柴黄清胰活血颗粒对重症急性胰腺炎模型大鼠JAK2/STAT3信号通路的影响

Effect of Chaihuang Qingyi Huoxue Granules on JAK2/STAT3 Signal Pathway in Rats with Severe Acute Pancreatitis

目的观察柴黄清胰活血颗粒对重症急性胰腺炎(severe acute pancreatitis,SAP)模型大鼠炎症反应、病理损伤以及JAK2/STAT3信号通路的影响,为柴黄清胰活血颗粒的临床应用提供理论依据。方法将64只大鼠随机分为假手术组16只、模型复制组48只。模型复制组采用胰胆管注射5%牛磺胆酸钠进行模型复制。将模型复制成功的大鼠随机分为模型组、柴黄清胰活血颗粒组及阳性对照(AG-490)组,每组16只。假手术组、模型组、阳性对照组予以生理盐水灌胃,柴黄清胰活血颗粒组大鼠予以1.6 g·kg^(-1)柴黄清胰活血颗粒溶液灌胃,每8 h 1次。阳性对照组大鼠术前30 min腹部皮下注射8 mg·kg^(-1)的AG-490注射液。术后各组按照术后12、24 h的时间点分别取8只大鼠,暴露腹主动脉并采血、剪取胰腺组织。HE染色观察胰腺组织病理变化并进行病理评分;生化法检测血清淀粉酶、脂肪酶活性;ELISA法检测血清白细胞介素6(IL-6)、白细胞介素8(IL-8)以及肿瘤坏死因子α(TNF-α)水平;免疫组化法检测胰腺组织p-JAK2、p-STAT3的活性;Western Blot法检测胰腺组织JAK2、p-JAK2、STAT3、p-STAT3蛋白的表达水平。结果与相应时点的假手术组比较,模型组胰腺组织HE染色可见胰腺腺泡结构被明显破坏,有明显充血、水肿、白细胞浸润,病理评分升高,血清淀粉酶、脂肪酶含量增加,血清IL-6、IL-8、TNF-α水平升高,p-JAK2、p-STAT3免疫组化阳性表达量升高,JAK2、p-JAK2、STAT3、p-STAT3蛋白表达量上升(均P<0.05)。与相应时点的模型组比较,柴黄清胰活血颗粒组胰腺组织HE染色改善,病理评分下降,血清淀粉酶、脂肪酶活性下降,血清IL-6、IL-8、TNF-α水平下降,p-JAK2、p-STAT3免疫组化阳性表达量下降,JAK2、p-JAK2、STAT3、p-STAT3蛋白表达量下降(均P<0.05)。与柴黄清胰活血颗粒组比较,阳性对照组胰腺组织HE染色无明显变化,且病理评分、血清淀粉酶、脂肪酶、IL-6、IL-8、TNF-α及p-JAK2、p-STAT3免疫组化阳性表达量,JAK2、p-JAK2、STAT3、p-STAT3蛋白表达量的差异无统计学意义(P>0.05)。结论柴黄清胰活血颗粒可减轻重症急性胰腺炎模型大鼠炎症反应,其机制可能是通过抑制JAK2/STAT3信号通路,从而发挥对重症急性胰腺炎的治疗作用。

Objective To observe the effects of Chaihuang Qingyi Huoxue Granules on inflammatory response,pathological injury and JAK2/STAT3 signal pathway in rats with severe acute pancreatitis(SAP),and to provide a certain theoretical basis for the clinical application of Chaihuang Qingyi Huoxue Granules.Methods Sixty-four rats were randomly divided into sham operation group(16 rats)and modeling group(48 rats).The modeling group was established by injecting 5%sodium taurocholate into pancreaticobiliary duct.After successful modeling,48 rats were randomly assigned to model group,Chaihuang Qingyi Huoxue Granules group and positive control(AG-490)group,with 16 rats in each group.Sham operation group,model group and AG-490 group were given normal saline by gavage,and rats in Chaihuang Qingyi Huoxue Granules group were given 1.6 g·kg^(-1) Chaihuang Qingyi Huoxue Granules solution by gavage,once every 8 hours.Rats in AG-490 group were abdominally and subcutaneously injected with 8 mg·kg^(-1) of AG-490 at 30 minutes before operation.Eight rats in each group were taken at 12h and 24h after operation.The abdominal aorta in AG-490 group was exposed and blood was collected,and the pancreatic tissue was cut.The pathological changes of pancreatic tissue were observed and scored by HE staining.Serum amylase(Amy)and lipase(LIP)were detected by biochemical method.Serum interleukin 6(IL-6),interleukin 8(IL-8)and tumor necrosis factorα(TNF-α)were detected by ELISA.The activities of p-JAK2 and p-STAT3 in pancreatic tissue were detected by immunohistochemistry.The protein expression levels of JAK2,p-JAK2,STAT3 and p-STAT3 in pancreatic tissue were detected by Western Blot.Results Compared with sham operation group at the same time,HE staining of pancreatic tissue in model group showed obvious destruction of pancreatic acinar structure,obvious congestion,edema and leukocyte infiltration.Pancreatic histopathological score of model group was increased;AMY and LIP increased;IL-6,IL-8 and TNF-αwere all increased;the immunohistochemical positive expression levels of p-JAK2 and p-STAT3 were increased;the protein expression levels of JAK2,p-JAK2,STAT3 and p-STAT3 were increased(all P<0.05).Compared with model group at the same time,HE staining of pancreatic tissue in Chaihuang Qingyi Huoxue Granules group was improved;pancreatic histopathological score was decreased;both AMY and LIP were decreased;serum IL-6,IL-8 and TNF-αwere decreased.The immunohistochemical positive expression levels of p-JAK2 and p-STAT3 were decreased.The protein expression levels of JAK2,p-JAK2,STAT3 and p-STAT3 were decreased(all P<0.05).Compared with Chaihuang Qingyi Huoxue Granules group,there was no significant change in HE staining of pancreatic tissue found in AG-490 group,and pancreatic histopathological score,immunohistochemical positive expression levels of AMY,LIP,IL-6,IL-8,TNF-α,p-JAK2 and p-STAT3,and protein expression levels of JAK2,STAT3,p-JAK2 and p-STAT3 were not statistically significant(P>0.05).Conclusion Chaihuang Qingyi Huoxue Granules may play a therapeutic role in the treatment of SAP by inhibiting JAK2/STAT3 signaling pathway and alleviating inflammatory response.