The Study Of The Theraputic Effect And Related Mechanism Of Phloroglucinol Derivatives On Parkinson’s Disease

Section 1. The study of the protective effects of phloroglucinol derivatives on Parkinson’s Disease modelsParkinson’s Diseases is one of the most common type of neurodegenerative disorders. The neuropathological hallmarks of PD are the progressive loss of dopamine-producing neurons in the substantia nigra pars compacta(SNc) and the formation of intraneuronal proteinaceous inclisions, called Lewy Bodies(LBs).These pathological changes lead to the functional impairment of the substantia nigra striatum system. Phloroglucinol derivatives WYD1-8 and WYD7-6 are new compounds synthesed by the Phytochemistry laboratory of instutute of Materia Medica, Chinese Academy of Medical Science & Peking Union Medical College. Previous studies demonstrated that WYD1-8 and WYD7-6 had strong anti-inflammatory property and good effects in PD models. Related data had been applied for patent. The main purpose of this thesis is to investigate the neuroprotection of WYD1-8 and WYD7-6 on MPTP-induced PD model mice and LPS-induced PD model by using midbrain was also applied.Results demonstrated that WYD1-8 and WYD7-6 treatment significantly improved the abnomal behavior of PD model mice and showed potent efficacy in protecting dopaminergic neurons. WYD1-8 and WYD7-6 also increased dopamine content in the striatum of PD model mice. ELISA results suggested that WYD1-8 and WYD7-6 could inhibit release of proinflammation cytokines including IL-1β and TNF-a. In the LPS-induced midbrain primary culture PD model, WYD1-8 and WYD7-6 significantly inhibited release of IL-1β and TNF-a and protected dopaminergic neurons.In conclusion, the present study demonstrates that WYD1-8 and WYD7-6 have strong anti-inflammatory and neuroprotective effects. The neuroprotective effects of WYD1-8 and WYD7-6 are closely related to the inhibition of microglial over-reaction. Our results also indicated that WYD1-8 and WYD7-6 might be potent drug candidates for the treatment of PD.Section 2. The Mechanic study of the anti-neuroinflammatory effects of phloroglucinol derivative WYD7-6In the first part of this thesis, we found that WYD7-6 could protect neurons through inhibiting neuroinflammation, however, the mechansm is still unclear. The main purpose of this part of the thesis is to investigate the mechanism of WYD7-6’s anti-neuroinflammatory effect. Proteomics was used to identify the possible target of WYD7-6 by comparing protein expression in the midbrain of PD model mice with or without WYD7-6 treatment. Western blot assay was used to validate the results of proteomic quantization. According to these results, we focused on phosphatase and tensin homolog on chromosome 10 (PTEN) which is a upstream regulator of Akt signal pathway.The role of PTEN in neuroinflammation was first studied. Microglial cell line BV2 was treated with LPS to induce neuroinflammation. Results demonstrated that the inhibition of PTEN lead to more microglial activation and releasing of inflammtory cytokines compared to LPS treated group, revealing that PTEN is a negative regulator of neuroinflammation. Then we explore the upstream regulator of PTEN. PTEN function is regulated mainly through phosphorylation by specific kinases, which frequently render PTEN functionally inactive. Among those kinases. Src tyrosine kinase was found to be the most obviously changed kinase by WYD7-6 treatment. The siRNA interference of Src lead to inhibition of LPS-induced neuroinflammation in BV2 cells, indicating that Src also plays a key role in regulating of neuroinflammation. Besides, interference of Src could decreased the phosphorylati-on of PTEN and Akt induced by LPS. Taken together, we conclude that Src regulates neuroinflammation through PTEN and its downstream Akt pathway.We also used BV2 cells to investigate the impact of WYD7-6 on Src/PTEN/Akt pathway in LPS-induced neuroinflammation. Results indicated that WYD7-6 could inhibit the activation of inflammation pathway mediated by Src kinase. To futher confirme this finding, more neurotoxins such as LPS, MPP+ and α-synuclein were used. Primary midbrain mixed cultures were treated with different neurotoxins and Src siRNA, then the neuroprotective effect of WYD7-6 was studied. Results demonstrate that inteference of Src attenuates WYD7-6’s protective effects, indicating that Src plays in important role in WYD7-6’s anti-neuroinflammatory effects. To futher study whether WYD7-6 directly inhibit the activity of Src, c-Src kinase assay/inhibitor screening kit was used. Results show that WYD7-6 didn’t inhibit the activity of c-Src directly, inhibitors of c-Src, indicating that WYD7-6 may inhibit Src/PTEN/Akt pathway by regulating its upstream molecules.The present study demonstrates that Src/PTEN/Akt pathway plays an imporant role in the anti-neuroinflammatory effect of WYD7-6. However, WYD7-6 couldn’t directly inhibit the activity of Src, it may regulate Src through other upstream proteins.