A Study On The Mechanism Of Modified SanJiaSan In The Treatment Of Senile Dementia By Regulating The Relationship Between Neuroinflammation And Autophagy And Apoptosis

Senile dementia or Alzheimer's disease is a common neurodegenerative disease,whose clinical characteristics are the onset hidden,unknown etiology and pathogenesis of complex and diverse symptoms and cognitive dysfunction,abnormal mental and behavior change and social function impairment,there is no effective treatment.Traditional Chinese Medicine(TCM)has a long history and unique characteristics in the treatment of this disease.In the first part,the theoretical study part of this paper reviews understanding of the ancient and modern medical cause of the disease,the symptom,pathogenesis,syndrome differentiation and treatment.The ancient physicians thought this disease is deficiency in origin and excess in superficiality,as well as decreased pulp brain empty,god machine malfunction,five zang-organs deficient,gasification deficiency,five-spirits disuse,damaged by excess of seven emotions,obsessed with the phlegm,obstruction of collaterals by blood stasis is the basic pathogenesis of dementia.Treatments focus on invigorating heart and kidney and strengthening Spleen and Supplementing heart,filling lean pulp,resolving phlegm and removing blood stasis,mainly valuing to invigorate heart and kidney.Modern subjects inheriting and developing the previous understanding of dementia,have been clear about the designation and symptoms of dementia,in syndrome differentiation deficiency in origin and excess in superficiality are still the main lines,the deficiency in origin emphasizes the kidney and marrow,brain,excess in superficiality emphasizes on phlegm,blood stasis and toxin,treatment is given priority to consolidate the Kidney and supplement Essenceto and nourish blood,as well as resolve phlegm and remove blood stasis and detoxification.Article further reviewes the use of single taste TCM,Chinese medicine compound preparation and acupuncture method for the treatment of senile dementia clinical and basic research in the modern TCM,summarizes the medicine understanding of the disease and treatment,combing the relationship between autophagy and senile dementia,in order to provide help for Chinese medicine treatment of senile dementia.Then discussing the theoretical mechanism of scattered treatment for Alzheimer's disease and combing its clinical and basic research,GAILIANGSANJIASAN might improve the basis of marrow depletion due to kidney deficiency,phlegm-accumulation stasis and can reinforce liver and kidney,obsessed with the brain essence,remove phlegm-accumulation stasis,has a good application prospect.In the second part,we investigate the relationship between neuroinflammation and autophagy and the effects of autophagy on the cytotoxicity and apoptosis of neurons in the primary cultured microglia,BV2 cells and PC 12 cells.First,the original generation of microglia is cultivated and the purity is determined by immunofluorescence staining technique,the glial cell activity is determined by MTT to detect the effection of LPS,determining concentration and time of BV2 cells autophagy produced LPS though western blotting.The glial cell activity medicated cerebrospinal fluid influenced on is detected by CCK 8 method,the expression of LC3,Beclinl and P62 in BV2 cells which are improved by GAILIANGSANJIASAN medicated cerebrospinal fluid is detect by western blotting;the level of inflammatory factors like IL-1?,IL-6,TNF-?,IL-4,IL-10 in the supernatant of microglia are detected by ELISA.The changes of autophagysome related protein LC3 are observed by immunofluorescence.The ultrastructure changes of autophagic corpuscles are observed by transmission electron microscopy.PI3K/AKT/mTOR signaling pathways protein expression of BV2 cells is detected by western blotting.PC 12 cells is induced by microglial cells culture supernatant as conditioned medium for 24 hours,then APP,BACE1,BAX,BCL-2,Caspase-3 protein and mRNA level are detected respectively with western blotting and RT-PCR methods,cells apoptosis of PC12 are detected by flow cytometry.The results show that the purity of the primary microglia cells is 99%,which could be used for subsequent experiments.Toxicity of LPS to microglia increases with concentration dependence,When LPS induces microglia for 24 hours at 5 ?g/ml,autophagy protein LC3? is in the stability quadratic peak level,which can be the follow-up mold conditions.GAILIANGSANJIASAN medicated cerebrospinal fluid has protective effect on the cells,which can obviously improve the survival rate of cells and alleviate the cytotoxic with LPS.Compared with model group,GAILIANGSANJIASAN medicated cerebrospinal fluid can raise the expression of P62,p-mTOR,IL-4 and IL-10 level and decreasing LC3,Beclin1,PI3K,p-AKT,IL-1?,IL-6,TNF-? level,reducing autophagic vacuole generation,and the number of autophagosome,and regulating nerve inflammation of microglia and excessive autophagy reaction.The level of APP,BACE1,Bax,Caspase 3 protein and mRNA are reduced by GAILIANGSANJIASAN medicated cerebrospinal fluid conditioned medium,which also increase the expression of Bcl-2 protein and mRNA,alleviate cell apoptosis caused by nerve inflammation and excessive autophagy reaction.Above all,these observation show that GAILIANGSANJIASAN medicated cerebrospinal fluid may protect neurons by regulating nerve inflammation and excessive autophagy though adjusting PI3K/AKT/mTOR signaling pathways relying on Beclinl,thus improve the protection of neurons,which provide direction for further research of mechanism of GAILIANGSANJIASAN to the treatment of senile dementia.