Department of Integrated Traditional Chinese and Western Medicine, Sichuan Provincial Pancreatitis Centre and West China-Liverpool Biomedical Research Centre, West China Hospital, Sichuan University, Chengdu 610041, China; West China-Washington Mitochondria and Metabolism Center, West China Hospital, Sichuan University, Chengdu 610041, China; Applied Surgery and Metabolism Laboratory, School of Biological Sciences, University of Auckland, Auckland 1010, New Zealand.
Department of Integrated Traditional Chinese and Western Medicine, Sichuan Provincial Pancreatitis Centre and West China-Liverpool Biomedical Research Centre, West China Hospital, Sichuan University, Chengdu 610041, China.
Phytomedicine. 2020 Dec;79:153328. doi: 10.1016/j.phymed.2020.153328. Epub 2020 Sep 1.
Chaiqin chengqi decoction (CQCQD) is a Chinese herbal formula derived from dachengqi decoction. CQCQD has been used for the management of acute pancreatitis (AP) in the West China Hospital for more than 30 years. Although CQCQD has a well-established clinical efficacy, little is known about its bioactive ingredients, how they interact with different therapeutic targets and the pathways to produce anti-inflammatory effects.
Toll-like receptor 4 (TLR4) and the nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome-mediated pro-inflammatory signaling pathways, play a central role in AP in determining the extent of pancreatic injury and systemic inflammation. In this study, we screened the bioactive ingredients using a pharmacological sub-network analysis based on the TLR4/NLRP3 signaling pathways followed by experimental validation.
The main CQCQD bioactive compounds were identified by UPLC-QTOF/MS. The TLR4/NLRP3 targets in AP for CQCQD active ingredients were confirmed through a pharmacological sub-network analysis. Mice received 7 intraperitoneal injections of cerulein (50 μg/kg; hourly) to induce AP (CER-AP), while oral gavage of CQCQD (5, 10, 15 and 20 g/kg; 3 doses, 2 hourly) was commenced at the 3rd injection of cerulein. Histopathology and biochemical indices were used for assessing AP severity, while polymerase chain reaction, Western blot and immunohistochemistry analyses were used to study the mechanisms. Identified active CQCQD compounds were further validated in freshly isolated mouse pancreatic acinar cells and cultured RAW264.7 macrophages.
The main compounds from CQCQD belonged to flavonoids, iridoids, phenols, lignans, anthraquinones and corresponding glycosides. The sub-network analysis revealed that emodin, rhein, baicalin and chrysin were the compounds most relevant for directly regulating the TLR4/NLRP3-related proteins TLR4, RelA, NF-κB and TNF-α. In vivo, CQCQD attenuated the pancreatic injury and systemic inflammation of CER-AP and was associated with reduced expression of TLR4/NLRP3-related mRNAs and proteins. Emodin, rhein, baicalin and chrysin significantly diminished pancreatic acinar cell necrosis with varied effects on suppressing the expression of TLR4/NLRP3-related mRNAs. Emodin, rhein and chrysin also decreased nitric oxide production in macrophages and their combination had synergistic effects on alleviating cell death as well as expression of TLR4/NLRP3-related proteins.
CQCQD attenuated the severity of AP at least in part by inhibiting the TLR4/NLRP3 pro-inflammatory pathways. Its active ingredients, emodin, baicalin, rhein and chrysin contributed to these beneficial effects.
柴芩承气汤（CQCQD）是一种源自大承气汤的中药方剂。华西医院 30 多年来一直将 CQCQD 用于治疗急性胰腺炎（AP）。尽管 CQCQD 具有良好的临床疗效，但对于其生物活性成分、它们如何与不同的治疗靶点相互作用以及产生抗炎作用的途径知之甚少。
Toll 样受体 4（TLR4）和核苷酸结合寡聚化结构域样受体家族富含亮氨酸重复蛋白 3（NLRP3）炎性小体介导的促炎信号通路在确定胰腺损伤和全身炎症的程度方面在 AP 中起核心作用。在这项研究中，我们通过基于 TLR4/NLRP3 信号通路的药理学子网络分析筛选生物活性成分，并通过实验验证进行了确认。
通过 UPLC-QTOF/MS 鉴定 CQCQD 的主要生物活性化合物。通过药理学子网络分析确定 CQCQD 活性成分治疗 AP 的 TLR4/NLRP3 靶点。在第 3 次注射亮氨酸酶（50μg/kg；每小时）诱导 AP（CER-AP）时，小鼠接受 7 次腹腔注射，同时在第 3 次注射亮氨酸酶时开始口服 CQCQD（5、10、15 和 20g/kg；3 剂，每 2 小时 1 次）。组织病理学和生化指标用于评估 AP 的严重程度，而聚合酶链反应、Western blot 和免疫组织化学分析用于研究机制。进一步在新鲜分离的小鼠胰腺腺泡细胞和培养的 RAW264.7 巨噬细胞中验证鉴定出的 CQCQD 活性化合物。
CQCQD 的主要化合物属于黄酮类、环烯醚萜类、酚类、木脂素类、蒽醌类及其糖苷。子网络分析显示，大黄素、大黄酸、黄芩苷和白杨素是与直接调节 TLR4/NLRP3 相关蛋白 TLR4、RelA、NF-κB 和 TNF-α最相关的化合物。体内，CQCQD 减轻了 CER-AP 的胰腺损伤和全身炎症，与 TLR4/NLRP3 相关 mRNA 和蛋白表达减少有关。大黄素、大黄酸、黄芩苷和白杨素显著减轻胰腺腺泡细胞坏死，并对抑制 TLR4/NLRP3 相关 mRNAs 的表达产生不同的影响。大黄素、大黄酸和白杨素还降低了巨噬细胞中的一氧化氮产生，它们的组合对减轻细胞死亡和 TLR4/NLRP3 相关蛋白的表达具有协同作用。
CQCQD 通过抑制 TLR4/NLRP3 促炎途径至少部分减轻了 AP 的严重程度。其活性成分大黄素、黄芩苷、大黄酸和白杨素对这些有益作用有贡献。
