Effects Of Triptolide On The Senile Plaques Formation And Inflammatory Response In The Hippocampus Of APP/PS1 Double Transgenic Mice

BACKGROUNDThe pathological changes seen in Alzhemier's disease(AD) brains are a series of chronic inflammation processes. The chronic inflammation response is characterized by the presence of abundant activated microglia, and reactive astrocytes proliferation associated with deposition of fibrillarβ-amyloid(Aβ), and increases expression of pro-inflammatory cytokines and complement components. Non-steroidal anti-inflammatory drugs(NSAIDs) may not only reduce the risk of AD, moreover, they may delay AD progression. Animal experimental studies confirmed that NSAIDs may 1. suppress Aβdeposition and plaque formation, 2. Inhibit microglia activation and reactive astrocytes proliferation, and 3. decrease the expression and secretion of pro-inflammatory cytokines such as TNFα,and IL-1βin the AD brain. Therefore, the anti-inflammation effect of NSAIDs in AD seems to have an important value. However, as a result of the NSAIDs treatment there is serious and poisonous side effects, which limits its clinical usage.The Triptolide(T10) compound is one of the active components from an extract of Traditional Chinese Medicine Tripterygium wilfordii hook F. It possesses anti-inflammation and immuno-suppression functionality and has been used to treat rheumatism and rheumatism arthritis, systematic lupus erythematosus and other auto-immunity and inflammatory responsive diseases. Previous in vitro research has indicated that T10 anti-inflammation and immunosuppression effects is via it's suppression of the expression and releasing of pro-inflammatory cytokines like IL-1β, TNFα, and as well as suppression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2(COX-2) induction. Current research suggested that T10 has a neurotrophic function and may protect the DOPAminergic neurons in the mesencephalon from lipopolysaccharide (LPS)injury. The suggested T10's neuronal protective mechanism is involved in inhibiting microglia activation and suppresses the inflammatory reaction. There has been found no literature that reports on the effect of T10 on the inflammatory and immune cascade seen in the AD brain. The present research is the first that used T10 to treat APP/PS1 double transgenic mice model of AD(APP/PS1 dtg). This is a preliminary study to assess the effects of T10 on APP/PS1 dtg mice and discusses the possibility and the possible mechanism for T10 to treat or prevent AD.OBJECTIVESTo examine the effects of T10 using the APP/PS1 dtg mouse model of AD on: 1. Aβdeposition and senile plaques formation. 2. Microglia and astrocytes activation and proliferation. 3. COX-2 induction and possible mechanism of T10 inhibits expression of COX-2. METHODS1. Research in vivo: 18 Male APP/PS1 dtg mice aged at 4.5 months were used in this study. The mice were divided into 3 groups randomly, High-5μg/kg.d T10(T10 H, n=6), Low-1μg/kg.d T10(T10 L, n=6) and placebo(PLC, n=6). Intraperitoneal administration of T10 and capacity solvents (PLC group) was in a period of a total of 45 days. In addition, five male wild type non-transgenic mice of comparable age and receiving no treatment were used for comparison. After 45 days of treatment, the mice were sacrificed and the brains removed for processing. The brains were separated along the middle sagittal sulcus. The left side was used for the histological study and the right half was used for protein analysis. Histological research: According to unbiased stereological system SUR (Systematic-Uniform-Random) principle, every tenth section was collected for immunohistochemistry. 6E10, GFAP, MAC1, COX-2 Immunohistochemical staining combined with unbiased stereological methods was used to quantitative analyze Aβ,astrocytes,microglia cells and COX-2 as well as changes of Congo red positive senile plaques(SP) in hippocampus. Double immunofluorescence staining to showed the relationship between microglia cells and astrocytes with Aβand COX-2. Western blot protein analysis assessed changes of protein level for Aβand COX-2.2. Research in vitro: (1) Cultured human A172 astrocytes cell lines were pretreated with a low, middle and high dose of T10(0.2, 1 and 5μg/L T10) for 1h subsequent to use of 1mg/L LPS processing cells for 3 h and 6 h. Reverse transcription PCR(RT-PCR), and Western blot methods were