Elucidation of the Hemostatic and Anti-Inflammatory Effects of Walnut Shell Ethanol Extract by Network Pharmacology and Experimental Verification

Effective hemostatic and anti-inflammatory measures are essential for wound healing. In this study, Ultra-performance liquid chromatography (UPLC) combined with network pharmacology was used to analyze the composition of walnut shell ethanol extract (WSEE), and in vivo and in vitro experiments elucidated its potential mechanisms of hemostatic and anti-inflammatory effects. The results showed that 45 components were identified from WSEE, and the NF-κB signaling pathway, IL-17 signaling pathway, etc., could play significant roles in this context. In vitro, WSEE exhibited procoagulant activity including shortened serum APTT (activated partial thromboplastin time, 40.4 ± 3.05 s), TT (thrombin time, 26.13 ± 1.6 s), and PT (prothrombin time, 14.6 ± 2.1 s) (p < 0.01), and increasing FIB levels (Fibrinogen, 3.7 ± 1.01 s). In vivo, WSEE significantly shortened the clotting time in the WSEE group (05:26 s) compared with the control group (13:25 s). Comparing to the lipopolysaccharide alone treatment group, the expression of pro-inflammatory factors such as TNF-α (61.63%, 73.06%, and 66.03%), IL-1β (94.78%, 95.40%, and 88.99%), and IL-6 (91.37%, 97.02%, and 89.00%) were inhibited at the corresponding concentrations (10, 30, and 50 mg/mL) in RAW264.7 cells stimulated by lipopolysaccharide. Moreover, ADRB2 and PTPN2 were the key target proteins associated with bleeding-related and inflammatory-related diseases, which were obtained from the results of network pharmacology analysis. WSEE also alleviated the expression of ADRB2 (50.53%, 56.11%, and 83.41%) and PTPN2 (95.87%, 96.56%, and 91.49%) in RAW264.7 cells stimulated by lipopolysaccharide, findings that align with the results of network pharmacology. These findings provided evidence for considering WSEE as a promising candidate for wound healing.