文献类型：期刊文献

中文题名：基于p38MAPK/NF-kB信号通路探讨柴胡加龙骨牡蛎汤干预广泛性焦虑模型大鼠的作用机制

英文题名：Chaihu Longgu Mulitang Relieves Generalized Anxiety Disorder in Rats via p38 MAPK/NF-κB Signaling Pathway

作者：韦春昕[1];胡亦明[1];黄仕琦[1];谭国伟[1];安耀荣[1]

第一作者：韦春昕

机构：[1]甘肃中医药大学,甘肃省中医方药挖掘与创新转化重点实验室,兰州730000

第一机构：甘肃中医药大学科研实验中心（甘肃省中医药标准化技术委员会秘书处）

年份：2024

卷号：30

期号：4

起止页码：114

中文期刊名：中国实验方剂学杂志

外文期刊名：Chinese Journal of Experimental Traditional Medical Formulae

收录：CSTPCD;;Scopus;北大核心:【北大核心2023】；CSCD:【CSCD2023_2024】；

基金：甘肃省自然科学基金项目(22JR11RA121);甘肃中医药大学岐黄英才导师专项基金项目(3016190124)。

语种：中文

中文关键词：广泛性焦虑症;柴胡加龙骨牡蛎汤;炎症反应;p38丝裂原活化蛋白激酶/核转录因子-κB(p38 MAPK/NF-κB)信号通路;小胶质细胞

外文关键词：generalized anxiety disorder;Chaihu Longgu Mulitang;inflammatory response;p38 mitogen-activated protein kinase/nuclear factor-κB(p38 MAPK/NF-κB)signaling pathway;microglia

