Effect And Mechanism Of Bajijiasu Nuezhenoside And Icariin Having Action Of Reinforcing Kidney On Alzheimer’s Disease

Objective:Alzheimer’s disease (Alzheimer’s disease, AD) is a primary neurodegenerative disease, which is characterized by amyloid pathology (Amyliod-protein, Aβ) deposition formation of senile plaques (senile plaques, SPs), Neurons neurofibrillary tangles (neurofibrillary tangles, NFTs) and specific cholinergic neurons in a large number of death and loss, etc.The imbalance of Aβ with production and degradation mechanism is now more recognized in the pathogenesis of Alzheimer’s disease. At present, it reported that Bajijia su can improve Aβ25-35 injury model memory in mice; less literature on privet glycosides with anti-oxidative stress.This study aims to investigate protective effect of nuezhenoside Bajijiasu and Icariin on Aβ2-induced SH-SY5Y cell damage and the underlying mechanism.Methods1. Screening for optimum damage Aβ42 concentration SH-SY5Y cells, and then establish a stable cell injury.2. Endothelial brain cells and SH-SY5Y cells were cultured with BaJiJiaSu Nuezhenoside and Icariin of different concentration to choose the appropriate concentration for subsequent experiments.3. screened the effect of BaJiJiaSu, Nuezhenoside and Icariin by MTTand Changing of cell morphology.4. choose the protection of three of active components, and explore mechanisms.4.1 exploring mechanisms of protection of BaJiJiaSu on Alzheimer’s disease4.1.1 It is verified that the protective effect of Bajijiasu on Aβ42-induced SH-SY5Y cells neurotoxicity by MTT assay.4.1.2 SH-SY5Y cells were cultured with Bajijiasu for 12h,then LRPland APP expression was determined by Western Blot, and changes in real-time PCR detection of SH-SY5Y cells LRP1 mRNA expression. Aβ42 in intracellular levels of extracellular was determined by ELISA; it was observed the translation of Aβ42 after entering cells and co-localization with the lysosomal by confocal.4.1.3 Select 7-month-old SPF male APPswe/PSldE9 58 double transgenic mice [strain name: B6C3-Tg (APPswe, PSENldE9) 85Dbo/J (abbreviation: APPSWe/ PSldE9), C57BL mice of the same background/6J. Double transgenic mice were randomly divided into model group, Bajijiasu low-dose (20mg/kg) group, Bajijiasu high dose (80mg/kg) groups 10 only; background with non-transgenic C57BL/6J as wild mice in the control group, n=14; fed for 1 month. Take the end of the administration of the hippocampus of mice, the expression of LRP1 is detected by Western Blot.4.2 exploring mechanisms of protection of nuezhenoside on Alzheimer’ s disease.Screen nuezhenoside safe dose in SH-SY5Y cells, and appropriate concentration is used on the Aβ42-induced of SH-SY5Y cell damage model, protection of nuezhenoside on Aβ42-induced neurotoxicity SH-SY5Y cells is detected by MTT assay. Aβ42 in intracellular levels of extracellular was determined by ELISA; NF-KB, Bcl-2, LC3 expression was determined by Western Blot.Results1. SH-SY5Y cells was cultured with different concentrations of Aβ42 (5μmol · h-1,10μmol·L-1,15μmol·L-1,20μmol·L-1,40μmol·L-1),and the viability of cells were statistically significance (P<0.05).However ,there is need to select the appropriate concentration, concentrations of Aβ42 (5μmol·L-1,10 μmol·L-1) damage instability SH-SY5Y. With concentrations of 20μmol·~L-1,40 μmol·L-1make cell death, so choosing 15μmol·L-142 concentrations were as the best subsequent experiments.2. Screening safe dose of BaJiJiaSu, Nuezhenoside and Icariin on both mouse brain microvascular endothelial cells and human neuroblastoma cells (SH-SY5Y), results show that at a concentration of BaJiJiaSu, Nuezhenoside and Icariin 500μmol·L-1 did not effect cell viability. There was no significant difference (P> 0.05); Similarly, screening appropriate drug concentration in SH-SY5Y cells, results showed that there is no effect of the concentration of Bajijiasu (500μmol·L-1)on cell viability, it was no significant difference (P>O. 05).3. The concentration of Icariin(5、50、500μmol·L-1) have no significant protective effect on Aβ42-induced SH-SY5Y cells neurotoxicity (P>0.05)4. Discussion the protective effect of Bajijiasu on Aβ42-induced SH-SY5Y cells and mechanisms.4.1 The concentration of Bajijiasu (50,100,500μmol·L-1) have a protective effect on Aβ42-induced SH-SY5Y cells neurotoxicity (P <0.05).4.2 mechanisms of Bajijiasu on Aβ42-induced SH-SY5Y cells neurotoxicity.4.2. 