The Effect Of Carnosic Acid On Mouse Model Of Chronic Restraint Stress And The Research Of Occurrence Mechanism

Objective:Chronic stress event is an important factor in the onset of depression.It is a hot spot to study the mechanism of stress induced depression and find new markers of depression,then develop new antidepressant drugs.Fibroblast growth factor?FGF?family is a kind of cytokines which play a wide range of biological functions through FGF receptors,and FGF9 has been reported to be closely related to depression.In this study,we established a model of depression induced by chronic restraint stress?CRS?in mice,and to explore whether chronic stress leads to the change of FGF9 and its receptor FGFR3 pathway.On the basis of this,we evaluated whether the anti depression effect of the natural medicine CA were related to the regulation of FGF9-FGFR3 pathway.Methods:?1?The establishment of chronic restraint stress mouse model:52 male ICR mice,weight 18.2?21.5g,except the 20 mice of normal control group,the rest of the mice are subjected to restraint stress in the 50ml centrifuge tube.The diameter of the tube is 3.0cm and about10cm long,the wall of the tube have 6?7 vents which diameter are 0.5mm,restraint for 10h everyday,during the restraint time the animal fasting water,the rest of the time feeding.28 days after modeling,according to the sucrose preference results animals are randomly divided into restraint model group and remove restraint group,The restraint model group continued to be subjected to restraint and separated until the 56^th day of modeling,remove restraint group no longer restraint and separated.After 28 days and 56 days respectively,the animals in each group were subjected to behavior test include autonomic activity,preference of sugar syrup and tail suspension test.ELISA method was used to detect the content of fibroblast growth factor 9?FGF9? and fibroblast growth factor2?FGF2? in serum and hippocampus,RT-PCR method was used to detect the expression of FGF9 in hippocampus,immunohistochemical and histopathological examination of the hippocampus was performed,Westernbotting detection of the growth factor receptor 3?FGFR3? in hippocampus tissue.?2?The effect of carnosic acid on the model of chronic restraint stress in mice:100 male ICR mice,weight 18.5?21.8g,except the 20 mice of normal control group,the rest of the mice were subjected to restraint?Method is the same as before?,28 days after modeling,according to the sucrose preference results animals are randomly divided into restraint model group,fluoxetine group,low?25mg/kg?,medium?50mg/kg?,high?100mg/kg?dose groups of carnosic acid,continue subjecting to restraint and sepatating after grouping.The normal control group and the restraint model group were given distilled water every day after the grouping,and the other groups were given the corresponding medicine respectively.The volume was 20ml/kg,and the drug was given for 28 days.After grouping and the last administration,the animals were tested with the same behavioral and physiological indexes.Results:?Weight:Compared with the normal control group,the weight of the restraint model group was significantly reduced after 28days and 56 days?P<0.01?;Compared with the normal control group,there was no significant difference in the weight of the mice.?The spontaneous activity test:After modeling for 28 days and 56 days,the wall climbing number and activity time of restraint model group mice was significantly reduced?P<0.05 or P<0.01?;Compared with the normal control group,the number of wall climbing and activity time increased significantly?P<0.05 or P<0.01?in the 56^th day of remove restraint group.? Preference of sugar syrup:28 days and 56 days after modeling,the degree of preference of sugar in the restraint model group was decreased significantly?P<0.01?,and compared with the restraint model group,there was no significant difference in the preference index of sugar syrup in the remove restraint model group for 28 days and 56 days.? Tail suspension test:Compared with the normal control group,the activity ratio of the model was significantly reduced at 28^th and 56^th day?P<0.01 or P<0.05?;Compared with the normal control group,there was no significant difference in the activities of the remove restraint group on 28^th day and 56^th day.? At the 56^th day after modeling,the hippocampus cells of normal control group were closely arranged,and the cells of the vertebral body were normal.The restraint group in hippocampus cells arranged in disorder,part of vertebral cell necrosis.In the mice of the remove restraint group,the hippocampal cells were arranged in disorder and a small amount of necrosis was observed.? The mouse serum,hippocampus FGF9,FGF2 content:compared with normal control group,serum and hippocampus FGF9 in 28^th day and 56^th day of restraint model group increased significantly?P<0.01?,the content of FGF2 was decreased?P<0.01 or P<0.05?;And compared with restraint model group,mice in 28^th day,56^th day serum and hippocampus FGF9,FGF2 content had no significant difference.? The expression of FGF9 in hippocampus was detected in the RT-PCR:Compared with normal control group,the expression level of FGF9 of restraint model group and remove restraint group animal on 28^th and 56^th day was increased significantly?P<0.05? on 56^th day;Compared with restraint group,the expression of FGF9 had no significant difference on 56^th day.? The hippocampus immunohistochemical examination:Compared with normal control group,the expression of FGFR3 decreased significant in the restraint model group?P<0.01?,and compared with restraint model group,FGFR3 content in hippocampus of mice in remove restraint group were not changed significantly.? The expression of FGFR3 Western blotting detection in the hippocampus:Compared with normal control group,after56 days of modeling,the expression of FGFR3 protein in restraint model group in hippocampus decreased?P<0.05?;And compared with restraint model group,hippocampal FGFR3 protein expression level of remove restraint group is up-regulated,but no significant difference.?2?The effect of carnosic acid on the model of chronic restraint stress in mice:High dose of 100mg/kg could significantly increase the body weight of mice?P<0.05?;The number of wall climing in the low?25mg/kg?,medium?50mg/kg?and high?100mg/kg? dose groups was increased significantly?P<0.01?;The activity time in high?100mg/kg?dose groups was increased significantly?P<0.05?;The preference index and activity ratio of medium?50mg/kg? and high?100mg/kg? dose mice were increased significantly?P<0.05?;The content of FGF9 in serum and hippocampus of medium?50mg/kg? and high?100mg/kg? dose group mice decreased significantly,and the content of FGF2 in serum and hippocampus was significantly higher?P<0.05?;The expression of FGF9 mRNA in middle and high dose of carnosic acid decreased significantly?P<0.05?;In the low,middle and high dose groups,the cells in the hippocampus of the mice in the nomal control group were more compact than those in the restraint model group;The expression of FGFR3 in hippocampus was significantly increased?P<0.05?.Conclusion:depression induced by chronic restraint stress is related to the change of FGF9-FGFR3 pathway in hippocampus,and FGF9 may be a new biomarker of depression.the natural medicine carnosic acid can obviously improve the depression state of mice induced by chronic restraint stress,and its antidepressant effect is related to the regulation of FGF9-FGFR3 pathway.In this study,we propose a new mechanism of depression induced by chronic stress,which provides a new idea for the development of antidepressant drugs.