| ObjectivesAs society is increasing phenomenon of aging, dementia is becoming an important issue of world attention, and its prevalence and incidence rates increase exponentially with age increased, the high prevalence of physical, psychological and economic impact, not only affects Patients, but also the nursing staff, the patient's family and friends, health care systems and society as a whole. Therefore, the strengthening of AD etiology, pathogenesis research, development of effective prevention measures and drugs has important clinical value and practical significance. We seeks to explain the basic law of AD treatment based on the basic pathogenesis of AD.And based on successfully establishing in vitro model of AD,we investigated the effects and mechanisms on anti-inflammatory of AD by Bushen-Huatan-Yizhi Recipe(BSHTYZR), as AD in-depth studies provide a theoretical basis and experimental evidence.Methods1.Theoretical Discussion:Basiced on systematic analysis of ancient and modern literature on AD, summarized in medicine's understanding of AD pathogenesis, discuss the relationgship between kidney deficiency, phlegm and theosophy (Chi),exlain the basic pathogenesis and treatment principles of AD,and screen of effective treatment of AD prescriptions, to investigate the mechanism of treatment by BSHTYZR.2.Experimental Study:BV-2 and PC12 cells were cultured as in vitro microglia and neuron model. (1) BV-2 cells effected by 2.5mmol/L Aβ1-42 incubated for 3d, were producted for microglia model of AD,and then they were intervened by the serum containing-BSHTYZR,compared to the serum containing-Indometacin Enteric-Tablets(Indometacin). The protein expression of IL-1β, IL-6, TNF-αwas examined by ELISA,and IL-1βmRNA was detected by RT-PCR.Observing the BV-2 cells secreting inflammatory cytokines, and its IL-1βexpression on mRNA levels,by using BSHTYZ method,to explore the method's anti-inflammatory mechanism.Using Immunocytochemistry and Western Blot to observe the expression of NF-κB protein,immunofluorescence to detect the expression of COX-2,to investigate the influence on inflammatory signaling pathway and its important protein NFκB, COX-2 expression by BSHTYZ method, then to explore its mechanism,from morphology and molecular biology of inflammation. (2) PC12 cells effected by 2.5mmol/L Aβ1-42 incubated for 3d, were producted for microglia model of AD,and then they were intervened by the serum containing-BSHTYZR,compared to the serum containing-Indometacin. We observed the cell morphology, and assessed neuronal survival by counting MTT assay,used RT-PCR to detect the expression of APPmRNA,to observe BSHTYZ recipe's protective effect on neuronal cells and neurotoxic effects of anti Aβ, and its mechanism.Results1. Influence on the release of inflammatory factors on cell model of AD by BSHTYZ methodIn the model group, concentrations of IL-1β, IL-6 and TNF-αwere significantly increased with significant statistical difference, compared to the normal group (P<0.01). Compared to the model group, treatment groups can reduce the expression of IL-1β, IL-6 and TNF-α, and there was significant difference (P<0.01). Compared to the indomethacin group, the BSHTYZ group can more effectively inhibit the expression of IL-1β(P<0.05); but in reducing the expression of IL-6, as effective as the indomethacin group (P<0.05); for the expression of TNF-a, there was no significant difference (P>0.05).2. Influence on the expression of IL-1βmRNA on cell model of AD by BSHTYZ methodRT-PCR was used to determine the BV-2 cells' expression of IL-1βmRNA.Gray analysis showed that the expression of IL-1βmRNA in the model group significantly increased compared to the normal group, there was a significant difference (P<0.01). Compared to the model group, treatment groups can reduce the expression of IL-1βmRNA, with significant differences (P<0.01).Compared to the indomethacin group, BSHTYZ method was more significantly in regulating the expression level of IL-1βmRNA gene (P<0.05), which is consistent with the results of ELISA assay.This indicated that BSHTYZ Method can regulate the expression of IL-1βmRNA from the level of gene,then to suppress the secretion of inflammatory factors on BV-2cells.3.Pathologic effects on cell model of AD by BSHTYZ methodThe BV-2 cells in model group effected by Aβ1-42 appeared obviously morphological changes after 2 hours, with larger cell body, large and circular nucleus, prominent nucleolus.After 12 hours, majority of cells gathered to a group.Cells' bodies changed into Fusiform,and were out of one or more long dendrites, or even intertwined, winding and Interconnected processes and fusion between cells.Cytoplasm got yellow largely and deeply.There were brown particles around nucleus. Some cells Showed strong positive expression, and individual cells were swallowed like swelling, and some cells suspended off the wall even to death. This role has been maintained to 24 hours. In the BSHTYZ group,BV-2 cells effected by Aβ1-42 adhered after 2 hours,with singleand uniform size.Cell body were circular, cytoplasm were clear, nucleus were blue or light blue, branch-like protrusions were abvious.There were no significant deformed cells.4.Effects on the expression of protein of NF-κB on cell model of AD by BSHTYZ methodThe results by using immunocytochemistry and RT-PCR showed that BV-2 cells in model group are high expression of NF-κB protein, compared to the normal group, there was significant difference (P<0.01). Compared to the model group, both of the treatment groups can reduce the expression of NF-κB proteins significantly (P<0.01). Compared to the indomethacin group.Chinese medicine group with which they have the same rats in reducing NF-κB protein in BV-2 cells (P>0.05).5. Effects on the expression of protein of COX-2 on cell model of AD by BSHTYZ methodBV-2 cells in model group are high expression of COX-2 protein, compared to the normal group, there was significant difference (P<0.01). Compared to the model group, both of the treatment groups can reduce the expression of COX-2 proteins significantly (P<0.01). Compared to the indomethacin group.Chinese medicine group with which they have the same rats in reducing COX-2 protein in BV-2 cells (P>0.05).6. Effects on neuronal survival on cell model of AD by BSHTYZ methodIn the model group, the PC12 cells adhered slowly, and the number was significantly reduced after 24 hours, the size changed smaller.The cell rounded off, or into a piece of floating in the culture medium.The medium were not clear,with fragments, particulate matter increased. In BSHTYZ group,a clear background, cells grew up on the wall.Culture plate were covered with spindle-shaped or flat cells after 24hours.2.5mmol/L Aβ1-42 can significantly decrease cell's viability.Treatment groups had varying degrees of protection to neuronal cell caused by Aβ1-42 cytotoxicity.7. Effects on the expression of protein of APPmRNA on cell model of AD by BSHTYZ methodUsing RT-PCR to semi-quantitative analysis expression of APPmRNA,we found that expressions increased in each group, compared to the normal group, the difference was statistically significant (P<0.01). Compared to the model group, treatment groups can reduce the expression on different levels (P<0.01),and the BSHTYZ group was more obvious, there were significant differences (P<0.05).Conclusions1. Kidney-essence weak and phlegm stasis is the most basical pathogenesis of AD.The therapy of replenishing kidney-essence and removing phlegm to benefit wisdom is the basical and effective trestment for AD.2.There is correlationship between Aβneurotoxicity and immune inflammatory response in AD.3.BSHTYZ method can effectively surpress the expression of inflammatory signaling pathway related protein NF-κB and COX-2,block the inflammatory response process in AD.4.BSHTYZ method can regulate inflammatory cytokine IL-1β,IL-6,TNF-αand IL-1βmRNA's expression,to inhibit the inflammatory response in AD from the genetic level.5. BSHTYZ method can surpress the expression of APPmRNA,reduce the toxic effects of Aβin AD.6. BSHTYZ method can reduce the damage from inflammatory in AD to protect the nerve cells. |
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