摘要：目的:研究柴胡加龙骨牡蛎汤是否可以通过调控抑制p38丝裂原活化蛋白激酶/核转录因子-κB(p38 MAPK/NF-κB)信号通路抑制广泛性焦虑(GAD)大鼠下丘脑区炎症反应,改善GAD大鼠的焦虑样行为,缓解GAD大鼠的焦虑症状。方法:74只Wistar大鼠随机选择12只作为空白组,剩余大鼠采用慢性束缚应激法制造GAD模型大鼠,造模14 d后进行旷场实验(OFT)和高架十字迷宫实验(PMT)检测各组大鼠焦虑样行为,筛选造模成功的大鼠60只,随机分为模型组、柴胡加龙骨牡蛎汤低、中、高剂量组(6、12、24 g·kg^(-1))和地西泮组(1 mg·kg^(-1)),每组12只,连续14 d灌胃相应剂量柴胡加龙骨牡蛎汤和地西泮,给药14 d后继续通过旷场和十字迷宫实验观察大鼠焦虑样行为的变化;酶联免疫吸附测定法(ELISA)检测大鼠下丘脑和血清中肿瘤坏死因子-α(TNF-α)、白细胞介素IL-6(IL-6)、白细胞介素-1β(IL-1β)水平;实时荧光定量聚合酶链式反应(Realtime PCR)检测p38 MAPK、NF-κB p65、NF-κB抑制因子α(IκBα)、钙结合蛋白(Iba-1)mRNA的水平;蛋白免疫印迹法(Western blot)检测大鼠下丘脑p38 MAPK、磷酸化(p)-p38 MAPK、NF-κB p65、p-NF-κB p65、IκBα蛋白的表达水平;免疫荧光法检测大鼠下丘脑小胶质细胞激活蛋白Iba-1表达情况。结果:与空白组比较,模型组大鼠体质量较轻且皮毛散乱黯黄、易躁易怒、喜在角落蛰伏,OFT边缘运动距离和边缘运动时间显著增加(P<0.01),PMT开臂运动距离、开臂运动时间及进入开臂次数显著减少(P<0.01),下丘脑室旁核小胶质细胞激活标志蛋白Iba-1荧光强度显著升高(P<0.01),血清和下丘脑中IL-1β、IL-6、TNF-α显著增多(P<0.01),下丘脑p38 MAPK、NF-κB p65、p-p38 MAPK、p-NF-κB p65、Iba-1蛋白及mRNA表达明显升高(P<0.05,P<0.01),IκBα蛋白及mRNA表达显著减低(P<0.01)。与模型组比较,柴胡加龙骨牡蛎汤中、高剂量组和地西泮组大鼠体质量较重且皮毛较为光滑、活动较为正常,OFT边缘运动距离和时间明显减少(P<0.05,P<0.01),PMT开臂运动距离及开臂运动时间明显增加(P<0.05,P<0.01),下丘脑小胶质细胞激活标志蛋白Iba-1荧光强度降低(P<0.05,P<0.01),血清和下丘脑组织中IL-1β、IL-6、TNF-α明显降低(P<0.05,P<0.01),下丘脑p38 MAPK、NF-κB p65、p-p38 MAPK、p-NF-κB p65、Iba-1蛋白及mRNA表达明显降低(P<0.05,P<0.01),IκBα蛋白及mRNA表达明显升高(P<0.05,P<0.01)。结论:柴胡加龙骨牡蛎汤可通过抑制p38 MAPK/NF-κB信号通路,降低GAD大鼠下丘脑内小胶质细胞活化度、下调促炎因子水平,发挥改善大鼠焦虑样行为、缓解大鼠焦虑状态的作用。
Objective:To study whether Chaihu Longgu Mulitang can inhibit hypothalamic inflammation,mitigate anxiety-like behavior,and alleviate anxiety symptoms by regulating the p38 mitogenactivated protein kinase/nuclear factor-κB(p38 MAPK/NF-κB)signaling pathway in the rat model of generalized anxiety disorder(GAD).Method:Twelve out of 74 Wistar rats were randomly selected as the blank group,and the remaining rats were subjected to chronic restraint stress for the modeling of GAD.The open field test(OFT)and elevated Porteus maze test(PMT)were conducted 14 days after modeling to detect the anxietylike behaviors.Sixty successfully modeled rats were selected and randomized into model,low-,medium-,and high-dose(6,12,and 24 g·kg^(-1),respectively)Chaihu Longgu Mulitang,and diazepam(1 mg·kg^(-1))groups(n=12)and administrated with corresponding drugs for 14 consecutive days.OFT and PMT were then carried out to examine the anxiety-like behaviors of the rats.The levels of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),and interleukin-1β(IL-1β)in the hypothalamus and serum of rats were determined by the enzymelinked immunosorbent assay(ELISA).Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)was conducted to determine the mRNA levels of p38 MAPK,NF-κB p65,nuclear factorκB inhibitorα(IκBα),and ionized calcium binding adaptor molecule 1(Iba-1).The protein levels of p38 MAPK,phosphorylated(p)-p38 MAPK,NF-κB p65,p-NF-κB p65,and IκBαin the hypothalamus of rats were determined by Western blot.The expression of Iba-1 in the hypothalamic microglia was detected by immunofluorescence assay.Result:Compared with the blank group,the model group had decreased body weight,scattered dark yellow fur,increased irritability,and preference to hibernation in the corner.In addition,the modeled rats showed increased edge movement distance and time in OFT(P<0.01)and decreased movement distance and time and the number of entries in the open arm in PMT(P<0.01).The modeling increased the fluorescence intensity of Iba-1 in paraventricular nucleus of hypothalamus(P<0.01),elevated the levels of IL-1β,IL-6,and TNF-αin the serum and hypothalamus(P<0.01),up-regulated the protein and mRNA levels of p38 MAPK,NF-κB p65,p-p38 MAPK,p-NF-κB p65,and Iba-1(P<0.05,P<0.01),and down-regulated the protein and mRNA levels of IκBα(P<0.01)in the hypothalamus.Compared with the model group,mediumand high-dose Chaihu Longgu Mulitang and diazepam increased the body weight,improved the fur and behaviors,decreased the edge movement distance and time in OFT(P<0.05,P<0.01),and increased the movement distance and time in the open arm in PMT(P<0.05,P<0.01).Furthermore,they decreased the fluorescence intensity of Iba-1 in hypothalamic microglia(P<0.05,P<0.01),lowered the levels of IL-1β,IL-6,and TNF-αin the serum and hypothalamic tissue(P<0.05,P<0.01),down-regulated the mRNA and protein levels of p38 MAPK,NF-κB p65,p-p38 MAPK,p-NF-κB p65,and Iba-1(P<0.05,P<0.01),and up-regulated the mRNA and protein levels of IκBα(P<0.05,P<0.01)in the hypothalamus.Conclusion:Chaihu Longgu Mulitang can mitigate anxiety-like behaviors and relieve anxiety in GAD rats by inhibiting the p38 MAPK/NF-κB signaling pathway and reducing the activation of microglia and the levels of pro-inflammatory cytokines in the hypothalamus.