## 4 Discussion  
_C. japonicum_, a widely accessible and cost-effective Chinese herbal medicine, possesses various functions such as bacteriostasis, anti-inflammation, blood coagulation and hemostasis, blood pressure reduction, and anti-tumor effects [40]. One study has shown that the combined treatment of _Analia elata_ and _C. japonicum_ has anti-inflammatory effects on RAW 264.7 cells and demonstrates protective effects against dextran sulfate sodium (DSS)-induced colitis in mice and acetic acid-mediated colitis in dogs [41]. However, the  
Figure 6: Effects of vanillin on inflammatory factors in RAW264.7 cells stimulated by LPS. Mouse macrophages were divided into five groups: CON group, LPS group, LPS + 0.4 mg/mL vanillin group, LPS + 0.2 mg/mL vanillin group, and LPS + 0.1 mg/mL vanillin group. After 24 h of DMEM culture, vanillin was added into cells stimulated by LPS (1 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) after 3 h, and the supernatants and proteins were taken after 24 h. (**–d**) The expressions of cytokines. (**a**) IL-6; (**b**) IL-1\(\beta\); (**c**) TNF-\(\alpha\); (**d**) IL-10. The levels of IL-6, IL-1\(\beta\), TNF-\(\alpha\), and IL-10 were detected by ELISA kit. The data are expressed by the average standard deviation (n = 3). *: \(p<0.05\); **: \(p<0.005\); **: \(p<0.001\); ***: \(p<0.0001\). ***: \(p<0.0001\). precise mechanism underlying its anti-inflammatory action remains unclear. This study employs network pharmacology and molecular docking to investigate the anti-inflammatory effects of _C. japonicum_, followed by in vitro experimental verification at the cellular level. Flavonoids, widely distributed in _Cirsium_, are also the most abundant component and the main active ingredient of _Cirsium_. They possess various biological activities, such as anti-oxidation, anti-tumor effects, anti-inflammation, and liver, cardiovascular, and cerebrovascular protection [42; 43; 44]. In this study, 14 effective components of _C. japonicum_ were obtained from the TCMSP website, primarily including methyl linoleicate, oleic acid, stigmasterol, and sitosterol. Previous studies have demonstrated that stigmasterol exhibits potent anti-cancer properties, while also showing effects against osteoarthritis and inflammation. Moreover, it displays potent activity against parasites, fungi, and bacteria, while also exhibiting immunomodulatory and neuroprotective effects through its antioxidant properties [45; 46; 47; 48; 49; 50]. One study conducted by [PERSON] et al. [51] has revealed that stigmasterol significantly inhibits colon shortening and reduces colitis severity by suppressing pro-inflammatory IL-1\(\beta\), IL-6, and cyclooxygenase-2 (COX-2) monocyte chemotactic protein release. Additionally, stigmasterol improves intestinal function and regulates fat metabolism to alleviate hepatic steatosis in rats by fortifying the intestinal barrier and enhancing bile acid metabolism [52]. \(\beta\)-sitosterol, a phytosterol with anti-inflammatory properties, exerts regulatory effects on blood glucose metabolism [53]. [PERSON] et al. [54] demonstrated that \(\beta\)-sitosterol reduces serum TNF-\(\alpha\) levels in rats, thereby decelerating the progression of gastric mucosa damage through a decreased release and aggregation of inflammatory factors within the gastric mucosa. Moreover, the combination of \(\beta\)-sitosterol and aspirin can enhance the anti-inflammatory efficacy of aspirin [55]. Based on network pharmacology, we conducted a systemic analysis on the active components, targets, related pathways, and biological processes of _C. japonicum_. Through analyzing the relevant database of network pharmacology, a total of 14 active components and their corresponding 171 gene targets were identified for _C. japonicum_. A drug-component-target-disease network diagram was constructed to reveal that 14 effective active components in _C. japonicum_ can synergistically act on 32 anti-inflammatory targets (Figure 2).