Tetrandrine Attenuates Cerebral Ischemia/Reperfusion Injury by Regulating Th17/Treg Balance and Mediating PI3K/Akt...

Stroke has found to be the second leading cause of death in China. Research indicates that the immune imbalance of Th17/Treg is an important pathophysiological alteration and the cause of poor prognosis after ischemic stroke, the most common type of stroke. Tetrandrine (Tet), a bisbenzyl isoquinoline alkaloid and potential immunoregulator, has been reported to possess therapeutic effect on ischemic stroke; however, the specific mechanism remains to be clarified. This research aims to explore the molecular mechanisms underlying Tet's effects on cerebral ischemiareperfusion injury (CIRI) through bioinformatics methods and experimental validation.

Using public databases, we predicted potential therapeutic targets of Tet against CIRI. Common targets were then used to build a protein-protein interaction (PPI) network, from which key gene targets were identified. Functional and pathway enrichment analyses were conducted via Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Afterwards, molecular docking and experimental validation were performed to confirm the predictions. The middle cerebral artery occlusion and reperfusion (MCAO/R) mice model was established. Twenty-four hours after Tet injection (intraperitoneally, 25 and 50 mg/kg), behavior index evaluation, cerebral blood flow, and tetrazolium chloride (TTC) staining were performed. Pathological changes on cortex, striatum, and hippocampus were observed using hematoxylin-eosin (HE) staining. Percentages of Th17 cells and Treg cells were determined by flow cytometry analysis. The expressions of mRNAs of Th17/Treg markers were measured by quantitative real-time polymerase chain reaction (qPCR). And the expressions of proteins PI3K/Akt Signaling Pathway were measured by Western blot analysis.

Our study identified 52 potential therapeutic targets of Tet for CIRI, with 10 key gene targets selected from PPI network analysis. GO and KEGG enrichment analyses indicated that these targets were primarily associated with the PI3K/Akt signaling pathway and Th17 cell differentiation. Molecular docking suggested potential interactions between Tet and Trp53, Cdk4, and Pik3ca. Experimental validation demonstrated that Tet ameliorated neurological deficits and reduced cerebral ischemic damage. Flow cytometry revealed that, compared to the model group, Tet treatment increased Treg cells while decreasing Th17 cells. Additionally, Tet downregulated RORγt and upregulated FoxP3 at the mRNA level. Western blot analysis further confirmed that Tet reduced the p-Akt/Akt and p-PI3K/PI3K protein expression ratios.

Our integrated approach demonstrates that tetrandrine alleviates CIRI by restoring Th17/Treg balance via PI3K/Akt signaling - a mechanism previously unreported for this alkaloid. The downregulation of RORγt and upregulation of FOXP3 confirm Tet's immunomodulatory specificity, while reduced p-Akt/Akt ratios mechanistically link its efficacy to PI3K/Akt pathway inhibition. This dual regulation positions Tet as a multi-target therapeutic candidate distinct from single-pathway agents.

Through the combination of bioinformatics and experimental validation, our study revealed that tetrandrine regulates Th17/ Treg cells balance and mediates PI3K/Akt pathway, presenting a potential therapeutic agent for treating cerebral ischemic injury.