1.FLZ Inhibits HMGB1-mediated Neuroinflammatory Responses In Microglial Cells 2.Study On The Anti-neuroinflammatory Effects Of Dehydromiltirone And Related Mechanisms

High mobility group box 1 (HMGB1) is a ubiquitous and conserved chromosomal protein, located in all mammalian nuclei and plays a vital role in gene transcription, nucleosome stabilization, and neurite outgrowth. HMGB1 can be actively secreted from macrophage cells or passively released from necrotic cells to initiate inflammatory responses. Extracellular HMGB1 binds to its corresponding receptors and up-regulates the production of pro-inflammatory mediators via activating intracellular signaling pathways. The release of HMGB1 has been reported in various diseases such as septic shock, arthritis, acute myocardial infarction and acute pancreatitis. Therefore, attenuating aberrant macrophage activation by use of anti-HMGB1 agents might have therapeutic potential for the treatment of inflammation-related disorders.Compound FLZ is a novel synthetic derivative of squamosamide from a Chinese herb (formulated as:N-(2-(4-hydroxy-phenyl)-ethyl)-2-(2,5-dimethoxyphenyl)-3-(3-methoxy-4-hydroxy-phenyl)-acrylamide, the code name:FLZ). Previous studies have demonstrated that FLZ shows potent neuroprotective effects in both in vivo and in vitro PD models. Previous mechanistic studies revealed that the anti-inflammatory effect of FLZ contributed to its neuroprotective effect, while the underlying mechanisms need to be futher clarified. In this study, we investigated the important role of HMGB1 in the neuroinflammation and FLZ’s effect on HMGB1-mediated inflammatory responses in BV2 cells, a murine microglial cell line.The effects of FLZ on cytoplasmic localization and extracellular release of HMGB1 were first explored by Western blot and ELISA. Next, Co-immunoprecipition was used to study FLZ’s effect on the interaction between HMGB1 and its primary receptors. Finally, we employed recombinant HMGB1 to simulate microglial activation in vitro to study whether FLZ has inhibitory effect on HMGB1 induced neuroinflammatory responses. The results showed that HMGB1 existed mainly in nucleus in resting state, translocated to cytoplasm and was subsequently released into culture media after LPS stimulation. FLZ inhibited the HGMB1 translocation and release in a dose-dependent manner. The CO-IP result showed that HMGB1 bound to TLR4 while FLZ showed no effect on the bingding of HMGB1. After binding to TLR4, TLR4/MyD88/NF-κB signaling pathway was activated and thus triggered neuroinflammatory responses. FLZ effaciously blocked the activation of NF-κB and attenuated TNF-α production. These results suggested that FLZ could inhibit HMGB1 translocation and release during neuroinflammation as well as HMGB1-mediated inflammatory responses. HMGB1 might be one of the anti-inflammatory targets of FLZ. This study also revealed that targeting at this inflammatory mediator may have benefit effects on neuroinflammation related disorders, such as PD.Neuroinflammation is the innate immune response that mediated by microglial and astrocyte cells in brain. Under normal conditions, microglial and astrocyte cells are quiescent and serve important immune surveillance function in protecting neurons. Upon subtle changes in the micro-environment, or as a consequence of pathological insults, these immune cells rapidly transform into active state. Activated glial cells produce proinflammatory cytokines and neurotoxic mediators, such as tumor necrosis factor (TNF)-α、interlukin (IL)-6 and nitric oxide (NO), which have deterious effects on neighboring neurons. Accumulating evidence indicates that neuroinflammation is closely associated with the pathogenesis of several neurodegenerative diseases such as Alzheimer’s disease and Parkinson’ disease, and inhibition of microglia activation and suppression of pro-inflammatory mediators production has been reported to delay the disease progression. So compounds with anti-neuroinflammatory effects may offer potential therapeutic strategy for these diseases. DanShen is one of the most popular traditional Chinese medicines that has been widely used for cardial and vascular diseases such as ischemia and myocardial infarction. Dehydromiltirone is one of the main bioactive components of DanShen, previous studies revealed that Dehydromiltirone has strong anti-inflammatory activity and it might explain the reason DanShen shows therapeutic effect. However, whether it has anti-neuroinflmmatory effect has not been clarified. In this experiment, we used BV2 cells, a murine microglia cell line that is commonly used as a substitute for primary microglia in experimental studies, to investigate the anti-neuroinflammatory activities of Dehydromiltirone and the underlying mechanisms. BV2 cells were pre-treated with Dehydromiltirone and then stimulated by LPS. The levels of nitric oxide (NO) were measured by Griess assay and the concentrations of several pro-inflammatory cytokines were measured by ELISA. Confocal fluorescence microscopy was used to measure the expression of CD 11b, the biomarker of activated BV2 cells. The expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, NF-κB, PI3K and AKT were determined by Western blot analysis. The results showed that the treatment of Dehydromiltirone significantly inhibited the production of NO, TNF-α and IL-6, attenuated the expression of iNOS and COX-2 protein, dampened the microglial activation in LPS-stimulated BV2 cells. Futher mechanistic study revealed that Dehydromiltirone inhibited the phosphorylation of PI3K and AKT in LPS-stimulated BV2 cells, decreased NF-κB activation by suppressing the degradation of IκB. Taken together, these results indicated that Dehydromiltirone had significant anti-neuroinflammatory effects through inhibiting PI3K/AKT phosphorylation, inhibiting NF-κB signaling activation and then cytokine production. These results also suggested that Dehydromiltirone has the potential to be developed as a new neuroprotection agent, while the mechanisms need to be futher studied.