tsRNA-5006c regulates hippocampal neurons ferroptosis to ameliorate perioperative neurocognitive disorders in aged male mice

Abstract

The aim of this research is to investigate whether ferroptosis occurs in the pathogenesis of perioperative neurocognitive disorders (PND), and to explore the function and underlying molecular mechanism of tsRNA in the regulation of ferroptosis in PND. A PND aged mice model was established and behavioral changes and ferroptosis occurrence were confirmed. The effect of ferroptosis inhibitor ferrostatin-1 (Fer-1) on PND mice was detected. tsRNA expression profile in PND mice and the effect of tsRNA on ferroptosis in vitro were perfomed. We found that anxious exploration behavior and short-term working memory was declined in PND mice compared with control mice, and the levels of S100β and IL-6 were increased. Meanwhile, hippocampal neurons of PND mice were damaged and accompanied by ferroptosis. Fer-1 can improve cognitive impairment in PND mice, as reflected by improved anxious exploration behavior and short-term working memory, and the levels of S100β and IL-6 were decreased. The expression profile of tsRNA in PND mice is disordered, and the dysregulated tsRNAs were mainly enriched in biologic functions related to neuronal development and ferroptosis. The tsRNA-5006c, identified as a pivotal player, significantly suppressed ferroptosis in primary mice neurons. This study shows for the first time that the pathophysiological process of PND is accompanied by ferroptosis of neurons, and reveals that tsRNA-5006c regulates ferroptosis of hippocampal neurons to ameliorate PND, which is of great significance for the development of new treatment strategies.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13205-024-04176-3.

Introduction

Perioperative neurocognitive disorders (PND) is a common postoperative central nervous system complication, usually occurs 5–7 days after surgery and is characterized by confusion, anxiety, personality change, and memory impairment, which seriously affects the postoperative rehabilitation of patients (Evered et al. 2018; Qiu et al. 2020). Advanced age can lead to degenerative changes of the central nervous system, and the incidence of PND in elderly patients undergoing surgical treatment can be as high as 60% (Evered and Silbert 2018; Mahanna-Gabrielli et al. 2019). In the context of the current aging society, how to prevent and reduce PND after surgery in elderly patients is of great significance.

Ferroptosis, a recently identified form of cell demise, exerts a significant influence on a multitude of cellular activities (Chen et al. 2021). Emerging research underscores a profound link between the process of ferroptosis and the realm of cognitive performance. For example, iron exposure significantly increases lipid peroxidation and inflammation, disrupts brain iron homeostasis, induces ferroptosis in cognition-related brain regions, and aggravates cognitive impairment (Liu et al. 2022; Tripathi et al. 2019a, b). Insamgobonhwan can regulate the expression of ferroptosis-related proteins GPX4, HO-1, COX-2, inhibit amyloid-β induced lipid peroxidation, and ameliorate cognitive dysfunction (Tripathi et al. 2024; Yang et al. 2022). However, whether ferroptosis is involved in the regulation of PND is unclear.

tRNA-derived small RNA (tsRNA) is a newly discovered family of non-coding RNA, also known as transfer RNA-derived fragments (tRFs), which is related to a variety of cellular functions such as protein translation and the regulation of gene expression. Recent literatures report that tsRNA takes participate in the regulation of ferroptosis. Macrophages-derived exosomal tRF-22-8BWS7K092 induces ferroptosis by activating Hippo signaling pathway, which is involved in the pathogenesis of acute lung injury (Wang et al. 2022a, b). Increased tsRNA production regulates stress responses, protease stabilization, and neuronal survival during disease, playing a key role in nervous system function and dysfunction (Fagan et al. 2021). tsRNA is specifically present in different neuronal subdomains and selectively integrated into extracellular vesicles, equips neurons with critical molecular tools for intercellular communication (Mesquita-Ribeiro et al. 2021). Some tsRNA in primitive neuron necrosis in rats induced by glutamate concentration, the concentration depends on the increase of H3K4me3, it is worth noting that tsRNA induced neuron swelling and death (Cao et al. 2021). In neurodegenerative diseases, abnormal splicing and accumulation of tsRNAs may cause damage to neurons. For example, CLP1 gene mutation may lead to the accumulation of precursor tRNA and the failure of mature tRNA to connect properly, leading to the accumulation of tiRNAs, which in turn affect protein translation and neuronal cell survival (Qin et al. 2020). The expression pattern of tsRNAs in different brain regions may be different, which may be related to the development and function of specific brain regions. For example, tRF5 CysGCA has been suggested to delay hippocampal neuronal atrophy (Blanco et al. 2014), suggesting that tsRNAs may play a role in neuronal development and maintenance in specific brain regions. It is well known that tsRNA can regulate gene expression, and studies have shown that tsRNA can regulate apoptosis and autophagy (Wang et al. 2024; Zhu et al. 2022), suggesting that tsRNA may also regulate ferroptosis by mediating gene expression. The current literature lacks reports on the potential role of tsRNA in mediating ferroptosis and its regulatory effects on PND within neuronal cells.

The aim of this research is to investigate whether ferroptosis occurs in the pathogenesis of PND, to identify the cells that undergo ferroptosis, and to explore the function and underlying molecular mechanism of tsRNA in the regulation of ferroptosis in PND.

