The Mechanism Of Microglia On Alzheimer’s Disease Induced By Aβ

Alzheimer’s disease(AD) is a degenerative disease of the central nervous system, characterized by progressive cognitive dysfunction and memory impairment. Though its pathogenesis is complex, amyloid β-protein(Aβ) as the initiate factor has been accepted. The main pathological changes is the formation of Senile plaque extracellular, which is surrounded by masses of actived microglia(MG) and inflammatory factors. Research in domestic and abroad showed the "double-edged sword" effect of MG on AD, while the mechanism and whether there is a relation between "double-edged sword" effect and Aβ dose are unclear.Objective:To explore the mechanism of microglial cell in AD and relation with Aβdose. To analyze MG activation and elimination to Aβ. To analyze the relation between expression of Scavenger receptor A(SRA), Toll-like receptor2(TLR-2), Nuclear Factor Kappa B(NF-κB), matrix metalloproteinase-9(MMP-9), tissue inhibitor of metalloproteinase-1(TIMP-1), tumor necrosis factor alpha(TNF-α), interleukin 1 beta(IL-1β) and Aβ dose, as well as to neuron damage, providing experimental basis for new target-therapy to AD.Methods:50 male Wistar rats were randomly divided into five groups(A, B, C, D,E), 10 cases each group. The gelstate 0.9% Na Cl was injected into the rat’s hippocampus for group A. While Aβ 5ul(0.5, 1, 5, 10ug) was injected in group B-E. One week after injection Aβ Morris water maze was performed to record the latency of rats finding the hidden platform within 60 s.On the 6th day, the platform was moved to record the times crossing the platform area,time staying in the platform quadrant, ratio of path in platform quadrant to the whole path during 2min. On the 14 th day, blood was collected, enzyme linkedimmunosorbent assay(ELISA) was used to detect the tumor necrosis factor alpha(TNF-α), interleukin 1 beta(IL-1β) of serum. Six rats of each group were used for taking fresh hippocampus quickly, the m RNA level of CD11 b,MMP-9, TIMP-1, TLR-2, NF-κB were detected using Real-Time quantitative Polymerase Chain Reaction(q RT-PCR). Four rats of each group were infused with paraformaldehyde, brain tissue was performed external fixation, paraffin imbedding, HE staining was used to observe the neuron morphology,immunohistochemical was used to detect the expression of Aβ、CD11b、SRA、TLR-2 in hippocampus.Results:1 Morris test result The latency finding the platform of D, E groups in10 th, 11 th, 12 th, 13 th were significantly longer than those of A, B, C groups(P<0.01). On the 13 th day, the times crossing the platform of D(7.78±1.33),E(5.32 ±1.25)groups were significantly less than the groups of A(13.64±1.85),B(12.97±1.58), C(12.48±1.61) groups(P<0.01).The staying time in the platform quadrant of D(44.28±2.96s), E(41.32±3.12s)groups were significantly shorter than those of A(59.25±2.20s), B(57.38±3.66s), C(57.27±3.05s) groups(P<0.01).The ratio path in platform quadrant and the whole path during 2min of D(44.68±2.61%), E(41.52±2.72%)groups were significantly less than those of of A(62.21±1.82%), B(61.50±2.56%), C(60.45±2.48%)groups(P<0.01).2 Effect of Aβ on rat hippocampal neurons(HE) The HE stainning showed that there were 3-4 layers cell in hippocampus CA1 region in A, B, C group, and the shape of cell were clear, integrity with dentrite, nucleus staining was deep,and the axons of neurons were visible. While in D, E groups,there were obvious neuron loss in CA1 region with only 1-2 layers cells, large numbers of micrglial cells filling in the deletion part.Cell quantity of CA1 region in D(84.75), E(66.63) groups were significantly less compared with those of A(126.25), B(120.63), C(114.75)groups(P<0.01).3 IHC detection results Aβ is mainly distributed in the extracellular nerve and cytoplasm of MG. Expression of Aβ in the D(0.0202±0.0057),E(0.0257±0.0074)groups were significantly higher than those of A(0.0022±0.0004), B(0.0041±0.0010), C(0.0061±0.0016)groups. The results showed that CD11 b in hippocampus of rats in the D(0.0460±0.0083, E(0.0615±0.0078)groups were significantly higher than those in the A(0.0166±0.0048),B(0.0216±0.0034), C(0.0236±0.0044)groups(P<0.01). And the two groups of D, E had a significant difference(P<0.05). Expression of SRA in the D(0.0430±0.0113), E(0.0609±0.0175) groups were increased significantly compared with the A(0.0109±0.0036), B(0.0212± 0.0098), C(0.0264±0.0091)groups(P<0.01).TLR-2 mainly gathered around Aβ plaques. Expression of TLR-2 in the D(0.0366±0.0073), E(0.0448±0.0108) groups were increased significantly compared with the A(0.0187±0.0058), B(0.0100±0.0034),C(0.0151±0.0060)groups(P<0.01). And there were an signifant difference between A and C, D and E(P<0.05).4 TNF-α, IL-1β in serum TNF-α in serum of D(568.65±44.66),E(685.06±29.92)groups were higher than those of A(426.866±55.91),B(432.99±28.09), C(488.14±39.04)groups(P<0.01). IL-1β in serum of D(104.60±9.32), E(143.38±17.09) groups were higher than those of A(49.13±8.46), B(60.89±14.71), C(79.81±9.94)groups(P<0.01).5 q RT-PCR result Expression of m RNA on CD11 b in rats hippocampus of D(1.9052±0.2580), E(2.3619±0.4195)groups were higher than those of A(1.0000±0.0000), B(1.1803±0.0606), C(1.2795±0.1821)groups(P<0.01);Expression of MMP-9 in D(4.2696±1.4891), E(7.8016±1.2583)groups were higher than A(1.0000±0.0000), B(1.4696±0.3082), C(1.8371±0.4122)groups(P<0.01). Expression of TIMP-1 in D(3.4530±0.8926), E(5.5269±1.3717)groups were higher than those of A(1.0000±0.0000), B(1.1314±0.1143),C(1.3905±0.1419)groups(P<0.01). Expression of TLR-2 in D(2.1859±0.3814), E(2.9777±0.3907)groups were higher than those of A(1.0000±0.0000), B(1.1785±0.1066), C(1.4633±0.1273)groups(P<0.01).Expression of NF-κB in D(1.7470±0.1256), E(2.2719±0.5332)groups were higher than those of A(1.0000±0.0000), B(1.2743±0.1651), C(1.4291±0.0777)groups(P<0.01).And there was an significant difference between D and E(P<0.05).There were an significant difference betwen A and C in the expression of TLR-2, NF-κB(P<0.05).Conclusions:1 Aβ could activate the MG, the activated MG could eliminate Aβ.2 Low dose of Aβ could activate SRA, TLR-2 on the surface of MG,improve the elimination of MG to Aβ. Besides activating MG, high dose of Aβ could also activate NF-κB, improve more TNF-α, IL-1β release, induce neuron damage.