粉防己碱通过调控SOCE通路对L-精氨酸诱发的小鼠重症急性胰腺炎的防治作用

Effect of Tetrandrine on L-arginine Induced Severe Acute Pancreatitis in Mice by Regulating SOCE Pathway

目的观察粉防己碱(Tetrandrine,Tet)对L-精氨酸(L-arginine)诱发的重症急性胰腺炎(SAP)小鼠胰腺组织钙库操纵的Ca2+通道(SOCE)的关键效应蛋白基质相互作用分子1(STIM1)及钙释放激活钙通道蛋白1(ORAI1)表达的影响,探讨粉防己碱防治重症急性胰腺炎的作用机制。方法将健康昆明小鼠40只随机分为4组:对照组、重症急性胰腺炎模型组、粉防己碱高剂量组及粉防己碱低剂量组,每组10只。除对照组外,其余小鼠均给予腹腔注射20%L-精氨酸复制重症急性胰腺炎模型(3.5 g·kg^(-1),共2次,间隔1 h),在模型复制4 h后粉防己碱治疗组给予腹腔注射粉防己碱高、低剂量治疗(120、60 mg·kg^(-1),每日2次,共3 d)。于模型复制72 h后麻醉动物,收集血清,检测血清淀粉酶水平;完整摘取小鼠胰腺及肺组织,胰腺称质量并计算胰腺脏体比;HE染色观察各组小鼠胰腺及肺组织形态学改变;免疫组化法观察胰腺组织STIM1及ORAI1的阳染表达;Western Blot法检测胰腺组织STIM1及ORAI1蛋白的表达;RT-qPCR法检测胰腺组织STIM1及ORAI1 mRNA的表达。结果与对照组比较,重症急性胰腺炎模型组小鼠血清淀粉酶及胰腺脏体比均明显增加(P<0.01);胰腺组织出现明显水肿,腺泡细胞大面积坏死伴胰腺间质出血及大量炎症细胞浸润,肺组织可见肺泡间隔明显水肿、增宽,并伴大量炎症细胞浸润及充血;胰腺组织STIM1及ORAI1的蛋白和mRNA表达水平均明显增高(P<0.01)。与重症急性胰腺炎模型组比较,粉防己碱高、低剂量组小鼠血清淀粉酶及胰腺脏体比明显降低,胰腺及肺组织损伤程度减轻,胰腺组织水肿、出血及坏死程度明显减轻,炎性细胞浸润减少,胰腺组织STIM1及ORAI1的蛋白和mRNA表达水平均明显降低(P<0.01)。结论重症急性胰腺炎小鼠胰腺组织SOCE信号通路活化,参与重症急性胰腺炎的病程进展;粉防己碱可通过抑制SOCE信号通路活化,进而减轻重症急性胰腺炎胰腺损伤程度。

ObjectiveTo observe the effect of tetrandrine(Tet)on the expression of stromal interaction molecule 1(STIM1)and calcium release-activated calcium channel protein l(ORAI1)of store-operated calcium entry(SOCE)in pancreas of mice with L-arginine induced severe acute pancreatitis(SAP),and to explore the detailed mechanism of Tet in the prevention and treatment of SAP.MethodsForty KM mice were randomly divided into control group,SAP group,Tet low-and high-dose groups,and each group consisted of 10 mice. Mice in the SAP and Tet groups were all intraperitoneally injected with 20% L-arginine(3.5 g·kg^(-1),once an hour,twice a day). Mice in the Tet group were intraperitoneally injected with Tet(twice a day for 3 days)in different dose(120 mg·kg^(-1),60 mg·kg^(-1))at 4h after SAP induction. The mice were anesthetized and sacrificed at 72h after modeling, the amylase level in serum and the ratio of pancreas weight to body weight were assayed. The morphological changes of the pancreas and lung tissue were observed using HE staining. The positive staining rates of STIM1 and ORAI1 in the pancreas were observed by immunohistochemistry. The mRNA expressions of STIM1 and ORAI1 were determined using real-time quantitative PCR. The protein of pancreas was extracted to detect the expression of STIM1 and ORAI1 by Western blot.ResultsCompared with the control group,serum amylase level and the ratio of pancreas weight to body weight increased significantly in SAP group(P<0.01), The mice in SAP group appeared obvious edema of pancreatic tissue, inflammatory cell infiltration, acinar cells necrosis and interstitial hemorrhage. It was also found that the alveolar septum was edema and widened in lung tissue, which was accompanied by marked inflammatory cell infiltration and congestion, The mRNA expression and protein level of STIM1 and ORAI1 in pancreas were all increased significantly(P<0.01). Compared with SAP group, after the administration of Tet, the injuries of pancreatic and lung tissues were significantly improved,the level of serum amylase and the ratio of pancreas weight to body weight were decreased, the degree of edema, bleeding, necrosis in pancreatic tissue was relieved,inflammatory cell infiltration was decreased,and the mRNA and protein levels of STIM1 and ORAI1 in the pancreas were obviously reduced(P<0.01).ConclusionThe activation of SOCE signaling pathway is involved in the progress of SAP. Tet was effective in ameliorating the degree of pancreatic injury of SAP by inhibiting the SOCE signaling pathway.