Unraveling the anti-primary dysmenorrhea mechanism of Ainsliaea fragrans Champ. extract by the integrative approach of network pharmacology and experimental verification

The plant Ainsliaea fragrans Champ. (A. fragrans) named "Xingxiang Tuerfeng", is a traditional herb with a long history of therapeutic practice in southern China in the treatment of gynecological diseases. The anti-inflammatory extract of Ainsliaea fragrans Champ. (AF-ext) exhibited anti-primary dysmenorrhea (PD) activity in oxytocin-induced mice. This study aimed to unravel the underlying mechanisms of AF-ext on PD by the integrative approach of network pharmacology and experimental verification. First, the therapeutic targets of AF-ext are predicted using network pharmacology and molecular docking methods. Second, activity screening and immunoblotting methods were used for target validation. Then, the therapeutic effect of AF-ext on PD was evaluated using oxytocin-induced mice and uterine strips model. AF-p1, and AF-p2, the active ingredients of AF-ext, showed inhibitory effects on COX1/2 and EGFR, and all five active components showed antagonistic activity on TRPV1. AF-ext (25, 50, 100 mg/kg) could significantly reduce the number of writhing times and prolong writhing latencies in a dose-dependent manner. AF-ext inhibited spasmolytic activity in uterine strips induced by oxytocin and Ca2+ stimulation. AF-ext inhibited NF-κB/COX-2/PG pathway and activation of the NLRP3 inflammasome in PD mice. It significantly downregulated the PD-induced overexpression of p-p65/p65, p-IκBα, and COX-2 by inhibiting the NF-κB pathway. Moreover, the overexpression of NLRP3, p20/pro-Caspase 1, and p17/pro-IL-1β was greatly downregulated. AF-ext demonstrated anti-inflammatory, analgesic, and spasmolytic activity in the treatment of PD. It inhibited the NF-κB/COX-2/PG pathway and NLRP3 inflammasome activation in PD mice with a multi-target approach.