基于p38 MAPK/iNOS信号通路探讨加味散偏汤对硝酸甘油诱导的大鼠偏头痛作用机制  

Modified Sanpiantang Treats Nitroglycerin-induced Migraine in Rats via p38 MAPK/iNOS Signaling Pathway

作　　者：汪坤 马鸣 杨艳华 任权娜 陈雨晗 岳孟茹 赵旭 WANG Kun;MA Ming;YANG Yanhua;REN Quanna;CHEN Yuhan;YUE Mengru;ZHAO Xu(Henan Provincial Hospital of Traditional Chinese Medicine/The Second Affiliated Hospital of Henan University of Chinese Medicine,Zhengzhou 450002,China;School of Pharmacy,Henan University of Chinese Medicine,Zhengzhou 450000,China)

机构地区：[1]河南省中医院/河南中医药大学第二附属医院,郑州450002 [2]河南中医药大学药学院,郑州450000

出　　处：《中国实验方剂学杂志》2023年第22期64-70,共7页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：河南省重点研发与推广专项(科技攻关)(212102311114);中央本级重大增减支项目(2060302);河南省中医药科学研究专项(2022ZY1093);河南省中医药科学研究重大专项(20-21ZYZD08);河南省中医药拔尖人才培养项目(豫卫中医函[2021]15号)。

摘　　要：目的:探讨加味散偏汤对硝酸甘油诱导的大鼠偏头痛的作用机制。方法:将72只SPF级Wistar大鼠随机分为空白组、模型组(硝酸甘油,10 mg·kg^(-1))、利扎曲坦组(0.89 mg·kg^(-1))、加味散偏汤高、中、低剂量组(12.96、6.48、3.24 g·kg^(-1))。腹腔注射硝酸甘油建立大鼠偏头痛模型。测试大鼠眼眶和足底痛觉敏感[机械痛阈值(MPT)]行为学实验;酶联免疫吸附测定法(ELISA)检测大鼠血清一氧化氮(NO)、肿瘤坏死因子-α(TNF-α)、γ干扰素(IFN-γ)和白细胞介素-1β(IL-1β)等炎症因子水平;免疫组化法(IHC)检测大鼠三叉神经脊束核尾核(TNC)诱导性一氧化氮合酶(iNOS)的表达;蛋白免疫印迹法(Western blot)检测大鼠TNC区磷酸化p38丝裂原活化蛋白激酶(p-p38 MAPK)/p38丝裂原活化蛋白激酶(p38 MAPK)和iNOS蛋白表达;实时荧光定量聚合酶链式反应(Real-time PCR)检测大鼠TNC区iNOS、p38 MAPK和IL-1βmRNA表达。结果:与空白组比较,模型组大鼠机械痛阈值显著降低(P<0.01);血清NO、TNF-α、IFN-γ和IL-1β炎症因子水平显著升高(P<0.01),TNC区脑组织p38 MAPK、iNOS和IL-1β等炎症因子表达明显升高(P<0.05,P<0.01)。与模型组比较,各给药组大鼠MPT水平均明显提升(P<0.05,P<0.01),且加味散偏汤中剂量组效果最为显著(P<0.01)。与模型组比较,经加味散偏汤治疗后,大鼠TNC区组织iNOS、p38 MAPK和IL-1βmRNA及p-p38 MAPK、iNOS蛋白表达水平明显降低(P<0.05,P<0.01),血清中NO、TNF-α、IFN-γ和IL-1β等表达明显降低(P<0.05,P<0.01)。结论:加味散偏汤可通过抑制p38 MAPK/iNOS通路,减少NO、TNF-α、IFN-γ和IL-1β等促炎因子表达,减轻神经源性炎症反应,发挥治疗偏头痛的作用。Objective:To decipher the mechanism of modified Sanpiantang in the treatment of nitroglycerin-induced migraine in rats.Method:Seventy-two Wistar rats were randomized into the control,model(nitroglycerin,10 mg·kg^(-1)),positive control(rizatriptan,0.89 mg·kg^(-1)),and high-(12.96 g·kg^(-1)),medium-(6.48 g·kg^(-1)),and low-dose(3.24 g·kg^(-1))modified Sanpiantang groups.The rat model of migraine was established by intraperitoneal injection of 10 mg·kg^(-1) nitroglycerin.The behavioral test was carried out to measure the mechanical pain thresholds(MPT)of the periorbital region and hindpaw after successful modeling.The serum levels of nitric oxide(NO),tumor necrosis factor-α(TNF-α),interferon-γ(IFN-γ),and interleukin-1β(IL-1β)in rats were determined by enzyme-linked immunosorbent assay(ELISA).Immunohistochemistry(IHC)was employed to determine the expression of inducible nitric oxide synthase(iNOS)in the trigeminal nucleus caudalis(TNC).Western blot was employed to determine the protein levels of iNOS and phospho-p38 mitogen-activated protein kinase(p-p38 MAPK)in the TNC.Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)was performed to measure the mRNA levels of iNOS,p38 MAPK,and IL-1βin the TNC.Result:Compared with the control group,the model group showed decreased MPT(P<0.01),elevated serum levels of NO,TNF-α,IFN-γ,and IL-1β(P<0.01),and up-regulated expression levels of p38 MAPK,iNOS,and IL-1βin the TNC(P<0.05,P<0.01).Compared with the model group,modified Sanpiantang increased the MPT(P<0.05),and the medium-dose group showed the most significant effect(P<0.01).In addition,modified Sanpiantang down-regulated the mRNA levels of iNOS,p38 MAPK,and IL-1βand the protein levels of p-p38 MAPK and iNOS in the TNC of migraine rats(P<0.05,P<0.01)and lowered the serum levels of NO,TNF-α,IFN-γ,and IL-1β(P<0.05,P<0.01).Conclusion:Modified Sanpiantang may treat migraine by down-regulating the expression of pro-inflammatory factors such as NO,TNF-α,IFN-γ,and IL-1βin the p38 MAPK/i

关 键 词：加味散偏汤 偏头痛 p38丝裂原活化蛋白激酶/诱导性一氧化氮合酶(p38 MAPK/iNOS)信号通路 神经源性炎症 

分 类 号：R2-0[医药卫生—中医学] R33R747.2R289