| Microglia abnormal activation mediated nerve inflammation is associated with a variety of diseases of the central nervous system. Abnormal activation microglia can release interleukin–1beta(IL-1?), interleukin-6(IL-6), and other inflammatory factors. The above inflammation factors sustained release in great quantities can directly damage neurons, or further activated microglia, that will be avicious cycle, and eventually lead to neuronal degenerative changes gradually or death, lead to Alzheimer's disease(AD), Parkinson's disease(PD) and other neurodegenerative diseases. Therefore, the abnormal microglia activation suppression effectively has become an AD and PD potential targets for drug research.Our topic research pilot study first found, the traditional Chinese medicine dragon dragon's blood(LXJ) has significant activation to inhibition activity of microglia, and under the concentration of play activity showed no obvious toxicity. Furthermore, our activity oriented from the active site identified 20 monomer compounds, the activity of preliminary evaluation results showed that the activity of 8 compounds(LXJ-8) the stronger and the content is higher. In this paper, we adopt the model of Lipopolysaccharide(LPS) induced microglia activation, comprehensive utilization Real time PCR, Western blot, ELISA and Immunofluorescence and other experimental technology, proposed system research LXJ-8 to inhibit the action of the abnormal activated microglial cells and its possible mechanism. Therefore, our laboratory according to a series of dragon dragon's blood monomer has carried on preliminary screening, eventually determine that LXJ-8 have significant inhibition of the microglia NO release, and showed obvious concentration-response relationship. In this paper, we using fluorescence quantitative PCR, Western blot and ELISA, Immunofluorescence staining technology, further study LXJ-8 inhibit the action of the microglia activation mechanisms.Griess experimental results show that LXJ-8(10, 100 mM) can significantly inhibit LPS induced N9 microglia release of NO, ELISA experimental results show that LXJ-8(1, 10, 100 mM) can significantly inhibit LPS induced N9 microglia IL-1? release, LXJ-8(10, 100 mM) can significantly inhibit LPS induced N9 microglia release of IL-6. The Real time PCR experimental results show that the LXJ-8(1, 10, 100 mM) can significantly inhibit LPS induced N9 microglia induced nitric oxide synthetase(iNOS) mRNA expression, LXJ-8(10, 100 mM) can significantly inhibit LPS induced N9 microglia IL-1? mRNA expression, LXJ-8(1, 10, 100 mM) can significantly inhibit LPS induced N9 microglia IL-6 mRNA expression. Western blot experimental results show that LXJ-8(100 mM) can significantly inhibit LPS induced N9 microglia expression of TLR4 protein. And then we study the Mitogen-activated protein kinases(MAPKs) family and Akt pathway, the results show that the LXJ-8(1, 10, 100 mM) can significantly inhibit LPS induced N9 microglia p-JNK, p-p38 protein expression, the expression of p-ERK has no obvious effect. LXJ-8(1, 10 mM) can significantly inhibit LPS induced N9 microglia expression of c-Jun protein in the nuclei. LXJ-8(1, 10, 100 mM) on the LPS induced N9 microglia p-Akt protein expression had no obvious effect. Western blotexperimental results show that LXJ-8(1, 10, 100 mM) can significantly inhibit LPS induced N9 microglia p-I?B? protein expression, and significantly reverse I?B? protein degradation; Immunofluorescence experiments show that LXJ-8(100 mM) can significantly inhibit LPS induced N9 microglia NK-?B p65 protein nuclear transfer. The above results suggest that LXJ-8 inhibit LPS induced N9 microglia activation may related to the inhibition of NF-?B, MAPKs signaling pathway.To sum up, this study first found that LXJ-8 can significantly inhibit LPS caused microglia NO, IL-6, IL-1 beta release and iNOS, IL-6, IL-1? mRNA expression and its mechanism may be by inhibiting TLR4 mediated NF-?B, JNK-MAPK signaling pathway. |
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