| Recent research has focused on soluble oligomeric assemblies ofβ-amyloid peptides (Aβ) as the proximate cause of neuroinflammation, synaptic loss and the eventual dementia associated with Alzheimer's disease (AD). Oligomeric AP (oAβ), which may be the major pathogenic form of Aβin the early stages of AD, can stimulate glia to induce a more profound inflammatory response and neurotoxicity than fibrillar AP (fAp). The inhibition of Ap-dependent activation of microglia could be an effective therapeutic approach to delay the progression of AD. Additionally, an aberrant expression of mitogen-activated protein kinases (MAPKs) and activation of nuclear factor-κB (NF-κB) in Aβ-induced microglia is directly correlated with pathogenic events of AD. Accordingly, strategies to suppress MAPKs and/or NF-κB activation may attenuate neuroinflammation and neuronal damage, which will be of benefit in treatment of AD.Extracts of natural herb Tripterygium wilfordii Hook. F (TWHF) have been found to have potent anti-inflammatory and immunosuppressive functions and widely used in China for treatment of rheumatoid arthritis. Our previous findings demonstrated that tripchlorolide (T4), an extract of TWHF, even at nanomolar level exerted potent anti-inflammatory effects on LPS-stimulated microglial cells and protected neuronal cells from neuroinflammatory toxicity. It suggest that T4 could be a potential neuroprotective agent against neuroinflammatory process, such as AD.Here we firstly tested the effects of oAβ(1-42) versus fAβ(1-42) on the cells viability, the expression of inflammatory mediators, as well as phagocytosis function in microglial cells, and explored the activations of MAPKs and NF-κB signaling pathways in oA(3-induced microglia. Secondly, we investigated whether T4 suppressed oAβ-induced microglial activation and protected against microglia-mediated oAβneurotoxicity. Also, we gained insights into the anti-inflammatory molecular mechanisms of T4 in oAβ-stimulated microglial cells. Lastly, we chose senescence-accelerated mouse Prone 8 (SAMP8) as a model of sporadic AD to testify if T4 ameliorated learning and memory deficits.The results showed that oAβincreased interleukin-1β(IL-1β) level in a manner that was rapid, potent and transient. Also, it induced higher levels of tumor necrosis factor-α(TNF-α), nitric oxide (NO), prostaglandin E2 (PGE2) and intracellular SOA than fAβ. Interestingly, pretreatment of microglia with oAβfor 6～12h attenuated the capacity of microglial phagocytosis following fAβexposure. The further data was shown that a well inverse correlation between microglial phagocytosis and inflammatory mediators. Also, IL-1β, LPS and tert-butyl hydroperoxide (t-BHP) all decreased phagocytosis levels in fAβinduced-microglia, which could be relieved by NF-κB inhibitor, pyrrolidone dithiocarbamate (PDTC) and N-Acetyl-L-cysteine (NAC). Meanwhile, in microglia, the expressions of MAPKs (p38MAPK, ERK and JNK), I-κB and NF-κB were induced by oAβin a time-dependent manner. A peak activity of MAPKs and NF-κB was observed at 30-120min, and the expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were significantly elevated after oAβstimulation for 24h. Inductions of inflammatory mediators, iNOS and COX-2 were attenuated by JNK inhibitor (SP600125), ERK inhibitor (U0126) and PDTC, but not by p38 inhibitor (SB203580), in oAβ-stimulated microglia.Further, the conditioned media from oAβ-treated microglial cells (Aβ-CM) was applied to treat Neuro-2A and primary cortical neurons. The findings confirmed that the neuronal toxic effect of oAp was preferentially mediated by microglia. Therefore, Aβ-CM was applied to the examination of the neuroprotective effect of T4. As expected, T4 protected neuronal cells from microglia-mediated oAβtoxicity. It significantly attenuated oAβ-induced release of inflammatory productions, including TNF-α, IL-1β, NO and PGE2, in a dose-dependent manner (P<0.05). It also reversed oAβ-mediated microglial phagocytosis injury, as well as down-regulated the protein levels of iNOS and COX-2 in microglia. Further molecular mechanism study demonstrated that T4 inhibited the nuclear translocation of NF-κB without affecting I-κBαphosphorylation. It repressed AP-induced JNK phosphorylation but not ERK or p38 MAPK.Lastly, in order to testify if T4 would ameliorate cognitive deficits, the 7-month-old SAMP8 mice were chronically treated with T4 (0.25,1.0,4.0μg/kg per day, injected intraperitoneally for 2.5 months, respectively). The SAMR1 mice with the same age were used as "normal aging" control. A NF-κB inhibitor, PDTC-treated SAMP8 mice (50mg/kg per day, i.p.) were used as the drug treatment controls. The beneficial role of T4 was manifested to improve learning and memory function in 10.5-month-old SAMP8 mice by Y-maze and Morris water maze behaviour tests. The optimal dose of T4 was 0.25～1.0μg/kg per day, which did not show significant side-effects of T4 on the blood routine test, blood biochemical assays, as well as the survival of mice.Collectively, these results suggest that Aβoligomers induces a potent inflammatory response, subsequently exacerbates microglial phagocytosis in the early AD pathological affairs, and that activation of microglia by Aβoligomers is through NF-κB, JNK and ERK signaling which subsequently lead to the upregulation of inflammatory mediators. T4 protects neuronal cells by blocking inflammatory responses of microglia to Aβoligomers and that it acts on the signaling of NF-κB and JNK which are involved in the modulation of this process. Also, T4 ameliorates cognitive deficits in senescence-accelerated AD mice. Therefore, our findings may provide new insight for developing the clinical application of tripchlorolide to Alzheimer's disease.
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