Shexiang Tongxin Dropping Pills attenuate ischemic microvascular dysfunction via suppressing P66Shc-mediated mitochondrial respiration deficits.

ETHNOPHARMACOLOGICAL RELEVANCE

Ischemic stroke (IS) disrupts mitochondrial energy metabolism, leading to cerebral microvascular dysfunction (CMD). Shexiang Tongxin Dropping Pills (STDP) is a traditional Chinese medicinal formulation that has been clinically used for treating microcirculatory dysfunction. We have previously reported its ability to improve cerebral microcirculatory abnormalities. Nevertheless, the protective effects of STDP on cerebral microvascular mitochondria in the context of energy metabolism repair remain underinvestigated.

AIM OF THE STUDY

This study aims to investigate the potential mechanisms by which STDP ameliorates IS-induced CMD through the restoration of mitochondrial function.

MATERIALS AND METHODS

An ischemic stroke/reperfusion model was established by occluding and subsequently reperfusing the middle cerebral artery (MCAO/R) in C57BL/6 J mice. Laser speckle contrast imaging, Y-maze, rotarod tests and TTC staining were employed to evaluate the anti-ischemic stroke effects of STDP. Histological examination of cell adhesion proteins (ICAM 1, VCAM 1) and tight junction proteins (VE-cadherin, occludin) was conducted to assess the effects of STDP on the cerebral microvascular endothelium. In vitro, a bEnd.3 cell model was established through oxygen-glucose deprivation followed by reoxygenation (OGD/R). The cytoprotective capability of STDP was assessed by quantifying endothelial permeability, reactive oxygen species (ROS) levels, and cell viability. Mendelian randomization (MR) analysis and bioinformatic studies were performed to elucidate the causal associations between mitochondrial biological function and IS. Mitochondrial membrane potential (MMP) was assessed using a tetramethylrhodamine ethyl ester perchlorate fluorescent probe, while ATP production was quantified using a commercially available assay kit. Mitochondrial respiration was evaluated by measuring the oxygen consumption rate (OCR). Finally, the verification of important targets in mouse brain slices and bEnd.3 cells was conducted through immunoblotting and immunofluorescence.

RESULTS

STDP significantly restored cerebral blood flow and neurological function, and reduced infarct volume in MCAO/R mice. Furthermore, STDP markedly alleviated inflammation and hyperpermeability of the cerebral microvascular endothelium in MCAO/R mice, as evidenced by the suppression of ICAM-1 and VCAM-1 expression, along with the upregulation of VE-cadherin and occludin protein levels. Moreover, STDP not only mitigated hyperpermeability and excessive production of ROS induced by OGD/R in bEnd.3 cells but also enhanced the protective effects of the ROS scavenger N-acetylcysteine on bEnd.3 cells. Results of MR analysis and bioinformation studies demonstrated that the disruption of mitochondrial respiration is a critical pathogenic factor in IS-induced CMD. Our data confirmed that STDP effectively restored MMP and ATP production in OGD/R-treated bEnd.3 cells. Furthermore, STDP significantly enhanced basal respiration, maximal OCR, and spare respiratory capacity in bEnd.3 cells compared to the OGD/R group. Mechanistically, STDP markedly increased endothelial cystathionine γ-lyase (CSE)-mediated hydrogen sulfide (HS) production and S-sulfhydration of P66shc, resulting in reduced protein expression and phosphorylation levels of P66Shc. This inhibition prevented its translocation into mitochondria, thereby restoring mitochondrial respiration.

CONCLUSION

STDP facilitated CSE expression and promoted HS production, contributing to the inactivation of P66shc by suppressing its expression and increasing its sulfhydration. This process impeded P66Shc translocation to mitochondria, subsequently restoring mitochondrial respiration and alleviating IS-induced cerebral microvascular endothelial dysfunction.