Studies On Neuropeptides Related Feeding Change In Arcuat Nucleus Of Hypothalamus Of Liver Depression And Spleen Deficiency Syndrome Resulted From The Chronic Immobilization Stress Model Rats

Objective: Based on the early pathophysiological research, this study established animal models of syndrome of liver-depression and spleen-deficiency with method of chronic immobilization stress, and adopted the technology and methods of dual immunofluorescence, laser confocal imaging,and the real-time quantitative PCR(RT-q PCR), to detect the expression of ob-R, CART, POMC, α-MSH with function of inhibiting ingestion in ARC of hypothalamus, to provide the experimental basis for the malfunction of transpoting and transforming of the spleen and stomach with manifestations of appetide decreasing, body weight loss and loose stool, etc. in the syndrome of liver-depression and spleen-deficiency. Meanwhile, Xiaoyaosan was used as the interference medicine, to explore the target and mechanism of it’s effect on relieving the depressed liver, and invigorating the spleen for regulating appetide.Methods: SD rats were randomly divided into 4 groups, that is the normal group, the 7-days-model group, the 21-days-model group, and the Xiaoyaosan group. Rats in the normal group were bred normally without stress for 21 days. Rats in the 7-days-model group and 21-days-model group were adopted with stress of immobilization with 3h a day for 7 days and 21 days, to establish models. Rats in the Xiaoyaosan group were adopted with stress of immobilization meanwhile intragastric administration with Xiaoyaosan for 21 days. Rats of the 7-days-model group were anesthetized for drawing material on the 8th day, and rats of the normal group, the 21-days-model group and the Xiaoyaosan group were anesthetized for drawing material on the 22 th day. To observe changes of weight, food intake, drinking water, external appearance and signs, behaviors in open field test, and elevated Plus-maze test.Methods of double immunofluorescence, laser confocal imaging, and RT-q PCR were adopted to detect the expression of ob-R and CART, POMC, α-MSH in ARC.Results:1 General appearance characterization and behaviors changeCompared with the normal group, the weight, food intake, and drinking water increased slowly down in the control group rats; the appearance characterization, such as, sleepiness, moving slowly, unresponsive, hair dried and withered, auricle radical and becomes weak, stool becoming more dry or wet. At the zero day of open-field test, the total cross number in five minitues, central grilletime, stand number and modifynumber were basic on the same level in every group. With the experimental process, the central grille time increased significantly, but the total cross number, stand number and modifynumber decreased significantly in the model group. At the elevated plus-maze test, every group had hardly any difference in the open arm entry and open arm time, close arm entry and close arm time. As the experimental process, the open arm entry and open arm time of the rats in model group decreased significantly, but the close arm entry and close arm time increased significantly. The Xiaoyaosan group showed significant improvement to the weight, food intake, and drinking water, behavior test, also in the general appearance and characterization.2 Expression of ob-R, CART, POMC, α-MSH in ARC detected with method of dual immunofluorescenceIn the whole experiment process, as the constraints keep up, the area, the mean optical density(MOD), integral optical density(IOD) and positive cells(SUM) of the ob-R, CART, POMC, α-MSH expression in ARC in the model groups increased than the normal group, the co-localization area of CART, POMC, α-MSH and ob-R also increased than the normal group. And with the downtrend after xiaoyaosan decoction applied, especially for the ob-R and CART.3 Expression of ob-R m RNA, CART m RNA, POMC m RNA, α-MSH m RNA in ARC detected with method of RT–q PCRThe expression of ob-R m RNA increased in the 21-days-model group compared with the control group(P<0.01), the expression of CART m RNA, POMC m RNA, α-MSH m RNA also increased in the 21-days-model group compared with the control group, but there was no statistically significant(P>0.05) among groups. Xiaoyaosan could decrease the expression of ob-R m RNA, CART m RNA, POMC m RNA, α-MSH m RNA.Conclusion:1 ob-R, CART, POMC, α-MSH had high expression in ARC of 21-day model group, maybe the ob-R, CART, POMC, α-MSH had relationship with the spleen and stomach with manifestations of appetite decreasing, body weight loss and loose stool, etc. The 7 day model group had not significantly increased, indicating that chronic brake bound for 7 days, mainly in the liver like change model.2 Xiaoyaosan showed the decreased expression of ob-R, CART, POMC, α-MSH. We could conclude the function of xiaoyaosan might be related to inhibit the expression of ob-R, CART, POMC, α-MSH, showed ob-R in ARC and downware path of POMC/CART maybe is the foundation of xiaoyaosan decoctionat.