上皮-间质转化在胰腺纤维化中的作用及大柴胡汤的干预机制

Role of epithelial-mesenchymal transformation in pancreatic fibrosis and intervention mechanism of Dachaihu decoction

目的探索上皮-间质转化(epithelial mesenchymal transition,EMT)与慢性胰腺炎(chronic pancreatitis,CP)胰腺纤维化的关系及大柴胡汤防治CP的机制。方法健康昆明小鼠78只,随机分为:空白组(n=18)、CP组(n=30)及大柴胡汤组(n=30)。CP组和大柴胡汤组腹腔注射20%左旋精氨酸(L-arginine,3 g/kg,每周1次),空白组腹腔注射等体积的生理盐水。大柴胡汤组在注射L-arginine 1周后,同时给予注射L-arginine和大柴胡汤灌胃治疗5周(14 g/kg,每日一次)。从给药开始计时,各组分别于第2,4,6周麻醉处死小鼠,摘取小鼠胰腺组织,通过HE染色观察胰腺组织的病理改变,免疫组化观察胰腺组织中E-cadherin、N-cadherin、α-SMA的表达变化,Western blotting检测胰腺组织中E-cadherin、N-cadherin蛋白含量的改变。结果与空白组相比,CP组小鼠胰腺组织中胶原纤维沉积随着造模时间延长不断增加,α-SMA表达逐渐增多,E-cadherin表达逐渐减少,在第4,6周时最为显著(P<0.01),而N-cadherin表达逐渐增多,在第6周最为显著(P<0.01)。与CP组相比,大柴胡汤组第4,6周仅见部分腺泡萎缩及少量炎细胞浸润,胶原纤维沉积明显减轻,α-SMA表达显著减少,E-cadherin在第6周显著升高(P<0.01),而N-cadherin在第6周显著下降(P<0.01)。结论在20%L-arginine刺激作用下,小鼠胰腺实质细胞转化为间质细胞,表明EMT参与CP胰腺纤维化的进展。大柴胡汤可通过抑制EMT转化,减轻胰腺纤维化,对CP有明显治疗作用。

Objective To explore the relationship between the epithelial-mesenchymal transition(EMT) and the pancreatic fibrosis in chronic pancreatitis(CP) and the mechanism of Dachaihu decoction in prevention and treatment of CP. Methods Seventy-eight KM mice were randomly divided into three groups: control group(n=18), CP group(n=30) and Dachaihu decoction group(n=30). The mice in CP group and Dachaihu decoction group were intraperitoneally injected with 20% L-arginine(3 g/kg, once a week), while the mice in control group were intraperitoneally injected with the same amount of normal saline. After one week of injecting L-arginine, the mice in Dachaihu decoction group were simultaneously given L-arginine and intragastric administration of Dachaihu decoction(14 g/kg, once a day) for 5 weeks. The beginning of drug administration was recorded as the beginning of the experiment. The mice in each group were anesthetized and sacrificed at week 2, 4 and 6, respectively, and their pancreas tissues were collected. The morphological changes of the pancreas were observed by HE. The expression levels of E-cadherin, N-cadherin and α-SMA were observed by immunohistochemistry. The protein levels of E-cadherin, N-cadherin were determined by Western blotting. Results Compared with control group, the collagen fiber deposition in pancreatic tissues in CP group were increased with the extension of modeling time, the α-SMA expression was increased gradually, E-cadherin expression was decreased gradually and most significantly at week 4 and 6(P<0.01), and N-cadherin expression was increased gradually and most significantly at week 6(P<0.01). Compared with CP group, only part of acinar atrophy and a small amount of inflammatory cell infiltration were observed in Dachaihu decoction group at week 4 and 6, the collagen fiber deposition was reduced, α-SMA expression was decreased, E-cadherin was increased significantly at week 6(P<0.01), and N-cadherin was decreased significantly at week 6(P<0.01). Conclusion Epithelial cells are transformed into mesenchymal cells in response to 20% L-arginine stimulation, which suggests that EMT is involved in the progression of pancreatic fibrosis in CP. The Dachaihu decoction can have a therapeutic effect on CP through inhibiting EMT and relieving the pancreatic fibrosis.