Changes Of Appetite Factors In The Arcuate Nucleus Of The Hypothalamus In Rats With Liver Depression And Spleen Deficiency Syndrome

ObjectiveThe study established rat model of syndrome of liver-depression and spleen-deficiency induced by chronic immobilization stress,and adopted the technology and methods of immunosorbent(ELISA),the real-time quantitative PCR(RT-qPCR),and Western-blot to detect the expression of GHSR,NPY and a-MSH in ARC of hypothalamus,to provide the experimental basis for manifestations of appetite decreasing,body weight loss and loose stool,etc.with the syndrome of liver-depression and spleen-deficiency.Meantime,Xiaoyaosan was used as the interference formular,to explore the mechanism of its effect on relieving the depressed liver,and invigorating spleen for regulating appetite.Methods1.Establishment of rat model of syndrome of liver-depression and spleen-deficiency induced by chronic immobilization stress.44 male SD rats were randomly divided into the control group,the model group,Fuxiting group and Xiaoyaosan group.Rats in the control model were bred without stress for 21 days,and administrated with deionized water.Rats in the model group were adopted with stress of immobilization with 3 hours everyday for 21 days and intragastric administration half hours before restraint stress.Rats in Fuxiting group were adoperd with stress of immobilization and intragastric administration with Fuxiting half hours before restraint stress for 21 days.Rats in Xiaoyaosan group were adopted with stress of immobilization and intragastric administration with Xiaoyaosan half hours before restraint stress for 21 days.Rats of 4 groups were anesthetized for drawing materials on the 22th day.We observe the external appearance and signs,changes of weight,food intake,behaviors in open field test of rats every week,and the regulation effect of Xiaoyaosan.2.The expression of GHSR,NPY and a-MSH in ARC of hypothalamus and plasma of rats with syndrome of liver-depression and spleen-deficiency induced by chronic immobilization stress.44 male SD rats were randomly divided into the control group,the model group,Fuxiting group and Xiaoyaosan group.Duplicating model method was the same as above.Rats of 4 groups were anesthetized for drawing materials on the 22th day.Methods of real-time quantitative PCR(RT-qPCR)and Western-blot were adopted to detect the expression of GHSR,NPY and a-MSH in ARC of hypothalamus,and the method of elisa was used for detecting expression of Ghrelin and NPY in plasma.Results1.On the 21 day of chronic immobilization stress,on the hand of external appearance and signs,rats in the model group did not struggle to free themselves,hair dried and withered,the eyes were not net,loose stools,and the syptoms of spleen deficiency were obvious.Various indicators such as weight,food intake and the open field test were decreased in the rats of the model group.Xiaoyaosan group could improve above changes.2.Contents of Ghrelin and NPY in plasma detected with the method of ELISA Ghrelin:Compared with the control group,contents of Ghrelin in the model group decreased obviously,with significant difference.Compared with the model group,Fuxiting group and Xiaoyaosan group increased,and there was statistical difference between Fuxiting group and the model group.NPY:Compared with the control group,the model group has the tendency of descending.Compared with the model group,Fuxiting group increased with statistical difference,and Xiaoyaosan group increased with significant difference.Fuxiting group is close to the control group.3.Expression of GHSR,NPY and a-MSH in ARC of hypothalamus detected with method of RT-qPCR.GHSR:Compared with the control group,expression of GHSR in the model group decreased obviously,with significant difference.Compared with the model group,expression of two drug groups increased obviously,with significant difference.NPY:Compared with the control group,expression of NPY in the model group decreased obviously,with significant difference.There was statistical difference between Xiaoyaosan group and the control group,and Fuxiting group was close to the control group.a-MSH:Compared with the control group,expression of a-MSH in the model group increased obviously,with significant difference.Compared with the model group,expression of two drug groups decreased obviously,with significant difference.There was statistical difference between Fuxiting group and the control group,and Xiaoyaosan group was close to the control group.4.Expression of GHSR,NPY and a-MSH in ARC of hypothalamus detected with method of Western Blot.GHSR:Compared with the control group,expression of GHSR in the model group decreased with statistical difference.Compared with the model group,expression of two drug groups have the tendency of ascending,and was close to the control group.NPY:Compared with the control group,expression of NPY in the model group decreased obviously,with significant difference.Compared with the model group,expression of two drug groups have the tendency of ascending,but there was significant difference compared to the control group,so both of their regulation effects were not ideal.a-MSH:Compared with the control group,expression of a-MSH in the model group increased obviously,with significant difference.Compared with the model group,expression of two drug groups have the tendency of descending,but Fuxiting group was more close to the control group.Conclusions1.Rat model of syndrome of liver-depression and spleen-deficiency induced by chronic immobilization stress could be duplicated.The external appearance and signs combined with weight,food intake and the open field test,comprehensively showed that the chronic immobilization stress with 21 days could successfully reproduce the rat model of liver-depression and spleen-deficiency.2.The protein expression and gene expression of GHSR,NPY and a-MSH in ARC of rats of syndrome of liver-depression and spleen-deficiency with chronic immobilization stress have the same accord,mainly showed the protein expression and gene expression of GHSR and NPY decreased,and the protein expression and gene expression of a-MSH increased.With methods of RT-qPCR and Western Blot,the protein and gene expression of GHSR and NPY in ARC of model rat of syndrome of liver-depression and spleen-deficiency with chronic immobilization stress decreased,expression of a-MSH in ARC of model rat increased obviously;With the method of ELISA,content of Ghrelin in plasma of model rat of syndrome of liver-depression and spleen-deficiency with chronic immobilization stress decreased obviously,expression of NPY in plasma of model rat showed a downtrend.The study showed that the protein and gene expression of GHSR and NPY decreased,and the expression of the protein and gene expression of a-MSH increased.It could be acted as the biological basis for manifestations of appetite decreasing,body weight loss and loose stool,etc.with the syndrome of liver-depression and spleen-deficiency.To reveal the central neuroendocrine mechanism on malfunction of transporting and transforming of the spleen of syndrome of liver-depression and spleen-deficiency,and the mechanism of Xiaoyaosan on relieving the depressed liver,and invigorating spleen for regulating appetite.Expression of GHSR,NPY and a-MSH could be brought into the evaluation system,and provide the experimental basis for the establishment and evaluation on the rat model of syndrome of liver-depression and spleen-deficiency,and further illustrate the scientific connotation on syndrome of liver-depression and spleen-deficiency.