Treatment Of Diabetic Dementia By Reversing Neurovascular Depolarization And

1. An oxidative injury model in cultured macrophageObjective:To establish culture oxidative injury model in macrophage.Methods:Culture mediums contain in different concentrations of D-gal (10-103 mM, k=0.5) for 48-72h,k-0.56. MTT,LDH were detected. Median effect duration (ED50) of D-gal induced injury in macrophage was calculated to confirm an optimum observing window. And the median effect dose (IC50) of D-gal induced injury in macrophage was analyzed at various endpoints.Results:D-gal leads to oxidative injury in RAW246.7.The values of IC50 were 171.7 mM,62.5-471.6mM,R=0.97, y=0.7213+0.9747/(1+10(5.5964-2.504x))] at 48h endpoint. Conclusion:A model of oxidative stress in macrophage with D-gal was successfully established for evaluating in diabetic dementia.2.A diabetic dementia model in vitroObjective:To set up a diabetic dementia (DD) model in endothelia injury from co-cultured macrophage.Methods:A co-culture of endothelia and macrophages was used in this study. (1)Co-culture:At various primary culture densities and durations, the co-cultured were established with HUVEC (1035-106/ml, k=0.316; 36-384h,k≈0.75)and RAW246.7 (102-5-105/ml, k=0.316;36-384h,k≈0.75), respectively. The contact inhibition of each culture was evaluated through nuclear count in HE staining, cells were counted, the median primary culture density or duration (y) was regressed from the contact inhibition (x). (2)Direct endothelial injury:In 96 well plate, HUVEC direct injury (1.68×104/ml) was induced with D-gal at various concentrations (10-103mM, k=0.56) and culture durations (24-72h, k≈0.56). MTT was detected. The designed observing window of culture endothelia might be ranged from the linear equation regressed from the values of MD50(/ml) and MD50(h). (3)Indirect endothelial injury:In 96 well plate, after HUVEC (1.06×104/ml) being cultured for 24 hours, RAW246.7 at various densities (1.7×10-1.0×105/ml, k=0.56) was further added into each well previous containing endothelia with fixed oxidative stress (171.7mM D-gal) for 96h. With cellular activity was detected.With the level of ET-1 or NO was detected as biomarkers in neurovascular uncoupling. The median duration (DD50) to induce indirect endothelial injury (x) was regressed.The density of culture macrophage might be designed from the linear equation regressed from the values of MD50 and the durations.Results:(1) Co-culture:HUVEC primary culture density is 3.16×104/ml. (2)Direct endothelial injury:The midian concentration of D-gal (EC50) to induce endothelial injury was 277.3 mM [y=-0.02509+1.1191/(1+10(5.245-2.147x)),R=0.99] at 48h as its endpoint. (3)Indirect endothelial injury:The median density (ED50) is 1.33×104/ml [y=0.1104+1.0846/(1+10(0.15933-1.559x)), R=0.96] at 144h as its endpoint. As a DD model in vitro, after HUVEC (1.06×104/well) being cultured for 24 hours, RAW246.7 at density of 1.33×104/ml was further added into each well previous containing endothelia with fixed oxidative stress (171.7mM D-gal) for 96h.Conclusion:A DD model in vitro was defined as the endothelial injury from the expression of aldose reductase in co-cultured macrophage, which increased release of factors to contract vessels.3. Restoring neurovascular uncoupling of Gouteng Yin for diabetic dementiaObjective:To evaluate the effective molecule from Gouteng Yin for DD.Methods:Using the oxidative model in co-culture with macrophages and endothelia for evaluating DD candidates,6 molecules from Gouteng Yin were added into the DD model at 7 dosages, respectively, apigenin (102-104nM, k=0.56), rhynchophylline, chlorogenic acid, luteolin, paeonol (102-105nM, k=0.56), rutin (104-106nM, k=0.56). Cell activity was detected with MTT assay, the levels of NO or ET-1 in supernatant was determined by Griess Reagent or ELISA. The median effective concentration (IC50) of each molecule to protect DD model in co-culture was analyzed with GraphPad Software (GraphPad Prism V5.01).Results:Basing in the oxidative model in co-culture, the effect of 6 molecules from Gouteng Yin was confirmed as dose-effective relationship. Each of 6 molecules has been effective acting on ET-1 release from injured endothelia in co-culture with oxidative macrophages. The value of IC50 on NVUC was apigenin 0.6147 nM [y= 0.2783+0.601/(1+10(-1.09+5.156x)),R=0.97], rhynchophylline 1.322 nM [y=0.9324/(1+ 10(0.1558-9.544x)),R=0.99], chlorogenic acid 1.766nM [y=0.080+0.818/(1+10(0.7921-3.208x)),R=0.98], paeonol 3.339nM [y=0.0673+0.867/(1+10(8.0844-15.44x)), R=0.99], luteolin 9.387nM [y=0.1762+0.8238/(1+10(15.5989-16.04x)),R=0.97], or rutin 103.7nM [y=0.1415+0.8494/(1+10(24.2726-12.04x)), R=0.97]Conclusion:Six molecules from Gouteng Yin were effective on DD in co-culture, especially on ET-1 released level, such as apigenin, rhynchophylline, chlorogenic acid, paeonol, luteolin and rutin.4.Target-guiding mechanism of Gouting Yin on diabetic dementiaObjective:To study AR-guiding mechanisms of Gouteng Yin on DD.Methods:(1)In vitro:Basing in the oxidative model in co-culture, the regulation of each effective molecule [chlorogenic acid, luteolin, paeonol, rhynchophylline, apigenin, or rutin] from Gouteng Yin on AR molecule dynamics, was analyzed as its existed content in situ with immunohistochemical staining, genic expression with real time RT-PCR, and protein level with Western blot, respectively. The median inhibitory concentration (IC50) of each molecule to protect DD model in co-culture was analyzed with GraphPad Software (GraphPad Prism V5.01). (2)In vivo:db-/db-mice (7 week) were divided into nine groups, as control mice were fed standard diet, model mice did fatty diet, therapy mice did Gouteng Yin at each of seven dosages(10-104 mg/kg/d, k=0.316). The pathological changes of hippocampus were observed in HE staining, AR and SOD3 were qualified with the morphometry in immunohistochemical staining sections. The median effective dose (ED50) of Gouteng Yin to restore AR and SOD3 existed content in situ was analyzed with GraphPad Software (GraphPad Prism V5.01).Results:(1) Six molecules from Gouteng Yin have some inhibitory effect on AR expression, as decreasing existed level in situ, down-regulating gene expression, and reducing protein amounts of AR on the diabetic dementia model in vitro. (2)Gouteng Yin decoction restored AR and SOD3 molecule dynamics in db-/db- mice fed with fatty diet. It decreased AR existed levels, increased SOD3 existed levels in the early phage of diabetic dementia model in vivo.Conclusion:(1) Six effective molecules from Gouteng Yin relive the diabetic dementia via suppressing AR levels. (2)Gouteng Yin prevents diabetic dementia via decreasing oxidation, moderating AR/SOD3 of neurovascular unit in db-/db- mice.