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Alzheimer's disease(AD) patients in China have been surging, and the resultant medical burden and care demand have a huge impact on the development of individuals, families, and the society. The active component compound of Epimedii Folium, Astragali Radix, and Puerariae Lobatae Radix(YHG) can regulate the expression of iron metabolism-related proteins to inhibit brain iron overload and relieve hypofunction of central nervous system in AD patients. Hepcidin is an important target regulating iron metabolism. This study investigated the effect of YHG on the expression of a disintegrin and metalloprotease-17(ADAM17), a key enzyme in the hydrolysis of β amyloid precursor protein(APP) in HT22 cells, by mediating hepcidin. To be specific, HT22 cells were cultured in vitro, followed by liposome-mediated siRNA transfection to silence the expression of hepcidin. Real-time PCR and Western blot were performed to examine the silencing result and the effect of YHG on hepcidin in AD cell model. HT22 cells were randomized into 7 groups: control group, Aβ25-35 induction(Aβ) group, hepcidin-siRNA(siRNA) group, Aβ25-35 + hepcidin-siRNA(Aβ + siRNA) group, Aβ25-35+YHG(Aβ+YHG) group, hepcidin-siRNA+YHG(siRNA+YHG) group, Aβ25-35+hepcidin-siRNA+YHG(Aβ+siRNA+YHG) group. The expression of ADAM17 mRNA in cells was detected by real-time PCR, and the expression of ADAM17 protein by immunofluorescence and Western blot. Immunofluorescence showed that the ADAM17 protein expression was lower in the Aβ group, siRNA group, and Aβ+siRNA group than in the control group(P<0.05) and the expression was lower in the Aβ+siRNA group(P<0.05) and higher in the Aβ+YHG group(P<0.05) than in the Aβ group. Moreover, the ADAM17 protein expression was lower in the Aβ+siRNA group(P<0.05) and higher in the siRNA+YHG group(P< 0.05) than in the siRNA group. The expression was higher in the Aβ+siRNA+YHG group than in the Aβ+siRNA group(P<0.05). The results of Western blot and real-time PCR were consistent with those of immunofluorescence. The experiment showed that YHG induced hepcidin to up-regulate the expression of ADAM17 in AD cell model and promote the activation of non-starch metabolic pathways, which might be the internal mechanism of YHG in preventing and treating AD.
Subject(s)
Humans , ADAM17 Protein , Alzheimer Disease/genetics , Amyloid beta-Peptides , Drugs, Chinese Herbal/pharmacology , Hepcidins/genetics , PuerariaABSTRACT
Objective::To observe the effect of modified Dihuang Yinzi on the learning and memory ability and on the neurons in CA1 area of hippocampus of rats suffering from vascular dementia. Method::The 84 male SD rats were randomly selected to form the sham operation group of 12 rats, and the other 72 rats were chosen for the vascular dementia model by means of Bivascular occlusion, and among which 60 were chosen randomly into 6 groups of 12 rats, namely, the model group, the nimodipine group (0.011 g·kg-1), and the high, medium and low dose modified Dihuang Yinzi (4.54, 2.27, 1.14 g·kg-1) respectively. After 30 days of continuous gavage, Morris water maze was used to detect the learning and memory ability of rats, hematoxylin eosin (HE) was used to observe the morphological changes of hippocampal CA1 neurons, transmission electron microscopy was used to observe the ultrastructural changes of hippocampal CA1 neurons, TUNEL was used to detect the apoptosis level of hippocampal CA1 neurons, immunohistochemical(IHC) was used to detect the expression level of phosphatidylinositol-3(PI3K), protein kinase B (Akt), Caspase-3 in hippocampal CA1 tissues. Result::Compared with sham operation group, the escape latency of the model group was significantly prolonged, the number of times of crossing the original platform was significantly reduced (P<0.01), the neuronal morphology of hippocampal CA1 area was damaged to varying degrees, the apoptosis rate was significantly increased (P<0.01), the integral optical density and average optical density of PI3K, Akt were significantly reduced (P<0.01), and the integral optical density and average optical density of Caspase-3 were significantly reduced (P<0.01). Compared with model group, the escape latency was shortened (P<0.05, P<0.01), the number of crossing the original platform was increased (P<0.05, P<0.01), the integrated optical density and average optical density of PI3K and Akt were significantly increased (P<0.01), and the integrated optical density and average optical density of Caspase-3 were decreased in different degrees (P<0.05, P<0.01). Conclusion::Modified Dihuang Yinzi can improve the learning and memory ability of vascular dementia model rats and the damaged neurons in the hippocampal CA1 area. The potential mechanism may be related to the activation of PI3K/Akt signal transduction pathway and the inhibition of apoptosis of neurons in hippocampal CA1 area of rats.
