藏药如意珍宝丸介导PKA-CREB信号通路对偏头痛模型大鼠PACAP、PAC1的影响

Effect of Tibetan Medicine Ruyi Zhenbao Pills Mediated PKA-CREB Signal Pathway on PACAP and PAC1 in Migraine Model Rats

目的研究藏药如意珍宝丸介导偏头痛模型大鼠下行痛觉调节通路中蛋白激酶A(Protein kinase A,PKA)-反应元件结合蛋白(CAMP response element-binding protein,CREB)变化来探讨垂体腺苷酸环化酶激活肽(Pituitary adenylate cyclase activating polypeptide,PACAP)及受体PAC1的影响,以探究其干预偏头痛的有效机制。方法SPF级SD雄性大鼠90只随机等分为6组:空白组、模型组、阳性药组(琥珀酸舒马普坦片;9.72 mg/kg)、藏药如意珍宝丸组(预防组、治疗1组、治疗2组,500 mg/kg)。藏药如意珍宝丸(治疗1组、治疗2组)、空白组及模型组灌胃等体积蒸馏水,阳性药组、藏药如意珍宝丸预防组分别灌胃琥珀酸舒马普坦片、藏药如意珍宝丸进行药物干预,连续7 d;于末次给药1 h后,除空白组大鼠颈部皮下注射生理盐水外,其余各组同部位注射硝酸甘油(Nitroglycerin,NTG,10 mg/kg)以制备偏头痛模型,治疗1组造模1 h后灌胃藏药如意珍宝丸,各组均进行标本采集。而治疗2组需连续灌胃2 d藏药如意珍宝丸后再收集样本。酶联免疫吸附测定(Enzyme-linked immunosorb-ent assay,ELISA)法检测血清中PACAP和PAC1的蛋白含量,应用实时荧光定量聚合酶链反应(Real-time quantitative polymerase chain reaction,RT-q PCR)检测PACAP、AC、PAC1、CREB、PKA m RNA表达变化。结果与正常组比较,模型组大鼠血清PACAP、PAC1的蛋白表达显著升高(P<0.05)。与模型组比较,阳性药组PAC1蛋白表达明显降低(P<0.05),而PACAP含量仅呈下调趋势;藏药如意珍宝丸(预防组、治疗1组)PACAP蛋白水平差异性减少,且预防组PAC1蛋白表达显著下降(P<0.05,P<0.01)。与正常组比较,模型组大鼠中脑PACAP、AC、PAC1、CREB、PKA基因表达含量均明显上调(P<0.01),提示造模成功。与模型组比较,阳性药组、藏药如意珍宝丸(预防组、治疗1组、治疗2组)CREB、PKA基因表达变化均不同程度地下调,而PACAP、AC基因表达差异性降低见于藏药如意珍宝丸(预防组、治疗2组),PAK基因水平变化明显减少见于阳性药组、藏药如意珍宝丸预防组(P<0.05,P<0.01)。结论藏药如意珍宝丸可通过调节中脑内PKA-CREB信号通路以介导PACAP、PAC1含量变化来缓解偏头痛的发作。

Objective To study the changes of protein kinase A(PKA)-reactive element binding protein(CREB)in the descending pain regulation pathway of migraine model rats mediated by Tibetan medicine Ruyi Zhenbao Pills,and to explore the effect of pituitary adenylate cyclase activating polypeptide(PACAP)and receptor PAC1,in order to explore its effective mechanism of intervention in migraine.Method 90 SPF SD male rats were randomly and equally divided into 6 groups:blank group,model group,positive group(sumatriptan succinate tablets;9.72 mg/kg),Tibetan medicine Ruyi Zhenbao Pill group(prevention group,treatment group 1,treatment group 2,500 mg/kg).Tibetan medicine Ruyi Zhenbao Pill(treatment group 1,treatment group 2),blank group and model group were given distilled water of the same volume by gavage,positive control group and Tibetan medicine Ruyi Zhenbao Pill prevention group were given sumatriptan succinate tablets and Tibetan medicine ruyi zhenbao pills by gavage respectively for 7 consecutive days;One hour after the last administration,Nitroglycerin(nitroglycerin,NTG,10 mg/kg)was injected into the same part of the other groups except the normal saline subcutaneous injection into the neck of the rats in the blank group to prepare migraine model.The Tibetan medicine Ruyi Zhenbao Pills were injected into the stomach 1 hour after the model was established in the treatment group,and specimens were collected in each group.However,in the treatment group 2,the Tibetan medicine Ruyi Zhenbao Pills should be continuously administrated for 2 d before samples are collected.Enzyme-linked immunosorbent assay(ELISA)was used to detect the protein content of PACAP and PAC1 in serum.Real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect the expression levels of PACAP,AC,PAC1,CREB and PKA protein and mRNA.Results Compared with the normal group,the protein expressions of PACAP and PAC1 in serum of rats in the model group were significantly increased(P<0.05).Compared with the model group,the expression of PAC1 protein in the positive control group was significantly decreased(P<0.05),while the PACAP content only showed a downward trend.The difference of PACAP protein level in Tibetan medicine Ruyi Zhenbao Pill(prevention group and treatment group 1)was reduced,and PAC1 protein expression in prevention group was significantly decreased(P<0.05,P<0.01).Compared with the normal group,the expression levels of PACAP,AC,PAC1,CREB and PKA genes in the midbrain of the model group were significantly up-regulated(P<0.01),suggesting that the model was successfully established.Compared with model group,CREB and PKA gene expression changes in positive control group,Tibetan medicine Ruyi Zhenbao Pill(prevention group,treatment group 1 and treatment group 2)are all down-regulated to varying degrees,while PACAP and AC gene expression differences are reduced in Tibetan medicine Ruyi Zhenbao Pill(prevention group and treatment group 2),and PAK gene level changes are significantly reduced in positive control group and Tibetan medicine Ruyi Zhenbao Pill prevention group(P<0.05,P<0.01).Conclusion Tibetan medicine Ruyi Zhenbao Pills can alleviate migraine attacks by regulating PKA-CREB signaling pathway in midbrain to modulate changes in PACAP and PAC1 content.