摘要
目的探讨以四逆汤+桂甘龙牡汤为基础方的温阳法调控失眠大鼠认知功能、海马炎症反应及神经元凋亡的作用机制。方法将12只雄性SD大鼠随机分为空白组、模型组、温阳组和地西泮组,每组3只。除空白组外,其余组大鼠连续2 d腹腔注射氯苯丙氨酸诱导建立失眠模型。造模成功后,温阳组给予四逆汤+桂甘龙牡汤10 g/kg灌胃,地西泮组给予地西泮1 mg/kg灌胃,空白组和模型组给予等量生理盐水灌胃。连续灌胃7 d后,通过Morris水迷宫及旷场实验检测大鼠认知记忆能力,HE和TUNEL染色观察海马组织神经元形态与结构,ELISA法检测血清中白细胞介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)水平,qRT-PCR法检测海马组织中Caspase-3及Bcl-2 mRNA表达情况,Western blot法检测海马组织中Caspase-3及Bcl-2蛋白表达情况。结果与空白组比较,模型组大鼠目标象限时间、目标象限的游泳距离、距离比均明显缩短(P均<0.05),找到目标的活动时间和距离均明显延长(P均<0.05),水平穿越方格数和站立次数均明显增多(P均<0.05);海马区神经元损伤较为严重;血清IL-1β和TNF-α水平和海马组织中Caspase-3 mRNA及蛋白相对表达量均明显升高(P均<0.05),海马组织中Bcl-2mRNA及蛋白相对表达量均明显降低(P均<0.05)。与模型组比较,温阳组和地西泮组大鼠目标象限时间、目标象限的游泳距离、距离比均明显延长(P均<0.05),找到目标的活动时间和距离均明显缩短(P均<0.05),水平穿越方格数(地西泮组变化不明显)和站立次数均明显减少(P均<0.05);海马区神经元形态和结构损害相对减轻;血清IL-1β和TNF-α水平和海马组织中Caspase-3 mRNA及蛋白相对表达量均明显降低(P均<0.05),海马组织中Bcl-2 mRNA及蛋白相对表达量均明显升高(P均<0.05)。结论以四逆汤+桂甘龙牡汤为基础方的温阳法可能通过抑制神经炎症,调控Caspase-3和Bcl-2的表达,减轻失眠大鼠海马神经元损伤和凋亡,从而改善睡眠和认知记忆功能。
Objective It is to explore the mechanism of regulating cognitive function,hippocampal inflammation and neuronal apoptosis in rats with insomnia by warm Yang method based on Sini Decoction and Gui Gan Long Mu decoction.Methods Twelve SPF male SD rats were randomly divided into blank group,model group,warm Yang group and diazepam group,with 3 rats in each group.The rats of all groups except for the blank group were treated with chlorophenylalanine by intraperitoneal injection for 2 days to establish models of insomnia.After successful modeling,the warm Yang group was given Sini Decoction and Gui Gan Long Mu decoction 10 g/kg by gavage,the diazepam group was given diazepam 1 mg/kg by gavage,and the blank and model groups were given an equal amount of normal saline by gavage.After 7 d of continuous gavage,the cognitive memory ability of the rats was tested by Morris water maze and open field test,the morphology and structure of neurons in hippocampal tissues were observed by HE and TUNEL staining,the levels of interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)in serum were determined by ELISA,the expressions of Caspase-3 and Bcl-2 mRNA in hippocampal tissues were detected by qRT-PCR,and the expressions of Caspase-3 and Bcl-2 protein in hippocampal tissues were detected by Western blot.Results Compared with the blank group,the target quadrant time,swimming distance in the target quadrant,and distance ratio of the rats in the model group were significantly shortened(all P<0.05),the activity time and distance to find the target were significantly increased(all P<0.05),and the number of horizontal traversal of squares and number of times of standing were significantly increased(all P<0.05);the neuronal damage in hippocampal area was more severer;the levels of serum IL-1βand TNF-αand the relative expressions of Caspase-3 mRNA and protein in hippocampal tissues were significantly higher(both P<0.05),and the relative expressions of Bcl-2 mRNA and protein in hippocampal tissues were significantly lower(both P<0.05).Compared with the model group,the target quadrant time,swimming distance in the target quadrant,and distance ratio of the rats in the warm Yang group and diazepam group were significantly increased(all P<0.05),the activity time and distance to find the target were significantly shortened(all P<0.05),and the number of horizontal traversal of squares(the change was not significant)and number of times of standing were significantly decreased(all P<0.05);the neuronal damage in hippocampal area was less severer;the levels of serum IL-1βand TNF-αand the relative expressions of Caspase-3 mRNA and protein in hippocampal tissues were significantly lower(both P<0.05),and the relative expressions of Bcl-2 mRNA and protein in hippocampal tissues were significantly higher(both P<0.05).Conclusion The warm Yang method based on Sini Decoction and Gui Gan Long Mu decoction may alleviate the damage and apoptosis of hippocampal neurons by inhibiting neuroinflammation and regulating the expression of Caspase-3 and Bcl-2,thus to improve sleep and cognitive memory function in rats with insomnia.
作者
徐添
黄春华
官磊瑶
刘浪辉
黄梅
吴国燕
XU Tian;HUANG Chunhua;GUAN Leiyao;LIU Langhui;HUANG Mei;WU Guoyan(Jiangxi University of Traditional Chinese Medicine,Nanchang 330004,Jiangxi,China;The Affiliated Hospital of Jiangxi University of Traditional Chinese Medicine,Nanchang 330006,Jiangxi,China)
出处
《现代中西医结合杂志》
CAS
2023年第11期1496-1503,1510,共9页
Modern Journal of Integrated Traditional Chinese and Western Medicine
基金
江西省教育厅科学技术研究项目(GJJ211222)
第五批全国中医临床优秀人才研修项目(国中医药人教函〔2022〕1号)
江西省中医药中青年骨干人才(第三批)培养计划(赣中医药科教字〔2021〕4号)
饶旺福江西省国医名师传承工作室(赣中医药科教字〔2022〕3号)
江西省研究生创新专项资金项目(YC2021-S521)。
关键词
温阳法
失眠
海马
神经元凋亡
炎症水平
认知障碍
warm yang method
insomnia
hippocampus
neuronal apoptosis
inflammation levels
cognitive impairment