Astragaloside IV (AS-IV) is a bioactive compound derived from Radix Astragali, a traditional Chinese herb widely used as a dietary supplement to enhance immune function. Modern pharmacological studies have demonstrated that AS-IV exhibits anti-inflammatory and immunomodulatory properties. In this study, we investigated the effects of AS-IV on motor dysfunction, microglial polarization, and immune regulation mechanisms in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease (PD) mouse model. Our results showed that AS-IV (40 mg/kg) significantly improved motor function in PD mice, as evidenced by reduced descent time in the pole test, increased hanging score in the hanging test, increased stride lengths, and reduced paw angle in the gait test. Furthermore, AS-IV administration attenuated the loss of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra pars compacta (SNpc), promoted microglial polarization from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype, suppressed the levels of pro-inflammatory cytokines including interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNFα), enhanced the levels of anti-inflammatory cytokines including interleukin-4 (IL-4) and interleukin-10 (IL-10) in the SNpc of PD mice. Mechanistically, AS-IV significantly downregulated the expression and phosphorylation levels of TLR4/p38 (JNK)/NF-κB pathway-related proteins, including Toll-like receptor 4 (TLR4), Myeloid differentiation primary response protein 88 (MyD88), Apoptosis signal-regulating kinase 1 (ASK1), Mitogen-activated protein kinase 3/6 (MKK3/6), Phosphorylated-MKK3/6 (p-MKK3/6), Phosphorylated-mitogen-activated protein kinase 4/7 (p-MKK4/7), p38 mitogen-activated protein kinase (p38), Phosphorylated-p38 (p-p38), c-Jun N-terminal kinase (JNK), Phosphorylated-JNK (p-JNK), nuclear factor kappa-B (NF-κB), and Phosphorylated-NF-κB (p-NF-κB). To further validate the targeting effect of AS-IV, 1 mg/kg of LPS-EB Ultrapure was utilized as a specific TLR4 agonistwe to selectively activated the TLR4/NF-κB signaling pathway without triggering other inflammatory pathways, leading to elevated mRNA levels of TLR4, NF-κB, IL-1β, IL-6, TNFα and protein expression of TLR4, p-JNK, p-p38, p-NF-κB, IL-1β, IL-6, TNFα in the SNpc of PD mice. Importantly, AS-IV pretreatment can't counteract these LPS-EB Ultrapure-triggered effects, demonstrating its dependence on the TLR4/NF-κB signaling pathway. In conclusion, our findings indicate that AS-IV modulates microglial polarization and attenuates neuroinflammation by inhibiting the TLR4/NF-κB pathway, thereby ameliorating motor dysfunction and neuronal loss in PD mice.
