Mechanism Responsible For The Effect Of Exercise On Diabetes-related Depressive-like Behavior

Objective:Depression is one of the complications of diabetes.Up to now,about one third of diabetic patients in the world are accompanied with depression.However,the specific mechanism of diabetic depression has not been clarified.Although evidences have been suggested that the activation of NLRP3 inflammasome is involved in diabetic cardiomyopathy and depressive behavior,it is not known whether the activation of NLRP3 inflammasome is associated with the pathogenesis of diabetic depression.Exercise,a simple and easy way of intervention,has a certain therapeutic effect on diabetes and depression.Whether exercise can delay the pathological process of diabetic depression remains to be further explored.The main purpose of this paper is to explore the role of NLRP3 inflammasome in the process of diabetic depression and its specific pathogenesis,and explain whether exercise can improve diabetic depression.Methods:In the first part,STZ was used to induce type I diabetes mellitus.Four weeks later,?1?Behavioral tests were carried out on wild type and NLRP3 knockout diabetic mice;?2?The expression of NLRP3 inflammasome-related protein and apoptotic/pyroptotic proteins in hippocampus tissue of mice were detected by Western Blot and Elisa;?3?In situ,co-staining of neuronal apoptotic protein was observed by immunofluorescence histochemistry;?4?TUNEL kit was used to detect apoptosis in hippocampus of mice.At the cellular level:HT22 cells cultured with high glucose was used,?1?Methods of Western Blot and Elisa were used to detect the expression of NLRP3 inflammasome-related protein,and apoptotic/pyroptotic proteins in HT22cells.?2?TUNEL kit was used to detect the apoptosis of HT22 cellsThis part mainly explores the activation of NLRP3 inflammasome and its downstream in the process of diabetic depression.In the second part,we will carry out treadmill exercise training and exogenous NaHS supplementation on diabetic mice on the basis of the above model.At the end of the model cycle,?1?behavioral tests were conducted on diabetic mice with exercise intervention and NaHS intervention;?2?Western Blot were used to detect the related factors of inflammation/IDO pathway or the expression of H2S-related enzymes with exercise intervention,and the expression of IDO in the hippocampus of diabetic mice with NaHS intervention in the hippocampus of diabetic mice.At the cellular level:Western Blot was used to detect the expression of IDO in glial cells after NaHS intervention We will further to explore whether exercise could improve diabetic depression-like behavior in the hippocampus by restoring the expression of H₂S.Results:Part ?:The e?ffect of diabetes-induced inflammation in the hippocampus on depressive behavior and its mechanism?.Diabetes induce depressive-like behavior with activation of NLRP3inflammasome1.STZ induces depressive behavior in diabetic miceThe model of STZ-induced type I diabetes mellitus was successfully constructed for 4 weeks.Then,the depressive behavior of diabetic mice was detected.During open field test,the central distance,central/peripheral distance,central time,central/peripheral time and the number of times of crossing the center in diabetic mice were decreased,while the immobility time in forced swimming and tail suspension experiment were increased.2.NLRP3 inflammasome was activated during diabetic depressionWestern and Elisa was used to detect the expression of NLRP3 inflammasome in hippocampus of diabetic mice.The proteins of NLRP3 and caspase 1.and the expressions of IL-1?and IL-18 in hippocampus of diabetic mice increased.3.The expression of NLRP3 inflammasome increased in HT22 cells induced by high glucoseWestern and Elisa were used to detect the expression of NLRP3 inflammasome in HT22 cells.With the increase of high glucose concentration,the expression of NLRP3and caspase 1 increased in a dependent manner.Elisa results also showed that the expression of IL-1beta and IL-18 increased in a concentration-dependent manner.?.High glucose induces activation of NLRP3 inflammasome in hippocampal neurons by oxidative stress1.High glucose increases ROS expressionOn animal model,ROS production in hippocampus of diabetic mice was detected by intravenous injection of ethidium dihydro.At the cell level,ROS level in neurons was detected by fluorescent probe 2,7-Dichlorofluorescein diacetate.The results showed that the expression of ROS positive results increased in the high glucose group compared with the control group.2.High glucose activates NLRP3 inflammasome in hippocampal neurons through oxidative stressThe cells were treated with oxidative stress scavengers,and the expression of NLRP3 inflammasome in HT22 cells was detected by Western.Compared with the high glucose group,the expression of NLRP3 and caspase 1 in HG+NAC group was significantly decreased,and the expression of IL-1?and IL-18 was also decreased,which indicated that high glucose induced the activation of NLRP3 inflammasome through oxidative stress.?.NLRP3 knockout reverses the increase of pyroptosis/apoptosis in hippocampus of STZ-induced diabetic mice1.The levels of apoptosis and pyroptosis were increased hippocampus of diabetic miceWestern assay was used to detect apoptotic proteins such as p53,Bax,Puma and pyroptotic proteins such as GSDMD.The expression of apoptotic protein and apoptotic protein increased.At the same time,immunofluorescence was used to detect the co-staining of neuron-labeled protein NeuN and apoptotic effector protein cleaved caspase 3 in situ.The co-staining expression increased in hippocampal slices of diabetic mice.DNA fragmentation in hippocampus was detected by TUNEL kit.The results showed that TUNEL positive cells increased significantly in diabetic mice.2.NLRP3 knockout can alleviate pyroptosis and apoptosis