Bushen Yizhi Formula regulates the IRE1 alpha pathway to alleviate endoplasmic reticulum stress in an Alzheimer's disease rat model

Background: While the Bushen Yizhi Formula can treat Alzheimer's disease (AD), the yet to be ascertained specific mechanism of action was explored in this work. Methods: Different concentrations of the Bushen Yizhi Formula and amyloid-beta peptide (A(3) were used to treat rat pheochromocytoma cells (P12) and human neuroblastoma cells (SH-SY5Y). Cell morphological changes were observed to determine the in vitro cell damage. Cell Counting Kit (CCK)-8 assay and flow cytometry were employed to identify cell viability and apoptosis/cell cycle, respectively. Western blotting and immunohistochemistry were employed to measure the expressions of endoplasmic reticulum stress (ERS)-related proteins (GRP78 and CHOP), p-IRE1a, IRE1a, ASK1, p-JNK, JNK, Bax, Bcl-2, XBP-1, and Bim. Fura 2-acetoxymethyl ester (Fura-2/AM) was used to determine the intracellular calcium (Ca2+) concentration. Also, an AD model was constructed by injecting A(3 into the CA1 area of the hippocampus in Sprague Dawley rats. AD model rats were gavaged with different concentrations of Bushen Yizhi Formula for 14 consecutive days. The Morris water maze experiment was conducted to test the learning and memory of rats. Hematoxylin & Eosin (H&E) and Terminal-deoxynucleotidyl Transferase (TdT)-mediated dUTP Nick-End Labeling (TUNEL) staining were done to determine histopathological changes in the brain. Results: Bushen Yizhi Formula relieved the A(3-induced effects including cell injury, decreased viability, increased apoptosis, G0/G1 phase cell cycle arrest, up-regulation of GRP78, CHOP, p-IRE1a, p-JNK, Bax, XBP-1 and Bim, as well as down-regulation of Bcl-2. These results were also seen with IRE1a silencing. While A(3 suppressed the learning and memory abilities of rats, the Bushen Yizhi Formula alleviated these effects of A(3. Brain nerve cell injury induced by A(3 could also be treated with Bushen Yizhi Formula. Conclusion: Bushen Yizhi Formula could influence ERS through the IRE1a signaling pathway to achieve its therapeutic effects on AD.