为探究姜黄素(CUR)对慢性偏头痛(CM)大鼠的治疗作用,并研究其作用机制.随机将SD大鼠分为6组:对照组、模型组、CUR低、中、高剂量组、H-89组.采用硝酸甘油(NTG)腹腔注射构建CM大鼠模型,在造模第1、3、5、7、9天对大鼠进行疼痛阈值检测、行为学测试.分离大鼠神经元细胞,筛选CUR最佳浓度,将细胞分为NC组、NTG组、CUR低剂量组、CUR中剂量组、CUR高剂量组、H-89组,CCK-8法检测细胞活力,流式细胞术检测细胞凋亡;ELISA检测三叉神经脊束尾侧核(TNC)组织和神经元细胞环磷酸腺苷(cAMP)、白细胞介素1β(IL-1β)、肿瘤坏死因子-α(TNF-α)含量,Western Blot检测TNC组织和神经元细胞中蛋白激酶A(PKA)、p-PKA、cAMP反应元件结合蛋白(CREB)、p-CREB蛋白表达.结果显示,与对照组相比,模型组大鼠眶周机械痛阈、足底机械痛阈、热缩足潜伏期、cAMP含量、p-PKA/PKA、p-CREB/CREB蛋白表达显著降低,IL-1β、TNF-α、挠头次数、爬笼次数显著升高(P＜0.05);与模型组相比,CUR低、中、高剂量组大鼠眶周机械痛阈、足底机械痛阈、热缩足潜伏期、cAMP含量、p-PKA/PKA、p-CREB/CREB蛋白表达显著升高,IL-1β、TNF-α、挠头次数、爬笼次数显著降低(P＜0.05);在NTG诱导神经元细胞中,与0 μmol/L CUR 比较,5、10、20、40 μmol/L CUR显著提高细胞活力(P＜0.05),选用 5、10、20 μmol/L浓度CUR做后续实验;与NC组相比,NTG组细胞活力、cAMP含量、p-PKA/PKA、p-CREB/CREB蛋白表达水平明显降低,IL-1β、TNF-α、凋亡率(％)明显增加(P＜0.05);相比于NTG组,CUR低、中、高剂量组IL-1β、TNF-α、凋亡率明显降低(P＜0.05),细胞活力、cAMP含量、p-PKA/PKA、p-CREB/CREB蛋白表达水平明显增加.PKA抑制剂H-89能逆转CUR对CM大鼠的治疗作用(P＜0.05).表明CUR能缓解CM大鼠症状,降低炎症反应,减少神经元细胞凋亡,其作用机制可能与cAMP/PKA/CREB信号通路的激活有关.
The Therapeutic Effect of Curcumin on Chronic Migraine Rats by Regulating cAMP/PKA/CREB Signaling Pathway
In order to explore the therapeutic effect of curcumin(CUR)on chronic migraine(CM)rats and its mechanism.In animal experiments,SD rats were randomly grouped into 6 groups:control group,model group,CUR low dose group,CUR medium dose group,CUR high dose group,and H-89 group.CM rat models were constructed by intraperitoneal injection of nitroglycerin(NTG),and pain threshold and behavioral tests were performed on the 1st,3rd,5th,7th,and 9th days of modeling.Rat neuronal cells were isolated,the optimal concentration of CUR was screened,and the cells were divided into NC group,NTG group,CUR low dose group,CUR medium dose group,CUR high dose group,and H-89 group,CCK-8 method was applied to detect cell viability,flow cytometry was applied to detect cell apoptosis;ELISA was applied to detect the contents of cyclic adenosine monophosphate(cAMP),interleukin(IL)-1β and tumor necrosis factor-α(TNF-α)in the tissue and neuron cells of the caudal nucleus of the trigeminal neural crest tract(TNC),Western Blot was applied to detect protein kinase A(PKA),p-PKA,cAMP responsive element binding protein(CREB),and p-CREB protein expression in TNC tissue and neuronal cells.The results showed that compared with the control group,the periorbital mechanical pain threshold,plantar mechanical pain threshold,thermal contraction latency,cAMP content,p-PKA/PKA,and p-CREB/CREB protein expression in rats in the model group obviously reduced,the IL-1β,TNF-α,head bending frequency,and cage climbing frequency obviously increased(P＜0.05);compared with the model group,the periorbital mechanical pain threshold,plantar mechanical pain threshold,thermal contraction latency,cAMP content,p-PKA/PKA,and p-CREB/CREB protein expression in rats in the CUR low,medium,and high dose groups obviously increased,the IL-1β,TNF-α,head bending frequency,and cage climbing frequency obviously reduced(P＜0.05);in NTG induced neuronal cells,compared with 0 μmol/L CUR,5,10,20 and 40 μmol/L CUR obviously increased cell viability(P＜0.05),5,10,and 20 μmol/L CUR concentrations were selected for subsequent experiments;compared with the NC group,the cell viability,cAMP content,p-PKA/PKA,and p-CREB/CREB protein expression levels in the NTG group obviously decreased,IL-1β,TNF-α,and apoptosis rate obviously increased(P＜0.05);compared with the NTG group,IL-1β,TNF-α,and apoptosis rate in the CUR low,medium,and high dose groups obviously decreased(P＜0.05),the cell viability,cAMP content,p-PKA/PKA,and p-CREB/CREB protein expression levels obviously increased.PKA inhibitor H-89 was able to reverse the therapeutic effect of CUR on CM rats(P＜0.05).The results demostrated CUR can alleviate symptoms,reduce inflammatory response,and reduce neuronal apoptosis in CM rats,and its mechanism of action may be related to the activation of the cAMP/PKA/CREB signaling pathway.
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