| Objective: To investigate the changes of Orexin A(OXA)and Orexin receptor 1(OX1R)in the lateral hypothalamic area(LHA)of rats after chronic immobilization stress and the regulation of Xiaoyaosan decoction,and explore the center neuroendocrine mechanism of inappetence induced by chronic immobilization stress and function mechanism of Xiaoyaosan decoction.Methods: Healthy adult male Sprague Dawley(SD)rats(n=72)were randomly divided into three groups,that's control group,model group and Xiaoyaosan group,with 24 rats in each group.The animals were housed in a standard animal room at 21±1°C,40% to 60% relative humidity,and 07:00 to19:00 light and 19:00 to 07:00 dark conditions.All the rats were fed adaptively for a week,and ad libitum access to feed and water.The rats in the model group and Xiaoyaosan group were subjected to immobilization stress for 21 days(3 h/day)to establish the models of chronic immobilization stress with the syndrome of liver-depression and spleen-deficiency.Meantime,the rats in the Xiaoyaosan group were administered Xiaoyaosan decoction(3.85g-1·kg-1·d-1)while the model rats with the normal saline.The control group were fed routinely for 21 days and administered normal saline.The dosage was regulated on the basis of body weight.Before administering intragastrically,the body weight,food intake and water intake were weighed,and the appearance of the rats such as the fur,faeces and restraint response were observed.Before the study started and on the 7th and 21 st day of the study,the behavioral tests,including the open field test(OFT)and the elevated plus maze(EPM)were conducted.At the end of the experiment,6 rats in each group were anesthetized with an intraperitonealinjection of 2% pentobarbital sodium(4 mg/100 g body weight).Then,left ventricular ascending aortic perfusion fixation of the heart was performed,and immunofluorescence(IF)assay was used to study the expression of OXA,OX1 R and ob-R(Leptin receptor)in LHA.The others were decapitated for the enzyme-linked immunosorbent assay(ELISA)analysis to measure the OXA content in the LHA and the serum content of Leptin.Serum D-xylose was determined by phloroglucinol method,and quantitative real-time reverse transcription PCR(q RT-PCR)to measure the m RNA expression of OXA,OX1 R and ob-R of LHA.Results:1 The general appearance of the three groupsAll the rats survived during the experiment.There is no change within the rats of the control group,the auricle and nose is ruddy,the bulbus oculi are flexible,canthi is clean,fur is bright and smooth,stools are normal and moderate in dry and wet.After stress,the rats of the model group are in the condition of irritability at the first week.The rats scratch and bite when captured,and present attack-stance such as hunped back and horripilation.The rats tend to break away from the wooden shelf or bite the belt and the shelf when bounded.A week later,the stress rats present tachypragia,and in high vigilance.But the resistance,bite and struggle were weakened when captured.After two weeks,resistance was weakened when captured,and prefer to stay together and lazy dynamic.In addition,fur is gloomy,knotting and rough,and stool is constipation or diarrhea.Canthi had secretion,auricle and nose is pale.When bound to the shelf,there is virtually no resistance,the animals behave sleepy but easy to be awakened.As released from the restrictions,rats aggregate and crouch.The Xiaoyaosan group conform to the model group at the first week.About a week later,the fur luster,faeces and restraint response are superior to the model group.The animals emerge more flexible,stools are normal,the auricle and nose is ruddy though slightly pale,canthi is cleaner,occasionally have secretion,fur is slightly gloomy but lustre and neat.2 Results of the body weight,food intake and water intake in threegroupsCompared with the control group,the body weight of the model group grow slowly,food intake and water intake present decrease tendency in general(P < 0.05 or P < 0.01).Compared to the model group,the body weight,food intake and water intake of Xiaoyaosan group are improved(P < 0.05 or P<0.01).3 Results of behavior experiment among three groupsCompared with the control group,on the 7th and 21 st day of the study,the total mobile distance,as well as the mobile distance and residence time of the model rats in the central zone in the OFT within 5 minutes are decreased significantly(P < 0.01).In the EPM experiment,residence time and entrance numbers in the open arms of model group are reduced compare with the rats in the control group within 5 minutes(P < 0.05 or P < 0.01),and the time in the closed arms lengthened while the entries reduced(P < 0.01).Compared with the model group,the indicators mentioned above of the Xiaoyaosan group are ameliorated(P < 0.01).4 Results of serum D-xylose by phloroglucinol methodCompared with the control group,the D-xylose content of the model rats is declined(P < 0.01).Compared with the model group,the content of serum D-xylose content of Xiaoyaosan group is ascended(P < 0.01).5 Contents of serum Leptin and OXA in LHA by ELISACompared with the control group,contents of serum Leptin of the model rats elevated while the OXA in LHA diminished(P < 0.01).Compared with the model group,contents of serum Leptin is downregulated(P < 0.05)whereas OXA in LHA upregulated(P < 0.01)in Xiaoyaosan group..6 OXA,OX1 R and ob-R expression in LHA with immunofluorescent(IF)double stainingOXA and OX1 R are labeled red fluorescence while ob-R labeled green.Statistics show that compared with the control group,the expression of positive cell number and area as well as the integrated optical density(IOD)of the model rats are dropped(P < 0.01),whereas ob-R raised(P < 0.01).Compared with the model group,the indicators mentioned above of Xiaoyaosan group are ameliorated(P < 0.05 or P < 0.01).Results of Co-localization reveal that correlation coefficient(Pearson's correlation)of OXA and ob-R in the model group higher than that of the control group(P < 0.01);compared with the model group,correlation coefficient is descend in the Xiaoyaosan group(P < 0.05).Correlation coefficient of OX1 R and ob-R in the model group is lower than that of the control group(P < 0.01),meanwhile,there is no significant statistical differences between the model and Xiaoyaosan group(P > 0.05).7 OXA m RNA,OX1 R m RNA and ob-R m RNA expression with q RT-PCR detectionCompared with the control group,the relative expression levels of OXA m RNA and OX1 R m RNA in the LHA for the model group is downregulated(P < 0.01),while ob-R m RNA upregulated(P < 0.01).Compared with the model group,the expression levels of OXA m RNA and OX1 R m RNA for the Xiaoyaosan group raised whereas ob-R m RNA downregulated(P < 0.01).Conclusion:1 The reason for the immobilization stress induced the spleen and stomach dysfunction may be related to the expression of OXA and OX1 R descended,ob-R expression up-regulated in LHA,simultaneously the content of serum Leptin raised.The mechanism may be relevant to the peripheral excessive Leptin penetrate blood-brain barrier thus binding to ob-R resulting in the synthesis and secretion of OXA and OX1 R inhibited.2 Xiaoyaosan can regulate the expression of OXA,OX1 R and ob-R in LHA and downregulate the serum Leptin to exert the function of smoothing the liver and tonifying spleen,hereby accommodate the function of spleen and stomach.The regulation may accomplish through the feeding circuit of Leptin-ob-R-OXA/OX1 R. |
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