急性胰腺炎“从肠论治”新靶点：小肠损伤的Nfr2-ROS-NLRP3炎症通路探索及疗效验证

Small intestine is one of the most affected organs that are highly voluntary to ischaemia-reperfusion injury (IRI) during stage of acute pancreatitis (AP). The manifestation of small intestine injury during AP-IRI has been characterised as Yang Ming Heat and Dry Intestine, a typical Yangming Fushi syndrome. Based on “gut-centred therapy” concept in treating AP, we use "Tongfu" principle to remove the excess heat thus to restore the Yin by Chai-Qin-Cheng-Qi Decoction (CQCQD) and its clinical efficacy has been validated. In murine models of mesenteric IRI and AP-IRI, it is found that small intestine injury appears early in the disease and is associated with oxidative stress and pro-inflammatory pathway activation. CQCQD improves AP-IRI-induced small intestine injury by up-regulating small intestine nuclear factor-erythroid 2-related factor 2 (Nrf2), a key factor that modulates antioxidant response element (ARE) gene to counteract reactive oxygen species (ROS) which otherwise causes maturation of NLRP3 inflammasome, followed by release of interleukin-1β and activation of pro-inflammatory pathway. Based on these findings, we hypothesise that: small intestine is a major organ included in the “gut-centred therapy” and up-regulating Nrf2-AREs to antagonise ROS-induced activation of NLRP3 inflammasome is a potential protective mechanism; CQCQD counters oxidative stress and inflammation to alleviate small intestine injury and severity of AP via regulating Nrf2-ROS-NLRP3 pathway. This project seeks to use Nrf2 specific activator and Nrf2-siRNA to test the aforementioned hypothesis in a rat small intestine cell line (IEC-6) and in a mouse alcoholic AP-IPI model. Successful completion of this project will enrich the “gut-centred therapy” theory and identify novel therapeutic targets.

由于急性胰腺炎（AP）时小肠对缺血再灌注损伤（IRI）的高敏感低耐受特征，导致其成为AP早期器官功能受损的核心靶器官之一，并表现出阳明内热、肠燥津枯的阳明腑实典型证候。我们在运用通腑泄热、急下存阴临床验方柴芩承气汤（CQCQD）“从肠论治”AP取得显著疗效的基础上，根据IRI诱发氧化应激并激活炎性通路的分子机制，结合CQCQD减轻AP-IRI小肠损伤，上调小肠组织Nrf2表达，抑制IL-1β促炎因子释放的抗炎作用等预试验结果，提出假说：小肠是AP“从肠论治”的主体靶器官，Nrf2上调拮抗ROS介导的氧化应激损伤及其激活的NLRP3炎性通路是保护AP小肠IRI的潜在作用机制；CQCQD通过调控Nrf2-ROS-NLRP3新机制发挥抗氧化和抗炎双重作用，减轻小肠损伤，缓解AP病情。通过选择性激动、干扰技术调节Nrf2通路等研究验证该假说，从而丰富完善AP中医理论，获得 “从肠论治”创新靶点。

急性胰腺炎（AP）是病情凶险的急腹症之一。肠道功能受损是AP发生多器官功能障碍病情发展的扳机点。AP早期引起小肠损伤主要机制是缺血再灌注损伤（IRI）诱发氧化应激和炎症反应，导致小肠上皮细胞损伤和粘膜屏障功能破坏，加重小肠损伤。以活性氧族（ROS）蓄积的氧化应激和活化炎性信号通路是AP早期小肠损伤的可能机制。核转录E2相关因子（Nrf2）所调控的抗氧化反应元素（AREs）是调控ROS的主要信号通路，并且可选择性通过AREs调控抑制炎性反应。本研究采用TLCS和TNF-α刺激小肠上皮细胞株IEC-6构建的体外模型和酒精性AP代表的IRI模型，首先证实AP造成小肠损伤与胰腺损伤程度一致。研究发现Nrf2通路激活诱导小肠上皮细胞ROS生成增加，伴随caspase3/7表达升高，以细胞凋亡为主要细胞死亡方式。AP引起的小肠损伤与Nrf2表达水平下调，NLRP3炎性信号通路激活IL-1β水平升高相关。第二，前期研究发现CQCQD可减轻IRI模型胰腺病理损伤，减轻胰腺MPO水平、血清IL-6和血清IFABP水平。CQCQD可通过上调小肠Nrf2表达水平，抑制IL-1β水平减轻小肠损伤，进一步体外实验研究发现CQCQD可能通过调控IEC-6细胞Nrf2抑制ROS生成，抑制细胞凋亡，减轻TNF-α引起的细胞坏死而发挥抗氧化作用和抗炎作用保护小肠损伤。利用Nrf2 KO小鼠，发现Nrf2基因敲除可能通过促进POAEE诱导的胰腺腺泡细胞坏死向凋亡转化而发挥一定程度的保护作用。但是否通过拮抗ROS氧化应激通路活化的NLRP3通路发挥作用有待进一步证实。此外，本研究还通过多个小鼠AP模型证实CQCQD系列方剂可显著减轻胰腺损伤，且其水溶性提取物（AE）的作用显著优于复方和氯仿提取物。进一步筛选出AE最佳治疗AP有效浓度，并通过体内和体外实验研究发现该保护作用可能通过抑制腺泡细胞坏死和凋亡，抑制TLR4/NF-κB炎性信号通路相关。此部分研究为本课题的扩展部分。该研究为进一步探明AP小肠损伤的机制以及中药减轻AP机制提供了思路和潜在靶点，丰富了AP“从肠论治”科学内涵。