used to analyze the effects of the different concentrations of T10 on the COX2 expression induced by LPS; electrophoretic mobility shift assay(EMSA) studied the effects of T10 on the NF-κB/DNA binding activity in the cells. (2) Cultured high expresses the human COX-2 protein(hCOX-2) sf-9 cell lines were processed with T10, COX-2 activity inhibitor NS398 and indomethine. Radioimmunoassay method was used to analyses PGE2 level in the media to study the effects of T10 on the COX-2 enzyme activity.RESULTS1. Results in vivoA. Findings indicate(1) T10 may suppress Aβdeposition in the hippocampus, and reduces senile plaques formation. Unbiased stereology quantitative analysis showed that the total area of 6E10 positive Aβplaques in the hippocampus compared with the PLC group's, Aβplaques of T10 H was reduced 35％(P＜0.001), and that of T10 L reduced 18％(P＜0.05). In addition, the T10 H group compared with the T10 L group showed a reduction of 21％(P＜0.05). (2) Western blot protein analysis also displayed that T10 H group Aβprotein levels in the hippocampus was lowest, Aβprotein levels in PLC group was highest, and Aβprotein levels in T10 L group was moderate; (3) T10 also may reduce the number of 6E10 positive neuron in the CA1 region of hippocampus; (4) T10 may suppress Aβaggregation and fibrils, and reduces SP formation. Compared with PLC group's Congo red positive SP, the total area of SP in the hippocampus of T10 H group was reduced 32％(P＜0.001), and in the T10 L reduced 27％(P＜0.05).B. T10 may also inhibit neuroglia cell activation and proliferation: (1) T10 may suppress the microglia cell activation by reducing the cluster area of microglia cell. Compared with the PLC group, the total number of microglia cells in hippocampus of T10 H was reduced 30％(P＜0.01), the T10 L was reduced 18％(P＜0.05). When comparing T10 H with the T10 L a reduction of 17％(P＜0.05) was found. These results indicate that T10 assumes a dosage dependent inhibition of microglia proliferation. (2) T10 may suppress astrocytes activation, reduces GFAP expression as well as the suppression of astrocytes proliferation. Compared with the PLC group, the total number of astrocytes in the hippocampus of T10 H was reduced 20％(P＜0.01), the T10 L reduced by 13％(P＞0.05).Co T10 may suppress COX-2 expression/activation in the hippocampus of APP/PS1 dtg mice model of AD: (1) T10 may suppresses COX-2 expression/activation in hippocampus of APP/PS1 dtg; reduces the total number of COX-2 positive neurons in the CA2-4 region of hippocampus; (2) In the hippocampus of the wild type non-transgenic controls astrocytes was not seen to express COX-2, but astrocytes in the hippocampus of APP/PS1 dtg mice showed COX-2 expression. After 45 days T10 treatment, the total number of COX-2 positive astrocytes in hippocampus was reduced by 28％(T10H, P＜0.01) and 12％(T10L, P＞0.05) compared to the group PLC respectively; (3) Western blot protein analysis showed that COX-2 protein level was lowest in the hippocampus of wild type non-transgenic control, followed by the T10 H, the T10 L, and highest in the PLC group.2. Results in vitroA. LPS stimulated the A172 human astrocytes resulting in COX-2 expression enhancement; the level of PGE2 in the media was significant increased, and T10 showed the dosage-dependent manner to reduce COX-2/PGE2 production;B. T10 when administered to the high expressing human COX-2 sf-9 cell lines didn't change the COX-2 enzyme activity which suggested that T10 may have an effect on the transcription level for COX-2 expression.C. LPS stimulates cultured A172 human astrocytes line resulting in the increased binding of NF-kB/p50/P65; T10 assumes the dosage dependence response to suppress the NF-kB active binding.CONCLUSIONS:The results in present research indicated that T10 suppresses Aβproduction and deposition and inhibits its aggregation and plaques formation in the hippocampus of APP/PS1 dtg mouse, and inhibits microglia and astrocytes activation and proliferation. And T10 may also suppress activated astrocytes and neurons COX-2 induction. Theses results suggested that T10 possibly possesses some of NSAIDs identities and may suppress the chronic inflammation in the AD brain and/or may be useful in AD prevention and treatment. Because of T10 therapy there is no serious and poisonous side effects of NSAIDs, which may has advantage over the NSAIDs to be used for AD patients.