Betulinic acid protects SH-SY5Y cells exposed to lipopolysaccharide and ferrous sulfate through p38MAPK/NF-κB/GPX4/Nrf2/keap-1/HO-1 signaling axis.

Betulinic acid (BA) is a pentacyclic triterpenoid with broad pharmacological potential and widely recognized for its neuroprotective effects. This study investigated the potential protective effects of this compound on in vitro differentiated human neuroblastoma SH-SY5Y cells against LPS and FeSO₄-induced ferroptosis, apoptosis, neuroinflammation, and dopaminergic cell death, and explored the underlying mechanisms. Differentiated human neuroblastoma SH-SY5Y cells were exposed to LPS and FeSO₄, and the cellular viability was evaluated using the MTT assay. Flow cytometry was performed to assess apoptotic cell death. Additionally, the expression levels of key markers associated with ferroptosis, apoptosis, and other relevant signaling proteins were analyzed through western blotting and Immunocytochemical staining techniques. However, co-exposure with LPS and FeSO₄ resulted in a dose-dependent reduction in cell viability, which was significantly reversed by pretreatment with BA (0.3-30μM). Exposure to LPS and FeSO₄ increased the DMT1, Bax, caspase-3, and alpha-synuclein, and decreased the GPX4, FTH1, SLC7A11, Nrf2, Keap1, HO-1, PARK7, Bcl-2, NeuN, and TH levels, resulting in cell ferroptosis, apoptosis, and dopaminergic cell death. Furthermore, LPS and FeSO₄ significantly increased the expression of IL-6, TNF-α, and phosphorylation of p38, pMAPK, and pNFkB in the cells. Pretreatment with BA markedly suppressed LPS and FeSO₄-induced upregulation of pro-inflammatory cytokines, ferroptosis, apoptosis, and dopaminergic cell death markers. These findings suggest that BA exerts neuroprotection by modulating the GPX4/Nrf2/Keap-1/HO-1 antioxidant defense and p38MAPK/NF-κB inflammatory signaling pathways, highlighting its potential as a therapeutic agent for oxidative stress-related neurodegenerative conditions, such as Parkinson's disease (PD).