世界中医药

基金项目:国家科技重大专项“重大新药创制”项目（2018ZX09721005-004-007）；陕西省科技厅计划重点研发计划项目（2018ZDXM-SF-003、2021ZDLSF-04-06、2022ZDXM-SF-06、2024SF-YBXM-479）；陕西省中医医院苗圃培优项目（2021-13）；陕西省科技计划青年基金项目（2023-JC-QN-0820）；西安市科技计划项目（23Y XY J0165）

作者	单位
支文冰1，刘洋1，宗时宇1，王春柳1，孙婷婷1，李晔1，刘峰2，张红1	1 陕西省中医药研究院/陕西省中医医院，西安，710003； 2 陕西步长制药有限公司，西安，710075

摘要点击次数: 303

全文下载次数: 0

中文摘要:

目的：探讨头痛宁胶囊（TTN）对偏头痛大鼠模型和脂多糖（LPS）诱导神经小胶质细胞的影响及作用机制。方法：将21只无特定病原体(SPF)级斯泼累格·多雷(SD)雄性大鼠按照随机数字表法分为3组，分别为空白组、模型组、头痛宁给药组（TTN给药组）。TTN给药组大鼠给予TTN 640 mg/kg灌胃，空白组和模型组给予等体积生理盐水灌胃，均1次/d，预给药7 d。末次给药后30 min，TTN给药组和模型组大鼠给予皮下注射硝酸甘油注射液10 mg/kg造模，空白组给予皮下注射等体积生理盐水。用酶联免疫吸附试验法测定大鼠血清5-HT的水平、脑组织中DA。脂多糖（LPS）诱导神经小胶质细胞（BV2）模拟中枢敏化，将细胞分为空白组、脂多糖组和头痛宁高、低剂量给药组，噻唑蓝实验检测TTN和LPS对BV2细胞活力的影响；硝酸还原酶法检测细胞上清中一氧化氮（NO）；酶联免疫吸附试验法检测细胞上清中脑源性生长因子（BDNF）、5-HT、白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)水平；免疫组织化学法和蛋白质印迹法检测BV2细胞中Toll样受体4（TLR4）和磷酸化p65（p-p65 NF-κB）蛋白表达。结果：模型组大鼠血清5-HT、脑组织中DA水平低（P<0.05），TTN给药组血清5-HT、脑组织中DA水平高（P<0.05）。TTN（1.25~20 μg/mL）及LPS（1、2、4和8 μg/mL）对BV2细胞活力无显著影响。与空白组比较，LPS组细胞上清液中BDNF、5-HT水平低（P<0.05），NO、IL-6、TNF-α水平高（P<0.05），细胞中TLR4、p-p65 NF-κB蛋白表达高（P<0.05）；与LPS组比较，TTN给药组细胞上清液中BDNF、5-HT水平高（P<0.05），NO、IL-6、TNF-α水平高低（P<0.05），细胞中TLR4、p-p65 NF-κB蛋白表达低（P<0.05）。结论:头痛宁胶囊通过抑制TLR4/NF-κB信号通路，减轻了LPS诱导的BV2细胞炎症反应，缓解大鼠偏头痛症状。

English Summary:

To investigate the effect and mechanism of Toutongning Capsules(TTN) on a migraine rat model and lipopolysaccharide(LPS)-induced microglial cells.Methods:Twenty-one specific pathogen-free(SPF) grade male Sprague-Dawley(SD) rats were randomly divided into three groups according to a random number table：blank group,model group,and TTN treatment group(TTN group).Rats in the TTN group were administered TTN at 640 mg/kg by gavage,while those in the blank and model groups were given an equivalent volume of saline,once daily for 7 days.Thirty minutes after the last administration,the TTN and model groups received a subcutaneous injection of nitroglycerin injection at 10 mg/kg to induce a migraine model,while the blank group received a subcutaneous injection of saline.The serum levels of 5-HT and DA in brain tissues were measured using enzyme-linked immunosorbent assay(ELISA).LPS-induced microglial cells(BV2) were used to simulate central sensitization and were divided into a blank group,an LPS group,and high-and low-dose TTN treatment groups.The MTT assay was used to detect the effects of TTN and LPS on BV2 cell viability.Nitric oxide(NO) levels in the cell supernatant were detected using the nitrate reductase method,while brain-derived growth factor(BDNF),5-HT,interleukin-6(IL-6),and tumor necrosis factor-α(TNF-α) levels in the supernatant were measured using ELISA.Immunohistochemistry and Western blotting were used to detect the expression of Toll-like receptor 4(TLR4) and phosphorylated p65(p-p65) NF-κB proteins in BV2 cells.Results:The model group showed lower serum 5-HT and brain tissue DA levels(P<0.05),while the TTN group showed higher levels of these indicators(P<0.05).TTN(1.25 to 20 μg/mL) and LPS(1,2,4,and 8 μg/mL) had no significant effect on BV2 cell viability.Compared to the blank group,the LPS group had lower levels of BDNF and 5-HT in the cell supernatant(P<0.05) and higher levels of NO,IL-6,and TNF-α(P<0.05),as well as higher expression of TLR4 and p-p65 NF-κB proteins in cells(P<0.05).Compared to the LPS group,the TTN treatment groups had higher levels of BDNF and 5-HT in the cell supernatant(P<0.05),lower levels of NO,IL-6,and TNF-α(P<0.05),and lower expression of TLR4 and p-p65 NF-κB proteins in cells(P<0.05).Conclusion:Toutongning Capsules alleviate migraine symptoms in rats by inhibiting the TLR4/NF-κB signaling pathway,thereby reducing the inflammatory response induced by LPS in BV2 cells.

查看全文查看/发表评论下载PDF阅读器