Genistein Modifies Glutamate-evoked Masticatory Muscle Pain in Ovariectomized Rats

Objectives: To investigate whether 17β-estradiol (E2) affected glutamate-evoked masseter muscle pain in ovariectomized rats and whether genistein could modify this effect.
Methods: In the first part, 24 ovariectomized rats were subcutaneously injected with E2 respectively at dosages of 0μg, 20μg, 80μg, or 200μg for 10 days. All rats received glutamate injections at masseter muscles on the last day. In the second part, 48 ovariectomized rats were divided into 8 groups (n=6): no drug treatment group, genistein vehicle/E2 vehicle group, genistein vehicle/80μg E2 group, and 2, 7.5, 15, 30, or 60mg/kg genistein/80μg E2 groups. Genistein was injected subcutaneously once a day for 12 days and E2 administration started two days after genistein. At the final day, all rats received glutamate injections. Head withdrawal thresholds of masseters were measured on both sides of rats using a modified electronic von-Frey anesthesiometer. Expression ratios of tyrosine phosphorylation of NR2B subunits of NMDA receptor (pNR2B) and phosphorylation of ERK1/2 (pERK1/2) in hippocampal CA3 region were detected by immunofluorescence. Western blotting was used to examine the protein level of pNR2B and pERK1/2 in hippocampus. Differences between groups were examined by two-way ANOVA.
Results: Application of 1M glutamate 40μl induced a mechanical hyperalgesia in awake rats. The head withdrawal threshold was decreased with increasing dosage of estrogen（P<0.05）and increased at 7.5 and 15mg/kg dosages of genistein pretreatment (P<0.05), however low and high dosages of genistein were not analgesic. pNR2B and pERK1/2 expression were upregulated in hippocampus by glutamate injection and further potentiated by E2 (P<0.05). The 15mg/kg genistein pretreatment attenuated E2 enhancing glutamate-evoked upregulation of pNR2B and pERK1/2 (P<0.05) in hippocampus.
Conclusions: Our study suggests that E2 aggravates glutamate-evoked masticatory muscle pain and a certain dosage of genistein can attenuate this effect probably through modulating the expression of pNR2B and pERK1/2 in rat hippocampus.