The Improvement Of Movement Function By WYD1-8 And WYD7-6 In A-synuclein Transgenic Mice The Role Of Heat Shock Protein 70 In Regulation Of Inflammation In Astrocytes

Part 1. The improvement of movement function by WYD1-8 and WYD7-6 in a-synuclein transgenic miceParkinson’s disease (PD) is the most prevalent neurological disorder, characterized by accumulation of a-synuclein with Lewy body and the progressive loss of dopaminergic (DAergic) neurons in the substantia nigra (SN). The cardinal motor symptoms of PD include tremor at rest, bradykinesia, muscular rigidity, stooped posture and instability. Recently study revealed the involvement of neuroinflammation and microglial activation in the pathogenesis of PD. WYD1-8 and WYD7-6 were Phloroglucinol derivatives synthesised by Natural Pharmaceutical Chemistry group. Previous stuidies demonstrated that WYD1-8 and WYD7-6 have great anti-inflammatory effects and neuroprotective effects on several PD animal models including MPTP-induced PD mice and MPTP+probenecid-induced PD mice. The aim of the present study was to futher evaluated the neuroprotective effect of WYD1-8 and WYD7-6 on a-Syn (A53T) transgenic mice and the mechanism. We used 4 month a-Syn (A53T) transgenic mice, which already developed motor deficit. a-Syn (A53T) mice were treated with WYD1-8 and WYD7-6 daily for 12 weeks. The motor ability was tested with rod climbing and rotarod test. The results showed that treatment with WYD1-8 and WYD7-6 improved the motor deficit of the mice significantly. Immunohistochmical and western blot showed that tyrosine hydroxylase (TH)-positive neurons in the midbrain of WYD1-8 and WYD7-6-treated mice increased significantly and the expression of phospho-synuclein decreased as compared with the a-Syn (A53T) control group. Meanwhile, the concentration of DA in the striatum of WYD1-8 and WYD7-6 treated mice increased as compared with the a-Syn (A53T) control group. Further studies found that WYD 1-8 and WYD7-6 inhibited microglial activation and reduce the production of neurotoxic inflammatory mediators such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in midbrain of mice. WYD1-8 and WYD7-6 also decreased the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), two important neuroinflammation related enzymes. In addition, we found WYD 1-8 and WYD7-6 inhibited neuronal apoptosis by increasing Bcl-2 expression. Taken together, these results suggested that compounds WYD 1-8 and WYD7-6 have significant neuroprotective effects and their anti-inflammation effects were involved.Part 2. The role of heat shock protein 70 in regulation of inflammation in astrocytesDegenerative diseases are characterized by progreesive loss of neurons. Although the underlying mechanism remain unclear, recent study showed that neuroinflammation plays important roles in neuronal regeneration. Most of the studies of neuroinflammation focus on microglia, while recent studies revealed that astrocytes were induced in neuroinflammation, while the exact mechanism was still unknown. Heat shock protein 70 (Hsp70) is originally described as intracellular chaperone, which plays an important role on protein quality control in the cells. Recent study showed that up-regulation of HSP70 had anti-inflammatory effects in the brain. Hsp70 was reported to protect neurons from damage and improve neurological function by decreasing inflammatory response as indicated by inactivation of glial cells and inhibition of pro-inflammatory cytokine release. Whether Hsp70 is involved in the neuroinflammation induced by astrocyte is not known yet. In the present study, primary rat astrocytes were treated with a-synuclein (a-Syn) to induce inflammation, and the role of Hsp70 was studied. Inflammation induced by a-Syn in primary rat astrocytes resulted in increasing expression of cyclooxygenase-2 (COX-2)、 inducible nitric synthase (iNOS) and glial fibrillary acid protein (GFAP) as well as production of tumor necrosis factor-α (TNF-a) and interleukin-1β (IL-1β). Overexpression of Hsp70 could significantly prevent the accumulation of proinflammatory proteins and cytokines. Meanwhile, inhibition of HSP70 exacerbated the inflammatory responses. Further studies indicated that phosphorylated inhibitory kappa B (IκB), phosphorylated c-Jun N-terminal kinase (JNK) and activation of inflammatory transcription factor nuclear factor-kappa B (NF-κB) was inhibited after overexpression of Hsp70, indicating that the JNK/NF-κB pathway were inhibited. In accordance with this result, we found phosphorylated IκB, phosphorylated JNK and activation of NF-κB was elevated significantly after Hsp70.inhibition. These findings indicated that Hsp70 could regulate the neuroinflammatory signal pathway. Hsp70 may be a new target to protect neuron cells through inhibiting neuroinflammation.