Regulation of microglia inflammation and oligodendrocyte demyelination by Engeletin via the TLR4/RRP9/NF-κB pathway after spinal cord injury
Wang Chen, Leshu Zhang, Guangdi Zhong, Shuang Liu, Yuxuan Sun, Jiayun Zhang, Z. Q. Liu, Lichun Wang
Abstract
Microglia polarization is crucial for neuroinflammatory response after spinal cord injury (SCI). Small molecule compounds and hub genes play an important role in regulating microglia polarization, reducing neuroinflammatory response and oligodendrocyte demyelination after SCI. In this study, suitable data sets were used to screen hub genes, and Western blot and Immunofluorescence (IF) experiments were used to confirm the expressions of proteins related to SDAD1, RRP9 and NF-κB pathways under LPS/SCI conditions. Engeletin (ENG) reduces microglia polarization and inflammation in vivo and in vitro via the SDAD1, RRP9 or NF-κB signaling pathways. In addition, ENG binds to the membrane receptor Toll-like receptor 4 (TLR4) through small molecule-protein docking. COIP experiment and protein-protein docking revealed protein-protein interaction (PPI) between RRP9 and SDAD1. By gene knock-down (KD) / overexpression (OE) and Western blot experiments, RRP9 and SDAD1 can regulate inflammatory response through NF-κB signaling and ribosome biogenesis pathway. Western blot analysis showed that CU increased the expression of SDAD1, RRP9 and NF-κB pathway related proteins through TLR1/2, while C34 decreased the expression of SDAD1 and RRP9 proteins through TLR4. These results suggest that ENG can reduce inflammation through TLR4/RRP9(SDAD1)/NF-κB signaling pathway. In addition, we demonstrated that oligodendrocyte apoptosis and demyelination can be influenced by the regulation of microglia and tissue inflammation. In conclusion, this study found the gene Rrp9/Sdad1 and the small molecule compound ENG, which control the inflammatory response of microglia, and further explored the related mechanism of oligodendrocyte demyelination, which has important theoretical significance. LPS can activate the transcription and translation of Inos/Tnf-α/Il1β genes by introducing P-p65 into the nucleus through TLR4/NF-κB pathway: iNOS activation activates microglia to M1 phenotype. TNF-α and IL-1β are released extracellularly as inflammatory factors. TNF-α binds to the TNFR1 receptor on oligodendrocytes, cleaved Caspase-3 and Bax/Bcl-2 increase and activate the apoptotic pathway, leading to demyelination of oligodendrocytes. LPS can increase the expression of SDAD1 and RRP9 proteins through TLR4, while ENG can directly bind to TLR4 to reduce the expression level of SDAD1 and RRP9 proteins and weaken the LPS-activated NF-κB pathway. CU can increase the expression of SDAD1, RRP9 and NF-κB pathway related proteins through TLR1/2, while C34 can decrease the expression of SDAD1, RRP9 protein through TLR4. There is a PPI relationship between SDAD1 and RRP9. On the one hand, RRP9 can affect the transcription and translation of Inos/Tnf-α/Il1β genes through the ribosomal biogenesis. On the other hand, RRP9 regulates the expression of proteins associated with the NF-κB pathway by affecting the transcription and translation of Iκbα and p65, thereby affecting M1 polarization and the release of inflammatory factors.