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Aim To explore the best method of neural stem cellextraction and culture, and provide a technical reference for thebasic research of neural stem cells. Methods Different extractionand culture methods of neural stem cells were compared.The rate of ball of neural stem cells and the stability of neuralstem cells in undifferentiated state were observed by extraction offetal and neonatal rats cortex, using different types of medium,different inoculation density, different culture methods, differentmethods of changing liquid and different passage methods. Neuralstem cells' activities were detected by Varioskan LUX MultimodeMicroplate Reader. Results ① The brain cortex of fetalrats of 14 d had higher proportion of neural stem cells, less othercells and more neurospheres than newborn rats of 24 h. ② Neuralstem cells could be stabilized in undifferentiated state by usingserum-free medium, while most of the neural stem cellswere differentiated into neurons and glial cells by using serummedium. ③ Neural stem cells, seeding at 1. 0 ×109 ·L-1 , hada large number of neurosperes and were in good condition. ④Suspension culture was beneficial to form a stable neurosphereand keep the neural stem cells in an undifferentiated state thanadherent culture. ⑤ The state of neurosphere by changing halfof the medium and adding medium without discarding was betterthan that by replacing all medium. ⑥ The neurospheres couldbe separated into single cells by mechanical blow in primary generationand the second generation of neurospheres cultured in serum-free medium. While the percentage of viable cells in neuralstem cells was the highest digested with stem cell lysates afterthree generations and the neurospheres re-formed were better. ⑦Neural stem cells' activity of 14 d fetal rat in Accutase digestiongroup was significantly higher than that of the other threegroups, and the difference was significant (P <0. 05). ConclusionsNeural stem cells proliferate and divide well, with highrate of ball formation and good neurosphere condition, which canmaintain a stable undifferentiated state by extracting the cerebralcortex of 14 d fetal rats, using serum-free medium, inoculatinginto a 25 cm2 flask at a density of 1. 0 × 109 ·L-1 , and takingthe suspension culture (adding the medium 1 ~ 1. 5 mL every 2~3 d and passage every 6 ~8 d ).
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Stroke,especially ischemic stroke,is a serious threat to human health due to its high morbidity,le-thality and disability.Cerebral ischemia-reperfusion injury is an important pathophysiological process of ischemic stroke.As an important cell stress protein,DNA damage-inducible transcript 4(DDIT4)protein plays an important role in cerebral is-chemia-reperfusion injury,and it provides a new target for the study and treatment of cerebral ischemia -reperfusion injury. This review will focus on the structure and expression of DDIT 4,and its latest research progress on the regulation of auto-phagy in cerebral ischemia-reperfusion injury.
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The study is to investigate the effect of angiotensin II (Ang II) and its receptor blockers on migration and endothelin-1 (ET-1) expression of rat vascular adventitial fibroblast subpopulations. Vascular adventitial fibroblasts were individually expanded by using cloning rings, and the effects of Ang II on the migration of adventitial fibroblast subpopulations were evaluated by Transwell. Fluorescence quantitative-PCR detected the expression of preproET-1 mRNA induced by Ang II, and its receptor antagonists losartan and PD-123319. The concentration of ET-1 was determined by ELISA. It showed that spindle shaped and epithelioid shaped cells were isolated by using cloning rings, named as spindle cells and round cells. RT-PCR showed that fibroblast subpopulations did not have leukocytes, endothelial cells and smooth muscle cells, namely pure cell lines. Compared with respective control cells, two subpopulations had transferring ability. Ang II significantly improved round cells migration in a concentration-dependent manner, and had no obvious influence on spindle cells migration. Ang II (1 x 10(-8) - 1 x 10(-6) mol x L(-1)) significantly increased the expression of preproET-1 mRNA in round cells (P < 0.01), and had no significant effect on the expression of preproET-1 mRNA in spindle cells. Losartan blocked the expression of preproET-1 mRNA induced by Ang II in round cells, and had no significant effect on the expression of preproET-1 mRNA in spindle cells. The effects of Ang II and ET-1 receptor inhibitors on the release of ET-1 were similar to the expression of preproET-1 mRNA. The results indicate that there are two cell subpopulations: round cells and spindle cells in rat vascular adventitial fibroblasts. Ang II significantly improved cells migration, and increased the expression of ET-1 in round cell subpopulation. It suggested that there may be different migratory mechanisms in two cell subpopulations, and the two subpopulations may play a different role in vascular remodeling and reparative process.