The Changes Of Ob-R,CART,α-MSH In Hypothalamic Arcuate Nucleus Of Chronic Immobilization Stress Rats With Liver Depression And Spleen Deficiency And The Effect Of Xiaoyaosan Decoction

Objective: To observed the change of Leptin receptor(Ob-R),Cocaine and amphetamine regulated transcript(CART),α-melanocyte stimulating hormone(α-MSH)expression in ARC of chronic immobilization stress(IBS)rats with liver-stagnation and spleen-deficiency syndrome,to explore the biological basis of spleen and stomach dysfunction showed by the loss of appetite,weight loss,loose stools and other.At the same time,Xiaoyaosan decoction(《Taiping Huimin Heji Jufang》)as the drug intervention,to reveal the target and mechanism of soothing the liver and invigorating spleen to regulate appetite.Methods:Constructed NPY RNA interference plasmid of adenovirus vector(pAd-NYi),adenovirus of the non transfected plasmid was used as control(Ad-NT),and the effect of p Ad-NYi interference was verified in vitro.Healthy SD male rats,weighting 180-200 g,were raised in clean level animal house: room temperature of(21±1)℃,relative humidity of 40% ~ 60%,light 7:00~19:00 and dark 19:00~7:00.Free drinking and eating.After one week of adaptive feeding,SD rats were randomly divided into seven groups: the normal group,the model group,the xiaoyaosan group,the pAd-NYi group,the pAd-NYi+xiaoyaosan group,the Ad-NT group,and the jiashoushu group,each group has 25 rats.The pAd-NYi group、the pAd-NYi+xiaoyaosan group and the Ad-NT group rats were respectively injected with pAd-NYi or Ad-NT in bilateral ARC under the stereotaxic apparatus,and the needle of jiashoushu rats retained for 2min,not injected any substance.After injection,the rats recovered for one week,the rats in the normal group without stress,and with normal feeding;The animals of the model group,the xiaoyaosan group,the pAd-NYi group,the pAd-NYi+xiaoyaosan group,the Ad-NT group,the jiashoushu group were imposed measures of chronic immobilization stress with 3 hours each day for 21 consecutive days.The drug group(xiaoyaosan group,pAd-NYi+xiaoyaosan group)were given xiaoyaosan decoction(3.85 g/ day per kilo of body weight)before stress.The rats in the normal group,the model group,the p Ad-NYi group,the Ad-NT group and the jiashoushu group were fed with normal saline.Before injection,the body weight,food intake and behavioral experiments(open-field test and elevated plus-maze test)of rats in the normal group,the model group and the xiaoyaosan group were measured as the day 0 data.After the injection and immobilization stress,to observe the body weight,food intake and appearance at the 7th,14 th,21st day and the behavioral changes at the 7th,21 st day of the normal group,the model group,and the xiaoyaosan group;the rats were decapitated at the 22 nd after stress,the serum D-xylose were detected by phloroglucin method,in order to comprehensive evaluated the animal models.At the same time,the expression of ob-R、CART、α-MSH、NPY gene and protein in ARC of hypothalamus in each group were detected by immunofluorescence double staining(IF)and Quantitive Real-time PCR(qRT-PCR)method after twenty-second day of immobilization stress.Results:1 The general state,weight,ingestion and ethological changes of ratsBefore stress,the mental status of rats in the normal group,the model group,and the xiaoyaosan group were good,and no significant difference in body weight,food intake and behaviors.After twenty-one days of immobilization stress,phenomenon including listless,crouching in the corner,hunchbacked to move,unresponsive,dull fur,canthus and auricula lackluster,duck stools occurred in model rats;The total distance of the open field test within 5min in the model group showed a decreasing trend than the normal group;The enter times and residence time in open-arms of elevated plus-maze test of model group rats showed decreased,and the enter times and residence time in close-arms increased;and the serum D-xylose content of model group was significantly lower than that of normal rats(P <0.01).Compared with the model group,the body weight,food intake and general state of the xiaoyaosan group were improved;the ability of independent activity and the exploratory behaviour of the xiaoyaosan group rats in the open field test and elevated plus-maze test were enhanced,and serum D-xylose content was significantly higher than that of model group(P <0.01).The results showed that 21 days of continuous immobilization stress caused emotions of frustration,depression and fear in rats,accompanied by gastrointestinal dysfunction,and there was a liver depression and spleen deficiency syndrome.2 The pAd-NYi interference of NPY expression in the hypothalamus ARC of rats with chronic immobilization liver depression and spleen deficiency model.The immunofluorescence double staining and qRT-PCR results showed: With immobilization stress factors were continuously applied,the positive neurons expression of NPY Area,SUM,IOD,MOD and the NPY mRNA in hypothalamus ARC of model group,pAd-NYi group rats were decreased than the normal group;Compared with the model group,NPY protein and gene expression of pAd-NYi group was significantly decreased(P <0.05);Compared with p Ad-NYi group,NPY protein and gene expression in Ad-NT group,Jiashoushu group,pAd-NYi+xiaoyaosan group were increased.These results suggested that the NPY expression of ARC in hypothalamus could be reduced by stress factors,and the constructed adenovirus p Ad-NYi plasmid could inhibit the expression of NPY.3 The Expression of ob-R,CART,α-MSH protein and gene expression in hypothalamus ARC of rats with chronic immobilization liver depression and spleen deficiency model.Compared with normal group,ob-R,CART,and α-MSH protein and gene expression in hypothalamus ARC of model group,p Ad-NYi group were significantly increased.(P <0.05 or P <0.01),Compared with the model group,the protein and gene expression of ob-R 、 CART and α-MSH in hypothalamus ARC of Ad-NT group,Jiashoushu group have no significantly difference.The expression of ob-R、CART and α-MSH in pAd-NYi group increased(P <0.05 or P <0.01).The expression of xiaoyaosan group were significantly decreased(P <0.05 or P <0.01).Compared with the pAd-NYi group,the expression ob-R、CART、α-MSH in ARC of the model group,the Ad-NT group,and the jiashoushu group were decreased(P <0.05 或 P <0.01);The pAd-NYi+xiaoyaosan group were also reduced,and the CART integral optical density(IOD)decreased obviously(P <0.01).Conclusion:1 The adenovirus NPY interference plasmid can obviously inhibited the expression of NPY in hypothalamus ARC of rat.2 The pathway of ob-R-CART/α-MSH(POMC)in hypothalamus ARC could be used as one of the central neuroendocrine mechanisms of chronic stress liver stagnation and spleen deficiency animal model of spleen and stomach dysfunction.3 Xiaoyaosan decoction had a corresponding regulatory effect,especially for the obvious regulation effect on CART.It suggested that the pathway of ob-R-CART/α-MSH(POMC)in hypothalamus ARC could also be used as one of the targets of the traditional Chinese medicine-Xiaoyaosan decoction for soothing the liver and invigorating spleen to regulated appetite.