The Role Of MGluR5 In The Antidepressant Effects Of Exercise And Metformin

Background:About 3.8%of people worldwide suffer from depression,according to a 2021 report by the World Health Organization.In China,the prevalence of depression is about 95 million,among them,the number of cases is as high as 30%under the age of 18,Studies have found that depression caused by chronic social stress is common.Meanwhile,a growing number of researchers have found that stress causes changes in metabotropic glutamate receptor 5(mGluR5)in the brain.It regulates synaptic plasticity by regulating the number and function of other receptors and activating downstream signaling pathway after activation,which is very important for inhibiting the occurrence and development of depression.In fact,the expression of mGluR5 is significantly decreased in the brain of depressed patients,and the level of mGluR5 in the hippocampus and prefrontal cortex of stress-susceptible mice is significantly decreased,but some studies have shown the opposite.How mGluR5 mediates the occurrence of depression is not clear.A large number of studies have demonstrated that Chronic unpredictable mild stress(CUMS)mice exhibit depressive behavior,and stress causes postsynaptic mGluR5 density imbalance.Both exercise and metformin have the effect of improving depressive behavior.Whether they play the role of improving depressive behavior through mGluR5 and downstream signaling pathways and whether the mechanism of action is the same needs to be further explored and analyzed.Objective:The purpose of this study was to explore and compare the behavioral changes of CUMS mice under treadmill running,metformin and their combined intervention,and to explore and compare the expression and molecular mechanism of mGluR5 and downstream signaling molecules under treadmill running,metformin and their combined action.We propose that possible mechanism of exercise which improves depression.In addition,the effect and mechanism of metformin in the treatment of depression were compared and analyzed,providing reference for the diversity of treatment methods for depression.Methods:In this study,40 mice were used.About 4-5 weeks old,and their weights were 18-20g.In order to ensuring the accuracy of experiment results,the sex of experimental animals is male.In this experiment,C57BL/6 series mice were used,and the microbiology grade was SPF.Experiment groups:(1)CON group(n=8),namely control group,which was fed normally for 8 weeks;(2)CUMS group(n=8),8 weeks stress was accepted by experiment mice,randomly using one stimulus every day,the average frequency of each stimulus reach 6 times;(3)CUMS+EX group(n=8),after 3 weeks of CUMS,the experimental mice received exercise intervention at the fourth week at the same time.The intervention was divided into two parts:1 week of adaptive exercise,4 weeks of formal treadmill training,5 times/week,30min/time,the speed was controlled in 13m/min and the slope was 0°;(4)CUMS+MET group(n=8),after 3 weeks of CUMS,the experimental mice received drug intervention at the fourth week at the same time.Metformin was dissolved in drinking water at the concentration of 5mg/ml,and the mice were given metformin orally for 5 weeks;(5)CUMS+EX+MET group(n=8),after 3 weeks of CUMS,mice began to receive both exercise and drug intervention at the fourth week at the same time.The exercise intervention included 1 week of adaptive exercise and 4 weeks of formal treadmill training.The drug intervention was orally administered metformin at 5mg/ml for 5 weeks.After the experiment,three behavioral methods were used to detect the depressive behavior of the experimental mice.Then,the mice were killed and hypothalamus,cerebral cortex,midbrain and hippocampus tissues were taken.Real-time PCR was used to detect the mRNA expression levels of mGluR5 in hypothalamus,cerebral cortex and midbrain,Homer 1/ERK/CREB and Gadd45g/MEKK4/JNK signaling pathway molecules downstream.The expression levels of mGluR5 and its downstream Homer 1/ERK/CREB and Gadd45g/MEKK4/JNK signaling pathways were detected by Western Blotting.Results:(1)Depressive behavior results:The sucrose preference of CUMS group of sucrose preference test(SPT)significantly decreased compared with CON group,(P<0.05);the immobility time of forced swimming test(FST)significantly increased(P<0.01),the strugglling time significantly decreased(P<0.05);and distance in center of open field test(OFT)highly significant decreased(P<0.01).The immobility time of FST in CUMS+EX group significantly decreased(P<0.05)compared to the CUMS group;the number of poking of OFT significantly increased(P<0.05),the distance in center highly significant increased(P<0.01).The sucrose preference of mice in CUMS+MET group significantly increased compared with CUMS(P<0.05);the immobility time of FST significantly decreased P<0.05);and the number of poking of OFT significantly increased(P<0.05).Compared with CUMS,the number of poking of OFT in CUMS+EX+MET group was significantly increased(P<0.05).(2)mGluR5 mRNA and protein levels:Compared with CON group,mGluR5 mRNA levels of CUMS group were significantly increased(P<0.01).The mRNA level of mGluR5 in the midbrain of CUMS+EX group was significantly decreased in comparison to CUMS group(P<0.01),and the protein level of mGluR5 in the hippocampus was significantly increased(P<0.01).The mRNA level of mGluR5 in the midbrain of CUMS+MET group was significantly decreased in comparison to CUMS group(P<0.01),and the protein level of mGluR5 in the hippocampus was significantly increased(P<0.01).mGluR5 protein expression level of CUMS+EX+MET group highly significant increased(P<0.01)in the hippocampus compared with CUMS group.