Regulation of NR4A1 by Taohong Siwu decoction inhibits endothelial cell apoptosis in cerebral Ischemia-Reperfusion injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Taohong Siwu Decoction (THSWD), a classic prescription, is commonly used to treat ischemic stroke. However, the effects of THSWD on endothelial cells (ECs) in cerebral ischemia-reperfusion injury (IRI) are unclear.

AIM OF THE STUDY: This study investigated whether THSWD can protect ECs from cerebral IRI by regulating NR4A1 to mitigate mitochondrial fission and reduce cell apoptosis.

METHODS

We established the rat MCAO model and the OGD/R injury model of human BMECs cells. Observation of the protective effect of THSWD against IRI using Zea-Longa score, TTC staining, and HE staining; ELISA to detect ET-1, eNOS level; immunofluorescence to detect vWF, claudin-5, occludin in brain tissue; CCK8 to detect cell viability; transmission electron microscopy to observe mitochondrial morphology; Expression of mitochondrial division and apoptosis-related proteins and mRNAs detected by WB and RT-qPCR; immunohistochemistry to detect the expression of NR4A1 and Caspase9, ROS content was detected by flow cytometry; mitochondrial membrane permeability colorimetric assay kit was used to observe the degree of mitochondrial mPTP opening; and endothelial cell permeability was measured by FITC-dextran; Meanwhile, public database data analysis, chromatin immunoprecipitation and luciferase gene reporter techniques were used to analyze and validate the NR4A1-Mff binding relationship, and molecular docking techniques and molecular dynamics simulations were used to validate the docking activity of NR4A1 with THSWD active ingredients.

RESULTS

In the model group, vascular endothelial damage, mitochondrial structure impairment, increased opening of mPTP, elevated ROS levels, and upregulated NR4A1, Mff, and Drp1 expression at both the mRNA and protein levels, as well as increased apoptosis-related protein expression. After THSWD administration, vascular endothelial damage was improved. Mitochondrial damage was alleviated, degree of mPTP opening was reduced, ROS content was decreased, and NR4A1, Mff, and Drp1 expression at both the mRNA and protein levels was downregulated, along with a reduction in the expression of apoptosis-related proteins. NR4A1 was able to bind specifically to the R2 region of the Mff gene promoter. In microvascular endothelial cells with high expression of NR4A1, the transcriptional activity of the promoter region of the Mff gene was significantly enhanced, whereas THSWD-containing serum inhibited the activation of the Mff gene promoter region by NR4A1. Quercetin and kaempferol had a strong binding capacity to NR4A1, and the complexes remained stable during molecular dynamics simulations.

CONCLUSION

During cerebral IRI, NR4A1 promotes excessive mitochondrial fission in vascular ECs, leading to apoptosis. THSWD can protect vascular ECs by inhibiting NR4A1, reducing excessive mitochondrial fission, and suppressing cell apoptosis.