in hippocampus of diabetic miceOn the basis of the above part,we detected apoptosis and pyroptosis in hippocampus of diabetic mice of NLRP3 knockout.The results showed that compared with diabetic group,the expression of apoptotic-related proteins and the expression of GSDMDN significantly decreased in diabetes+NLRP3-/-group,the co-staining of NeuN and cleaved caspase 3 decreased in situ,the positive cells stained by TUNEL decreased.?.High glucose induces pyroptosis/apoptosis of HT22 in hippocampal neurons in NLRP3-dependent manner1.High glucose induces pyroptosis and apoptosis of hippocampal neuronal cellsWestern and Elisa were used to detect the expression of p53,Bax,Puma,GSDMD and other related apoptotic and apoptotic proteins,as well as IL-1?,IL-18and LDH.The expression of pyroptotic and apoptotic proteins increased in the high-glucose group.Meanwhile,TUNEL kit was used to detect DNA breakage in hippocampal neurons.The results showed that TUNEL positive cells in hippocampal neurons increased significantly in high glucose group.2.NLRP3 inhibitors reverse pyroptisis and apoptosis induced by high glucoseWe treated hippocampal neurons with NLRP3 inhibitor MCC950 and collected cells 48 hours after high glucose treatment.Western and Elisa results showed that the expression of apoptotic and pyroptotic proteins and the release of IL-18,IL-1?and LDH were significantly decreased in MCC950+high glucose group.Meanwhile,the number of TUNEL positive cells decreased.3.NLRP3 interference can reverse the apoptosis and pyroptosis by high glucoseTo further explore the role of NLRP3 in carbohydrate-induced apoptosis and pyroptosis,NLRP3 siRNA was used to treat cells,and the interference efficiency was about 70%.Western and Elisa results showed that the expression of apoptotic and pyroptotic proteins and the release of IL-18,IL-1?and LDH were significantly decreased in NLRP3 siRNA+high glucose group.Meanwhile,the number of TUNEL positive cells were decreased.These results suggest that NLRP3 inflammasome play an important role in the apoptosis and pyroptosis in hippocampal neurons induced by high glucose.?.NLRP3 knockout can alleviate depression-like behavior in diabetes mellitusSTZ was injected to wild-type mice and NLRP3 knockout mice,and the behavior was tested after 4 weeks of successful modeling.Compared with diabetic group,the immobility time of NLRP3-/-+diabetic mice in tail suspension test and forced swimming test decreased significantly,and the central distance,central/peripheral distance,central time,central/peripheral time and the times of crossing the center increased significantly in the open field test.Part?:The effect of exercise on depressive behavior of diabetes mellitus and its mechanism?.Exercise improves STZ-induced diabetic depression-like behaviorExercise was intervened after the establishment of STZ-induced diabetic model by for 4 weeks.After 4 weeks,the depression-like behavior of four groups of mice was detected.In the diabetes+exercise group,the central distance,central/peripheral distance,central time,central/peripheral time and the number of times of crossing the center were increased in the open field test,while the immobility time decreased in the forced swimming test and tail suspension test.?.Exercise inhibits activation of NLRP3/IDO pathway in hippocampus of STZ-induced diabetic miceWestern was used to detect the expression of IDO in hippocampus of diabetic mice and the proteins of NLRP3/IDO in hippocampus of mice after exercise.It was found that the expression of IDO in hippocampus of diabetic mice increased while the expression of NLRP3 and IDO in diabetic+exercise group decreased significantly.This suggests that exercise inhibits the activation of NLRP3/IDO pathway in the process of diabetic depression.?.Exercise improves the expression of H₂S-related enzymes in hippocampus of STZ-induced diabetic mice1.Reduction of H₂S-related enzymes expression in hippocampus of diabetic mice induced by STZThe expression of CBS in hippocampus of diabetic mice was detected by Western.The result shows that CBS in hippocampus of diabetic mice was decreased.2.Exercise improves the expression of H₂S-related enzymes in hippocampus of STZ-induced diabetic miceWestern detected CBS expression in hippocampus of diabetic mice after exercise.Compared with diabetic group,CBS expression in hippocampus of diabetic+exercise group was increased.?.Exogenous H₂S supplementary relief of STZ-induced diabetic depression-like behaviorExogenous H₂S supplementary was intervened after the establishment of STZ-induced diabetic model by for 4 weeks.After 4 weeks,the depression-like behavior of four groups of mice was detected.The results showed that compared with the diabetic group,the central distance,central/peripheral distance,central time,central/peripheral time and the times of crossing the center of diabetic+H₂S mice were increased in the open field test,while the immobility time in the forced swimming test and tail suspension test were decreased.?.Exogenous H₂S supplementation alleviated the increase of IDO expression induced by high glucoseWestern detected the expression of IDO in hippocampus of mice,and found that the expression of IDO in diabetes+H₂S group was decreased.Similarly,at the cellular level,we treated glial cells cultured in high glucose with different concentrations of NaHS,and found that the expression of IDO decreased in a concentration-dependent manner with the increase of NaHS concentration.Conclusion:NLRP3 inflammasome was activated in hippocampus by oxidative stress in STZ-induced type 1 diabetes.It leads to pyroptosis and apoptosis in hippocampal neurons,thus inducing depression-like behavior in diabetic mice.Exercise,an intervention,can inhibit the inflammation/IDO pathway in hippocampus by restoring the expression of H₂S,delaying the pathological process of diabetic depression.