Transcriptome Sequencing of Cecum Tissues Form Septic Rats Treated With or Without Tongfu Pingchuan Decoction

This study aimed to investigate the protective mechanisms of the Traditional Chinese Medicine Tongfu Pingchuan decoction (TFP) in the cecum of septic rats. Thirty rats were randomly divided into three groups: Sham, Model (sepsis induced by cecal ligation and puncture), and TFP (sepsis treated with TFP). Rats received 1 mL/kg TFP or PBS via gavage twice daily for one week, after which whole blood and cecum tissues were collected. Transcriptome sequencing was conducted on six cecum tissues per group. Bioinformatics analyses predicted the potential mechanisms of TFP, which were subsequently validated in cecum tissues and RAW264.7 cell cultures. We found that Pathways such as TNF signaling, complement and coagulation cascades, fatty acid metabolism, and PPAR signaling were activated, while cholinergic synapse, ECM-receptor interaction, and VSMC contraction were inhibited in the Model group compared to Sham. These changes were largely reversed in the TFP group. Chrm4 and Pygm were identified as potential TFP targets, as they were overlapping differentially expressed genes (DEGs) in the predicted TFP targets and an identified WGCNA module of interest. Chrm4 was downregulated in the Model group and restored by TFP treatment. In the Model group, M1 macrophages were induced, while M2 macrophages were suppressed. TFP inhibited M1 macrophages and further enhanced M2 macrophages. These effects were corroborated in LPS-stimulated RAW264.7 cells, where TFP also modulated cytokine levels (IL-6, IL-10, TNF-α). The effects of TFP were negated by Boc-2 (a PCTR1 inhibitor) or Chrm4 silencing. Additionally, TFP suppressed IL1RN, p-ERK2/ERK2, and p-AKT/AKT levels in LPS-treated RAW264.7 cells, but these effects were abolished upon Chrm4 silencing. Collectively, we show that TFP alleviates sepsis by promoting Chrm4/IL1RN-regulated macrophage polarization and facilitating inflammation resolution.本研究旨在探讨中药通腑平喘汤（Tongfu Pingchuan decoction, TFP）对脓毒症大鼠盲肠的保护作用机制。将30只大鼠随机分为三组：假手术组（Sham）、盲肠结扎穿刺脓毒症模型组（简称模型组）、TFP干预组（脓毒症大鼠给予TFP治疗）。大鼠每日两次经灌胃给予1 mL/kg的TFP或磷酸盐缓冲液（phosphate buffered saline, PBS），连续给药一周，随后采集全血及盲肠组织。每组选取6份盲肠组织进行转录组测序（transcriptome sequencing）。通过生物信息学分析（bioinformatics analyses）预测TFP的潜在作用机制，并分别在盲肠组织与RAW264.7细胞培养体系中验证该机制。 研究结果显示，与假手术组相比，模型组的TNF信号通路、补体与凝血级联反应、脂肪酸代谢及PPAR信号通路被激活，而胆碱能突触、细胞外基质-受体相互作用、血管平滑肌细胞收缩通路则受到抑制。上述异常通路变化在TFP干预组中基本得到逆转。 经分析发现，Chrm4与Pygm为TFP的潜在作用靶点：二者既属于预测得到的TFP靶点差异表达基因（differentially expressed genes, DEGs），同时也为本研究关注的加权基因共表达网络分析（Weighted Gene Co-expression Network Analysis, WGCNA）模块中的重叠差异表达基因。其中，Chrm4在模型组中表达下调，经TFP干预后其表达水平得以恢复。 在模型组中，M1型巨噬细胞被诱导激活，而M2型巨噬细胞则受到抑制；TFP可抑制M1型巨噬细胞极化，并进一步促进M2型巨噬细胞极化。上述效应在脂多糖（Lipopolysaccharide, LPS）刺激的RAW264.7细胞中得到验证，同时TFP可调节细胞因子分泌水平，包括IL-6、IL-10及TNF-α。 Boc-2（一种PCTR1抑制剂）或Chrm4基因沉默可抵消TFP的上述调控效应。此外，在LPS处理的RAW264.7细胞中，TFP可抑制IL1RN、p-ERK2/ERK2及p-AKT/AKT的表达水平，但该效应会因Chrm4基因沉默而被完全消除。 综上，本研究证实通腑平喘汤可通过调控Chrm4/IL1RN通路介导的巨噬细胞极化，促进炎症消退，从而改善脓毒症病症。