Mechanism Study On Yishen Huazhuo Decoction To Alleviat The Neuroinflammation Of Alzheimer’s Disease By Regulating Microglial Polarization

PurposeTo observe the effects of Yizen Huazhuo Decoction on the polarization of microglia in the hippocampus of 4-month-old SAMP8 mice and its related targets,and to study its mechanism reducing neuroinflammatory response and improving memory loss in Alzheimer’s disease.MethodsForty-five 4-month-old male SAMP8 mice randomly split into three groups:model group(P8),Yishen Huszhuo Decoction(YHD)group and positive control group(Ibuprofen),with 15 mice in each group.Fifteen 4-month-old SAMR1 mice were used as control(R1)group.Each experimental group was given 6.24g/kg YHD formula,0.026g/kg Ibuprofen granules,and R1 and P8 groups were given the same amount of distilled water.Mice in the four groups were given 10 m L/kg gavage once a day for 12 weeks.Morris water maze was used to analyze the swimming speed,escape latency time,times of crossing the platform and initial angular radian of each group,and the differences in learning and memory ability were compared between each group.Nissl staining was used to detect the array and count of neurons in hippocampal CA1 region of mice.The expression of i NOS and Arg-1 markers in M1/M2 microglia was observed by double-labeling immunofluorescence assay.The protein expressions of PU.1,TREM2,PPARγ and NF-κB were detected by Western blot.The expression of TNF-α and IL-10 was detected by ELISA.The gene expressions of transcription factors PU.1 and TREM2 were detected by q PCR.Results(1)Morris water maze:(1)Swimming speed:there was no statistical significance among all groups(P>0.05).(2)Escape latency time:all groups showed a downward trend,and the trend was roughly the same.Repeated measures ANOVA showed that compared with R1 group,the escape latency time of P8 group and Ibuprofen group was significantly longer(P<0.05).Compared with the P8 group,the escape latency time of the YHD group was significantly shortened(P<0.05),but there was no statistical difference between the P8 group and the Ibuprofen group(P>0.05).(3)The number of crossing platform:compared with R1 group,the number of crossing platform in P8 and Ibuprofen groups were significantly decreased(P<0.05);Compared with P8 group,the number of crossing platform in YHD group was significantly higher than P8 group(P<0.05),but there was no statistical significance in the number of crossing platform between Ibuprofen group and P8 group(P>0.05).(4)Initial angle radian:compared with R1 group,the initial Angle radian of P8 group and Ibuprofen group was significantly increased(P<0.05);Compared with the P8 group,the initial angular radian in the YHD group was significantly reduced(P<0.05),while there was no significant difference in the initial angular radian in the Ibuprofen group(P>0.05).(2)Results of Nissl staining:The neurons in the R1 group were arranged in a relatively compact and orderly way,with clear and regular cell boundaries,and fewer neurons were lost.In the P8 group,the neuronal arrangement was disorderly,and there were more dead cells with irregular shape or rupture,and more neurons were missing.Compared with the P8 group,the neurons in the YHD group arranged orderliness,the cells’ morphology was relatively regular,the boundary was relatively clear,and some areas of neurons were lost.Neurons in the Ibuprofen group were relatively compact but the cell boundaries were relatively fuzzy,and vacuoles appeared in some areas due to the absence of neurons.Neuron count in Haima CA1 region:compared with R1 group,the number of neurons in P8,YHD and Ibuprofen groups was significantly decreased(P<0.05);Compared with P8 group,the number of neurons in hippocampal CA1 region in YHD and Ibuprofen groups was significantly increased(P<0.05).There was no statistical significance between the YHD group and the Ibuprofen group(P>0.05).(3)Results of double-labeling immunofluorescence,Western blot,ELISA and qPCR:Double-labeling immunofluorescence shows that:compared with the R1 group,the expression of i NOS in the hippocampus of mice in P8 group was significantly increased,and Arg-1 was significantly decreased(P<0.05).Compared with the mice in P8 group,the expression of i NOS in hippocampus of mice in YHD group was significantly decreased,and the expression of Arg-1 was significantly increased(P<0.05).There was no statistical difference between the YHD group and the Ibuprofen group(P>0.05).Western blot results:compared with R1 group,the protein expression levels of Pu.1,TREM2 and PPARγ in P8 group were significantly decreased,while the protein expression level of NF-κB was significantly increased(P<0.05).Compared with the P8 group,the protein expressions of PU.1,TREM2 and PPARγ were significantly increased in YHD group and Ibuprofen group,while the protein expression of NF-κB was significantly decreased(P<0.05).There was no statistical difference between the YHD group and the Ibuprofen group(P>0.05).ELISA results showed that compared with R1 group,the expression of TNF-α in hippocampus of P8 group was significantly increased,and the expression level of IL-10 was significantly decreased(P<0.05).Compared with P8 group,the expression of TNF-α in YHD and Ibuprofen groups was significantly decreased,and the expression of IL-10 was significantly increased(P<0.05).There was no statistical difference between the YHD group and the Ibuprofen group(P>0.05).The qPCR results showed that compared with the R1 group,the expression of PU.1 in the hippocampus of P8 group was significantly decreased(P<0.05),and the expression of TREM2 was decreased.Compared with P8 group,the expression of PU.1 in YHD group and Ibuprofen group was significantly increased(P<0.05),and the expression of TREM2 had an upward trend.There was no statistical difference in the above two indexes between YHD and Ibuprofen groups(P>0.05).Conclusion(1)Yishen Huazhuo Decoction can effectively improve the learning and memory ability impairment of 4-month-old SAMP8 mice,which is related to reducing the neuroinflammatory response in the brain,and thereby reducing the loss of hippocampal neurons.(2)The mechanism of Yisen Huazhuo Decoction alleviating the neuroinflammatory response in 4-month-old SAMP8 mice may be related to the activation of PU.1/TREM2/PPARγ pathway and the inhibition of NF-κB expression,thus promoting the microglia polarization to M1-type and release anti-inflammatory factors,and reducing the polarization to M1-type and reducing pro-inflammatory factors.