The Neuroprotective And Anxiolytic Effects Of Sesamin And The Underlying Mechanisms

ObjectiveSesamin is one of major lignans found in sesame seeds and oil. Sesamin is recognized to have various pharmacological effects, such as anti-hypertension, hypocholesterolemia, and protective effects against oxidative stress as well as anti-nociception and anti-inflammation. However, less is known about the effects of Sesamin on neuronal injury induced by NMDA exposure and the development of anxiety in animals with chronic pain. The present study was designed to investigate the neuroprotective effects of sesamin against NMDA-induced neurotoxicity and the anxiolytic effects of sesamin in animals with chronic pain, and clarify the underlying mechanisms. Methods 1. MCAO modelA MCAO model of mouse was established to evaluate the neuroprotective effect of sesamin in vivo.2. Neurological scoring, infarct size and Nissl staining measurementTo assess the protective effect of sesamin on neuron and brain injury induced by MCAO, the neurobehavioral evaluation, infarct volume assessment, and Nissl staining were performed at 24 h after reperfusion. 3. Primary cortical neuronal culture and NMDA-induced exitotoxicity in vitro200μM NMDA or/and 0.1μM Sesamin were added into primary cultured cortical neurons to evaluate the neuroprotection of sesamin against NMDA-induced exitotoxicity in vitro. 4. MTT assay and Hoechst/PI double stainingMTT assay and hoechst/PI double staining were employed to detect the percentage of apoptosis and necrosis after the primary cultured cortical neurons were treated with 200μM NMDA or/and 0.1μM Sesamin. 5. Calcium imagingThe fluorescence ratio of the fluo-3/AM loaded neurons was measured by calcium imaging. Analyse the effect of Sesamin on Ca2+ elimination through the change of fluorescence ratio. 6. Expression of related proteins by Western blotThe expression of Bcl-2, Bax, and pro-caspase-3 were examed by Western blot. The underlying mechanism was analysed through the results. 7. Mouse model of chronic inflammatory painMouse model of chronic inflammatory pain was induced by an injection of 10μl of 50% complete Freund’s adjuvant(CFA) into the plantar surface of the left hind paws of mice. The same volume of saline 0.9% was injected into the plantar surface of the left hind paws of control animals. 8. Measurement of mechanical threshold and thermal thresholdThe mechanical hypersensitivity threshold was evaluated by using a set of von Frey filaments(0.008–2g) with the up-down method. Thermal threshold of mice was measured by hot plate experiment. The effect of Sesamin on reducing pain threshold was analysed after the chronic infrlmmatory pain. 9. Measurement of paw edemaA dial thickness gauge was used to measure the ipsilateral paw thickness in the mice. The anti-inflammatory effect of Sesamin was assessed by the results. 10. Elevated plus-maze and open field testThe number of entries in opened and closed arms as well as time spent in each arm were recorded by a camera in elevated plus maze(EPM). The distance travelled and time in the center areas of mice were recorded by a camera in open field. Analyse the anxiolytic effect of Sesamin in mice with chronic inflammatory pain. 11. Western Blot AnalysisThe expressions of GABAA-α2 and glutamate receptors were detected by Western blot in the amygdala of mice. Analyse the underlying anxiolytic mechanism of Sesamin in mice with chronic inflammatory pain.Results 1. Pretreatment of sesamin decreased ischemia-reperfusion injury in vivoThe neuroprotective effect of sesamin against ischemia-reperfusion injury was evaluated via neurological scoring, infarct size measurement, and Nissl staining. Sesamin(43.2 mg/kg) significantly decreased the infarct volume and neurological deficit score as compared with those of MCAO group. The Nissl staining showed that neuronal damage was obvious in the hippocampus CA1 region and prefrontal cortex after MCAO. Sesamin(43.2 mg/kg) markedly rescued the damaged neurons in the hippocampus and prefrontal cortex. 