5-羟色胺与其受体亚型在肝郁脾虚证大鼠的表达及逍遥散的影响

Expressions of 5-hydroxytryptamine and Its Receptor Subtypes in Rats with Liver-Stagnation and Spleen-Deficiency Syndrome and Effects of Xiaoyao Powder

目的：探讨慢性束缚应激肝郁脾虚证大鼠5-羟色胺（5-HT）与其受体亚型的表达变化及逍遥散的干预作用。方法：将175只雄性SD大鼠随机分为正常组（N）、7 d模型组（7 M）、7 d逍遥散组（7 XYS）、14 d模型组（14 M）、14 d逍遥散组（14 XYS）、21 d模型组（21 M）、21 d逍遥散组（21 XYS）,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 d、14 d、21 d结束后,应用放射免疫分析法测定下丘脑、结肠组织5-HT含量,实时荧光定量PCR方法检测下丘脑、结肠组织5-HTR1AmRNA表达,基因芯片检测下丘脑、胃和结肠组织5-HT受体相关基因的表达变化（其中结肠组织基因芯片检测7 d和21 d大鼠）,并用实时荧光定量PCR方法验证5-HT受体相关基因差异表达结果的可靠性。结果：①与正常组比较：21 d模型组大鼠下丘脑5-HT含量、5-HTR1AmRNA表达降低（P〈0.01）,21 d模型组大鼠结肠组织5-HT含量升高（P〈0.01）,7 d、14 d、21 d模型组大鼠结肠组织5-HTR1AmRNA表达均显著升高（P〈0.01）。逍遥散对下丘脑、结肠组织5-HT和5-HTR1AmRNA的异常表达均有改善作用（P〈0.01或P〈0.05）。②基因芯片结果显示,5-HTR1A、5-HTR2A、5-HTR2C、5-HTR3A、5-HTR4、5-HTR5A、5-HTR5B等基因在模型组大鼠下丘脑或胃或结肠组织出现了显著上调或下调表达,逍遥散能逆转这些基因的异常表达;实时荧光定量PCR方法检测这些差异基因的表达结果与基因芯片检测结果基本一致,证实了基因芯片数据结果的可靠性。结论：5-HT及其受体5-HTR1A、5-HTR2A、5-HTR2C、5-HTR3A、5-HTR4、5-HTR5A、5-HTR5B是肝郁脾虚证的重要物质基础,其参与了肝郁脾虚证的脑肠轴调控;逍遥散通过脑肠轴途径调节5-HT及其受体的表达可能是其防治肝郁脾虚证的机制之一。

Objective：To investigate the expression changes of 5-hydroxytryptamine（5-HT） and its receptor subtypes in rats with liver-stagnation and spleen-deficiency syndrome induced by chronic immobilization stress and the interferential effect of Xiaoyao Powder. Methods： A total of 175 male SD rats were randomly divided into normal control group,7-day model group,14-day model group,21-day model group,7-day Xiaoyao Powder group,14-day Xiaoyao Powder group and 21-day Xiaoyao Powder group.Except for those in the normal control group,the rats were subjected to chronic immobilization stress combined with lonely raising for 7,14 and 21 consecutive days and intervened with Xiaoyao Powder. The contents of 5-HT in hypothalamus and colon tissues were measured by radioimmunoassay. The expressions of 5-HTR1 AmRNA in hypothalamus and colon tissues were detected by real time qPCR and the whole-genome expression chips（Agilent） of rats were used to detect the expression changes of 5-HT receptor-related genes in the hypothalamus,stomach and colon tissues（colon tissues detect the rats in 7-day and 21-day groups）. The reliability of 5-HT receptor-related gene differential expression was verified by real time qPCR. Results： Compared with the normal control group,the 5-HT content of the hypothalamus in the 21-day model group was significantly decreased（P〈0.01）,and the 5-HT content of colon tissues was significantly increased（P〈0.01）. The expression of 5-HTR1 AmRNA in hypothalamus was significantly decreased in the 21-day model group,and that in colon tissues was significantly increased（P〈0.01） in the 7-day,14-day and 21-day model groups. Xiaoyao Powder on the 5-HT content,5-HTR1 AmRNA abnormal expression in the hypothalamus and colon tissues has improved the role（P〈0.01 or P〈0.05）. The results of gene microarray showed that 5-HTR1 A,5-HTR2 A,5-HTR2 C,5-HTR3 A,5-HTR4,5-HTR5 A and 5-HTR5 B significantly appeared up or down expressions in the hypothalamus or stomach or colon tissues of the model group rats. Xiaoyao Powder can reverse the abnormal expression of these genes. The results of these differential genes detected by real time qPCR were basically consistent with the results of gene chip detection,confirming the reliability of gene chip data results. Conclusion： 5-HT and 5-HTR1 A,5-HTR2 A,5-HTR2 C,5-HTR3 A,5-HTR4,5-HTR5 A and 5-HTR5 B are important material basis for liver-stagnation and spleen-deficiency syndrome,which is involved in the brain axis regulation of liver-stagnation and spleen-deficiency syndrome. Xiaoyao Powder on the regulation of 5-HT and its receptor expression through the brain axis pathway may be one of its preventions and treatments to the liver-stagnation and spleen-deficiency syndrome.