻胰腺组织中HIF-1α表达水平较低;与假伤+CCK8组大鼠伤后即刻相比,2个烫伤组大鼠伤后不同时间点胰腺组织中HIF-1α表达水平均有一定程度升高,表达位置从胰腺组织边缘向全胰腺转移,2个烫伤组大鼠伤后7天胰腺组织中HIF-1α表达水平趋于正常。与单纯假伤组大鼠伤后即刻相比,假伤+CCK8组大鼠胰腺组织腺泡细胞胞质比例增加;随着伤后时间延长,2个烫伤组大鼠胰腺组织出现水肿、出血、坏死及炎性浸润。与重度烫伤+CCK8组相比,极重度烫伤+CCK8组大鼠伤后各时间点胰腺组织水肿、炎性细胞浸润、出血及坏死评分均有不同程度升高,2个烫伤组大鼠胰腺组织评分伤后7天基本恢复正常。与单纯假伤组大鼠伤后即刻相比,假伤+CCK8组大鼠胰腺组织腺泡细胞酶原颗粒数量增加,随着伤后时间延长,2个烫伤组大鼠腺泡细胞酶原颗粒逐渐减少。严重烫伤大鼠早期胰腺外分泌功能如胰酶的合成和分泌降低。且烫伤面积越大,胰腺外分泌功能下降越显著。这种变化可能与严重烫伤后胰腺组织缺氧损伤及炎症有关。