ARHGEF-7在慢性应激肝郁脾虚证大鼠模型海马、胃组织RhoA/Rac1/CDC42/PAKs细胞骨架通路中的变化及逍遥散的作用机理

Based on the previous studies, the expression of ARHGEF-7 in the actin cytoskeleton pathway of hippocampus and gastric tissue of liver depression and spleen deficiency rat model changed, and Xiaoyao San had the function of regulating brain gut interaction, this study proposes the hypothesis that bidirectional regulating the expression of ARHGEF-7 of hippocampus and gastric may be one of the mechanism of Xiaoyao San prevents liver depression and spleen deficiency.we use the method of chronic immobilization stress to make the liver depression and spleen deficiency rat model, activate and inhibit ARHGEF-7 expression of hippocampus and stomach tissue of rat model by ARHGEF-7 over-expression lentivirus and shRNA lentivirus, adopt techniques of Real-time qPCR and Western blot to detect the gene and protein expression of the key factors in the RhoA/Rac1/CDC42/PAKs pathway and ARHGEF-7of hippocampus and gastric tissue.Trough the study, we can clear the correlation between ARHGEF-7, RhoA / Rac1 / CDC42 / PAKs Pathways and Liver Stagnation and Spleen Deficiency,and the mechanism of Xiaoyao San on ARHGEF-7 and RhoA/Rac1/CDC42/PAKs pathways. In our study, it reveals the biological foundation of the liver depression and spleen from the angle of brain-bowel-axis, explains the mechanism and effects target on Xiaoyao San for prevention and treatment of liver depression and spleen deficiency as well as it effect of anti-stress damage. It may provide a scientific experimental evidence for the guidance of clinical treatment and enrichment of the connotation of the Liver-Zang-Xiang Theory.

基于申请者前期研究发现ARHGEF-7在肝郁脾虚证大鼠模型海马和胃组织肌动蛋白细胞骨架通路中出现了不同的表达变化及逍遥散具有调节脑肠互动的功能，本研究提出“通过双向调节海马-胃ARHGEF-7的表达可能是逍遥散防治肝郁脾虚证的机制之一”的假说，采用慢性束缚应激方法制作肝郁脾虚证大鼠模型，通过DNA重组技术构建过表达慢病毒和shRNA慢病毒激动和抑制大鼠模型海马、胃组织ARHGEF-7的表达，运用PCR、WB等方法检测海马和胃组织ARHGEF-7及RhoA/Rac1/CDC42/PAKs通路中关键因子的基因和蛋白表达变化，通过研究明确ARHGEF-7及RhoA/Rac1/CDC42/PAKs通路与肝郁脾虚证的相关性及逍遥散的作用机理。从脑-肠轴角度揭示肝郁脾虚证的生物学基础，阐明逍遥散防治肝郁脾虚证及抗应激损伤的作用机制和靶点，为指导临床治疗及丰富中医肝藏象学说的内涵提供科学的实验依据。

课题组前期的基因芯片研究发现ARHGEF-7在肝郁脾虚证大鼠模型海马和胃组织RhoA/Rac1/CDC42/PAKs肌动蛋白细胞骨架通路中出现了异常的表达变化，逍遥散具有调节脑肠互动的功能。本研究采用慢性束缚应激方法制作肝郁脾虚证大鼠模型，通过DNA重组技术构建过表达和干扰ARHGEF-7腺病毒对大鼠模型海马、胃组织中ARHGEF-7进行干预，运用ELISA法测定血清MLCK、PI3K、LIMK1含量，免疫组化检测海马、胃组织中ARHGEF-7的蛋白表达，Real-time qPCR、Western blot 方法检测海马组织ARHGEF-7、Rac1、CDC42、PAK4、RhoA、ROCK2和胃体组织ARHGEF-7、Rac1、CDC42、PAK2、RhoA、ROCK1、ROCK2、MMP2、TIMP2的mRNA和蛋白表达变化。研究结果显示，与正常组比较，模型组大鼠血清MLCK含量降低，PI3K、LIMK1含量升高；模型组大鼠海马组织ARHGEF-7、Rac1、CDC42、PAK2 的mRNA和蛋白表达水平降低，RhoA、ROCK2的mRNA和蛋白表达水平升高；模型组大鼠胃体组织ARHGEF-7、Rac1、CDC42、PAK2的mRNA和蛋白表达水平降低，MMP2、TIMP2的mRNA和蛋白表达水平升高；与模型组比较，ARHGEF-7干扰模型组大鼠海马和胃体组织中ARHGEF-7、Rac1、CDC42、PAK2、PAK4的mRNA和蛋白表达水平降低，ARHGEF-7过表达模型组大鼠海马和胃体组织中ARHGEF-7、Rac1、CDC42、PAK2、PAK4的mRNA和蛋白表达水平升高；逍遥散对上述指标的异常变化均具有调节作用。本次研究揭示ARHGEF-7/RhoA/Rac1/CDC42/PAKs通路与肝郁脾虚证相关，逍遥散对该通路具有双向调节作用。本研究从脑-肠轴角度丰富了肝郁脾虚证的生物学基础，为逍遥散临床防治肝郁脾虚证及抗应激损伤提供了科学的实验依据。