Andrographolide safeguards neurons in an oxygen glucose deprivation (OGD) model through modulating the JNK3/Cyt-c/caspase pathway

Abstract

Ischemic stroke occurs due to an interruption in cerebral blood flow, leading to oxygen-glucose deprivation and subsequent neuronal injury. This triggers apoptotic and inflammatory pathways, exacerbating brain damage. Among key regulators of neuronal apoptosis, c-Jun N-terminal kinase 3 (JNK3) plays a crucial role in stroke-induced neurodegeneration. While JNK3 inhibition has been proposed as a neuroprotective strategy, no specific JNK3 inhibitor has been approved for stroke therapy. Moreover, no studies have explored the role of andrographolide in mitigating JNK3 protein expression and its neuroprotective potential. In this study, we systematically screened 190 terpenoids against JNK3 using virtual screening and identified andrographolide as the most promising candidate based on molecular docking (-8.486 kcal/mol) and MMGB/SA binding energy (-102.03 kcal/mol). A JNK kinase assay confirmed andrographolide’s preferential inhibition of JNK3 over its isoforms, JNK1 and JNK2. Under hypoxic conditions, andrographolide exhibited neuroprotective effects comparable to SP600125, a standard JNK3 inhibitor. Additionally, caspase-3 and −9 assays demonstrated a concentration-dependent reduction in apoptotic markers, corroborated by Western blot analysis showing JNK3 downregulation. Molecular interaction studies further validated andrographolide’s direct binding to JNK3. By integrating computational, biochemical, and molecular approaches, our study provides the first evidence that andrographolide mitigates hypoxia-induced neuronal apoptosis by inhibiting the JNK3/cyt-c/caspase pathway. These findings highlight andrographolide’s potential as a neuroprotective agent for ischemic stroke and warrant further preclinical validation.