Experimental Study On The Effect Of Liuwei Dihuang Pills On TLR4 / NF-κB Signaling Pathway In APP / PS1 Double Transgenic Mice

Purpose:To investigate the effects of Liuwei Dihuang Wan on learning and memory abilities and activity of cholinergic system,TLR4/NF-κB signal transduction pathway in APP/PS1 mice. Utilize in vitro cell experiment, investigated the effects of Liuwei Dihuang Wan Serum on TLR4/NF-κB signal transduction pathway of Aβ induced PC12. Discussing the effects and mechanism of Liuwei Dihuang Wan on preventing and treating of AD. Provided important theoretical support on treatment of AD from kidney.Material and method:The 3-month-old APP/PS1 mice were randomly divided into the APP/PS1 model group, Liuwei Dihuang Wan low dose group(0.59 g·kg-1·d-1),medium dose group(1.18 g·kg-1·d-1),high dose group(2.36 g·kg-1·d-1) and ibuprofen group(0.04g·kg-1·d-1),with 8 mice in each group,40 mice in total.Another 8 3-month-old mice(WT) were selected as the normal control group. A week before the end of oral administration(3months),Morris water maze was used to test the effects of Liuwei Dihuang Wan on learning and memory abilities. Determinating the time required for each group of mice to find the platform(latency), residence time in the quadrant of the platform, also calculate percentage of time, record the frequency of cross platform.And the content of Ach, Ch AT and Ach E in cortex were measured by ELISA after Morris water maze test, 4% hydrate anesthesia.Blood taken from the abdominal aorta. after the separation of serum,in accordance with Beijing Cheng Lin Biotechnology Co., Ltd. production of IL-1β,TNF-α ELISA kit instructions for operation. Blood taken from the abdominal aorta, the mice were sacrificed. Tested Aβ、GFAP and NF-κB with immunohistochemical detection,tested TLR4、My D88 and NF-κB m RNA with Real-Time PCR,tested TLR4、My D88 and p-NF-κB Protein expression with Western blot. 40 Wistar rats, natural lighting, room temperature 16-25℃, Standard environment for relative humidity 60%, free drinking water intake, adaptive feeding 2-3d, randomly divided into two groups, including normal control group 20, traditional Chinese medicine group 20.Liuwei Dihuang Wan aqueous solution gavage with mass concentration 1.18g·kg-1·d-1 in traditional Chinese medicine group,10 m L·kg-1 twice a day, normal control group were given equal volume of saline. Select the blood after continuous perfusion of 5d. 2 hours orso after gavaging, preparate serum. PC12 with high differentiation, 5×105/m L of density,25cm2 culture flask.Put 4m L culture medium in each culture flask. Divided into normal control group, model group, 10% serum group, My D88 Silence group, VE group.Cultured in 37℃,5% CO2 incubation box 24 h,PBS rinsed 2-3 times, hunger treatment for 24 hours, normal control group, model group, VE group change into 10% rat serum culture medium DMEM,pre protection for 24 h, put in final concentration 30 μM Aβ25-35 36 h. Before transfecting 24 h,inoculated in standard 24 hole plate with 5×105/hole. High glucose DMEM culture medium(without antibiotics), transfecting cell with growth abundance was 60%.Each hole is added Lipofectamine 2000 5μL +20μM si RNA 5μL. After transfection was performed 24 h,replace culture medium to 10% fetal bovine serum, Observed si RNA fluorescence color under the fluorescence microscope, determined transfection efficiency. When the cell transfection efficiency was more than90%, collecting cells. Control group, si RNA group, and negative control group Respectively carried by Real-Time PCR,to detect the expression of My D88 m RNA and protein,selected the highest silence efficiency si RNA. Contents of IL-1β and TNF-α in cell supernatant was determined in each group. Determinate each group’s TLR4,My D88 and NF-κB m RNA, Determinate each group’s TLR4,My D88,and p-NF-κB protein expression. Using SPSS 18.0 statistical software, Group data expressed by x ±s,comparison between groups using single factor analysis of variance, the difference was statistically significant by P<0.05.Results:1 The experimental study of the effect of Liuwei Dihuang Wan on the behavior of APP/PS1 double transgenic mice.1.1 Effect of Liuwei Dihuang Wan on navigation experiment of Morris water maze in rats In the experiment of navigation of water maze in each group,compared with the normal control group, latency was longer in the model group of each period(p<0.05), in Liuwei Dihuang Wan and ibuprofen group,the latency was shorten, and there was significant difference(P<0.05)Compared with the model group, the low dose group of Liuwei Dihuang Wan, the latency was shorten, in test 5, 6, 7d had significant differences(P<0.05).1.2 Effects of Liuwei Dihuang Wan on the experiment of Morris water maze space exploration in each group.Compared with the APP/PS1 model group, latency was shorten(p<0.05), residence time of swimming, time percentage in platform and frequency of crossingsincreased(p<0.05) in Liuwei Dihuang Wan low, medium and high dose group.1.3 Contents of Ach, Ch AT and Ach E in the hippocampus of each group of mice.the content of Ach and the activity of Ch AT declined(p<0.05) in the APP/PS1 model group.The contents of Ach and Ch AT increased(p<0.05),while Ach E was not significantly different(p>0.05).2 Effects of Liuwei