Dachengqi Decoction Attenuates Intestinal Vascular Endothelial Injury in Severe Acute Pancreatitis in Vitro and in Vivo
Dachengqi Decoction Attenuates Intestinal Vascular Endothelial Injury in Severe Acute Pancreatitis in Vitro and in Vivo
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大承气汤减轻重症急性胰腺炎肠血管内皮损伤的体内外研究
DOI：
10.1159/000486155
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发表时间：
2017-01-01
影响因子：
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通讯作者：
Feng, Dian-Xu
中科院分区：
文献类型：
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作者：
Pan, Li-Yun;Chen, Ya-Feng;Feng, Dian-Xu
背景/目的：大承气汤（DCQD）是一种著名的传统中药，具有很强的抗炎作用。血管生成素-1 (Ang-1) 在维持血管壁的稳定性和完整性以及防止炎症介质引起的血管渗漏方面发挥着至关重要的作用。我们之前的工作发现 DCQD 可以保护患有严重急性胰腺炎 (SAP) 的大鼠免受胰腺损伤。本研究旨在探讨 DCQD 对受损人脐静脉内皮细胞 (HUVEC) 和 SAP 大鼠肠内皮损伤的影响。方法：将HUVEC随机分为4组：对照组、TNF-α组、TNF-α+Ang-1组（Ang-1组）、TNF-α+DCQD组（DCQD组）。收集前将细胞孵育 6 小时、12 小时和 24 小时。 DCQD 的处理浓度根据细胞计数试剂盒-8 (CCK-8) 测定确定。通过测量跨内皮电阻（TEER）来评估 HUVEC 的单层渗透性。通过流式细胞术分析细胞凋亡。通过 RT-PCR、免疫细胞荧光和蛋白质印迹评估水通道蛋白 1 (AQP-1)、基质金属蛋白酶 9 (MMP9) 和连接粘附分子-C (JAM-C) 的 mRNA 和蛋白表达。 40只雄性Sprague-Dawley大鼠随机分为对照组、SAP组、SAP+Ang-1组（Ang-1组）、SAP+DCQD组（DCQD组）。 SAP通过腹腔注射雨蛙素和脂多糖（LPS）诱导，而对照组则注射0.9%盐水溶液。通过阴茎静脉注射伊文思蓝，造模12小时后处死大鼠。采用 ELISA 法测定血清淀粉酶、TNF-α、IL-1β、IL-2 和 IL-6 水平。通过组织学分析肠组织，并通过伊文思蓝外渗测定评估组织中的毛细血管通透性。通过免疫组织荧光、蛋白质印迹和 RT-PCR 评估 AQP-1、MMP9 和 JAM-C 的蛋白和 mRNA 表达。结果：DCQD 降低了体外 TNF-α 诱导的 HUVEC 的通透性。此外，在 TNF-α 诱导后，DCQD 改变了 HUVEC 中 JAM-C、MMP9 和 AQP-1 的 mRNA 和蛋白质水平。雨蛙素联合脂多糖诱导 SAP 肠道损伤时，肠组织中 JAM-C 和 MMP9 表达增加，AQP-1 表达减少。 DCQD预处理可减轻SAP肠道损伤，并有效降低血清淀粉酶、TNF-α、IL-1β、IL-2和IL-6的水平。我们的研究表明，DCQD 在体外和体内均可降低 JAM-C 和 MMP9 的表达，并增加 AQP-1 的表达。结论:DCQD可减轻急性胰腺炎相关肠损伤时毛细血管内皮损伤,其机制可能与调节内皮屏障功能相关蛋白AQP-1、MMP9、JAM-C有关。
Background/Aims: Dachengqi decoction (DCQD) is a well-known traditional Chinese herbal drug with strong anti-inflammatory effects. Angiopoietin-1 (Ang-1) plays a vital role in maintaining the stability and integrity of the vascular wall and prevents vascular leakage due to inflammatory mediators. Our previous work found that DCQD protects against pancreatic injury in rats with severe acute pancreatitis (SAP). This study aims to investigate the effects of DCQD on intestinal endothelial damage in both damaged human umbilical vein endothelial cells (HUVECs) and SAP rats. Methods: HUVECs were randomly divided into four groups: control group, TNF-α group, TNF-α plus Ang-1 group (Ang-1 group), and TNF-α plus DCQD group (DCQD group). Cells were incubated for 6 h, 12 h, and 24 h, before collection. The treatment concentration of DCQD was decided based on a Cell Counting Kit-8 (CCK-8) assay. The monolayer permeability of the HUVECs was assessed by measuring the transendothelial electrical resistance (TEER). Apoptosis was analyzed by flow cytometry. mRNA and protein expression of aquaporin 1 (AQP-1), matrix metalloproteinase 9 (MMP9), and junctional adhesion molecule-C (JAM-C) was evaluated by RT-PCR, immunocytofluorescence, and western blot. Forty male Sprague-Dawley rats were randomized into a control group, SAP group, SAP plus Ang-1 group (Ang-1 group), and SAP plus DCQD group (DCQD group). SAP was induced by intraperitoneal injection of cerulein and lipopolysaccharide (LPS), while the control group received 0.9% saline solution. Evans blue was injected through the penile vein and the rats were then sacrificed 12 h after modeling. Levels of serum amylase, TNF-α, IL-1β, IL-2, and IL-6 were determined by using ELISA. Intestinal tissue was analysed by histology, and capillary permeability in the tissues was evaluated by Evans blue extravasation assay. Protein and mRNA expression of AQP-1, MMP9, and JAM-C were assessed by immunohistofluorescence, western blot, and RT-PCR. Results: DCQD reduced the permeability of HUVEC induced by TNF-α in vitro. Furthermore, DCQD altered the mRNA and protein levels of JAM-C, MMP9, and AQP-1 in HUVECs after TNF-α induction. SAP intestinal injury induced by cerulein combined with lipopolysaccharides was concomitant with increased expression of JAM-C and MMP9, and reduced expression of AQP-1 in intestinal tissue. Pretreatment with DCQD attenuated SAP intestinal injury and lowered the levels of serum amylase, TNF–α, IL-1β, IL-2, and IL-6 effectively. Our study demonstrated that DCQD decreased the expression of JAM-C and MMP9 and increased the expression of AQP-1 both in vitro and in vivo. Conclusion: DCQD can reduce capillary endothelial damage in acute pancreatitis-associated intestinal injury and the mechanism may be associated with the regulation of endothelial barrier function-associated proteins AQP-1, MMP9, and JAM-C.