| Objective: The establishment of diabetes mellitus with depression(DD)and co-culture model mimetic DD condition,on two aspects of in vivo and in vitro,from behavior,biochemistry,molecular biology and pathology,are used to explore the protective effects and mechanisms of Zuogui Jiangtang Jieyu prescription(ZJJP)on hippocampal synaptic damage "Q uad Partite" under DD(state)and to provide new ideas and method for the prevention and treatment of the disease.Methods: 1.To investigate the effects of ZJJP on the damage of "Q uad Partite" synapse in hippocampus of DD rats in vivo: SD rats were randomly divided into 8 groups: normal group,diabetes mellitus group,depression group,DD model group,positive group[fluoxetine(1.8 mg·kg-1)+metformin(0.18 g·kg-1)],and ZJJP of high,medium and low dose(40.5 g·kg-1,20.25 g·kg-1,10.125 g·kg-1)group.The animal model of diabetes mellitus with depression was established by Using STZ intravenous injection(38 mg·kg-1)combined with 28 d in chronic unpredictable mild stress(CUMS),and all drugs were given by intragastric administration during C UMS;changes in body weight and blood glucose level were detected weekly,Morris water maze and Open field test were used to detect behavioral changes,ELISA to tes t the plasm levels of insulin,glycosylated hemoglobin,TNF-α and IDO,HPLC-ECD to detect the contents of NA,DA and 5-HT in the rats hippocampus,immunohistochemistry to show the expression of SYN,PSD-95,GFAP and Iba-1;transmission electron microscope to display ultrastructure of hippocampal synapse,Tunel staining to detect the apoptosis of hippocampal neurons and Western blot to exhibit the expression of NR2 A and NR2 B in hippocampus.2.To explore the effects of ZJJP on the damage of "Quad Partite" synapse under mimetic DD condition in vitro: the co-culture model of "Q uad Partite" synapse was established by mixed culture combined with modified Banker method using neurons,astrocytes and microglia,and the mimetic DD condition was simulated by high glucose(150 m M)associated with corticosterone(200 μM).All established co culture models were randomly divided into 7 groups: normal group,high glucose group,corticosterone group,DD model group,blank serum group,positive drug(fluoxetine + metformin)serum group,ZJJP serum group.the normal group and DD model group were given the same amount of medium,the rest group give the corresponding volume fraction of 10% drug serum or blank serum.After 18 h intervention,light microscope was used to observe the morphological changes directly,high content analysis to detect and analysis the expression of SYN,PSD-95,GFAP and Iba-1 and ELISA to detect TNF-α and IDO levels in the supernatant of the co culture system.Besides,RT-PCR was used to test NR2 A and NR2 B m RNA level and the apoptosis of neurons was detected by Tunel staining.Results: 1.Compared with the normal group,the body weight of DD model rats decreased significantly,which learning and memory ability,times of spontaneous activity and insulin level in plasm were significantly decreased,while blood glucose level and plasm contents of TNF-α and IDO were significantly increased;NE nucleus showed significant shrinking and staining,organelles serious lose,synapse dissolution and blurring boundaries,synaptic vesicles ruptured and the positive expression rates of SYN and PSD-95 were significantly decreased;AS cell body was swelling,chromatin distributed in a lump,and the positive rate of GFAP increased significantly;MG cell expansion,which protuberances reduced or disappeared and the expression rates of Iba-1 rising.The positive area of Tunel staining enlarged,while the expression of NR2 A and NR2 B protein increased significantly.Compared with the model group,ZJJP can alleviate the above trend to some extent,and has a significant protective effect on the Quad Partite synapse in hippocampus of DD rats.2.Compared with the normal group,N E dendrites ruptured or decreased,neural network connection decreased in model group,and the average fluorescence intensity of SYN and PSD-95 protein decreased significantly;there were irregular shape,low degree of integration,massive cell debris and higher fluorescence intensity of GFAP in AS cells;most of MG were circular or merged into a flake,and the fluorescence intensity of Iba-1 was obviously increased.In addition,the positive area of Tunel staining was prominentlyincreased,but the expression of NR2 A and NR2 B m RNA increased significantly.Compared with the model group and blank control group,the serum of ZJJP can relieve the above trend to a certain extent,and has a significant protective effect on the "Q uad Partite" synapse of hippocampus in mimetic DD condition.Conclusions: 1.The activation of glial may be the initial factor of diabetes mellitus with depression;2.There are obvious damages at the morphology,structure and function of hippocampal "Q uad Partite" synapse or its co-culture system in the state of diabetes with depression.3.ZJJP can repair the damages of hippocampal "Q uad Partite" synapse in the simulated DD environment by ma intaining cell morphological structure,protecting neuronal plasticity and antiapoptosis. |
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