Xingnao jianshen prescription reduces mitochondrial cristae breakage by protecting GPX1 from oxidation against cerebral ischemia-reperfusion injury

Background: Ischemic stroke (IS) is a common cerebrovascular disease. Inhibiting oxidative stress is a recognized therapeutic mechanism for this disease. Xingnao Jianshen prescription (Xnjs) has been clinically applied since its creation in the 1990s, and it has a significant effect in the conservative treatment of acute IS. However, due to a lack of objective studies, the mechanism underlying its pharmacological effects has not been elucidated.

Methods: LC‒MS and GC‒MS were employed to analyze the chemical components of Xnjs. A rat model of middle cerebral artery occlusion (MCAO) was constructed to explore the mechanism underlying its pharmacological efficacy. Concurrently, we examined the possible pathways and targets of Xnjs using network pharmacological analysis and proteomics. This included methods such as transmission electron microscopy, Western blotting, flow cytometry, fluorescent probe live cell labeling, and lentiviral stable cell lines, which aims to validate mitochondrial oxidative stress both in vivo and in vitro. Molecular docking analysis and surface plasmon resonance were applied jointly to determine the binding affinity between the potential active ingredients and target proteins

Results: Xnjs was found to be composed of a total of 398 natural monomer products by a chemical composition test. In the MCAO model, Xnjs has demonstrated efficacy in alleviating cerebral ischemia-reperfusion-induced neurological dysfunction and enhancing mitochondrial integrity, particularly in the high-dose group. This has been achieved by inhibiting excessive oxidative stress responses in the central nervous system and mitigating apoptotic damage. This study has successfully identified GPX1 as the core target of Xnjs, which has the potential to promote active expression of GPX1, thereby contributing to the maintenance of a more complete mitochondrial cristae structure. In GPX1-knockdown cell lines, Xnjs was observed to protect neuronal cells from entering the damaging apoptotic stage, reduce GSH depletion and inhibit ROS production on mitochondria to mitigate mitochondrial cristae rupture. The results of this study also screened out six active monomeric components in Xnjs, all of which appeared to bind to GPX1 to varying degrees. While detecting their relative content in Xnjs compounds, the antioxidant stress effects of the mixed active ingredient group (6CP) were also preliminarily verified.

Conclusion: The findings of the research indicate that Xnjs has the capacity to enhance the biological activity of the GPX1 protein, thereby ensuring the preservation of mitochondrial cristae. This results in the augmentation of antioxidative stress and the mitigation of the extent of pathological apoptosis in cells. Based on the above drug target proteins, potential active ingredients in Xnjs were screened to lay the foundation for further pharmacological research.