姜黄素介导TLR4/NF-κB信号通路对重症急性胰腺炎的作用及机制研究

Effect and Mechanism of Curcumin-Mediated TLR4/NF-κB Signaling Pathway on Severe Acute Pancreatitis

摘要: 目的：观察姜黄素(CUR)对重症急性胰腺炎(SAP)的影响，并基于Toll样受体4 (TLR4)/核因子κB (NF-κB)信号转导通路探究其可能的作用机制。方法：选取40只成年雄性Sprague-Dawley大鼠随机把其分为4组，分别为：假手术组(SO)、重症急性胰腺炎组(SAP)、SAP CUR预处理组(SAP CUR)、SAP CUR预处理 TLR4受体激活剂脂多糖(LPS)组(SAP CUR LPS)。SAP CUR组、SAP CUR LPS组的大鼠分别CUR (200 mg/kg)灌胃，SAP CUR LPS组在CUR灌胃干预前30 min腹腔注射TLR4受体激活剂LPS (0.1 mg/kg)，SO组和SAP组分别给予相同剂量的无菌生理盐水溶液灌胃及腹腔内注射。各组均给药1次/天，连续4周。采用胆胰管逆行注射5%牛磺胆酸钠的方法建立SAP大鼠模型，术后24 h采集胰腺组织以及下腔静脉血液以供后续分析。使用自动生化分析仪以检测大鼠血清中淀粉酶(AMY)、脂肪酶(LIPA)的活性水平；酶联免疫吸附试验(ELISA)检测血清肿瘤坏死因子-α (TNF-α)、白细胞介素6 (IL-6)的表达水平；苏木素-伊红(HE)染色用以观察胰腺的组织病理变化；实时荧光定量逆转录聚合酶链式反应(RT-qPCR)测定胰腺组织中TLR4、NF-κB的mRNA表达水平。结果：相较于SO组，SAP组大鼠的血清中AMY、LIPA、TNF-α、IL-6的表达水平显著升高(均P < 0.05)；表现出较严重的胰腺病理损伤；胰腺组织中TLR4/NF-κB信号通路相应mRNA水平显著升高(均P < 0.05)。相反，SAP CUR组与SAP组相比AMY、LIPA、TNF-α、IL-6的表达水平显著降低(均P < 0.05)；大鼠胰腺组织表现出较轻的病理损伤；胰腺组织中TLR4/NF-κB信号通路相应mRNA水平显著降低(均P < 0.05)。而与SAP CUR组相比，LPS进一步处理后则能够显著逆转CUR对SAP大鼠各检测指标的调控作用(均P < 0.05)。结论：CUR能够通过下调TLR4/NF-κB信号传导通路抑制炎症反应减轻SAP相关损伤。

Abstract: Objective: To observe the effects of curcumin (CUR) on severe acute pancreatitis (SAP) and explore its potential mechanism of action based on the Toll-like receptor 4 (TLR4)/nuclear factor κB (NF-κB) signaling pathway. Methods: Forty adult male Sprague-Dawley rats were divided into four groups: sham operation group (SO), severe acute pancreatitis group (SAP), SAP CUR pretreatment group (SAP CUR), SAP CUR pretreatment TLR4 receptor agonist lipopolysaccharide (LPS) group (SAP CUR LPS). The rats in the SAP CUR group and SAP CUR LPS group were administered CUR (200 mg/kg) by gavage. The SAP CUR LPS group was injected intraperitoneally with the TLR4 receptor agonist LPS (0.1 mg/kg) 30 minutes before CUR gavage intervention. The SO group and SAP group were given the same dose of 0.9% sodium chloride solution by gavage and intraperitoneal injection, respectively. All groups were administered once a day for 4 consecutive weeks. The SAP rat model was established by retrograde injection of 5% sodium taurocholate into the pancreatic duct, and pancreatic tissue and inferior vena cava blood were collected 24 hours postoperatively for subsequent analysis. An automatic biochemical analyzer was used to detect the activity levels of amylase (AMY) and lipase (LIPA) in rat serum; enzyme-linked immunosorbent assay (ELISA) was used to detect the expression levels of serum tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6); hematoxylin-eosin (HE) staining was used to observe the histopathological changes of the pancreas and perform pathological damage scoring; real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to determine the changes in mRNA expression levels of TLR4 and NF-κB in pancreatic tissue. Results: Compared with the SO group, the expression levels of AMY, LIPA, TNF-α, and IL-6 in the serum of the SAP group were significantly increased (all P < 0.05); the rats showed more severe pancreatic pathological damage and significantly increased pathological scores (all P < 0.05); the mRNA levels of the TLR4/NF-κB signaling pathway in pancreatic tissue were significantly increased (all P < 0.05). In contrast, compared with the SAP group, the expression levels of AMY, LIPA, TNF-α, and IL-6 in the SAP CUR group were significantly decreased (all P < 0.05); the rats showed less severe pathological damage and lower pathological scores in the pancreatic tissue (all P < 0.05); the mRNA levels of the TLR4/NF-κB signaling pathway in pancreatic tissue were significantly decreased (all P < 0.05). Compared with the SAP CUR group, further treatment with LPS was able to significantly reverse the regulatory effects of CUR on the measured indicators in SAP rats (all P < 0.05). Conclusion: CUR can inhibit the inflammatory response and alleviate SAP-related damage by down regulating the TLR4/NF-κB signaling pathway.