DataSheet3_Uncovering the active constituents and mechanisms of Rujin Jiedu powder for ameliorating LPS-induced acute lung injury using network pharmacology and experimental investigations.docx

Background: Acute lung injury (ALI) is a common clinical disease with high mortality. Rujin Jiedu powder (RJJD) has been clinically utilized for the treatment of ALI in China, but the active constituents in RJJD and its protective mechanisms against ALI are still unclear.Methodology: ALI mice were established by intraperitoneal injection of LPS to test the effectiveness of RJJD in treating ALI. Histopathologic analysis was used to assess the extent of lung injury. An MPO (myeloperoxidase) activity assay was used to evaluate neutrophil infiltration. Network pharmacology was used to explore the potential targets of RJJD against ALI. Immunohistochemistry and TUNEL staining were performed to detect apoptotic cells in lung tissues. RAW264.7 and BEAS-2B cells were used to explore the protective mechanisms of RJJD and its components on ALI in vitro. The inflammatory factors (TNF-α, IL-6, IL-1β and IL-18) in serum, BALF and cell supernatant were assayed using ELISA. Western blotting was performed to detect apoptosis-related markers in lung tissues and BEAS-2B cells.Results: RJJD ameliorated pathological injury and neutrophil infiltration in the lungs of ALI mice and decreased the levels of inflammatory factors in serum and BALF. Network pharmacology investigations suggested that RJJD treated ALI via regulating apoptotic signaling pathways, with AKT1 and CASP3 as crucial targets and PI3K-AKT signaling as the main pathway. Meanwhile, baicalein, daidzein, quercetin and luteolin were identified as key constituents in RJJD targeting on the above crucial targets. Experimental investigations showed that RJJD significantly upregulated the expression of p-PI3K, p-Akt and Bcl-2, downregulated the expression of Bax, caspase-3 and caspase-9 in ALI mice, and attenuated lung tissue apoptosis. Four active constituents in RJJD (baicalein, daidzein, quercetin and luteolin) inhibited the secretion of TNF-α and IL-6 in LPS-induced RAW264.7 cells. Among these components, daidzein and luteolin activated the PI3K-AKT pathway and downregulated the expression of apoptosis-related markers induced by LPS in BEAS-2B cells.Conclusion: RJJD alleviates the inflammatory storm and prevents apoptosis in the lungs of ALI mice. The mechanism of RJJD in treating ALI is related to the activation of PI3K-AKT signaling pathway. This study provides a scientific basis for the clinical application of RJJD.

背景：急性肺损伤（ALI）是一种临床常见疾病，其死亡率较高。我国临床已广泛应用如瑞金解毒粉（RJJD）治疗ALI，然而RJJD中的活性成分及其对ALI的保护机制尚不明确。方法：通过腹腔注射脂多糖（LPS）建立ALI小鼠模型，以测试RJJD在治疗ALI中的有效性。采用组织病理学分析评估肺损伤程度，通过髓过氧化物酶（MPO）活性测定评估中性粒细胞浸润。运用网络药理学探索RJJD对ALI潜在靶点的可能性。通过免疫组化和TUNEL染色检测肺组织中凋亡细胞。利用RAW264.7和BEAS-2B细胞探索RJJD及其成分对ALI的体外保护机制。采用ELISA方法检测血清、支气管肺泡灌洗液（BALF）和细胞培养上清液中的炎症因子（TNF-α、IL-6、IL-1β和IL-18）。通过Western blotting检测肺组织和BEAS-2B细胞中凋亡相关标记物。结果：RJJD显著改善了ALI小鼠肺部的病理损伤和中性粒细胞浸润，并降低了血清和BALF中炎症因子的水平。网络药理学研究提示，RJJD通过调节凋亡信号通路治疗ALI，其中AKT1和CASP3为关键靶点，PI3K-AKT信号通路为主要途径。同时，白藜芦醇、大豆苷元、槲皮素和木犀草素被确认为RJJD中的关键成分，针对上述关键靶点。实验研究表明，RJJD显著上调了ALI小鼠中p-PI3K、p-Akt和Bcl-2的表达，下调了Bax、caspase-3和caspase-9的表达，并减轻了肺组织凋亡。RJJD中的四种活性成分（白藜芦醇、大豆苷元、槲皮素和木犀草素）抑制了LPS诱导的RAW264.7细胞中TNF-α和IL-6的分泌。在这些成分中，大豆苷元和木犀草素激活了PI3K-AKT通路，并下调了LPS在BEAS-2B细胞中诱导的凋亡相关标记物的表达。结论：RJJD缓解了ALI小鼠肺部的炎症风暴并防止了凋亡。RJJD治疗ALI的机制与PI3K-AKT信号通路的激活有关。本研究为RJJD的临床应用提供了科学依据。