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 <i>in vitro</i> 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<b>-</b>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). BA protected SH-SY5Y cells against LPS and FeSO₄-induced ferroptosis, apoptosis, and neurotoxicity.BA inhibited LPS and FeSO₄-induced cell death and inflammatory signaling via suppression of IL-6, TNF-α, and p38MAPK/NF-κB/Nrf2/Keap-1/HO-1 signaling.BA may be a promising neuroprotective agent for therapeutic targeting of neuroinflammation and iron-induced neurodegeneration, like PD. BA protected SH-SY5Y cells against LPS and FeSO₄-induced ferroptosis, apoptosis, and neurotoxicity. BA inhibited LPS and FeSO₄-induced cell death and inflammatory signaling via suppression of IL-6, TNF-α, and p38MAPK/NF-κB/Nrf2/Keap-1/HO-1 signaling. BA may be a promising neuroprotective agent for therapeutic targeting of neuroinflammation and iron-induced neurodegeneration, like PD.

白桦酸（betulinic acid，BA）是一种五环三萜类化合物，具备广泛的药理学应用潜力，其神经保护作用已获得学界广泛认可。本研究探究了该化合物对体外分化的人神经母细胞瘤SH-SY5Y细胞的潜在保护作用，以对抗脂多糖（lipopolysaccharide，LPS）与硫酸亚铁（FeSO₄）诱导的铁死亡、细胞凋亡、神经炎症及多巴胺能细胞死亡，并对其潜在作用机制进行了探索。
将体外分化的人神经母细胞瘤SH-SY5Y细胞暴露于LPS与FeSO₄环境中，采用MTT法检测细胞活力；通过流式细胞术评估细胞凋亡情况。此外，通过蛋白质印迹法与免疫细胞化学染色技术，分析了与铁死亡、细胞凋亡及其他相关信号蛋白相关的关键标志物的表达水平。
然而，LPS与FeSO₄联合暴露可导致细胞活力呈剂量依赖性下降，而经0.3~30μM的BA预处理可显著逆转这一现象。LPS与FeSO₄处理可使二价金属转运体1（DMT1）、Bcl-2相关X蛋白（Bax）、半胱氨酸天冬氨酸蛋白酶3（caspase-3）及α-突触核蛋白（alpha-synuclein）的表达水平升高，同时使谷胱甘肽过氧化物酶4（GPX4）、铁蛋白重链1（FTH1）、溶质载体家族7成员11（SLC7A11）、核因子E2相关因子2（Nrf2）、Kelch样ECH相关蛋白1（Keap1）、血红素氧合酶1（HO-1）、帕金森病相关蛋白7（PARK7）、B细胞淋巴瘤2蛋白（Bcl-2）、神经元核抗原（NeuN）及酪氨酸羟化酶（TH）的表达水平降低，最终引发细胞铁死亡、细胞凋亡及多巴胺能细胞死亡。
此外，LPS与FeSO₄处理可显著提升细胞中白细胞介素6（IL-6）、肿瘤坏死因子α（TNF-α）的表达水平，以及p38、磷酸化丝裂原活化蛋白激酶（pMAPK）、磷酸化核因子κB（pNFκB）的磷酸化水平。经BA预处理后，可显著抑制LPS与FeSO₄诱导的促炎细胞因子上调、铁死亡、细胞凋亡及多巴胺能细胞死亡相关标志物的异常表达。
上述研究结果表明，BA可通过调控GPX4/Nrf2/Keap-1/HO-1抗氧化防御通路与p38MAPK/NF-κB炎症信号通路发挥神经保护作用，提示其有望成为治疗氧化应激相关神经退行性疾病（如帕金森病（Parkinson’s disease，PD））的潜在治疗药物。
BA可保护SH-SY5Y细胞对抗LPS与FeSO₄诱导的铁死亡、细胞凋亡及神经毒性。BA可通过抑制IL-6、TNF-α及p38MAPK/NF-κB/Nrf2/Keap-1/HO-1信号通路，缓解LPS与FeSO₄诱导的细胞死亡与炎症信号活化。BA有望成为针对神经炎症及铁诱导的神经退行性病变（如PD）的潜在神经保护治疗药物。
BA可保护SH-SY5Y细胞对抗LPS与FeSO₄诱导的铁死亡、细胞凋亡及神经毒性。BA可通过抑制IL-6、TNF-α及p38MAPK/NF-κB/Nrf2/Keap-1/HO-1信号通路，缓解LPS与FeSO₄诱导的细胞死亡与炎症信号活化。BA有望成为针对神经炎症及铁诱导的神经退行性病变（如PD）的潜在神经保护治疗药物。