Naodesheng Pills Ameliorate Cerebral Ischemia Reperfusion-Induced Ferroptosis via Inhibition of the ERK1/2 Signaling Pathway

Abstract

Background

Ferroptosis plays a crucial role in the occurrence and development of cerebral ischemia-reperfusion (I/R) injury and is regulated by mitogen-activated protein kinase 1/2 (ERK1/2). In China, Naodesheng Pills (NDSP) are prescribed to prevent and treat cerebrosclerosis and stroke. However, the protective effects and mechanism of action of NDSP against cerebral I/R-induced ferroptosis remain unclear. We investigated whether NDSP exerts its protective effects against I/R injury by regulating ferroptosis and aimed to elucidate the underlying mechanisms.

Methods

The efficacy of NDSP was evaluated using a Sprague-Dawley rat model of middle cerebral artery occlusion and an in vitro oxygen-glucose deprivation/reoxygenation (OGD/R) model. Brain injury was assessed using 2,3,5-triphenyltetrazolium chloride (TTC), hematoxylin and eosin staining, Nissl staining, and neurological scoring. Western blotting was performed to determine the expression levels of glutathione peroxidase 4 (GPX4), divalent metal-ion transporter-1 (DMT1), solute carrier family 7 member 11 (SLC7A11), and transferrin receptor 1 (TFR1). Iron levels, oxidative stress, and mitochondrial morphology were also evaluated. Network pharmacology was used to assess the associated mechanisms.

Results

NDSP (1.08 g/kg) significantly improved cerebral infarct area, cerebral water content, neurological scores, and cerebral tissue damage. Furthermore, NDSP inhibited I/R- and OGD/R-induced ferroptosis, as evidenced by the increased protein expression of GPX4 and SLC7A11, suppression of TFR1 and DMT1, and an overall reduction in oxidative stress and Fe²⁺ levels. The protective effects of NDSP in vitro were abolished by the GPX4 inhibitor RSL3. Network pharmacology analysis revealed that ERK1/2 was the core target gene and that NDSP reduced the amount of phosphorylated ERK1/2.

Conclusion

NDSP exerts its protective effects against I/R by inhibiting cerebral I/R-induced ferroptosis, and this mechanism is associated with the regulation of ferroptosis via the ERK1/2 signaling pathway.

Keywords:

• Naodesheng pills
• ferroptosis
• cerebral ischemia/reperfusion
• glutathione peroxidase 4
• mitogen-activated protein kinase 1/2

Abbreviations

ADME, absorption, distribution, metabolism and excretion; CCA, common carotid artery; DMT1, divalent metal-ion transporter-1; ECA, external carotid artery; ERK1/2, mitogen-activated protein kinase 1/2; GPX4, glutathione peroxidase 4; GSH, glutathione; H₂O₂, hydrogen peroxide; ICA, internal carotid artery; I/R, ischemia/reperfusion; MDA, malondialdehyde; NDSP, Naodesheng pill; NDSP-L, low-dose NDSP; NDSP-H, high-dose NDSP; OGD/R, oxygen glucose deprivation/reoxygenation; OD, optical density; ROS, reactive oxygen species; SEM, standard error of the mean; SLC7A11, solute carrier family 7 member 11; SOD, superoxide dismutase; TCMSP, Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform; TEM, transmission electron microscopy; TFR1, transferrin receptor 1; TTC, triphenyltetrazolium chloride.

Data Sharing Statement

The datasets used and/or analyzed in the current study are available from the corresponding author (Yong-Yu Yang) upon reasonable request. Uncropped gels and blots are provided in Supplementary Figure 1.

Ethics Approval and Consent to Participate

All experimental protocols were designed according to the Guide for the Care and Use of Laboratory Animals (NIH Publication, 8th edition, 2011) and approved by the Animal Ethics and Welfare Committee of the Second Xiangya Hospital of Central South University (No. 2021955).

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis, and interpretation, or in all these areas; took part in drafting, revising, or critically reviewing the article; gave final approval of the version to be published; agreed on the journal to which the article has been submitted; and agreed to be accountable for all aspects of the work.

Disclosure

The authors have no competing interest to declare in this work.

Additional information

Funding

This study was supported by the Changsha Science and Technology Major Project (No. kh2205035).