Overexpression of the circadian gene Bmal1 regulates the Nrf2/HO-1 oxidative stress pathway to alleviate inflammation and apoptosis in PC12 cells following cerebral ischemia-reperfusion injury.

The circadian clock gene brain and muscle Arnt-like 1 (Bmal1) plays a crucial role in cerebral ischemia-reperfusion injury. Therefore, we established stable transfections of Rat adrenal pheochromocytoma cells (PC12) to overexpress the Bmal1 gene and a negative control using lentivirus. An in vitro model of cerebral ischemia-reperfusion injury was created through oxygen-glucose deprivation/reoxygenation (OGD/R) induction. The cells were divided into 4 groups: control, OGD/R, OGD/R with Bmal1 negative expression, and OGD/R with Bmal1 overexpression. The mRNA expression level of Bmal1 was measured using quantitative reverse transcription polymerase chain reaction. Protein levels of Bmal1, Nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), BCL2-Associated X, B-cell lymphoma-2 (Bcl-2), and cysteine-dependent aspartate-directed protease 3 were assessed via Western blotting. The levels of inflammatory cytokines interleukin-6, and interleukin-1β in the cell supernatant were quantified using ELISA. Apoptosis rates were analyzed by flow cytometry, and intracellular reactive oxygen species levels were measured using a fluorescent probe. Following OGD/R induction, Bmal1 gene and protein expression levels were reduced in PC12 cells. After lentiviral transfection, mRNA and protein expression levels of Bmal1 significantly increased in the overexpression model. Bmal1 overexpression down-regulated apoptotic proteins BCL2-Associated X and cysteine-dependent aspartate-directed protease 3, up-regulated the antiapoptotic protein Bcl-2, reduced apoptosis, and inhibited the release of inflammatory factors interleukin-6 and interleukin-1β following OGD/R. Further experiments indicated that Bmal1 overexpression activated proteins in the Nrf2/HO-1 oxidative stress signaling pathway, reducing intracellular reactive oxygen species release. This study demonstrated that Bmal1 overexpression inhibits inflammatory responses and apoptosis in PC12 cells after ischemia/reperfusion injury by regulating the Nrf2/HO-1 oxidative stress signaling pathway.