BACKGROUNDTemporomandibular disorder (TMD) pain is more prevalent in females than in males, with high estrogen levels potentially being a risk factor. Research indicates that 17β-estradiol (E2) exacerbates experimental occlusal interference (EOI)-induced orofacial hyperalgesia, which can be reversed by genistein. This study aimed to explore the central mechanisms within the spinal trigeminal nucleus (Sp5) related to the pain-exacerbating effect of E2 and the antiestrogenic properties of genistein in a model of EOI-induced chronic masseter pain.METHODSFemale rats underwent ovariectomy (OVX), followed by pretreatment with genistein or genistin (a control drug for genistein that does not inhibit protein tyrosine kinases (PTKs)), E2 replacement, and EOI application. The head withdrawal thresholds (HWTs) of the bilateral masseters were measured to evaluate pain sensitivity. Expression levels of p-ERK and two PTKs (Yes-associated protein, YAP; Src kinase, Src) in bilateral Sp5 were assessed through immunofluorescent staining and/or Western blotting. The ERK inhibitor PD98059 or vehicle was administered via intrathecal injection (i.t.) to inhibit the ERK1/2 signaling pathway.RESULTSE2 intensified EOI-induced masseter mechanical hyperalgesia in OVX rats, and upregulated the phosphorylation of ERK1/2 in bilateral Sp5. Blocking phosphorylation of ERK1/2 in Sp5 reversed the exacerbating effect of E2. Genistein partially reversed the masseter hyperalgesia induced by E2 combined with EOI, possibly through the inhibition of PTKs and p-ERK1/2 upregulation in bilateral Sp5.CONCLUSIONGenistein alleviates the pain-exacerbating effect of E2 on EOI-induced chronic mechanical hyperalgesia by inhibiting YAP and Src tyrosine kinases as well as the downstream ERK1/2 signaling pathway in Sp5.