PUERARIN PLAYS AN ANTI-INFLAMMATORY ROLE BY DOWN-REGULATE THE PHOSPHORYLATION OF MAPKS SIGNAL AND UP-REGULATE THE LEVEL OF O-GLCNAC GLYCOSYLATION PROTEIN

Guoyi Liu¹, Shu Ma², Liyan Fu², Yan Wu¹, Jia Geng^{1, *}

¹Department of Neurology, The First Affiliated Hospital of Kunming Medical University, Kunming, PR China - ²Department of Neurology, the 920th Hospital of Logistics Support Force, People’s Liberation Army, Kunming, PR China

Objective: To investigate the mechanism by which puerarin inhibits inflammation damage in microglia and to further analyse the correlations of various signalling molecules. 

Methods: Lipopolysaccharide (LPS) induced inflammatory damage in microglia. Microglia were untreated or treated with a low concentration (25 µM) or a high concentration (100 µM) of puerarin, respectively. The nitric oxide (NO) content of the microglia was detected by flow cytometry, and microglia-associated eggs were detected by Western blot. The expression of related genes was detected by PCR. 

Results: The results of flow cytometry showed that the NO production of microglia stimulated by LPS was significantly higher than that of a control group (P<0.05). Compared with the untreated LPS group, the puerarin high concentration group could significantly inhibit NO production (P<0.05). The effect of the puerarin low concentration group on NO was not significantly different from that of the LPS group (P>0.05). Western blot analysis showed that the iNOS protein level increased significantly after LPS stimulation compared with the control group (P<0.05), decreased significantly after puerarin treatment compared with the LPS group, and was inhibited by puerarin in a dose-dependent manner (P<0.05). The results of real-time quantitative PCR showed that the change in the iNOS gene level was consistent with that of the protein level. Compared with the control group, the puerarin high concentration group significantly inhibited the activation of the NF-kB gene in microglia (P<0.05). Compared with the control group, LPS could significantly promote the phosphorylation of p38, ERK1/2 and JNK (P<0.05). Compared with the LPS group, puerarin at both high and low concentrations could inhibit the phosphorylation of ERK1/2 (P<0.05), while puerarin at the high concentration could significantly inhibit the phosphorylation of p38 and JNK (P<0.05). The level of O-GlcNAc glycosylated protein in microglia treated with LPS was significantly lower than that in the control group (P<0.05). The levels of O-GlcNAc glycosylated protein in microglia treated with different concentrations of puerarin were significantly higher than that in the LPS group (P<0.05). 

Conclusion: Puerarin can inhibit the activation of the NF-kB gene by reducing the release of NO and the expression of iNOS, then down-regulating the phosphorylation of the MAPKs signal and up-regulating the level of O-GlcNAc glycosylation protein, thus playing an anti-inflammatory role.