基于“双心同治”探讨解郁化痰中药对慢性不可预知应激下ApoE-/-小鼠血脂、HPA轴及海马BDNF表达的影响

Effect of Chinese Herbal Medicine for Relieving Depression and Resolving Phlegm on Blood Lipid,HPA Axis and BDNF Expression in Hippocampus of ApoE-/-Mice under Chronic Unpredictable Stress Based on"Double Heart Treatment"

目的探讨解郁化痰中药对慢性不可预知应激下ApoE^(-/-)小鼠血脂代谢、下丘脑-垂体-肾上腺轴(hypothalamic-pituitary-adrenal axis,HPA axis)应激激素及海马脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)表达的影响。方法选用雄性C57Bl6和ApoE小鼠随机分为A:空白对照组、B:AS模型组、C:应激模型组、D:中药治疗组、E:西药治疗组,每组8只。除空白组外,其余4组均给予高脂饲料喂养,C、D、E组同时给予慢性不可预知应激刺激16周。从第9周开始,D组给予解郁化痰中药煎剂,E组给予辛伐他汀片2.6mg/(kg·d)及路优泰156mg/(kg·d)灌胃治疗8周,ELISA法检验小鼠血清总胆固醇(total cholesterol,TC)、甘油三酯(triglyceride,TG)、低密度脂蛋白胆固醇(low density lipoprotein cholesterol,LDL-C)、高密度脂蛋白胆固醇(high density lipoprotein cholesterol,HDL-C)水平,及HPA轴促肾上腺皮质激素(adrenocorticotropic hormone,ACTH)、5-羟色胺(5-hydroxytryptamine,5-HT)含量,qrt-PCR方法检测小鼠海马组织促肾上腺皮质激素释放激素(corticotropin releasing hormone,CRH)基因表达,Western blot法检测海马组织BDNF蛋白含量。结果与空白对照组比较,B、C两模型组小鼠血清TC、TG及LDL-C含量均明显升高(P<0.01),HDL-C含量减低(P<0.01);与AS模型组比较,C组HPA轴激素ACTH含量增多(P<0.01)而5-HT表达下降(P<0.01),CRH mRNA表达明显上升(P<0.01),海马内BNDF蛋白含量减少(P<0.01);与应激模型组比较,血脂方面,D、E组小鼠血清血脂水平改善,TC、TG、LDL-C明显下降(P<0.01),HDL-C含量增加(P<0.05),且E组优于D组,TC、TG(P<0.05),LDL-C(P<0.01),HPA轴代谢方面,ACTH下降而5-HT上升(P<0.01),CRH mRNA表达减少(P<0.01),海马内BNDF蛋白增多(P<0.01),且E组较D组变化明显ACTH(P<0.01),CRH mRNA(P<0.05),BNDF(P<0.01)。结论慢性不可预知应激下ApoE^(-/-)小鼠HPA轴功能亢进,血脂代谢异常,解郁化痰中药能够有效调节ApoE^(-/-)小鼠血脂水平,缓解应激状态,改善HPA轴平衡,而调节BDNF表达可能是其作用机制之一。

Objective To investigate the effects of Chinese herbal medicine for relieving depression and resolving phlegm on lipid metabolism,hypothalamic-pituitary-adrenal(HPA)axis stress hormone and brain-derived neurotrophic factor(BDNF)expression in hippocampus of ApoE^(-/-)mice under chronic unpredictable stimulation.Methods Male C57 Bl6 and ApoEmice were randomly divided into A group(blank control group),B group[atherosclerosis(AS)model group],C group(stress model group),D group(traditional Chinese medicine treatment group)and E group(Western medicine treatment group),with 8 mice in each group.Except the group A,the other 4 groups were fed with high fat diet,and group C,D and E were simultaneously given chronic unpredictable stress stimulation for 16 weeks.From the 9 th week,group D was given Jieyu Huatan Decoction,group E was given simvastatin tablet 2.6 mg/(kg·d)and St.John’s grass extract tablets 156 mg/(kg·d)by gavage for 8 weeks.The serum levels of total cholesterol(TC),triglyceride(TG),low density lipoprotein cholesterol(LDL-C)and high density lipoprotein cholesterol(HDL-C)as well as the contents of HPA axis stress hormones adrenocorticotropic hormone(ACTH)and 5-hydroxytryptamine(5-HT)were determined by ELISA.Qrt-PCR was used to detect corticotropin releasing hormone(CRH)gene expression and Western blot was used to detect BDNF protein content.Results Compared with those of group A,the serum levels of TC,TG and LDL-C of mice in group B and C were significantly increased(P<0.01),while the HDL-C level was decreased(P<0.01).Compared with those of group B,the level of ACTH in HPA axis hormone of group C was increased(P<0.01),the 5-HT expression decreased(P<0.01),the CRH mRNA expression was increased significantly(P<0.01),and BNDF protein content decreased(P<0.01).Compared with those of group C,the serum lipid levels in group D and E were improved,the levels of TC,TG and LDL-C were decreased significantly(P<0.01),and the HDL-C level was increased(P<0.05).The levels of TC,TG and LDL-C of group E were better than those of group D(P<0.05,P<0.01).In terms of the HPA axis metabolism,the level of ACTH was decreased and the level of 5-HT was increased(P<0.01),the CRH mRNA expression was decreased(P<0.01)and the BNDF protein was increased(P<0.01),and ACTH,CRH mRNA and BNDF in group E changed significantly compared with those in group D(P<0.01,P<0.05).Conclusion Under chronic unpredictable stress,ApoE^(-/-)mice have hyperfunction of HPA axis and abnormal lipid metabolism.Jieyu Huatan Traditional Chinese medicine can effectively regulate lipid level of ApoE^(-/-)mice,relieve stress state and improve HPA axis balance,and regulation of BDNF expression may be one of its mechanisms.