To Explore Protective Mechanism Of Liu Wei Di Huang Tang On Neurons Injuryed By Aβ Protein From Microglia Activation

Objective:1.To discuss the relationship between kidney deficiency and cognition from the angle of"kidney brain correlation",to explain the basic pathogenesis and treatment method of senile dementia,and to discuss the theoretical basis of traditional Chinese medicine and the modern biological basis of Liuwei Dihuang decoction in treating AD.2.From the angle of small glial cell activation,the effect and possible mechanism of Liuwei Dihuang decoction in the treatment of senile dementia were revealed,which provided experimental basis and action target for traditional Chinese medicine to promote the functional rehabilitation of senile dementia patients.Methods:1.To analyze and sort out the ancient and modern literature about AD,sum up the understanding of the etiology and pathogenesis of senile dementia in traditional Chinese medicine,discuss the relationship between kidney deficiency and cognition,and explain the basic pathogenesis and treatment method of senile dementia from the angle of"the kidney connects to the brain".To clarify the theoretical basis of Liuwei Dihuang decoction for the treatment of senile dementia.2.The senile dementia model of mice was made by Aβ1-40.The animals were randomly divided into normal group,sham operation group,model group and Liuwei Dihuang decoction group.The morphology of microglia was observed by transmission electron microscope（TEM）in order to observe the degree of activatio n,the morphology of neurons and the degree of injury,and the degree of neuronal injury was observed by silver nitrate staining.The expression level of NF-κB signal in animal brain tissue was detected by ELISA,qPCR and immunohistochemistry.3.Aβ_1-40-40 were used to injure microglia in vitro was treated with different treatments.The changes of inflammatory cytokine secretion were observed by ELISA method at different time points at 1h 3h and 12h,and the survival and activation status of microglia cells were observed by immunofluorescence and flow cytometry.The intervention effect of Liuwei Dihuang decoction was discussed from NF-κB pathway.Results:1.In the part of theoretical study,the relationship between kidney and AD is discussed from two aspects:the physiological function of kidney and the relationship between meridian and meridian.Based on the classical articles and modern medical literature,it is considered that deficiency of kidney essence is due to deficiency of kidney essence and the generation of toxin due to deficiency.Toxic damage to myelin is the main etiology and pathogenesis of senile dementia.It is creatively proposed that the pathogenesis of AD is closely related to the neuronal inflammatory injury mediated by the activation of microglia.Treatment should be based on the use of kidney tonifying drugs intervention as the fundamental law,Liuwei Dihuang decoction is an effective clinical prescription.2.The results of animal experiments showed that Liuwei Dihuang decoction could improve the growth and aging of senile dementia mice,reduce the escape latency of dementia mice,increase the number of collapses and improve the spatial learning and memory ability of dementia mice.The results of electron microscope showed that Liuwei Dihuang decoction could improve the ultrastructure of neurons and reduce the damage of endoplasmic reticulum.At the same time,silver nitrate staining showed that Liuwei Dihuang decoction could improve the pathological morphology of neurons in the brain of C57BL/6 wild-type mice after Aβinjury.To reduce neuronal tangles and senile plaques,and to play a protective role in the brain,all of these confirmed the effect of Liuwei Dihuang decoction on senile dementia mice.CD11b positive staining showed that the damage of Aβcould lead to the increase of microglia activation in model mice.Thus,it can increase the expression of inflammatory factors such as IL-1βand TNF-a in brain and aggravate the inflammatory injury of neurons in brain.Liuwei Dihuang decoct ion can inhibit the expression of CD11b positive cells to a certain extent and reduce the secretion of inflammatory factors.All these indicate that Liuwei Dihuang decoction reduces the activation of microglia and reduces the secretion of inflammatory factors.At the same time,the expression of NF-κB/P65/NF-κB was increased and the expression of I-κB was decreased after the treatment of Liuwei Dihuang decoction,and the expression of NF-κB/P65-κ-κwas decreased and the expression of I-κB was upregulated after the treatment of Liuweidihuang decoction.3.The results of cell experiment showed that the BV-2 damage of Aβ1-40 was significantly higher than that of the normal control group,and its survival was decreased significantly.After treated with the drug serum of Liuwei Dihuang decoction,the cell activity increased obviously,and the apoptosis rate of BV-2 was increased by double staining flow cytometry,and the apoptosis rate of each group was lower than that of the model group.At the same time,we also found that the expression of Bcl-2 and Caspase-3 in BV-2 of Aβ_1-40 injured group was significantly lower than that of model group,while the expression of Bcl-2mRNA and Caspase-3mRNA in serum group were significantly higher than those in model group.The results showed that the serum of Liuwei Dihuang decoction could regulate the expression of Bcl-2 and Caspase-3 in BV-2 cells damaged by Aβ_1-40,inhibit apoptosis and play a protective role.Microglia were activated and increased after Aβ_1-40 intervention,and the activation of microglia was inhibited after intervention by Liuwei Dihuang decoction.IL-1βand TNF-a secreted by microglia began to increase significantly at 1 h after treatment with Aβ1-40.The secretion of TNF-a reached the peak at 6 h and the secretion of IL-1βreached the peak at 3 h（P<0.05）.Compared with the model group,the secretion of TNF-a and IL-Iⁱn each drug-containing serum group was significantly decreased（P<0.05）,and the secretion of IL-Iβwas significantly decreased（P<0.05）at 12 h after 3H（P<0.05）,while the secretion of IL-Iβdecreased significantly（P<0.05）at 612 h（P<0.05）.The results showed that Liuwei Dihuang decoction could reduce the activation of microglia induced by Aβ1-40 and increase the secretion of inflammatory factors.At the same time,the NF-κB/P 65、NF-κB/P50 expression increased and the NF-κB/P 65、P50 expression decreased after treatment with Liuwei Dihuang decoction.Conclusion:1.The basic cause of senile dementia is deficiency of kidney essence,the basic pathogenesis of dementia is caused by deficiency of kidney essence,and the basic pathogenesis is caused by deficiency,and the treatment should be based on the intervention of tonifying kidney medicine in the super early stage.2.Liuwei Dihuang decoction can down-regulate the expression of IL-1βand TNF-a protein and mRNA in brain tissue of AD mice,and regulate the activation of microglia and NF-κB signaling pathway.Regulating the release of inflammatory factors is the possible mechanism of its mental health and brain strengthening3.The serum of Liuwei Dihuang decoction can protect BV-2 damage,reduce cell apoptosis,inhibit its activation and release of inflammatory factors,and affect its NF-κB signal expression.