Mechanism For The Effect Of Licochalcone A On Lps-induced Microglia Activation

In normal brain tissue, microglia exhibit aramified morphology with resting state. Ramified microglial cells could maintain the dynamic balance of microenvironment of the nervous system and repairdamage. Activated microglia showed amoeboid-like morphology could phagocytize foreign materials and secret some cytokines andchemokines. Excessively activated microglia release pro-inflammatory cytokines(TNF-α, IL-6 and IL-1β) and pro-inflammatory enzymes(i NOS and COX-2) and result inneuronal death, then further trigger the activation of microglia. The vicious circle of inflammatory can result in sustaining degeneration and death of brain neurons, exacerbating the occurrence and development of inflammation.An increasing number of studies have proved the neuroinflammation resulted from the activation of microglia plays an important role in the development of neurodegenerative diseases such as Parkinson’s disease. Therefore, inhibiting the inflammation induced by the activation of microglia may prevent the further development of neurodegenerative diseases. Licochalcone A belongs to flavonoids in licorice, and has extensive pharmacological activities, including anti-inflammatory, anti-tumor, anti-bacterial and anti-parasitic and so on. Studies have shown that Licochalcone A can inhibit the expression of i NOS and COX-2 on LPS induced RAW 264.7 cells. Another study found that Licochalcone A can inhibit the activation of NF-κB singaling pathway. So, we speculated that Licochalcone A can inhibit the activation of microglia and have neuroprotective effect. Therefore, basing on the culture of microglial cell lines and primary mesencephalic neuron-glial cells in vitro, we explored the mechanism for the effect of Licochalcone A on LPS-induced microglial cells.Firstly, MTT was used to determine the optimal concentration range of Licochalcone A on BV-2 cells. BV-2 cells were pre-treated with different concentrative Licochalcone A for 4 hours, followed by LPS for 4 hours, then Western bloting and real-time PCR were applied to measure the dynamic expression of i NOS and COX-2 and determine the optimum concentration of Licochalcone A. In order to determine the effect of Licochalcone A of different concentrations on LPS-induced pro-inflammatory cytokines produced by microglial, RT-PCR and ELISA were used to detect the expressions of pro-inflammatory cytokines(TNF-α, IL-6 and IL-1β) pre-treated with Licochalcone A for 1hour and followed by LPS for 24 hours, respectively; Meanwhile, Western blot were used to detecte the effects of Licochalcone A on MAPK singaling pathway and NF-κB singaling pathway of LPS-induced microglia. To verify whether the protective effect of Licochalcone A on dopamine neuron was mediated by inhibiting inflammation of glial cells, model of Parkinson’s disease was based on the culture of LPS-induced primary mesencephalic mixed neuronal glial cell in vitro, and the number of TH-positive cells was determined by immunohistochemical staining to detect dopamine neuron degeneration. In order to explore the effect of Licochalcone A on dopaminergic neurons, the expression of inflammatory factors produced by nerve mixed glial cells was measured by real-time PCR.In BV-2 inflammatory models, we found that Licochalcone A could significantly inhibit the expression of LPS-induced i NOS, COX-2, TNF-α, IL-1β and IL-6 at gene and protein level. Western blot results showed that Licochalcone A can significantly inhibited LPS induced phosphorylation of ERK in MAPK singaling pathway and phosphorylation of p65 in NF-κB signaling pathway on BV-2 cells. However, the phosphorylation level of p38 and JNK had almost no effect. Based on our in vitro inflammatory cell model of Parkinson disease, we found that Licochalcone A significantly inhibited the LPS-induced degeneration of dopaminergic neurons and the expressions of inflammatory factors of glial cells.These results suggest Licochalcone A can significantly inhibit the LPS-induced activation of microglial and the inflammatory reaction in inflammatory cell model of Parkinson disease, revealing its neuroprotective pharmacological activity.