Establishment Of An In Vitro Model Of Neurovascular Unit In The Hippocampus Of Simulated Diabetes Complicated With Depression And Intervention Of Traditional Chinese Medicine

Objective:1. In vitro hippocampal NVU induced by mimetic DD condition established by glucose combined with corticosterone damage of NE, AS, BM consisting of hippocampal NVU, and model evaluation system from the cell morphology, barrier function, structure and secretory function of multi-angle.2. To explore the therapeutic mechanism of Zuo Gui Jiang Tang Jie Yu prescription, what was a traditional Chinese medicine compound prescription on the cell model of in vitro hippocampal NVU induced by mimetic DD condition, and to further clarify the effect of its treatment of specific mechanisms. This research will reveal the foundations of diabetes mellitus with depression for experimental study.Methods:1. To build a cell model of in vitro hippocampal NVU induced by mimetic DD condition. Isolating and purifing NE, AS, BM established NE + AS, AS + BM, NE +AS + BM three normal control group and NE + AS + G & P, AS + BM + G & P, NE +AS + BM + G & P three model group. Improve the experimental method in our previous work based on the use of glucose(150mmol/L) and corticosterone(200?mol /L) intervention 18 h.(1) morphological assessment: inverted phase contrast microscope to observe cell growth state;(2) Evaluationn of barrier function: cell resistance meter measuring transendothelial resistance value and 4h leak test;(3)Evaluationn of barrier function:western-blotting method to detect structural protein BM Occluding, AS Connexin CX43 expression and cytoskeletal proteins GFAP protein;(4) Evaluationn of secretory function: ELISA was used to detect BM and AS secreted proteins secreted protein LIF, TGF-?1, GDNF, bFGF, ANG1 content; ELISA was used to detect the neurotransmitter 5-HT content; flow cytometry(FCM)detection of apoptosis in each group hippocampal neurons.2. The effect of Zuo Gui Jiang Tang Jie Yu prescription. The in vitro hippocampal NVU induced by mimetic DD condition were randomly divided into five groups: normal group, model group, model + blank serum group, model +(metformin + Prozac) drug-containing serum group, model + Zuo Gui Jiang Tang Jie Yu prescription drug-containing serum group. After drug intervention 18 h,(1)morphological assessment: inverted phase contrast microscope to observe cell growth state;(2) Evaluationn of barrier function: cell resistance meter measuring transendothelial resistance value and 4h leak test;(3) Evaluationn of barrier function:western-blotting method to detect structural protein BM Occluding, AS Connexin CX43 expression and cytoskeletal proteins GFAP protein;(4) Evaluationn of secretory function: ELISA was used to detect BM and AS secreted proteins secreted protein LIF, TGF-?1, GDNF, bFGF, ANG1 content; ELISA was used to detect the neurotransmitter 5-HT content; flow cytometry(FCM) detection of apoptosis in each group hippocampal neurons.Results:1. Compared with normal control group, two or three-cell model cell culture systems group induced by glucose combined with corticosterone, NE, AS, BM cells grew significantly deteriorated; TEER significantly reduce, the level difference of 4h leak test was significantly increased(P <0.05); AS Connexin CX43 and cytoskeletal proteins GFAP and BM tight junction protein occluding significantly decreased(P<0.05); BM and AS secreted proteins secreted protein LIF, TGF-?1, GDNF, bFGF,ANG1 content significantly decreased(P <0.05); and the three cell model group is better the state of cells than and two cell model group the intracellular and cell supernatants 5-HT was decreased significantly(P <0.05); hippocampal neuron apoptosis rate increased(P <0.05).2. Compared with normal control and normal serum group, model group indicators are obvious abnormalities. Compared with the model group, the growth state of the cells ZGJTJYF drug-containing serum group was significantly changed for the better. TEER significantly incresased, the level difference of 4h leak test was significantly reduced(P <0.05); AS Connexin CX43 and cytoskeletal proteins GFAP and BM tight junction protein occluding significantly increased(P <0.05); BM and AS secreted proteins secreted protein LIF, TGF-?1, GDNF, bFGF, ANG1 content significantly incresased(P <0.05); the intracellular and cell supernatants 5-HT wasix incresased significantly(P <0.05); hippocampal neuron apoptosis rate decreased(P<0.05).Conclusions:1. Combined with previous studies by our group and study the results of this experiment, select glucose(150 mmol / L) and corticosterone(200 ?mol / L) as the intervention condition. And combined with DD clinical features and physiological effects of neurovascular unit, using the cell morphology, structure and function of multi-angle indicators may be established in vitro hippocampal NVU induced by mimetic DD condition.2. In vitro hippocampal NVU induced by mimetic DD condition cell morphology,barrier function, structure and secretory function injury, which may be the pathological mechanism of the development of DD.3. Zuo Gui Jiang Tang Jie Yu prescription can improve the hippocampal NVU the growth state of the cells, modulating neurotransmitter secretion disorder, increased hippocampus NVU barrier function, improve the function of the hippocampus NVU associated secretory protein LIF, TGF-?1, GDNF, bFGF, ANG1 expression, up high survival rate of hippocampal neurons. The treatment of Zuo Gui Jiang Tang Jie Yu prescription for diabetes mellitus with depression to be worthy of further research and development.