Dihuang Wan on signal transduction pathway of TLR4/ NF-κB in APP/PS1 double transgenic mice.2.1 immunohistochemistry method detected the protein expression of Aβ、GFAP and NF-κB.In normal control group,Aβ deposition was not observed in the brain of senile plaques,and the APP/PS1 model group, the number of senile plaques(Aβ) in the hippocampus, the total area is larger, the calculated value of the integral light density is higher. The value of Aβ,high dose group and Western medicine ibuprofen group were significantly decreased(p<0.01), the number of senile plaques was decreased, and the total area was decreased. The activation of astrocytes in the middle and high dose group and Western medicine ibuprofen group was significantly decreased. In APP/PS1 model group, the expression of NF-κB was significantly increased(p<0.01), and the expression of high expression was observed in the nucleus of the hippocampus.2.2 Comparison of TNF-α and IL-1β levels in serum of mice in each group.Compared with the normal control group, the TNF-α and IL-1β levels of serum in the APP/PS1 model group were higher than that in the control group(p<0.05). Compared with the model group of APP/PS1, Liuwei Dihuang pill, high dose group and Western medicine ibuprofen group mice serum TNF-α and IL-1β reduced(p<0.05), and Liuwei Dihuang Pill low dose group had no statistical difference(p>0.05).2.3 The m RNA expression of TLR4, My D88, and NF-κB in the hippocampus of mice in each groups In APP/PS1 model group, the expression of TLR4, My D88, and NF-κB m RNA was increased in the hippocampus of mice(p<0.05). Compared with the APP/PS1 model group, the expression of TLR4, My D88, and NF-κB m RNA were decreased in the hippocampus of the mice of the high dose group and western medicine ibuprofen group(p<0.05).2.4 The protein expression of TLR4, My D88 and NF-κB in the hippocampus of the mice in each groups.In APP/PS1 model group, the expression of TLR4, My D88, and NF-κB protein were increased in the hippocampus of mice(p<0.05). Compared with the APP/PS1 model group,the protein expression of TLR4, My D88, and p-NF-κB decreased in the hippocampus of the high dose group and western medicine ibuprofen group(p<0.05).3 Effects of Liuwei Dihuang Wan containing serum on TLR4/NF-κB signaling pathway in PC12 cells induced by Aβ.3.1 Effects of different concentration of Aβ25-35 on PC12 cells.With final concentrations of 10, 20, 30, 40 μM Aβ25-35 in PC12 cells respectively. After The effects of 24, 36, 48 and 72 hours, with the increase of Aβ25-35 concentration and action time, cell survival rate decreased gradually and has obvious dose and time dependent.Compared with the control group(with the volume of sterile water), the cell survival rate of10, 20μM Aβ25-35 group was no significant difference(p>0.05), and the effect was not obvious. The survival rates of the 30 and 40 μM Aβ25-35 were significantly decreased(p<0.05).Among them, 40 μM Aβ25-35 group had the lowest survival rate.3.2 Si RNA interference silenceg the expression of My D88 in PC12 cells.The chemically synthesized three My D88 si RNA sequences and negative control sequence were transfected into PC12 cells,by real time PCR screening silencing efficiency highest sequence for My D88-3, was detected by Western blot, the sequence of My D88 protein expression cut down 85±6.3%(p﹤0.01).3.3 Comparison of the contents of TNF-α and IL-1β in supernatant of PC12 cell culture medium in each groups Contents of TNF-α and IL-1β in model group PC12 cell culture medium increased significantly(P<0.05), drug containing serum group, My D88 silenced group and VE group TNF-α and IL-1β were significantly reduced.3.4 The m RNA expressions of TLR4, My D88, and NF-κB in PC12 cells in each group.The expression of TLR4, My D88, and NF-κB m RNA in model group was increased(P<0.05), compared with the model group, the serum group, My D88 silenced group and VE group, TLR4, My D88, and NF-κB m RNA expressiondecreased(P<0.05).3.5 The protein expressions of TLR4, My D88, and p-NF-κB in PC12 cells.The expression of TLR4, My D88, and NF-κB in model group were increased(P<0.05).Expressions of serum group, My D88 silenced group and VE group, TLR4, My D88, and p-NF-κB decreased(P<0.05).Conclusion:1 The latency was shorten by Liuwei Dihuang Wan of the APP/PS1 double transgenic mice in navigate experiment, the time in the quadrant of the platform, the percentage of the number of cross platform were increased. Liuwei Dihuang Wan can obviously improve the learning and memory impairment in APP/PS1 mice.2 APP/PS1 double transgenic mice hippocampus appeared a lot of senile plaque deposition caused by Aβ, Liuwei Dihuang Wan can effectively reduce its Aβ deposition, inhibit the excessive activation of As.3 Liuwei Dihuang Wan can inhibit TLR4/NF-κB signal transduction pathway, reduce the expression of TNF-α and IL-1β, so as to reduce the inflammatory response of the central immune system, and play the role of anti AD.4 After silenced My D88, TLR4/NF-κB downstream factor NF-κB m RNA and protein expression were decreased, the activation TLR4/NF-κB signaling pathway which mediated by My D88 is an important mechanism of AD central immune inflammatory response mechanism,provide experimental support for Liuwei Dihuang Pill treat AD from the kidney.