2. Pretreatment of sesamin protected neurons against NMDA-induced cell deathExposure to NMDA(200 μM) for 60 min markedly induced cell injury. Treatment with sesamin(0.1-1 μM) significantly protected neurons against NMDA injury. Sesamin(0.1 μM) alone did not affect cell viability. NMDA caused a significant amount of LDH release, and sesamin(0.1 μM) completely protected the cells from cell damage. The results of Hoechst 33258 and PI double-staining suggested 0.1 μM sesamin markedly attenuated apoptosis or death induced by NMDA exposure. 3. Sesamin reduced the NMDA-evoked Ca2+ increase in cortical neuronsNMDA(200 μM) treatment produced a persistent elevation of Ca2+ level in cultured neurons. However, treatment with sesamin(0.1 μM) reduced the NMDA-evoked increase of Ca2+ concentration as shown by decreased fluorescence intensity. 4. Pretreatment of sesamin affected expression of related proteinsNMDA exposure down-regulated the Bcl-2 expression, up-regulated the Bax expression, and thus increased the Bax/Bcl-2 ratio. However, 0.1 μM sesamin showed anti-apoptotic activity with increasing Bcl-2 expression, decreasing Bax expression, and decreasing Bax/Bcl-2 ratio. Cultures treated with NMDA produced a sustained cleavage of pro-caspase-3, which resulted in the decrease of pro-caspase-3. Sesamin(0.1 μM) significantly increased the levels of pro-caspase-3. 5. Effect of sesamin on CFA-induced mechanical hypersensitivity and thermal hypersensitivity in miceMechanical allodynia and thermal hyperalgesia were examined during the experimental period. Sesamin(80 mg/kg) did not reduce mechanical allodynia and thermal hyperalgesia. The paw withdrawal threshold or latency was not significantly in the changed in the contralateral hindpaw. 6. Effect of sesamin on CFA-induced long-term paw edema in miceA single treatment with CFA remarkably increased the thickness of ipsilateral hind paw. Sesamin did not reduce chronic inflammation, as shown by the unchanged edema in CFA-injected hindpaws. 7. Sesamin relieved anxiety-like behaviors following CFA-induced chronic inflammatory painMice showed a significant decrease in percentage of time spent and number of entries in open arms in the elevated-plus maze test and a significant decrease in the percentage of time spent in central squares in the open-field test on day 21 after CFA injection. Treatment of Sesamin(80 mg/kg) for 1 week notably increased the percentage of time spent and number of entries in open arms and the time spent in central squares. The number of entries in closed arms in the elevated-plus maze test and the total distance traveled in the open-field test were comparable among these groups, suggesting that the there was no deficit of locomotor activities by CFA injection or SE administration. 8. Sesamin reverses down-regulation of GABAA receptor in the amygdala of mice with inflammatory painThe levels of GABAA-α2 receptor was notably decreased in BLA at 21 days after CFA-injection. However, no significant difference was observed in the levels of GAD67(glutamate decarboxylase). Treatment with sesamin significantly reversed the down-regulation of GABAA-α2 expression in the BLA of CFA injected mice, but had no effects on the GAD67 expression. 9. Sesamin reverses up-regulation of glutamate receptor expression in the amygdala of CFA-injected miceThe levels of Glu R1 and phosphorylation of Glu R1 at Ser831(p-Glu R1-Ser831) significantly increased after CFA-injection. However, the CFA-injection had a different effect on the levels of NMDAR subtypes in the amygdala. There is a remarkably increased expression of NR2B-containing NMDARs while no difference of NR2A-containing NMDARs, suggesting the different roles of NMDAR subtypes in BLA circuits. Another critical excitatory synaptic protein Ca2+ /calmodulin-dependent protein kinase II-α(Ca MKII-α) also significantly increased after CFA injection. Treatment with sesamin(80 mg/kg, for 7 days) significantly reversed the alteration of above proteins in the BLA. ConclusionOur findings suggested that sesamin protected neurons against excitotoxicity through reversing down-regulation of Bcl-2 and pro-caspase-3 expression and up-regulation of Bax expression and relieving the calcium overload in cultured neurons after NMDA exposure. Sesamin has the ability to reduce chronic pain-induced anxiety-like behaviors at least partially through maintaining the balance between GABAergic and glutamatergic transmission in the amygdala of mice.