应激-ghrelin信号-疏肝抗抑郁促动力的关系

We found that α2-AR(adrenoceptor) is shared molecule mediated by antidepressive and prokinetc(A&P) induced by TCM formula and its absorbed compounds (ACs).According to the growing evidences, we hypotheses that Ghrelin(Ghr) signal is a better unifying pathogenesis than α2-AR and IL-1 for linking TCM-related A&P via gut/brain axis following stress. To test hypothesis, at firestly, the healthy C57BL/6J mice, Wistar rat, Ghsr-/-/IL-1ra-/- /α2(AR) -/- and their treatment groups with sham, water, Ghs, positive antidepressants or prokinetic, TCM formula and its ACs each are selected and groupted. Then, their responses to acute or chronic stress are detected, compared and evaluated according to causal index including depressive behaviour, gastric empting, AMPA pathway, HPA axis components, brain-gut appetides, cytokines, acyle Ghr concontration in curculation blood/hippocampus/hypothalomus/stomach, BDNF, neural plasticities in hippocampus and hypothamapus, BOLD-fNMI detection-response of neural cricuit, et al. Finally,the severities of responsing to same stressor targeting Ghsr-/-, α(2A)AR-/-,IL-1ra-/- mice from adult male C57Bl/6 mice and the latter WT mice are compared and evaluated. And interactios of stress, gut-brain axis, Ghs-signal, TCM-related A&P and neural circuit-based changes are elucidated and understood. From above results we shall have a conclusion from an unifying pathogenesis happened from the pivotal role played by Ghr and GHS-R1A 1a signals simultaneously to regulate gut-brain disorder, TCM-related A&P accompanying neural cuicuit of response by BOLD-fMRI，supporting the evidences for unified pathogenesis of patient with comorbidity of FD and depression and biomarker.

我们发现：疏肝方及其在海马/胃肠吸收成分(ACs)通过调节肠脑轴共享α2-AR(肾上腺素受体)抗抑郁和促动力(A&P)。依大量证据，本项目假设：与α2-AR与IL-1比，Ghrelin (Ghr)信号是更好的藉肠脑轴连接疏肝方药A&P的统一机制。为验证假说，拟选健康、Ghsr-/-/IL-1ra/-/C57BL/6J小鼠等及其用药组(疏肝方、ACs、Ghr、盐水与阳性药)，以抑郁样行为学、胃排空/肠张力、HPA轴、脑肠肽、循环血/海马/下丘脑/胃的乙酰化Ghr浓度、AMPA通路、ERK1/2、在/离体海马BDNF和BOLD-fMRI测定神经回路为指标，依各组对急/慢性应激反应：比较基因缺失对同一应激反应程度的贡献度；藉应激-肠脑互动-Ghr信号反应-疏肝A&P-神经回路反应相关性，验证假说。Ghr信号连接了应激、肠脑失调与疏肝A&P，从而奠定了Ghr成为生物标志物的科学基础。

我们采取吸收成分与母方药效相比较的模式。测定柴胡疏肝散中枳壳、芍药、川芎、厚朴中主要化学成分含量；建立慢性温和不可预知刺激造模方法并对SD雄性大鼠进行慢性抑郁造模，进行肠推进与胃排空促动力实验；通过PCR检测海马中IL-1β，IL-6，TNF-α的mRNA表达情况，从GHSR基因敲除小鼠的主要肠脑信号遴选出ghrelin信号，进一步阐述应激-ghrelin信号-疏肝抗抑郁促动力的关系。拟用 UPLC-qTOF-MS分析方法，遴选了A&P 疗效贡献度最大的4个ABCs，定量分析柴胡疏肝散汤剂中成分，定性分析灌胃(ig)柴胡疏肝散后在大鼠体内吸收的活性物质，观察柴胡疏肝散对雌雄大鼠经强迫游泳急性应激和慢性轻度不可预知应激(CUMS)刺激后的抗抑郁作用，同时比较不同性别的疗效差异，并揭示可吸收生物活性成分(absorbed bioactive compounds，ABCs)达到母方疗效的可行性。 SD大鼠在灌胃柴胡疏肝散(30g/kg)后血浆中的22个成分被成功分离、定性，为药效学研究提供了可吸收证据。雌雄大鼠抑郁程度出现明显差异，同时10 ABCs发挥了与母方柴胡疏肝散类似的抗抑郁疗效，初步阐明了柴胡疏肝散的药效物质。用LCMS/MS分析方法成功测定了枳壳厚朴汤剂中19个成分的含量，为药效学研究提供灌胃剂量依据及可重复疗效的证据。通过测试枳壳厚朴汤剂不同灌胃量及其可吸收成分对强迫游泳急性应激大鼠抑郁样行为的不动时间以及在体胃肠动力，探讨可吸收成分能否代替母方枳壳厚朴汤发挥抗A&P 作用。枳壳厚朴汤有明显的抗抑郁、促动力作用，而含有水合橙皮内酯的8个ABCs组分发挥了与母方枳壳厚朴汤剂类似的抗A&P作用，初步阐明枳壳厚朴汤抗A&P的药效物质。