| Objective: The exact mechanisms of migraine are not known, but the activated trigeminovascular system (TVS) has emerged as a critical efferent component involved in migraine pathophysiology. Activated satellite cells produce and release proinflammatory cytokines associated with migraine. Reducing the production of proinflammatory cytokines of migraine becomes a research priorities. NF-κB is a transcription factor implicated in the inflammatory response. Previous evidence indicated that GTN could induce NF-κB activation in Trigeminal nucleus caudalis (TNC) at neuronal level. Our study aimed to address whether NF-κB activation in satellite cells would trigger them releasing inflammatory cytokines involved in migraine. The focus on migraine's treatment has been a topic of great interest for a longtime. Finding a drug that protects both the neurons and the glial cells may offer a new direction in migraine's research and treatment. Tetrandrine (Tet), a traditional Chinese medicine as a calcium channel blocker, inflammatory antagonist, has been used in various neurologic diseases and has been demonstrated that tetrandrine was a potent inhibitor of NF-κB activation in cells. Therefore, we hypothesized that Tet has the ability to attenuate NF-κB activation in trigeminal ganglia and reduces the production of inflammatory cytokines of satellite cells to protect migraine. We used Nitroglycerin (GTN)-induced migraine model and activation of satellite cells to study the mechanisms of Tet on NF-κB, in order to find a novel and promising candidate for future treatment or prophylaxis of migraine.Methods: 1. Neonatal Rat satellite cells of trigeminal ganglia were cultured, and alarm blue assay detected cell viability after different concentrations of GTN acts on satellite cells, then filtered that can affect the viability of the appropriate concentration of stimulating; Reverse transcription-polymerase chain reaction (RT-PCR) detected the expression of inflammatory cytokines; 2. Alarm blue assay detected cell viability after different concentrations of Tet acts on the activated satellite cells, then filtered that can affect the viability of the appropriate concentration of stimulating; The cells were cultured with 0.55 mmol/L GTN for 4 h, 6 h and 12 h, then the expression of NF-κB and IL-1βmRNA were detected by Real-time fluorescence quantitative polymerase chain reaction (FQ -PCR) to determine the highest of NF-κB mRNA in different time; 3. Cells were separated into three groups as following: 1) Group CON: normal cultured cells without treating; 2) Group GTN: the cells were cultured with 0.55 mmol/L GTN; 3) Group Tet: the cells were treated with 0.55 mmol/L GTN and 10-7 mol/L Tet respectively. The mRNA and protein level of NF-κB and IL-1βwere detected by FQ-PCR and Fluorescent Immunohistochemistry; The concentration change of intracellular Ca2+ ([Ca2+]i) in single satellite cell loaded with Fluo-3/AM was determined by laser scanning confocal microscopy; 4. The study has two parts. Rats were separated into four groups as following: 1) Group CONT (n = 16): normal animals received an intraperitoneal injection of saline; 2) Group GTN (n =16): normal animals received hypodermic injection of GTN (10 mg/kg) in the back of the neck ; 3) Group Tet (1 mg/ml)+GTN (n = 16): normal animals received an intraperitoneal injection of Tet (10 mg/kg) for thirty minutes prior to receiving an intraperitoneal injection of GTN (10 mg/kg); 4) Group Tet (5 mg/ml)+G TN (n = 16): normal animals were receiving an intraperitoneal injection of Tet (50 mg/kg) for thirty minutes prior to receiving an intraperitoneal injection of GTN (10 mg/kg). The mRNA and protein level of NF-κB and IL-1βwere detected by FQ-PCR, Western blot and Fluorescent Immunohistochemistry; The change of [Ca2+ ] in trigeminal ganglia of the four groups were detected by means of a confocal laser scanning microscope using the Ca2+ indicator Fluo-3AM.Results: 1. Satellite cells activities decreased with 0.55,1.1 and 2.2 mmol/L GTN stimulating (P<0.05), but according to the viability and modality of the cells, 0.55 mmol/L GTN was quite suited; After the GTN stimulating, the expression of IL-1βand TNF-ɑmRNA in satellite cells increased (P<0.05). 2. 10-7 mol/L Tet was the suitable prophylaxis and the level of NF-?B and IL-1βmRNA at 4 h is the highest (P<0.05). 3. In the part of the cells, Tet can inhibit the elevation of cytosolic free calcium of rat satellite cells and decrease the mRNA and protein levels of NF-?B and IL-1β(P<0.05). 4. The rats pretreated with Tet decreased significantly compared with Group GTN (P<0.05); A significant increase of the level of NF-κB and IL-1βmRNA and protein in Group GTN compared with the control group (P<0.05). However, we noticed a prominent reduction in GTN-induced activation of NF-κB and IL-1βin rats pretreated with Tet(P<0.05); The changes of [Ca2+ ] in trigeminal ganglia were identical with the effect of Tet on NF-κB.Conclusion:1. In our study, GTN-induced activation of satellite cells in trigeminal ganglia was successfully and stably constructed, and can effectively simulate migraine attacked in vitro.2. The activation of NF-κB of satellite cells in trigeminal ganglia regulated the releasing of IL-1β.3. Tet via preventing Ca2+ influxion inhibited NF-?B activation of satellite cells which decreased IL-1βexpression. |
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