Paeoniae Radix Alba-Chuanxiong Rhizoma herbal pair alleviates trigeminal nucleus caudalis neuroinflammation in chronic migraine associated with P2Y12R/PPARγ-related NF-κB signaling.

Neuroinflammation in the trigeminal nucleus caudalis (TNC) plays an important role in the pathological process of chronic migraine (CM). The Paeoniae Radix Alba (Baishao, BS)-Chuanxiong Rhizoma (Chuanxiong, CX) herb pair (BSCX) is widely used in the treatment of migraine, but its mechanism of action and representative candidate constituents remain unclear. This study aimed to evaluate the effects of BSCX on TNC neuroinflammation and to explore its representative candidate constituents and potential mechanisms associated with microglial inflammatory phenotype-related changes and P2Y12R/PPARγ-related NF-κB signaling. A nitroglycerin-induced rat model of CM was used to evaluate the anti-migraine effects of BSCX through behavioral testing and molecular analyses. ELISA, RT-qPCR, immunofluorescence staining, and Western blot were used to evaluate changes in inflammatory factors, pro-/anti-inflammatory phenotype-related markers, and pathway-related proteins in blood samples and the TNC. Potential candidate constituents in the TNC were identified using ultrahigh-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS). The associations between the candidate constituents and their putative targets were further investigated by integrating molecular docking, molecular dynamics simulation, drug affinity responsive target stability (DARTS) assays, and cellular thermal shift assays (CETSA). A lipopolysaccharide (LPS)-induced BV2 cell model was established, with additional interventions using the P2Y12R agonist 2-MeS-ADP and the PPARγ inhibitor GW9662. Flow cytometry was used to assess CD86/CD206-positive cells and phagocytic activity, while immunofluorescence staining and Western blot were used to evaluate related protein expression and signaling changes. BSCX alleviated NTG-induced migraine-like behaviors and pain sensitization. Compared with the model group, the medium- and high-dose BSCX groups showed reduced spontaneous head-scratching episodes, increased periorbital mechanical withdrawal threshold, and prolonged thermal withdrawal latency. BSCX reduced the expression of CGRP and c-Fos in the TNC by 16.2%-21.6% and 19.6%-55.1%, respectively. BSCX also reduced serum TNF-α and IL-1β levels while increasing IL-10 and TGF-β levels. Immunofluorescence analysis of the TNC further showed that BSCX decreased the proportion of iNOS+/Iba1+ cells by 49.8%-86.2% and increased the proportion of Arg-1+/Iba1+ cells by 222%-271%. These changes were accompanied by reduced expression of P2Y12R/RhoA/ROCK2/NF-κB-related proteins and increased PPARγ expression. Among the constituents detected in the TNC, benzoylpaeoniflorin (Ben) and senkyunolide I (SENI) reduced LPS-induced TNF-α release by 13.7% and 21.6%, respectively, at 2.5 μM, and increased IL-10 release by 18.1% and 21.0%, respectively, at 5 μM. Molecular docking showed favorable binding energies for the P2Y12R-Ben and PPARγ-SENI complexes. Molecular dynamics simulations further showed that their binding free energies were -23.31 and -22.06 kcal/mol, respectively, which were more favorable than those of the corresponding cross-combinations. In DARTS and CETSA assays, Ben enhanced the protease resistance and thermal stability of P2Y12R, while SENI enhanced the protease resistance and thermal stability of PPARγ. In reversal experiments, 2-MeS-ADP increased the phagocytic activity and P2Y12R expression relative to the Ben group by 40.6% and 76.8%, respectively; GW9662 increased phagocytic activity by 41.7% and decreased PPARγ expression by 26.8% relative to the SENI group. BSCX alleviates TNC neuroinflammation in CM, accompanied by regulation of pro-/anti-inflammatory phenotype-related markers and changes in P2Y12R/RhoA/ROCK2/NF-κB signaling and PPARγ-related signaling. Ben and SENI may represent candidate constituents associated with the P2Y12R- and PPARγ-related signaling branches, respectively, and may partly contribute to the pharmacological effects of the BSCX herb pair against CM.