龟鹿二仙胶对创伤后应激障碍大鼠HPA轴负反馈功能的影响及其机制

Effect and mechanism of Guilu Erxian Jiao on negative feedback function of HPA axis in rats with post-traumatic stress disorder

目的探讨龟鹿二仙胶(Guilu Erxian Jiao,GEJ)对创伤后应激障碍(post-traumatic stress disorder,PTSD)大鼠下丘脑-垂体-肾上腺(hypothalamic-pituitary-adrenal,HPA)轴负反馈功能影响及其可能机制。方法采用单一延长应激(single prolonged stress,SPS)的方法制备PTSD大鼠模型。96只SD大鼠按照随机数字表分为正常组(Control)、模型组(SPS)、龟鹿二仙胶组(GEJ)和帕罗西汀组(paroxetine,PRX),每组24只。除正常组外,其余大鼠均进行造模。造模后第8天开始,龟鹿二仙胶组(3.6 g/kg)和帕罗西汀组(10 mg/kg)大鼠分别灌胃给药,连续21 d;正常组和模型组大鼠则给予等量纯水灌胃,1次/d。连续灌胃21 d后,每组随机选取12只大鼠用于地塞米松抑制实验(dexamethasone suppression test,DST),6只大鼠用于RT-PCR实验,6只大鼠用于免疫组化实验。采用Elisa法检测血浆促肾上腺皮质激素(adrenocorticotrophic hormone,ACTH)含量,RT-PCR和免疫组化法检测海马和杏仁核中糖皮质激素受体(glucocorticoid receptor,GR)、盐皮质激素受体(mineralocorticoid receptor,MR)、促肾上腺皮质激素释放因子Ⅰ型受体(corticotropin releasing factor 1 receptor,CRF1R)和促肾上腺皮质激素释放因子Ⅱ型受体(corticotropin releasing factor 2 receptor,CRF2R)的表达。结果(1)在DST中,模型组血浆ACTH含量较正常组明显下降[(145.89±19.41)μg/L,(203.59±35.78)μg/L,t=3.16,P<0.01];帕罗西汀组和龟鹿二仙胶组较模型组则显著升高[(218.47±37.55)μg/L,t=3.98,P<0.01;(205.33±66.54)μg/L,t=3.26,P<0.01]。(2)在RT-PCR实验中,模型组海马GR mRNA、MR mRNA表达较正常组明显升高[(1.29±0.02),(1.00±0.06);t=6.88,P<0.01;(1.38±0.02),(1.00±0.05),t=7.97,P<0.01];龟鹿二仙胶组较模型组则显著下降[(0.96±0.07),t=7.87,P<0.01;(0.86±0.13),t=11.03,P<0.01]。(3)免疫组化实验结果显示,模型组海马GR、MR阳性细胞表达较正常组明显升高[(84.33±12.82),(69.33±8.19),t=2.50,P<0.05;(77.33±6.65),(56.33±11.79),t=2.25,P<0.05];龟鹿二仙胶组较模型组则显著下降[(68.33±4.55),t=2.67,P<0.05;(59.50±4.18),t=2.25,P<0.05]。杏仁核中,模型组GR、MR和CRF1R阳性细胞表达较正常组均明显下降[(62.67±6.89),(77.17±10.70),t=3.10,P<0.05;(60.50±11.66),(91.83±15.63),t=3.43,P<0.05;(54.50±19.96),(88.17±22.43),t=2.31,P<0.05],龟鹿二仙胶组较模型组则显著上升[(74.33±5.85),t=2.11,P<0.05;(83.67±12.55),t=2.53,P<0.05;(88.67±16.28),t=2.35,P<0.05]。结论龟鹿二仙胶能抑制SPS引起的HPA轴负反馈功能增强,其作用机制可能和龟鹿二仙胶调节海马和杏仁核中GR、MR和CRF1R的表达有关。

Objective To investigate the effect of Guilu Erxian Jiao(GEJ)on the negative feedback function of hypothalamic-pituitary-adrenal(HPA)axis and its possible mechanism in rats with post-traumatic stress disorder(PTSD).Methods The PTSD rat model was established using single prolonged stress(SPS).Ninety six SD rats were randomly divided into control group(control),model group(SPS),GEJ group(GEJ)and paroxetine group(PRX)according to the random number table with 24 rats in each group.Except the control group,the rats in the other groups were constructed using the PTSD model.On the 8th day after the establishment of the model,the rats of the GEJ group(3.6 g/kg)and the PRX group(10 mg/kg)were respectively given the drug by gavage for 21 days.The rats in control group and SPS group were given the same amount of distilled water once a day for 21 days.After continuous administration for 21 days,12 rats were randomly selected from each group for the dexamethasone suppression test(DST),then 6 rats were selected for the RT-PCR,and the remaining 6 rats were used for immunohistochemistry.The contents of plasma adrenocorticotrophic hormone(ACTH)were measured by Elisa.The expression levels of glucocorticoid receptor(GR),mineralocorticoid receptor(MR),adrenocorticotropic hormone releasing factorⅠreceptor(CRF1R)and adrenocorticotropic hormone releasing factorⅡreceptor(CRF2R)were detected by RT-PCR and immunohistochemistry.Results(1)In DST,plasma ACTH level in SPS group was significantly lower than that in control group((145.89±19.41)μg/L,(203.59±35.78)μg/L,t=3.16,P<0.01),and that in the PRX group and GEJ group were significantly higher than that in SPS group((218.47±37.55)μg/L,t=3.98,P<0.01;(205.33±66.54)μg/L,t=3.26,P<0.01).(2)RT-PCR results showed that,in hippocampus,the GR mRNA and MR mRNA expressions in SPS group were significantly higher than those in control group((1.29±0.02),(1.00±0.06),t=6.88,P<0.01;(1.38±0.02),(1.00±0.05),t=7.97,P<0.01),and that in the GEJ group significantly decreased comparing to SPS group((0.96±0.07),t=7.87,P<0.01;(0.86±0.13),t=11.03,P<0.01).(3)Immunohistochemical results showed that,in hippocampus,the positive cell expressions of GR and MR in the SPS group were significantly higher than those in control group((84.33±12.82),(69.33±8.19),t=2.50,P<0.05;(77.33±6.65),(56.33±11.79),t=2.25,P<0.05),and that in the GEJ group significantly were lower than SPS group((68.33±4.55),t=2.67,P<0.05;(59.50±4.18),t=2.25,P<0.05).In amygdala,the positive cells expression of GR,MR and CRF1R in the SPS group significantly decreased compared with the control group((62.67±6.89),(77.17±10.70),t=3.10,P<0.05;(60.50±11.66),(91.83±15.63),t=3.43,P<0.05;(54.50±19.96),(88.17±22.43),t=2.31,P<0.05);and that in GEJ group significantly increased compared with the SPS group((74.33±5.85),t=2.11,P<0.05;(83.67±12.55),t=2.53,P<0.05;(88.67±16.28),t=2.35,P<0.05).Conclusion GEJ can inhibit the enhanced HPA axis negative feedback function induced by SPS,which may be related to regulating expression of GR,MR and CRF1R in the hippocampus and amygdala.