蟛蜞菊内酯在重症急性胰腺炎肠屏障功能中的作用及机制

The Role and Mechanism of Wedelolactone in Intestinal Barrier Function of Severe Acute Pancreatitis

摘要: 目的：探讨蟛蜞菊内酯(Wedelolactone)在重症急性胰腺炎(SAP)大鼠肠屏障损伤中的作用及机制。方法：选取SPF级健康雄性Wistar大鼠40只，随机分为4组：假手术组(SO组)、重症急性胰腺炎组(SAP组)、蟛蜞菊内酯25 mg/kg治疗组(Wed-L)和蟛蜞菊内酯50 mg/kg治疗组(Wed-H)。采用逆行胆胰管穿刺法，向胆胰管内注入5%牛黄胆酸钠溶液(1 ml/kg)诱导SAP模型。假手术组给予等量的生理盐水。Wed-L组和Wed-H组在SAP模型建立后在1小时和6小时后给予腹腔注射25和50mg/kg的蟛蜞菊内酯。制模24 h后，再次麻醉大鼠。使用自动生化分析仪检测血液中的血清淀粉酶和脂肪酶水平及血清肿瘤坏死因子-α、白细胞介素-6，白细胞介素-18和白细胞介素-1β均使用标准诊断试剂盒进行检测；HE用于评估胰腺、肠道病理变化，并进行病理评分；通过用Western blot技术测定肠组织中细胞焦亡相关蛋白GSDMD、Caspase-11和紧密连接蛋白ZO-1、Occludin、Claudins-1的表达；RT-PCR检测细胞焦亡相关基因GSDMD和Caspase-11的表达情况。使用免疫荧光染色法测定肠上皮细胞细胞焦亡相关蛋白GSDMD、Caspase-11和紧密连接蛋白ZO-1、Occludin、Claudins-1的分布情况。结果：HE染色结果示，与SO组相比，SAP组出现明显肠道病理损伤(P < 0.01)，而Wed-H组肠道损伤明显低于SAP组(P < 0.01)；同样，SAP组胰腺损伤明显高于SO组(P < 0.01)，Wed-H组的损伤较于SAP组的胰腺损伤减轻(P < 0.05)，Wed-L和Wed-H之间的差异无统计学意义。SAP组的血清淀粉酶和脂肪酶水平明显高于SO组(P < 0.01)，Wed-L和Wed-H组明显低于SAP组(P < 0.01)。SAP组TNF-α、IL-6、IL-1β、IL-18水平明显高于SO组(P < 0.01)，Wed-L和Wed-H组明显低于SAP组(P < 0.01)。RT-PCR结果显示SAP组肠道组织GSDMD和Caspase-11的表达水平较SO组明显升高(P < 0.01)，Wed-H组的表达水平明显低于SAP组(P < 0.01)。Western blot显示，SAP组肠道组织GSDMD和Caspase-11蛋白表达水平较SO组升高(P < 0.05)。与SAP组相比较，Wed-H组肠道组织GSDMD和Caspase-11蛋白表达水平明显下降(P < 0.05)。SAP组肠道组织ZO-1、Claudin-1、Occludin蛋白表达水平较SO组降低(P < 0.01)。与SAP组相比较，Wed-H组肠道组织ZO-1、Claudin-1、Occludin蛋白表达水平明显升高(P < 0.01)。免疫荧光结果显示SAP组GSDMD明显高于SO组和Wed-H组，而紧密连接蛋白ZO-1、Occludin、Claudin-1显示较SO组弱，Wed-H组较SAP明显改善。结论：蟛蜞菊内酯通过抑制Caspase-11下调GSDMD表达，减少其介导的细胞焦亡炎性因子的释放，减轻肠粘膜损伤。

Abstract: Objective: To explore the role and mechanism of Wedelolactone in intestinal barrier injury in rats with severe acute pancreatitis (SAP). Methods: Forty SPF healthy male Wistar rats were randomly divided into four groups: sham operation group (SO group), severe acute pancreatitis group (SAP group), wedelolactone 25 mg/kg treatment group (Wed-L) and wedelolactone 50 mg/kg treatment group (Wed-H). SAP model was induced by retrograde cholangiopancreatography by injecting 5% sodium taurocholate solution (1 ml/kg) into cholangiopancreatography. The sham operation group was given the same amount of normal saline. Wed-L group and Wed-H group were given intraperitoneal injection of 25 and 50 mg/kg wedelolactone 1 hour and 6 hours after the estab-lishment of SAP model. After 24 hours of modeling, the rats were anesthetized again. The levels of serum amylase and lipase in blood and serum tumor necrosis factor-α, interleukin-6, interleukin-18 and interleukin-1β were detected by automatic biochemical analyzer, and standard diagnostic kits were used. HE was used to evaluate the pathological changes of pancreas and intestine, and made pathological score. The expressions of apoptosis-related proteins GSDMD, Caspase-11 and tight junction proteins ZO-1, Occludin and Claudins-1 in intestinal tissues were determined by Western blot. RT-PCR was used to detect the expression of apoptosis-related genes GSDMD and Caspase-11. The distribution of apoptosis-related proteins GSDMD, Caspase-11 and tight junction proteins ZO-1, Occludin and Claudins-1 in intestinal epithelial cells was determined by immunofluorescence staining. Results: HE staining showed that compared with SO group, SAP group had obvious intestinal pathological injury (P < 0.01), while Wed-H group had significantly lower intestinal injury than SAP group (P < 0.01). Similarly, the pancreatic injury in SAP group was significantly higher than that in SO group (P < 0.01), and the pancreatic injury in Wed-H group was less than that in SAP group (P < 0.05). There was no significant difference between Wed-L and WED-H. Serum amylase and lipase levels in SAP group were significantly higher than those in SO group (P < 0.01), while those in Wed-L and Wed-H groups were significantly lower than those in SAP group (P < 0.01). The levels of TNF-α, IL-6, IL-1β and IL-18 in SAP group were significantly higher than those in SO group (P < 0.01), while those in Wed-L and Wed-H groups were significantly lower than those in SAP group (P < 0.01). RT-PCR results showed that the expression levels of GSDMD and Caspase-11 in intestinal tissues in SAP group were significantly higher than those in SO group (P < 0.01), and the expression levels in Wed-H group were significantly lower than those in SAP group (P < 0.01). Western blot showed that the expression levels of GSDMD and Caspase-11 protein in intestinal tissues of SAP group were higher than those of SO group (P < 0.05). Compared with SAP group, the expression levels of GSDMD and Caspase-11 protein in intestinal tissue of Wed-H group decreased significantly (P < 0.05). The expression levels of ZO-1, Claudin-1 and Occludin in intestinal tissues in SAP group were lower than those in SO group (P < 0.01). Compared with SAP group, the expression levels of ZO-1, Claudin-1 and Occludin in intestinal tissue in Wed-H group were significantly higher (P < 0.01). Immunofluorescence results showed that GSDMD in SAP group was significantly higher than that in SO group and Wed-H group, while tight junction proteins ZO-1, Occludin and Claudin-1 were weaker than that in SO group, and Wed-H group was significantly improved than that in SAP. Conclusion: Wedelolactone down-regulates the expression of GSDMD by inhibiting Caspase-11, reduces the release of inflammatory factors in pyroptosis mediated by Caspase-11, and reduces intestinal mucosal injury.