Study On The Regulation Mechanism Of Heixiaoy-ao Powder On Polarization Of Microglia In APP/PS1 Mice Based On NOX/ROS/NF-κB Signal Pathway

Objective : Study on the regulation mechanism of Heixiaoyao powder on polarization of microglia in APP/PS1 Mice by NOX2/ROS/NF-κB signal pathway.Methods : Fifty 4-month-old male APP/PS1 double transgenic mice were randomly divided into model group,MCC950 group(10 mg/kg)and low,medium and high dose groups of Heixiaoyao powder(6.45 g/kg,12.89 g/kg,25.78 g/kg).Thirty male C57BL/6J mice of the same age and strain were randomly divided into blank group,blank intragastric intervention group and blank intraperitoneal injection group.Each group was administered for 90 days.Morris water maze test learning and memory ability,Nissl staining and transmission electron microscopy were used to observe the pathological morphology and ultrastructure of hippocampal neurons.Immunofluorescence was used to detect the positive expression of microglia M1 type marker CD16/32+/Iba-1+,M2 type marker CD206+/Iba-1+and the expression of hippocampal ROS.Colorimetric method was used to detect the content of MDA and SOD in the hippocampus.ELISA was used to detect inflammatory factor of IL-6,IL-8,TNF-α content in the hippocampus.WB detection of Aβ,Iba1,CD16/32,CD206,NOX2,NF-κB,p-NF-κB,IKBα and p-IKBα protein expression in the hippocampus.Results:Compared with the blank group,the model group showed prolonged target quadrant movement distance and escape latency(P<0.01),shortened target quadrant retention time and percentage(P<0.01),disorganized hippocampal neuronal cells with swelling,nuclear disappearance or bias,reduced number of cells,dissolved or absent Nissan bodies,and a clear area in the cytoplasm,damaged and shrunk cell membrane with abnormal cell morphology,few organelles in the cytoplasm,reduced and swollen mitochondria,increased MG M1-type marker CD16/32+/Iba-1+(P<0.01),decreased M2-type marker CD206+/Iba-1+(P<0.01),increased ROS activity and MDA content(P<0.01),decreased SOD content(P<0.01),elevated inflammatory factors IL-6,IL-8,and TNF-α(P<0.01),up-regulated protein expression and phosphorylation of Aβ,CD16/32,Iba-1,NOX2,NF-κB,and IKBα(P<0.01),and down-regulated CD206(P<0.01).There was no statistically significant difference between the blank group,the blank + intragastric intervention group,and the blank + intraperitoneal injection group.After the intervention of Heixiaoyao Powder,the Heixiaoyao Powder groups showed shortened target quadrant movement distance and escape latency(P<0.01),prolonged target quadrant retention time and percentage(P<0.01),increased and neatly arranged cells with relieved swelling,increased Nissl bodies,regular cell morphology,and intact cell membrane,relieved swelling of mitochondria,slightly expanded endoplasmic reticulum decreased CD16/32+/Iba-1+(P<0.01),increased CD206+/Iba-1+(P<0.01),decreased ROS activity and MDA content(P<0.01),increased SOD content(P<0.01),decreased content of inflammatory factors IL-6,IL-8,and TNF-α(P<0.01),downregulated protein expression and phosphorylation of Aβ,CD16/32,Iba-1,NOX2,NF-κB,and IKBα(P<0.01),and up-regulated CD206(P<0.01).Conclusion: Heixiaoyao powder can alleviate neuronal damage and improve the learning and memory abilities of APP/PS1 mice.The mechanism of action may be related to the inhibition of NOX2/ROS/NF-κB signaling pathway,regulating the polarization of Microglia,increasing the expression of M2 type,inhibiting the expression of M1 type,and reducing the release of inflammatory factors.