Ginsenoside Rb3 represses CPZ-induced demyelination and neuroinflammation by inhibiting TRAF6 K63 ubiquitination

Multiple sclerosis is a chronic inflammatory and neurodegenerative disorder of the central nervous system. Despite ongoing research, effective treatments remain limited, especially during progressive phase. Saponins extracted from the stem and leaf of Panax notoginseng (PNSL) demonstrate a superior anti-inflammatory effect by inhibiting NO production in LPS-induced BV2 cells. Ginsenoside Rb3, the primary active and most abundant component in PNSL, has been demonstrated to mitigate inflammation-induced damage. However, whether Rb3 mitigates demyelination by inhibiting neuroinflammation had not been previously reported. In this study, biochemical and histological assays revealed that ginsenoside Rb3 effectively mitigated Cuprizone-induced demyelination and attenuated aberrant microglial activation and reactive astrogliosis within the demyelinated areas. Mechanistic investigations demonstrated that Rb3 suppresses glial cell activation and consequently mitigates inflammatory responses by inhibiting the secretion of TNF-α, IL-6, and IL-1β. TNF receptor-associated factor 6 (TRAF6) is activated by K63-linked polyubiquitination, which leads to downstream activation of the inhibitor of nuclear factor-κB kinase (IKK) complex and mitogen-activated protein kinases (MAPKs). Furthermore, Rb3 was found to inhibit the activation of nuclear factor-κB (NF-κB) and MAPKs, as evidenced by the dephosphorylation of NF-κB p65 and the MAPKs p38 and JNK. Further investigation revealed that Rb3 binds to TRAF6 at residues 69 and 88, thereby inhibiting its K63-linked polyubiquitination. Conversely, the TRAF6 mutation at E69Q or R88N abolished the inhibition effects of Rb3 on K63-linked ubiquitination of TRAF6 and subsequent downstream signaling activation. Meta-analysis showed that Rb3 exerts its anti-inflammatory effects primarily by inhibiting the NF-κB pathway. Collectively, it is concluded that Rb3 alleviates demyelination and inhibits inflammation through bound to TRAF6 to prevent its K63-linked ubiquitination and subsequent activation of NF-κB. In this study, we have for the first time elucidated that dual mechanism by which Rb3 inhibits both NF-κB and MAPK pathways to exert its anti-inflammatory effects. This study demonstrates that Rb3 shows promising preclinical therapeutic potential. Additionally, TRAF6 represents a potential therapeutic target for MS treatment.