1. The Effect And Mechanism Of Rab8a On Vesicle Anchoring In Astrocytes Stimulated By Lipopolysaccharide; 2. The Effect Of β-amyloid 25-35 On Autophagy In Rat Astrocytes

PART 1: THE ROLE AND MECHANISM OF RAB8 A PROMOTES VESICLE ANCHORING IN LIPOPOLYSACCARIDE-STIMULATED ASTROCYTEObjective: Neuropathic pain is the most common chronic.The release of inflammatory factors from vesicles of astrocytes may be an important cause of NPP.This study aims to explore the role of Rab8 a in promoting vesicle anchoring in NPP,and to explore its mechanism.Methods: The primary Sprague-Dawley(SD)neonatal rat astrocytes were randomly divided into a normal control group and an LPS group.The LPS group cells were treated with 100 ng/ml LPS for 24 hours as a pain model,and the normal control The group treated astrocytes with the same amount of PBS for 24 h.CCK-8 method was used to detect the cell viability of the two groups;RT-PCR was used to detect the levels of inflammatory factors in the two groups;immunofluorescence staining and western blotting were used to detect the intracellular vesicle marker proteins VAMP2,fusion anchor protein SNAP25,SNARE related The levels of protein Syntaxin16 and Rab8a;transmission electron microscopy to detect the secretion level of cell vesicles;immunoprecipitation to detect the formation of SNARE complexes.Results: Compared with the control group,the viability of astrocytes in the LPS group was increased;the expression of GFAP increased;the inflammatory factors IL-1β and TNF-α increased;the expression of vesicle marker protein VAMP2 was significantly increased;intra-astrocytic vesicles There was a significant increase in mitochondria,endoplasmic reticulum,and Golgi apparatus;the expression of Rab8 a protein increased significantly,and the co-expression of Rab8 a with VAMP2 and SNARE complexes increased;the results of co-immunoprecipitation experiments showed that the expression of SNARE complex-related proteins was obvious Increase,the formation of SNARE complex increases.Conclusion: The Rab8a/SNARE complex signaling pathway was obviously activated in activated astrocytes,which enhanced the release activity of astrocyte vesicles.It may be related to Rab8a’s promotion of the formation of SNARE complex in astrocytes,which in turn accelerated the release of vesicles and the secretion of biologically active molecules in the activated astrocytes.PART 2: EFFECTS OF Aβ25-35 ON AUTOPHAGY OF ASTROCYTES IN RATObjective: Alzheimer’s disease is the most common neurodegenerative disease.Astrocytes are the most abundant type of glial cells,and the autophagy disorder of astrocytes may play an important role in the occurrence and development of AD.This article aims to explore the effect and mechanism of β-amyloid 25-35(Aβ25-35)on autophagy in rat astrocytes.Methods: The primary Sprague-Dawley(SD)neonatal rat astrocytes were randomly divided into the normal control group and the Aβ25-35 group.The Aβ25-35 group cells were treated with 40 μmol/L Aβ25-35 for 24 h As an AD model,the normal control group used the same amount of PBS to treat astrocytes for 24 h.The viability of rat astrocytes was assessed by using the Cell Counting Kit-8 method(CCK-8).Western Blot analysis was used to detect the expression of LC3,Beclin1,Cathepsin B and PI3K/Akt/m TOR signaling pathway related proteins.Results: Compared with the blank control group,the cell activity of Aβ25-35 treatment group increased.The LC3-Ⅱ /LC3-Ⅰ ratio was increased.The expression of Beclin1 was increased and the expression of Cathepsin B was decreased.The expression of PI3 K was decreased.The p-AKT/AKT ratio,p-m TOR/m TOR ratio was obviously decreased.Conclusion: Aβ25-35 activate autophagy in rat astrocytes and attenuates autolysosom degradation,possibly through the PI3K/AKT/m TOR signaling pathway.