1SH-SY5Y cells was cultured with Bajijiasu after 12h. Compared with the normal group, in Bajijiasu group (50,100μmol·L-1) cell membrane transporter protein LRP1 protein expression was significantly increased (P<0.05),and β-amyloid protein (APP) showed no significant change (P>0.05). Bajijiasu can effectively increase the uptaking and degrading effect of SH-SY5Y cells, then level of Aβ42 in intracellular levels of extracellular was determined by ELISA. Compared with model group Aβ42 conditioned media content, the content of Aβ 42 in Bajijiasu group(50, 100μmol·L-1) is relative reduction (P<0. 05); compared with the model group content of intracellular Aβ42, the content of Aβ42 in Bajijiasu group (50 μmol·L-1) is relative increase(P <0.05), the Aβ42 intracellular content is not obvious in Bajijiasu group (100μmol·L-1) (P> O. 05).4.2.2 LRP1 gene silencing SH-SY5Y cells was cultured with Bajijiasu. Then Aβ42 in intracellular levels of extracellular was determined by ELISA. Bajijiasu group within cells and conditioned media content of Aβ42. The results showed that compared with the model group conditioned medium, the content of conditioned medium Aβ42 in Bajijiasu group is no significant difference (P> 0.05);Compared with the extracellular content of of Aβ42 on the normal SH-SY5Y cell model group, Aβ42 content in model group was significantly higher on LRP1 gene silencing (Si LRP1) SH-SY5Y cells (P <0.05);Compared with the Bajijiasu group extracellular content of Aβ42 on the normal SH-SY5Y cell, A~42 content in Bajijiasu group was significantly higher on LRP1 gene silencing SH-SY5Y cells (P <0.05) . On gene silencing group, campared with model group, Bajijiasu groups is decreased, but the difference was not statistically significant (P> 0.05).4.2.3 After feeding transgenic mice with Bajijiasu one month, detected LRP1 protein expression in hippocampus of mice by Western blot and found that LRP1 protein expression was no significant difference in Bajijiasu group, the normal group and the model group (P>0.05).5. Discussion the protective effect of nuezhenoside on Aβ42-induced SH-SYSY cells and mechanisms.5.1 The concentration of nuezhenoside (50, 100, 500μmol·L-1) have a protective effect on Aβ 42-induced SH-SY5Y cells neurotoxicity (P<0.05). It was observed by microscope that in the model group compared with control group, cells grew well, obvious dendritic axons, translucent cells under a microscope; cell morphology model set of edge blur, cell shrinkage axons disappeared; compared with the model group, nuezhenoside two treatment groups significantly reduced.5.2 nuezhenoside increase clearance of Aβ42 on SH-SY5Y cells. Elisa results showed that compared with the model group content Aβ42 conditioned medium, nuezhenoside (50,100μol ·L-1) of Aβ42 levels were decreased.detection of autophagosome marker LC3 was detected by Western blot, results showed that the model group LC3 II/LC3 I expression was significantly increased as compared with the normal group (P<0.05)。Compared with the model group, nuezhenoside(50,100μmol ·L-1) of LC3 Ⅱ/LC3 I ratio decreased significantly (P<0.05); compared with the model group, significantly reduce the detection privet glycosides (50,100μmol ·L-1) protein expression of NF-κB (P<0.05), the anti-apoptotic factor Bcl 2-protein expression was significantly increased (P<0.05).Conclusion1.The concentration of Icariin(5、50、500μmol ·L-1) have no significant protective effect on Aβ42-induced SH-SY5Y cells neurotoxicity (P>0.05)2. Bajijiasu protect Aβ42-induced SH-SY5Y cells from neurotoxicity injury.. The mechanism can effectively increase the LRP1 expression on human neuroblastoma SH-SY5Y cell, which can increase cell endocytosis and lysosomal transport, and reduce Aβ42 deposition and cytotoxic to protecting neurons in the extracellular. There is no significantly difference of LRP 1 protein expression in the transgenic mice hippocampus, may be mechanism caused inconsistent between animal models and cell model.3. Nuezhenoside can effectively protect SH-SY5Y cell from Aβ42 induced cytotoxicity and improve cell viability. The potential mechanism is that nuezhenoside may increase Aβ42 uptaking to reduce its extracellular aggregation and inhibit autophagy, inhibit NF κB activation and increased expression of anti-apoptotic factor Bcl-2 protein, so as to achieve the protective effect on cells.