| Objective:To observe the changes of leptin receptor（ob-R）-Janus family tyrosine kinases 2（JAK2）/Signal transducer and activator of transcription 3（STAT3）pathway in hypothalamic Arcuate nucleus（ARC）of rats with chronic unpredictable mild stress（CUMS）of liver stagnation and spleen deficiency and the regulatory effect of Xiaoyao Powder.Methods:Sixty rats were fed for one week for adaptation and then randomly divided into 4 groups:the control group,the model group,the Xiaoyao Powder group and the fluoxetine group,with 15 rats in each group and3 rats in each cage.The animal model was established by the method of CUMS,which concluded 9 kinds of stress methods,such as binding for 2 h,tail-clipping for 5 min,ice-water swimming for 5 min,hot water swimming for 5 min,wet environment for 24 h,diurnal reversal for 24 h,fasting for 24 h,tilting for 24 h,noise for 3 h.Two kinds of stress factors were applied in one day and not repeatedly used within the next 48 hours,and models were constructed in the model group,the Xiaoyao Powder group and the fluoxetine group.Every day,rats were given intragastric administration before stress stimulation with 19.27g·kg-1·d-1 of Xiaoyao Powder suspension(calculation according to the average body weight of adults 65 kg dosage,soaked in deionized water,boiled and concentrated,the Xiaoyao Powder suspension 1.927 g·m L-1)or fluoxetine aqueous solution 2 mg·kg-1·d-1（Conversion according to the average body weight of adult 65 kg,dissolved in deionized water）,and the rats in control group and model group were fed with the same volume of normal saline for 6weeks.The body weight and 24-hour food intake of rats were measured before the experiment（day0）and the 7th,14th,21st,28th,35th and 42nd day after the experiment（labeled with day7,day14,day21,day28,day35,day42）.Open field test and sucrose consumption test were also carried out.After the experiment,2%pentobarbital sodium 0.2 m L·100g-1 body weight for anesthesia in rats,then blood and ARC were collected,and the expression of ob-R,JAK2,STAT3 and Suppressor of cytokine signaling 3（SOCS3）in ARC were detected by Quantitative Real-time PCR（q RT-PCR）and Western blot（WB）.Results:1.Changes of body weightCompared with the control group,the body weight of rats in the model group decreased significantly on the 7th,14th,21st,28th,35th and 42nd day of the experiment（P<0.01）.Compared with the model group,the body weight of rats in the fluoxetine group increased significantly on the 7th and 14th day（P<0.01 or P<0.05）,and raised evidently on the 7th,14th,21st and 28th day in the Xiaoyao Powder group（P<0.01 or P<0.05）of the experiment.2.Changes of food intakeCompared with the control group,the food intake of rats in the model group declined obviously on the 7th,14th,21st,28th,35th and 42nd day of the experiment（P<0.01 or P<0.05）.Compared with the model group,the food intake of rats increased significantly on the 21st and 35th day in the Xiaoyao Powder group and the fluoxetine group（P<0.05）.3.Changes of the Open Field Test behaviorCompared with the control group,the total distance in open field of the model group lessened evidently on the 7th,21st and 42nd day of the experiment（P<0.01 or P<0.05）,and the total distance in the central area decreased clearly on the 42nd day（P<0.01）.Compared with the model group,the total distance in the central area and in the open field of each treatment group enhanced,with statistical difference on the 7th day of the experiment（P<0.05）.Compared with the control group,the crossing-grid numbers in the central area of the model group reduced significantly on the 7th,21st,28th and 42nd day（P<0.01 or P<0.05）.Compared with the model group,the crossing-grid numbers in the central area of two treatment groups were enhanced evidently,and there was a statistical difference between the Xiaoyao Powder group and the model group on the 7th day of experiment（P<0.05）.4.Changes of the sucrose consumptionCompared with the control group,the sucrose consumption ratios of the model group decreased significantly on the Day 7th,14th,21st,28th,35th and42nd day of the experiment（P<0.01）.Compared with the model group,the sucrose consumption ratios of Xiaoyao Powder group increased obviously on the 7th,21st,28th,35th and 42nd day of the experiment（P<0.01 or P<0.05）,and raised in the fluoxetine group on the 28th,35th and 42nd day（P<0.01 or P<0.05）.5.Changes of the content of serum D-xyloseThe content of serum D-xylose in rats in the model group was lower than that in the control group（P<0.01）.The content of serum D-xylose of two treatment groups was higher than that of model group with no statistical difference（P>0.05）.6.Changes of ob-R,JAK2,STAT3 and SOCS3 protein expression in hypothalamic Arcuate nucleusThe protein expression of ob-R,JAK2,STAT3 and SOCS3 in the Arcuate nucleus of hypothalamus in the model group was higher than that of the control group（P<0.01）,and the protein expression of ob-R,JAK2,STAT3 and SOCS3in each treatment group was lower than that of the model group（P<0.01）.7.Changes of ob-R,JAK2,STAT3 and SOCS3 m RNA expression in hypothalamic Arcuate nucleusCompared with the control group,the expression of ob-R,JAK2,STAT3and SOCS3m RNA in the ARC of the model group increased obviously（P<0.01 or P<0.05）.The expression of ob-R,JAK2 and STAT3 m RNA in treatment groups reduced significantly than that of the model group（P<0.01or P<0.05）.SOCS3 m RNA was lower than that of model group with no statistical difference（P>0.05）.Conclusion:1.After 6 weeks of chronic unpredictable mild stress,the behaviors in open field test,the sucrose consumption ratios,body weight,the food intake,and the content of serum D-xylose decreased,while Xiaoyao Powder has the regulating effect.Comprehensive analysis indicates chronic unpredictable mild stress for6 weeks can replicate the rat model with liver depression and spleen deficiency.2.The expression of ob-R,JAK2,STAT3,SOCS3 protein and gene in hypothalamic Arcuate nucleus of model rats increased,while Xiaoyao Powder had obvious down-regulation effect,suggesting that the anti-stress effect of Xiaoyao Powder may be related to the regulation of ob-R-JAK2/STAT3pathway in hypothalamic Arcuate nucleus. |
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