Porcine cardiac blood - Salvia miltiorrhiza root alleviates cerebral ischemia reperfusion injury by inhibiting oxidative stress induced apoptosis through PI3K/AKT/Bcl-2/Bax signaling pathway

by Selleckchem | Oct 17 2023

Ethnopharmacological relevance: Salvia miltiorrhiza Bge. mixed with porcine cardiac blood (PCB-DS) is mainly employed for the treatment of brain ischemia-induced mental disturbances, palpitations and phlegm confusion based on the traditional principle of Menghe medical sect. PCB is the guide to DS and enhances the effect of DS. However, the potential mechanism of PCB-DS preventing cerebral ischemia/reperfusion injury (CIRI) from the perspective of oxidative stress induced cell apoptosis remains unknown.

Aim of the study: To investigate the pharmacological activity and molecular mechanism of PCB-DS against CIRI.

Materials and methods: DS samples processed with different methods were prepared and UPLC-Q-TOF-MS/MS was employed for qualitative analysis of the respective processing product. The middle cerebral artery occlusion reperfusion model was then established to investigate the pharmacological activities of PCB-DS. Pathological changes in the rat brain were observed by triphenyl tetrazolium chloride (TTC), hematoxylin-eosin, and TUNEL staining. The levels of IL-6, IL-1β, and TNF-α were detected by ELISA to evaluate the inflammatory damage. Metabolomics of cerebrospinal fluid was further used to explore the potential mechanism of PCB-DS in preventing CIRI. Based on this, the levels of oxidative stress-related lactate dehydrogenase (LDH), reactive oxygen species (ROS), malondialdehyde (MDA), and superoxide dismutase (SOD) were determined. The protein levels of PI3K, AKT, Bcl-2, Bax, cleaved-caspase-3, and cleaved-caspase-9 proteins of the cerebral infarct zone were finally measured by western blotting.

Results: Forty-seven components were identified in four processing products. Compared to DS, the content of total aqueous components in PCB-DS was significantly increased including salvianolic acid B isomer, salvianolic acid D, salvianolic acid F, and salvianolic acid H/I/J. Among the DS, DS processed with wine, DS processed with pig blood, and DS processed with porcine cardiac blood, PCB-DS best alleviated the CIRI through the neurological score, brain infarct volume, brain histopathology and the levels of inflammatory factors in the brain. Twenty-five significant metabolites in the cerebrospinal fluid were screened out between the sham and I/R groups. They were mainly involved in the beta-alanine metabolism, histidine metabolism, and lysine degradation, which indicated that PCB-DS may inhibit oxidative stress-induced apoptosis to achieve treating ischemic stroke. The results of biomedical examination showed that PCB-DS could alleviate oxidative damage, significantly downregulate the expression of Bax, cleaved caspase-3 and cleaved caspase-9, and upregulate the expression of p-PI3K, p-AKT, and Bcl-2.

Conclusion: In summary, this study demonstrated that PCB-DS alleviated CIRI and the molecular mechanism may be related to inhibiting the oxidative stress induced apoptosis through PI3K/AKT/Bcl-2/Bax signaling pathway.

Comments:

The aim of the study was to investigate the pharmacological activity and molecular mechanism of Salvia miltiorrhiza Bge. mixed with porcine cardiac blood (PCB-DS) against cerebral ischemia/reperfusion injury (CIRI). PCB-DS is traditionally used for the treatment of brain ischemia-induced mental disturbances, palpitations, and phlegm confusion.

The researchers prepared DS samples processed with different methods and used UPLC-Q-TOF-MS/MS for qualitative analysis of the processing products. They then established a middle cerebral artery occlusion reperfusion model in rats to evaluate the pharmacological activities of PCB-DS. Pathological changes in the rat brain were observed, and the levels of inflammatory cytokines (IL-6, IL-1β, TNF-α) were measured to assess inflammatory damage. Metabolomics of cerebrospinal fluid was employed to explore the potential mechanism of PCB-DS in preventing CIRI. Additionally, oxidative stress-related parameters such as lactate dehydrogenase (LDH), reactive oxygen species (ROS), malondialdehyde (MDA), and superoxide dismutase (SOD) were measured. Finally, the protein levels of PI3K, AKT, Bcl-2, Bax, cleaved-caspase-3, and cleaved-caspase-9 in the cerebral infarct zone were assessed through western blotting.

The results showed that PCB-DS had significantly increased content of total aqueous components compared to DS alone. PCB-DS exhibited better alleviation of CIRI compared to DS processed with wine, DS processed with pig blood, and DS processed with porcine cardiac blood, as evidenced by neurological score, brain infarct volume, brain histopathology, and levels of inflammatory factors in the brain. Metabolomic analysis revealed significant changes in 25 metabolites involved in pathways such as beta-alanine metabolism, histidine metabolism, and lysine degradation, suggesting that PCB-DS may inhibit oxidative stress-induced apoptosis to treat ischemic stroke. Biomedical examination showed that PCB-DS could alleviate oxidative damage, downregulate the expression of Bax, cleaved caspase-3, and cleaved caspase-9, and upregulate the expression of p-PI3K, p-AKT, and Bcl-2.

In conclusion, the study demonstrated that PCB-DS alleviated CIRI, and its molecular mechanism may involve inhibiting oxidative stress-induced apoptosis through the PI3K/AKT/Bcl-2/Bax signaling pathway. These findings provide insights into the potential therapeutic effects of PCB-DS for the treatment of cerebral ischemia/reperfusion injury.