Protective Effects Of Rapamycin On Inflammation Model Of Alzheimer's Disease

Objective: To study the protective effects and mechanism of rapamycin on inflammation model of Alzheimer's Disease in vitro and in vivo.Method: We built inflammation models by using A?25-35 or LPS in vitro and in vivo, and used RAPA in the A?25-35-induced or LPS-induced inflammatory models;Western Blot was applied to detect the protein expression levels of m TOR ?p-m TOR(Ser2448)?Stat3?p-Stat3(Ser727)?NF(?)B?p-NF(?)B(Ser536)?TNF??IL-1? ? Bcl-2 and Bax; Cell apoptosis and viability were detected by flow cytometry and MTT; Cell and tissue immunofluorescence technique were performed to observe the expression and localization of m TOR/NF(?)B and the proliferation of GFAP.Result: At the cellular level, comparing with control group, the protein expression levels of m TOR ? p-m TOR(Ser2448) ? Stat3 ? p-Stat3(Ser727) ? NF(?)B ?p-NF(?)B(Ser536)?TNF??IL-1? and Bax increased and Bcl-2 decreased in N2 a cells treated with A?25-35 for 6h or LPS 1h(p<0.05), while combined treatment with A?25-35 /RAPA or LPS / RAPA completely abolished the stimulate effects mediated by A?25-35 or LPS on above-mentioned proteins(p<0.05); When N2 a cells were transfected with plasmid of NF(?)B, the protein expression levels of m TOR ?p-m TOR(Ser2448)?Stat3?p-Stat3(Ser727)?NF(?)B?p-NF(?)B(Ser536)?TNF?, IL-1?and Bax increased and Bcl-2 decreased.But the transfected cells with RAPA treatment for 12 h had almost the same protein expression levels of N2 a cells(p<0.05). Comparing with control group, m TOR and NF(?)B transferred from the cytoplasm to the nucleus in N2 a cells treated with A?25-35 for 6h, while combined treatment with A?25-35 /RAPA completely abolished the stimulate effects mediated by A?25-35; Comparing with control group, partial NF(?)B transferred from the cytoplasm to the nucleus in N2 a cells which transfected with plasmid of NF(?)B.Comparing with N2 a cells were transfected with I(?)B mutant plasmid, there were no significant differences in the protein expression levels of m TOR ?p-m TOR(Ser2448)?Stat3?p-Stat3(Ser727)?NF(?)B and p-NF(?)B(Ser536) in I(?)B and I(?)B+LPS groups. Flow cytometry showed that the apoptosis rate of untreated N2 a was 2.96%, while a 8.57% apoptosis rate was detected in cells cultured with A?25-35 for 6h and an approximately 3.91% apoptosis rate was observed in N2 a cells co-treated with A?25-35 and RAPA(p<0.05); MTT assay showed a significant reduction in the viability of N2 a cells following treatment with A?25-35 for 6h or LPS 1h as compared to controls(p<0.05), while the viability of N2 a cells co-treated with A?25-35+RAPA or LPS+RAPA increased(p<0.05). In vivo the protein expression levels of m TOR?p-m TOR(Ser2448)?Stat3?p-Stat3(Ser727)?NF(?)B?p-NF(?)B(Ser536)?TNF??IL-1? and Bax increased and Bcl-2 decreased in C57 mice treated with A?25-35 for 24 h as compared to control group, while combined treatment with A?25-35+RAPA completely abolished the stimulate effects mediated by A?25-35 on above-mentioned proteins(p<0.05); There had a same similar results on above-mentioned proteins in KM mice treated with LPS 24 h by tail vein injection, while KM mice administrated RAPA for 3 days and then treated with LPS for 24 h, had almost the same protein expression levels with control group(p>0.05). Immunofluorescence results showed that the number of Astrocytes increased in C57 and HM mice treated with A?25-35 or LPS, and treatment with A?25-35+RAPA or LPS+RAPA completely abolished the stimulate effects mediated by A?25-35 or LPS on Astrocytes.Conclusion: A? can cause neurons inflammation. The phosphorylation level of m TOR in inflammation models was significantly increased, indicating that m TOR signaling was activated and played an important role in pathogenesis of neurons inflammation. Rapamycin has a protective effects on neurons inflammation by using A?25-35 or LPS, and these anti-inflammatory effects of RAPA seems to be mediated by inhibiting m TOR, down-regulating Stat3 and inhibiting NF(?)B shuttle from the cytoplasm to the nucleus pathway.