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The Effect and Mechanism of Tetrandrine on the Satellite Cells of Trigeminal Ganglia in A Rat Model of Migraine

Author: CuiZhiWei
Tutor: ZengZhaoFang
School: Chongqing Medical University
Course: Clinical Laboratory Science
Keywords: Tetrandrine Migraine Satellite glial cells Ca2 NF-κB
CLC: R285.5
Type: Master's thesis
Year: 2011
Downloads: 34
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Abstract

Objective: The exact pathogenesis of migraine is not yet clear, but the activation of the trigeminal nerve system and the consequent release of neurochemicals still play a leading role in its mechanism. In recent years about the activation of satellite glial cells secrete the release of inflammatory factors influence the development of migraine is widely recognized, therefore, has become a hot research satellite glial cells in migraine. Nuclear transcription factors (nuclear factor-κB, NF-κB) play a central role in the inflammatory response, and its target gene in the brain vascular endothelial cells, nerve cells and glial cells. The study found that glyceryl trinitrate (nitroglycerin, GTN) induced NF-κB activation in rat trigeminal caudate nucleus neurons migraine, with the result that the transcriptional activation of cytokines, promote the inflammatory response when a migraine attack. Therefore, we speculate that the activation of NF-κB is also essential for satellite glial cells secrete the release of inflammatory cytokines. A long time for the treatment of migraine has been hot people to explore, looking for a side effect low for the the multiple pathogenesis of migraine are effective drugs become the primary task for the treatment of migraine. In powder tetrandrine (Tetrandrine,, referred to as Tet) is extracted from the root of Menispermaceae plant tetrandrine double-benzyl isoquinoline alkaloids, is a natural calcium channel blockers, and anti-inflammatory analgesic effect. Therefore, it is predicted that Tet may reduce the trigeminal ganglion (trigeminal ganglia, referred to as TG) inflammatory cytokine production, play a protective role in migraine. The topics use of GTN trigeminal ganglion satellite glial cell activation model and migraine model as an entry point to the role of NF-κB from regulating cytokines, research that Tet is how regulating the expression of NF-κB to migraine The clinical treatment to find an effective neuroprotective agent. Method: 1. Vitro purified and cultured 1-3 day Sprague-Dawley (SD) of rat trigeminal parts satellite glial cells with different concentrations of GTN role of glial cells in the satellite, the application Alarm blue detect cell survival, screening the most appropriate activated cell GTN concentration; cells using RT-PCR detection after activation of inflammatory factors generated; application of different concentrations of Tet role in the activation of the satellite glial cells, application Alarm blue detect cell survival, filter out the most excellent Tet therapeutic concentration. Optimum use of screening out 0.55 mmol / L GTN were acting on the the satellite glial cell 4, 6, and 12 h after application FQ-PCR detection of NF-κB and expression of inflammatory cytokines of IL-1βmRNA of NF-determine which point in time κB and express IL-1βmRNA, the strongest; 3 cells were divided into normal control group (CON group): normal cultured satellite glial cells, without any treatment; GTN group: 0.55 mmol / L GTN and satellite glial cells co-cultured 4 h; of Tet treatment groups the (Tet GTN group) :10-7 mol / L Tet and 0.55 mmol / L GTN ratio of 1:1 were added to the satellite glial cells co-cultured 4 h. Application FQ-PCR and immunofluorescence detection Tet from the mRNA and protein levels of GTN satellite glial cells induced activation of NF-κB and IL-1β expression. Detection satellite glial cells Fluo-3/AM probe load after calcium concentration changes in explore the Tet molecular mechanisms regulating inflammatory factors change; animals were divided into normal control group: normal rat abdominal injection of saline; the GTN group: the back of the neck of the normal rats injected GTN (10 mg / kg); high concentrations of the Tet GTN group: normal rats the abdominal injection Tet (50 mg / kg), injected 30-minute neck back GTN (10 mg / kg); low concentrations of Tet GTN group: normal rat abdominal injection Tet (10 mg / kg), 30-minute neck back injection GTN (10 mg / kg). The observed changes in the behavior of rats. Application FQ-PCR, immunofluorescence and Western blot detected from both the mRNA and protein levels of NF-κB and IL-1β in the trigeminal ganglion in each experimental group. Detection of intracellular calcium concentration in rat trigeminal ganglion probe load Fluo-3/AM, and to explore the molecular mechanisms of Tet regulating inflammatory factors change overall. Results: 1. Nitroglycerin stimulate optimal concentration screening: Alarm blue to detect cell survival, which 0.55,1.1 and 2.2 mmol / L after three concentrations of nitroglycerin stimulate astrocytes activity was significantly lower (P lt; 0.05 ), but the observed cell motility and morphology optimum concentration of 0.55 mmol / L nitroglycerin; RT-PCR results: 0.55 mmol / L nitroglycerin stimulate cells, IL-1β and TNF-αmRNA expression increased (P lt ; 0.05). Alarm blue screened Tet, the most appropriate therapeutic concentration of 10-7 mol / L; 0.55 mmol / L GTN with cells cultured for 4 h, 6 h, 12 h after 4 h detected by FQ-PCR NF-κB and express IL-1βmRNA, the highest (P lt; 0.05). 3. Cells Experimental results show that: found by FQ-PCR and immunofluorescence assay, GTN-induced activation of NF-κB in satellite glial cells, increased intracellular NF-κB and the expression of IL-1β (P lt; 0.05); Tet treatment group, NF-κB and the expression of IL-1β in cells decreased (P lt; 0.05). Tet can inhibit within GTN satellite glial cells induced [Ca ^{2 ] i increased (P lt; 0.05). 4 Animal experiments showed: Tet after treatment, the model group migraine significantly reduce the number of rats scratching their heads (P lt; 0.05); FQ-PCR, immunofluorescence and Western blot showed that NF-κB: the GTN group trigeminal ganglia within The expression of IL-1β and normal control group was significantly higher (P lt; 0.05), while the Tet the treatment group reduce migraine rat trigeminal ganglion NF-κB and the expression of IL-1β (P lt; 0.05). The expression of calcium in each experimental group is basically consistent with the expression of NF-κB and IL-1β. The the trigeminal ganglion inflammation model conclusions: 1. Experiment using the the GTN induced trigeminal ganglion satellite glial cell activation established stable, successful, can be easily and effectively simulation vitro migraine attack inflammation. 2. NF-κB activation in the trigeminal ganglion satellite glial cells regulate the release of inflammatory cytokines IL-1β. 3. Tet by blocking calcium in astrocytes flow regulation of the expression of NF-κB, thereby reducing the release of inflammatory cytokines IL-1β.}

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