灵芝补气安神功效对动物焦虑行为影响的实验研究

Influence of Lingzhi(灵芝)on Animal Anxiety Behavior:An Experimental Study

目的:观察灵芝水煎液及其粗多糖对动物焦虑行为的抗焦虑作用及机制。方法:建立装置焦虑模型小鼠和心气虚焦虑装置大鼠模型,除正常对照组、焦虑模型对照组,大小鼠分别灌胃地西泮2.5 mg/kg、灵芝水煎液2、4、6 g/kg、灵芝粗多糖0.04、0.08、0.12 g/kg,末次给药后1 h,观察大、小鼠于高架十字迷宫(EPM)实验中开臂进入次数百分比(OE%)、开臂滞留时间百分比(OT%)及明暗箱(LDT)实验中穿梭次数;行为学实验结束后取大鼠血及大、小鼠脑组织,ELISA法测定小鼠脑组织中小鼠γ-氨基丁酸(GABA)、谷氨酸(GLU)、5-羟色胺(5-HT)的含量;试剂盒测定大鼠脑组织中GABA、GLU含量,γ-氨基丁酸转氨酶(GABA-T)、谷氨酸脱羧酶(GAD)、谷氨酰胺合成酶(GS)活力,血清超氧化物歧化酶(SOD)活力、丙二醛(MDA),血浆环磷酸腺苷(cAMP)、环磷酸鸟苷(cGMP)、心钠素(ANP)含量;免疫组化法观察海马γ-氨基丁酸受体α1(GABAARα1)阳性表达。结果:随心气虚造模时间延长,各组大鼠体质量明显降低,19 d起,各给药组大鼠体质量降幅逐渐减小,22 d起各给药组大鼠体质量均大于心气虚焦虑模型组;灵芝水煎液及粗多糖均可显著升高大、小鼠的OE%、OT%、穿梭次数和脑组织中GABA含量,降低GLU含量及GLU/GABA的比值;降低小鼠脑中5-HT含量;降低大鼠脑中GABA-T活性;升高大鼠脑中GAD、GS活力,血清SOD活力、血浆cAMP含量,降低血清MDA、血浆cGMP、ANP含量,增强海马GABAARα1受体阳性表达;等质量灵芝饮片的水提取物、多糖提取物,表现出相似的抗焦虑效果。结论:灵芝水煎液及其粗多糖对动物焦虑行为有明显抗焦虑作用,抗焦虑主要活性部位可能为灵芝多糖。作用机制与通过调节GLU/GABA-Gln代谢环路中相关酶的活力,升高GABA含量和上调下游GABA/GABAARα1信号通路的表达,纠正兴奋-抑制性神经递质的失衡有关。

Objective:To observe the anti-anxiety effect and mechanism of Lingzhi decoction and its crude polysaccharides on animal anxiety behavior. Methods:The anxiety mouse model and anxiety rat model of heart-qi deficiency syndrome were established. Except for blank group and anxiety model group, all other rats and mice were administrated with diazepam suspension(diazepam suspension group),Lingzhi decoction(Lingzhi decoction group) and Lingzhi crude polysaccharide solution(crude polysaccharide group) separately. One hour after the last administration, the percentage of open arm entries(OE%),and the percentage of time spent in open arms(OT%) in elevated plus maze(EPM) test, and the number of transitions in light-dark transition(LDT) test were observed. After the behavioral experiment, the rat blood and rat and mouse brain tissues were taken. The contents of gamma-aminobutyric acid(GABA),glutamic acid(Glu) and 5-hydroxytryptamine(5-HT) in mouse brain were determined by enzyme linked immunosorbent assay(ELISA),while the contents of GABA and Glu in rat brain, superoxide dismutase(SOD) and malondialdehyde(MDA) in serum, cyclic adenosine monophosphate(cAMP),cyclic guanosine monophosphate(cGMP) and atrialnatriureticpeptide(ANP) in plasma as well as the activities of GABA transaminase(GABA-T),glutamic acid decarboxylase(GAD) and glutamine synthetase(GS) in rat brain were detected by kits. The expression of GABAARα1 in hippocampus was analyzed by immunohistochemical method. Results:The body weight of rats in each group was decreased with the modeling time of heart-qi deficiency syndrome increasing. From the 19th day, the treatment groups had gradually reduced weight loss. From the 22nd day, the body weight of rats in treatment groups was higher than that in anxiety model group. Both Lingzhi decoction and crude polysaccharide significantly increased OE%,OT%,the number of transitions and content of GABA in rat and mouse brain tissues, while decreased the content of GLU and GLU/GABA ratio. In both Lingzhi decoction and crude polysaccharide groups, the content of 5-HT in mouse brain was decreased;the activity of GABA-T in rat brain and the contents of MDA in serum and cGMP and ANP in plasma were down-regulated, while the activities of GAD and GS in rat brain, the activity of SOD in serum and the content of cAMP in plasma were up-regulated. Also the expression of GABAARα1 in hippocampus was enhanced. The water extract and polysaccharide extract of Lingzhi pieces of equal quality showed similar anti-anxiety properties. Conclusion:Lingzhi decoction and its crude polysaccharides have remarkable anti-anxiety effect on anxiety animal, and the active anti-anxiety part may be Lingzhi polysaccharides. The mechanism of action is related to increasing the content of GABA by regulating related enzymes in GLU/GABA-Gln metabolic loop, enhancing the downstream expression of GABA/GABAARα1 signal pathway, and balancing excitement-inhibitory neurotransmitters.