目的 采用链脲佐菌素（streptozotocin，STZ）诱导的妊娠糖尿病（gestational diabetes mellitus，GDM）模型，观察GDM模型大鼠孕期进行左归丸干预后其子代胰腺发育情况，并进一步探讨胰腺十二指肠同源框蛋白1（pancreatic and duodenal homeobox 1，Pdx1）路径对GDM模型子鼠胰腺β细胞超微结构及氧化应激的影响。方法 将孕鼠分为对照组、模型组、地特胰岛素（15 U/kg）组和左归丸低、中、高剂量（0.95、1.89、3.78 g/kg）组，每组6只。除对照组外，在大鼠妊娠第3天（E3d）时ip STZ构建GDM模型，对照组ip等体积柠檬酸钠缓冲液，通过测定空腹血糖判断孕鼠是否造模成功。除对照组和模型组外，其余各组孕鼠在E6d开始给药至分娩前，每日1次。待孕鼠自然分娩，子鼠由母鼠喂养至离乳第21天（B21d），从各组母鼠子代中随机选取1雌1雄子鼠作为对照组、模型组、地特胰岛素组、左归丸低、中、高剂量组，每组12只。在各组子鼠B22d时，采用血糖仪测定空腹血糖（fasting blood glucose，FBG），记录子鼠体质量、体长（测定尾根至鼻尖的距离），处死子鼠，分离血清。采用ELISA法检测空腹胰岛素水平（fasting plasma insulin，FINS）并计算胰岛素抵抗指数（homeostasis model assessment of insulin resistance，HOMA-IR）；采用苏木素-伊红（hematoxylin-eosin，HE）染色法观察子鼠胰腺病理结构；在透射电子显微镜下观察子鼠胰腺β细胞超微结构；采用ELISA法检测子鼠血清及胰腺组织中超氧化物歧化酶（superoxide dismutase，SOD）、丙二醛（malonaldehyde，MDA）、过氧化氢酶（catalase，CAT）水平；采用Western blotting法检测子鼠胰腺组织Pdx1、神经因子3（neurogenin 3，Ngn3）、肌筋膜性纤维肉瘤癌基因同源物A（v-maf avian musculoaponeurotic fibrosarcoma oncogene family protein A，Mafa）的蛋白表达。结果 与对照组比较，模型组子鼠体质量增加、体长减少（P＜0.01），FBG、FINS水平以及HOMA-IR升高（P＜0.01），胰腺组织病理结构及胰腺β细胞超微结构损伤明显，血清及胰腺组织氧化应激指标（SOD、MDA、CAT）水平降低（P＜0.05、0.01），胰腺组织中Pdx1、Ngn3、Mafa蛋白表达量显著降低（P＜0.01）。与模型组比较，各给药组体质量减少、体长增加（P＜0.01）；FBG、FINS水平以及HOMA-IR降低（P＜0.05、0.01），血清及胰腺组织氧化应激指标（SOD、MDA、CAT）水平升高（P＜0.05、0.01），胰腺组织病理结构及胰腺β细胞超微结构损伤减轻，胰腺发育关键蛋白（Pdx1、Ngn3、Mafa）表达升高（P＜0.05、0.01）。结论 左归丸能有效改善GDM模型子代胰腺发育，其作用机制可能与抑制氧化应激反应从而保护子鼠胰腺β细胞超微结构有关。

Objective To observe pancreatic development in the offsprings and investigate further effect of pancreaticoduodenal homeobox 1 (Pdx1) pathway on the ultrastructure of pancreatic β cells and oxidative stress in gestational diabetes mellitus (GDM) using a streptozotocin (STZ)-induced GDM model, Zuogui Pills (左归丸) were administered during pregnancy. Methods Pregnant rats were divided into control group, model group, detemir insulin (15 U/kg) group, and low, medium, and high-dose Zuogui Pills (0.95, 1.89, 3.78 g/kg) groups, with six rats in each group. Except for the control group, the others were established by intraperitoneal injection (ip) STZ on the third day of pregnancy (E3d) in pregnant rats. Pregnant rats in the control group were given an equal volume of sodium citrate buffer. The formation of the GDM model in pregnant rats was determined by measuring fasting blood glucose. Except for the control group and the model group, pregnant rats in the remaining groups were administered once daily from E6d until delivery. After pregnant rats delivered naturally, the offsprings were fed by their mothers until they were weaned at 21st d (B21d). One female and one male offspring from each group of mothers were randomly selected to form control group, model group, detemir insulin group, and low, medium, and high-dose Zuogui Pills groups, with 12 offsprings in each group. On the 22nd day of life (B22d), fasting blood glucose (FBG) levels were measured using a glucometer in offsprings from each group. Body weight and body length (measured from the base of the tail to the tip of the nose) were recorded. The offsprings were then euthanized, and serum was separated for the detection of fasting insulin (FINS) levels by ELISA, which was used to calculate the homeostasis model assessment of insulin resistance (HOMA-IR). The pathological structure of the pancreas was observed using hematoxylin-eosin (HE) staining. Ultrastructural changes in β-cells were examined under a transmission electron microscope. Serum and pancreatic tissue levels of superoxide dismutase (SOD), malondialdehyde (MDA), and catalase (CAT) were determined by ELISA. Western blotting was performed to assess the relative protein expression of Pdx1, neurogenin 3 (Ngn3), and v-maf avian musculoaponeurotic fibrosarcoma oncogene family protein A (Mafa) in the pancreatic tissue of offsprings. Results Compared with the control group, offsprings in the model group exhibited increased body mass, reduced body length (P < 0.01), and elevated levels of FBG, FINS, and HOMA-IR levels (P < 0.01). Notably, significant pathological damage and ultrastructural impairment were observed in pancreatic tissue and β-cells. Furthermore, serum and pancreatic tissue levels of oxidative stress markers (SOD, MDA, CAT) were decreased (P < 0.05, 0.01), accompanied by significantly reduced protein expression of Pdx1, Ngn3, and Mafa in pancreatic tissue (P < 0.01). In contrast, compared to the model group, all treatment groups showed decreased body mass and increased body length (P < 0.01). Additionally, there were reductions in FBG, FINS, and HOMA-IR levels (P < 0.01), along with increased serum and pancreatic tissue levels of oxidative stress markers (SOD, MDA, CAT) (P < 0.05, 0.01). The pathological damage and ultrastructural impairment in pancreatic tissue and β-cells were alleviated, and the expression of key pancreatic developmental proteins (Pdx1, Ngn3, Mafa) was upregulated (P < 0.05, 0.01). Conclusion Zuogui Pills can effectively improve the pancreatic development of offsprings in the GDM model, and its mechanism of action may be related to inhibiting oxidative stress responses, thereby protecting the ultrastructure of β-cells.

R285.5

国家自然科学基金面上项目(82274378);山西省卫生健康委员会方药配伍及功用重点研究室建设项目(zyyyjs2024023)