慢性应激肝郁脾虚证大鼠CRHR2的变化及逍遥散的影响

Changes of CRHR2 in rats with chronic stress syndrome of stagnation of liver qi and spleen deficiency and the effect of Xiaoyao Powder

目的:观察慢性束缚应激肝郁脾虚证大鼠下丘脑、胃、结肠组织CRHR2的变化及逍遥散的干预作用。方法:105只雄性SD大鼠随机分为正常组,7、14、21d模型组,7、14、21d逍遥散组,以慢性束缚应激结合孤养方法制作肝郁脾虚证大鼠模型并用逍遥散进行干预。造模7、14、21d结束后,应用基因芯片检测下丘脑、胃和结肠组织CRHR2 mRNA表达,并采用实时荧光定量PCR和原位杂交方法检测胃组织CRHR2 mRNA表达(其中结肠组织基因芯片与原位杂交实验检测7、21d大鼠),从组织和细胞层面对基因芯片结果进行验证。结果:与正常组比较,基因芯片结果显示CRHR2在21d模型组下丘脑表达下调但无显著性差异,在7、21d模型组胃组织显著下调(P<0.05,P<0.01),在21d模型组结肠组织显著上调(P<0.01);实时荧光定量PCR检测胃组织CRHR2 mRNA的表达情况与基因芯片结果趋势一致;原位杂交实验结果显示,7、21d模型组胃组织CRHR2 mRNA表达的阳性面积和积分光密度均减少(P<0.05,P<0.01);逍遥散能逆转胃组织CRHR2基因的异常表达(P<0.05,P<0.01)。结论:CRHR2与肝郁脾虚证的发生发展密切相关,调节CRHR2的基因表达可能是逍遥散治疗肝郁脾虚证的机制之一。

Objective:To observe the changes of CRHR2 in the hypothalamus,stomach and colon of rats with chronic restraint stress and stagnation of liver qi and spleen deficiency syndrome.Methods:A total of 105 male SD rats were randomly divided into normal group(N),7,14,21 d model group(M),7,14,21 d Xiaoyao Powder group(XYS),the rat model of liver stagnation and spleen deficiency syndrome was made by chronic restraint stress combined with orphan method and intervened with Xiaoyao Powder.At 7,14,21 d after the end of the modeling,the expression of CRHR2 mRNA in hypothalamus,stomach and colon tissues was detected by gene chip,and the expression of CRHR2 mRNA in gastric tissue was detected by real-time fluorescent quantitative PCR and in situ hybridization.The colon tissue microarray and in situ hybridization assay were used for7 d and 21 d rats.The results of gene chip were verified from the tissue and cell layer.Results:Compared with the normal group,the results of gene chip showed that CRHR2 was down-regulated in the hypothalamus of the 21 d model group but there was no significant difference.In the 7,21 d model group,the gastric tissue was significant decreased(P<0.05,P<0.01).The expression of CRHR2 mRNA in colon tissue was significantly up-regulated in the 21 d model group(P<0.01).The expression of CRHR2 mRNA in gastric tissues detected by real-time fluorescence quantitative PCR was consistent with the trend of gene chip results.The results of in situ hybridization showed that the positive area and integral optical density of CRHR2 mRNA expression in gastric tissues were decreased in both 7 d and 21 d model groups(P<0.05,P<0.01).Xiaoyao Powder can reverse the abnormal expression of CRHR2 gene(P<0.05,P<0.01).Conclusion:CRHR2 is closely related to the occurrence and development of stagnation of liver qi and spleen deficiency syndrome,and regulating the gene expression of CRHR2 may be one of the mechanisms of Xiaoyao Powder in the treatment of stagnation of liver qi and spleen deficiency syndrome.