The influence of Liuwei Dihuang Wan on the behavior and TLR4/NF-κB signaling pathway of Alzheimer's disease model mice
Objective To explore the effects of Liuwei Dihuang Wan on the behaviors and Toll-like receptor 4/nuclear factor kappa-B(TLR4/NF-KB)signal transduction pathway of amyloid β-precursor pro-tein/presenilin-1(APP/PS1)double transgenic mice.Methods Forty 3-month-old female APP/PS1 mice were randomly divided into model group,low-dose group(0.59 g/kg),medium-dose group(1.18 g/kg),high-dose group(2.36 g/kg)of Liuwei Dihuang Wan(gavaged according to grouped doses),and ibuprofen group(0.04 g/kg,gavage)using a random number table method,with 8 mice in each group.Eight 3-month-old wild-type female C57BL/6 mice with matched body weight were used as the control group.The mice in control group and model group were given an equal volume of 0.9％sodium chloride solution by gavage.The gavage administration was twice a day for a continuous period of 3 months.Morris water maze test was used to detect the learning and memory abilities of mice.ELISA was used to detect the serum levels of tumor necro-sis factor-α(TNF-α),interleukin-1β(IL-1β)and immunohistochemistry was used to detect the levels of amyloid β-protein(Aβ),glial fibrillary acidic protein(GFAP)and NF-κB in hippocampal tissue.Western blot was used to detect the expression levels of TLR4,myeloid differentiation primary response gene 88(MyD88),and phosphorylated NF-κB(p-NF-κB)proteins in hippocampal tissue.The SPSS 20.0 software was used for data analysis.Multiple group comparisons were conducted by repeated measure ANOVA or one-way ANOVA.Results The results of repeated measure ANOVA showed that as for the escape latency of the 6 groups of mice,the interaction effect between time and group was significant(Finteraction=117.219,P＜0.001).The escape latencies of mice in the 6 groups on the 5 th day were all lower than those on the 1st day(all P＜0.05).The escape latencies of mice in the ibuprofen group and the medium-dose and high-dose groups of Liuwei Dihuang Wan were lower than that in the model group from 1st day to 5th day(all P＜0.05).On the 3rd to 5th day,the escape latencies of mice in the medium-dose and high-dose groups of Li-uwei Dihuang Wan were lower than those in the low-dose group of Liuwei Dihuang Wan(all P＜0.05).There were statistically significant differences in the percentage of residence time in the platform quadrant and the numbers of crossing platform among the 6 groups of mice(F=5.451,4.824,both P＜0.05).The percentage of residence time in the platform quadrant(50.77±5.49)％,(54.39±5.71)％,(51.98± 6.12)％),and the numbers of crossing platform((5.9±1.1)times,(6.0±1.3)times,(5.1±0.8)times)in the high-dose and medium-dose groups of Liuwei Dihuang Wan and the ibuprofen group were all higher than those in the model group((27.32±3.22)％,(2.2±1.0)times)(all P＜0.05).The immunohisto-chemical results showed that there were statistically significant differences in the integrated optical density values of Aβ,GFAP and NF-κB in the hippocampal tissues of 6 groups of mice(F=57.52,45.37,79.10,all P＜O.05).The integrated optical density values of Aβ,GFAP and NF-κB in the high-dose and medium-dose groups of Liuwei Dihuang Wan and the ibuprofen group were all lower than those in the model group(all P＜0.05).And the integrated optical density values of Aβ,GFAP,and NF-κB in the high-dose and medium-dose groups of Liuwei Dihuang Wan were all lower than those in the low-dose group of Liuwei Dihuang Wan(all P＜0.05).There were statistically significant differences in the levels of serum TNF-α and IL-1β detec-ted by ELISA(F=3.996,6.395,both P＜0.05)and the proteins levels of TLR4,MyD88,and p-NF-κB in hippocampal tissue detected by Western blot among the 6 groups(F=15.710,3.522,4.119,all P＜0.05).The serum TNF-α and IL-1β levels in the high-dose and medium-dose groups of Liuwei Dihuang Wan and ibuprofen group were all lower than those in the model group(all P＜0.05).The serum TNF-α((18.90± 2.33)ng/L,(21.56±2.49)ng/L)and IL-1β((5.88±0.80)ng/L,(6.75±0.83)ng/L)levels in the high-dose and medium-dose groups of Liuwei Dihuang Wan were lower than those in the low-dose group((30.77±2.89)ng/L,(9.11±1.27)ng/L)(all P＜0.05).The protein expression levels of TLR4,MyD88,and p-NF-κB in the hippocampus of the high-dose and medium-dose groups of Liuwei Dihuang Wan and ibuprofen group were lower than those of the model group(all P＜0.05).The protein expression levels of TLR4((0.254±0.091),(0.318±0.122)),MyD88((0.229±0.077),(0.386±0.119)),and p-NF-κB((0.412±0.188),(0.358±0.119))in the hippocampus of the high-dose and medium-dose groups of Liuwei Dihuang Wan were lower than those of the low-dose group((0.617±0.172),(0.672±0.166),(0.799±0.227))(all P＜0.05).Conclusion Liuwei Dihuang Wan can significantly alleviate learning and memory impairment in Alzheimer's disease model mice,possibly by inhibiting TLR4/NF-κB signal pathway,reducing TNF-α and IL-1β expression,thereby alleviate central immune inflammatory response and exert anti Alzheimer's disease effects.
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