(3)mRNA and protein levels of Homer1/ERK/CREB signaling pathway molecules:Compared with CON group,the relative protein expression level of p-ERK/ERK highly significant decreased in hippocampus of CUMS group(P<0.01);the mRNA level of BDNF significantly decreased in hypothalamus(P<0.05),and the protein level of BDNF highly significant increased in hippocampus(P<0.01).Compared with CUMS group,Homer1 mRNA level significantly increased in both cerebral cortex(P<0.05)and midbrain(P<0.01)of CUMS+EX group;The relative protein expression level of p-ERK/ERK in hippocampus highly significant increased(P<0.01).ERK mRNA levels highly significant increased in hypothalamus and cerebral cortex of mice in CUMS+MET group in comparison to CUMS group(P<0.01),and the relative protein expression levels of p-ERK/ERK highly significant decreased in hippocampus(P<0.01).Compared with CUMS group,the mRNA level of ERK highly significantly increased in hypothalamus of CUMS+EX+MET group(P<0.01),the relative protein expression level of p-ERK/ERK significantly increased in hippocampus(P<0.05);and the mRNA level of CREB significantly increased in midbrain(P<0.05);The relative protein expression level of BDNF highly significantly decreased in hippocampus(P<0.01);Homer1 mRNA level of CUMS+EX+MET group significantly decreased in cerebral cortex in comparison to CUMS+EX group(P<0.05);and the mRNA level of ERK highly significantly increased in cerebral cortex of CUMS+MET group(P<0.01),The relative protein expression level of p-ERK/ERK highly significantly decreased in hippocampus of CUMS+EX+MET group(P<0.01),and the mRNA level of CREB significantly decreased in cerebral cortex of CUMS+EX+MET group(P<0.05);The mRNA level of BDNF significantly increased in midbrain(P<0.05).The mRNA level of ERK highly significantly decreased in cerebral cortex of CUMS+EX+MET group in comparison to CUMS+MET group(P<0.01),and the relative protein expression level of p-ERK/ERK significantly decreased in hippocampus of CUMS+EX+MET group(P<0.05);The relative protein expression level of BDNF highly significantly decreased in hippocampus(P<0.01).mRNA and protein levels of Gadd45g/MEKK4/JNK signaling pathway molecules:Compared with the CON group,The mRNA levels of Gadd45g and MEKK4(P<0.01)and the mRNA levels of JNK and c-Jun(P<0.05)in CUMS group significantly decreased in the hypothalamus.Compared with CUMS group,mRNA levels of MEKK4 in CUMS+MET group highly significant increased in hypothalamus(P<0.01),Gadd45g protein level increased in hippocampus(P=0.06),mRNA levels of JNK and c-Jun significantly increased in cerebral cortex(P<0.05).Compared with CUMS group,mRNA level of MEKK4 significantly increased in hypothalamus of CUMS+EX+MET group(P<0.05).(4)Correlation analysis results between mGluR5 level and depressive behavior:mGluR5 mRNA level was significantly negatively correlated with the immobility time of FST in hypothalamus(P<0.05).There was a significant negative correlation between the mRNA level of mGluR5 and the sucrose preference of SPT(P<0.05)in the cerebral cortex;and a significant positive correlation between the mRNA level of mGluR5 and the number of poking of OFT(P<0.05).The mRNA level of mGluR5 in midbrain was negatively correlated with the distance in center of OFT(P<0.05).There was a significant positive correlation between the protein level of mGluR5 in hippocampus and the immobility time of FST(P<0.05);a significant positive correlation with the number of poking of OFT(P<0.05),and a significant negative correlation with the distance in center of OFT(P<0.05).Correlation analysis results between mGluR5 level and Homer1/ERK/CREB signaling pathway molecules:There was a significant positive correlation between mGluR5 mRNA level and Homerl in hypothalamus(P<0.05)and in cerebral cortex(P<0.01).There was a significant negative correlation between mGluR5 and ERK mRNA level in midbrain(P<0.05).Homer1 and ERK mRNA levels were significantly positively correlated in hypothalamus(P<0.05)and in cerebral cortex(P<0.01),protein levels of Homer1 and ERK were highly significant positively correlated in hippocampus(P<0.01).There was a highly significant positive correlation between ERK mRNA level and CREB in hypothalamus,cerebral cortex and midbrain(P<0.01),and a highly significant positive correlation between p-ERK and CREB protein level in hippocampus(P<0.01).There was a highly significant positive correlation between CREB mRNA level and BDNF in hypothalamus and cerebral cortex(P<0.01).Correlation analysis results between mGluR5 level and Gadd45g/MEKK4/JNK signaling pathway molecules:mGluR5 mRNA levels were highly significant positively correlated with Gadd45g in hypothalamus,cerebral cortex and midbrain(P<0.01).The mRNA level of Gadd45g was highly significant positively correlated with MEKK4 in hypothalamus and cerebral cortex(P<0.01),and the protein level of Gadd45g was significantly positively correlated with MEKK4 in hippocampus(P<0.05).The mRNA level of MEKK4 was highly significant positively correlated with JNK in hypothalamus,cerebral cortex and midbrain(P<0.01),and the protein level of MEKK4 was significantly positively correlated with JNK in hippocampus(P<0.05).The mRNA level of JNK was highly significant positively correlated with c-Jun in hypothalamus,cerebral cortex and midbrain(P<0.01),and the protein level of P-JNK was highly significant positively correlated with c-Jun in hippocampus(P<0.01).Conclusion:(1)Chronic unpredictable mild stress cause mice’s depressive behaviour.Both exercise and metformin intervention could improve the depressive performance of CUMS mice,and the effect of the two alone was better than the combined effect.(2)Chronic and unpredictable mild stress may alter mGluR5 mRNA and protein levels in the brain.Both exercise and metformin correct abnormal mGluR5 levels in the brain of CUMS mice(3)The depressive behavior in mice caused by chronic unpredictable mild stress may be related to the disorder of Homer1/ERK/CREB and Gadd45g/MEKK4/JNK downstream of mGluR5.Exercise may exerts an antidepressant effect by restoring the abnormal Homer 1/ERK/CREB signaling pathway,and metformin may exert an antidepressant effect by activating the Gadd45g/MEKK4/JNK signaling pathway.