Mechanism of Shanhu Qishiwei pills in regulating endoplasmic reticulum stress through GRP78/CHOP signaling pathway for the treatment of cerebral ischemia.

ETHNOPHARMACOLOGICAL RELEVANCE: Stroke, a prevalent cerebrovascular condition, is characterized by high occurrence, disability, mortality, and recurrence rates. Shanhu Qishiwei pills (བྱུར་དམར་བདུན་ཅུ།, SHQSWP) are a Tibetan prescription, which were included in the national drug standard ZZ-8432 of the National Food and Drug Administration with approval number Z54020114. SHQSWP can relieve palpitation, tranquilize, and arrest convulsion and is mainly used for cerebral thrombosis, cerebral hemorrhage, and coronary heart disease. This study aimed to investigate the mechanism of SHQSWP for the treatment of cerebral ischemia.

AIM OF THE STUDY: This study aimed to screen the differential proteins of SHQSWP for the treatment of cerebral ischemia and verify its mechanism in regulating the endoplasmic reticulum stress signaling pathway.

MATERIALS AND METHODS: Triple quadrupole liquid chromatography-mass spectrometry (HPLC-QqQ-MS) was to quantitatively detect 25 chemical components in SHQSWP. TTC staining, HE staining, Nissl staining, and TUNEL staining were used to verify the effectiveness of SHQSWP for the treatment of cerebral ischemia in middle cerebral artery occlusion (MCAO) model rats. Deferentially expressed proteins were screened using a protein chip, and the potential targets and signaling pathways of SHQSWP in the treatment of cerebral ischemia were visually screened using protein-protein interaction (PPI) network analysis. Molecular docking technology was used to predict the binding affinity and binding mode of the key compounds in SHQSWP with related target proteins. RT-qPCR, WB and IF were used to verify the signaling pathways and targets regulated by SHQSWP in the treatment of cerebral ischemia.

RESULTS: HPLC-QqQ-MS was used to determine the contents of 25 chemical components in SHQSWP, including neochlorogenic acid, isorhamnetin, chlorogenic acid, gallic acid, hydroxysafflor yellow-A, and crocin I. Each SHQSWP dose can reduce the rate of cerebral infarction in MCAO rats, improve neurobehavioral and pathological changes in the cortical area of brain tissues, increase the number of Nissl bodies in the cortical area, and reduce the rate of positive apoptotic cells in the cortical area. Protein chip prediction showed that SHQSWP may play a therapeutic role by regulating EPhA5, IL-1α, fractalkine, IL-10, IL-7, and IL-3 proteins. Molecular docking revealed that GRP78 may be the common dominant target of 15 components in SHQSWP, and Rutin may be the representative component with the strongest binding force with 7 key targets. SHQSWP can significantly increase the mRNA expression levels of PARP1 and XBP1 in the brain tissue of MCAO rats, significantly reduce the protein expression levels of CHOP, GRP78 and BAX, and increase the protein expression level of XBP1. A medium SHQSWP dose can reduce the fluorescence intensity of NLRP3, BAX, and CHOP in rat brain tissues and enhance the fluorescence intensity of XBP1.

CONCLUSIONS: SHQSWP can inhibit endoplasmic reticulum stress-mediated apoptosis by regulating key proteins such as BAX, CHOP, GRP78, and XBP1, thus playing a neuroprotective role in cerebral ischemia-reperfusion injury.