This paper aimed to explore the protective effects of picroside II against the neuronal apoptosis and changes in morphology and structure that follow cerebral ischemic injury in rats. A focal cerebral ischemic model was established by inserting a monofilament thread to achieve middle cerebral artery occlusion (MCAO) in 60 Wistar rats, and intraperitoneal injections of picroside II (20 mg/kg) were administered. The neurobehavioral functions were evaluated with the modified neurological severity score (mNSS) test. The cerebral infarct volumes were measured with tetrazolium chloride (TTC) staining. The morphology and ultrastructure of the cortical brain tissues were observed with hematoxylin-eosin staining and transmission electron microscopy, respectively. The apoptotic cells were counted with terminal deoxynucleotidyl transferase dUTP nick-end labeling and flow cytometry, and pERK1/2 expression was determined by immunohistochemical assay and Western blot. The results indicated that neurological behavioral malfunctions and cerebral infarcts were present in the MCAO rats. In the model group, the damage to the structures of the neurons and the blood brain barrier (BBB) in the cortex was more severe, and the numbers of apoptotic cells, the early apoptotic ratio (EAR) and pERK1/2 expression were significantly increased in this group compared to the control group (P<0.05). In the treatment group, the neurological behavioral function and the morphology and ultrastructure of the neurons and the BBB were improved including the number of Mi increased and relative area of condensed chromosome and basement (BM) thickness descreased, and the cerebral infarct volume, the number of apoptotic cells, the EAR and pERK1/2 expression were significantly decreased compared to the model group (P<0.05). These results suggest that picroside II reduced apoptosis and improved the morphology and ultrastructure of the neurons and the BBB and that these effects resulted in the recovery of the neurobehavioral function of rats with cerebral ischemia.

Mitochondrial complex I (CI) is a major source of reactive oxygen species (ROS) in plant cells, and iron–sulfur (Fe-S) cluster proteins are critical for maintaining CI structure and electron transport. C1-FDX, a mitochondrion-localized ferredoxin containing a [2Fe-2S] cluster, acts as a structural component and assembly factor of mitochondrial CI and complex V (CV) in Arabidopsis. In this study, we further characterized the physiological and molecular functions of C1-FDX in regulating ROS production, NADH/NAD⁺ homeostasis, and iron metabolism. Histochemical staining demonstrated that loss of function of C1-FDX in Arabidopsis c1-fdx mutants accumulated excessive superoxide anions (O₂^•−) but exhibited reduced hydrogen peroxide (H₂O₂) relative to the wild type (WT). Biochemical assays further confirmed that there was an elevated NADH/NAD⁺ ratio in mutant seedlings, which facilitated O₂^•− overproduction at mitochondrial CI. Loss of C1-FDX also led to Fe³⁺ accumulation in mutant tissues. Immunoblot and qRT-PCR results showed that the abundance of MnSOD markedly increased, while other antioxidant proteins, including FeSOD, CAT1, and GR, were downregulated in c1-fdx mutants of Arabidopsis. The enhanced MnSOD activity accelerated the conversion of O₂^•− into H₂O₂, and the subsequently accumulated Fe³⁺ may be derived from the Fenton reaction, which consumes H₂O₂, ultimately resulting in decreased H₂O₂ levels. Additionally, deficiency of C1-FDX disturbed the abundance of mitochondrial chaperones, proteases, and alternative respiratory pathway proteins. Collectively, our findings demonstrate that C1-FDX participates in the regulation of mitochondrial ROS balance and iron homeostasis and suggest there is a novel regulatory link between ferredoxin, the electron transport chain, and the Fenton reaction in plant mitochondria.