Regulation of Calcitonin Gene-Related Peptide by a Methanol Extract of Theobroma Cacao

Release of calcitonin gene-related peptide (CGRP) from trigeminal sensory nerves contributes to neurogenic inflammation in migraine and temporomandibular joint (TMJ) disorders. Cocoa flavonoids inhibit pathophysiological mechanisms associated with inflammation. To determine whether cocoa bean extracts and the flavonoid catechin could inhibit CGRP gene expression, primary cultures and a human cell line were utilized. Treatment of primary rat trigeminal ganglia cultures with a depolarizing stimulus (KC1) or capsaicin caused a significant increase in CGRP release as determined by radioimmunoassay. The stimulatory effects were repressed by pretreatment for 30 min with a concentrated methanol extract of cocoa beans. Surprisingly, treatment with catechin, which is abundant in cocoa beans, did not repress stimulated CGRP release. Next, the effect of cocoa extract on stimulated human CGRP promoter activity and signaling pathway reporter genes were studied in a human neuronal-like cell line, DMS 153 cells, using transient transfection and luciferase activity measured. Overnight treatment with cocoa extract repressed basal human CGRP promoter activity and activity stimulated by MEK1 and PK4. Cocoa also inhibited basal levels of c-Jun, CHOP, ATF2, and NF-B, as well as PKA-stimulated CREB, MEK1-stimulated Elk1, and MEKK- stimulated NF-B reporter activity. In conclusion, these data provide evidence that cocoa extract can inhibit stimulated CGRP synthesis and secretion from trigeminal neurons, and repress MAP kinase and NF-B pathways. Additionally, this study validates the use of primary trigeminal neuron cultures and the human cell-line DMS 153 as cellular models for screening plant-based compounds for inhibitory effects on CGRP gene expression