Objective  To investigate whether Danshen injection (DSI) can protect human brain organoids from oxidative stress damage by regulating the Nrf2 signaling pathway and its mechanism.

Methods  A Nrf2 plasmid was constructed, lentivirus was packaged and transfected into human induced pluripotent stem cell (hiPSC) to form a stable cell line. Brain organoids with Nrf2 overexpression (LV-Nrf2 group) and knockdown (sh-Nrf2 group) were constructed using kit. The transfection efficiency was verified by real-time fluorescence quantitative polymerase chain reaction. An oxidative stress injury model was established by inducing hydrogen peroxide (H₂O₂). Intervention was performed either in combination or without DSI pre-treatment. Cell viability was detected by Cell Counting Kit-8 (CCK-8), and the expression levels of Nrf2, malondialdehyde (MDA), interleukin (IL)-1β, sex-determining region Y-box protein 2 (SOX2) and neuronal-specific nuclear protein (NeuN) were detected by Western blotting and immunofluorescence staining. Total RNA from human brain organoids was extracted for transcriptome sequencing and analysis.

Results  LV-Nrf2 group and sh-Nrf2 group brain organoid models were successfully constructed. The CCK-8 results determined that the oxidative stress model was established with 32 mmol/L H₂O₂ intervention conditions. The fluorescence expression of Nrf2 in LV-Nrf2 group was higher than that in control group, and the fluorescence expressions of MDA and IL-1β were lower than those in control group; sh-Nrf2 group showed a damage phenotype of low Nrf2 expression, high MDA and IL-1β expression (all P < 0.05). Compared with H₂O₂ group, the Nrf2 signal in DSI + H₂O₂ group was stronger, and the levels of MDA and IL-1β were lower. Among them, the protective effect of LV-Nrf2 was the most significant. H₂O₂ treatment could down-regulate the protein levels of Nrf2, SOX2 and NeuN, and DSI intervention could effectively reverse the downward trend of these indicators. Sh-Nrf2 could weaken the recovery effect of DSI on SOX2 and NeuN, while LV-Nrf2 could synergistically enhance the regulatory effect of DSI.

Conclusions  DSI may antagonize oxidative stress damage in human brain organoids through the Nrf2 signaling pathway, and maintaining the stability of the neural cell lineage is its core therapeutic characteristic.