Materials and methods

Animals

C57BL/6 J mice (male, 18 months of age, 33–35 g) were kept in a controlled environment (23 °C, 50% humidity, 12 h light–dark cycle) with free access to food and water. All animal experiments in this study were followed the principles of the National Institute of Health Guide for the Care and Use of Laboratory Animals.

PND mice model construction and treatment

Mice were allocated to two groups through a process of randomization: sham (n = 5) and PND (n = 5). For the PND model construction, we referred to the previous literature to expose the right carotid artery (Lin et al. 2020; Min et al. 2022). Mice were subjected to anesthesia using 1.4% isoflurane (792,632, Sigma, Germany) for at least 10 min, allowed to breathe spontaneously, and kept at 37 °C with a heating blanket (TCAT-2LV, Physitemp instruments Inc., Clifton, NJ). After local application of 0.25% bupivacaine (PHR1128, Sigma, Germany), a 1.5 cm longitudinal incision was made in the neck’s central region. The tracheal soft tissue was meticulously cleaned, and a 1 cm segment of the right common carotid artery was carefully dissected, taking care not to harm the vagus nerve. The wound was rinsed and sutured. The sterile surgery, approximately 15 min in duration, was followed by subcutaneous bupivacaine administration to all animals. The anesthesia lasted for 2 h, with no signs of pain response to toe pinch.

To investigate the role of ferroptosis inhibitor ferrostatin-1 (Fer-1), PND mice were divided into two groups: vehicle (n = 5) and Fer-1 (n = 5), and were intraperitoneally injected with Fer-1 (2.5 μmol/kg) or the same amount of normal saline at 30 min before anesthesia and on day 1–2 after surgery (Chen et al. 2022a, b). Hippocampal tissues were harvested from the cerebral hemisphere of individual mice at postoperative days 3 and 9 in PND group, and on day 9 in sham group. Figure 1 shows the flowchart of this study.

Fig. 1.

Experimental flow chart of this study. PND perioperative neurocognitive disorders, Fer-1 Ferrostatin-1

Behavioral test

Open field test (OFT)

Behavioral tests were performed continuously from day 5–8 after surgery. The OFT was employed to assess signs of anxiety in mice. In the OFT, mice will typically exhibit natural anxiety about open space, preferring marginal activity close to the field and less access to the central area. This behavior is thought to be related to the anxiety state of the mice. Mice with higher levels of anxiety tended to have shorter periods of activity in the central region, move more slowly, and stay more frequently in the limbic region (Pentkowski et al. 2021). Anxiety behavior can be quantified by analyzing the activity of the mice in the central and limbic areas of the open field. During the experimental phase, the mice were introduced into a square arena measuring 40 cm × 40 cm and allowed to explore for a duration of 5 min (Chen et al. 2022a, b). Behavioral analysis was conducted utilizing the ANY-maze (Stoelting, Wood Dale, IL). The immobility time of the mice in the designated central zone was analyzed.

Y-Maze test

Following a 6-h recovery period post the OFT, then the Y-maze test was administered to assess the short-term working memory capabilities in mice (Amoah et al. 2023; Kim et al. 2023). The Y-maze test is based on the animal’s natural exploratory curiosity about a novel environment and assesses the animal's short-term spatial memory by observing its spontaneous alternating behavior in the maze. The apparatus for this test included a Y-shaped maze, a camera, and tracking software. The maze featured three equal-length arms (30 cm × 15 cm × 8 cm) arranged in an equilateral triangular pattern. Mice were consistently positioned from the same height and angle at the start of each trial. They were allowed to explore the maze freely for 8 min, with a complete entry into an arm being defined as when all of the mouse’s limbs and body, excluding the tail. During the experiment, the animal needs to remember previously explored directions, which reflects its spatial working memory capacity. At the end of the experiment, the maze needed to be wiped with 70% alcohol to eliminate odor marks and prevent interference with subsequent experiments (Shang et al. 2023). The camera and tracking software captured each mouse's movement path, total arm entries, and the sequence of entries. The total number of correct entries and the spontaneous alternation behaviors were calculated using the formula. Spontaneous alternation correct rate = [number of correct arm entries/(total number of arm entries−2)] × 100%.

Nissl staining

Nissl staining was used to observe the distribution of neurons in the hippocampus (Wang et al. 2022a, b). In brief, hippocampal sections were routinely deparaffinized to water and washed three times with distilled water. Following the preparation, the tissue sections were dyed using the Nissl staining solution (C0117, Beyotime, China) for 10 min. After washing with distilled water, the dyes were dehydrated in different concentrations of ethanol. The slices were cleared with xylene for another 3 min and sealed with neutral gum. Finally, images were obtained for observation using a biomicroscope (Olympus DP80, Japan).

Enzyme-linked immunosorbent assay (ELISA)

Concentrations of S100β and IL-6 in hippocampal extracts were measured employing commercially available ELISA kits (mlbio, China). The procedures were strictly adhered to as outlined in the manufacturer's protocol, ensuring accurate and reliable measurements of these biomarkers (Sharma et al. 2024).

Western blot (WB)

Hippocampal tissue samples homogenates were centrifuged at 800 g, 4 °C for 5 min, and supernatants were resolved on SDS-PAGE and transferred to PVDF. After 3 h blocking with TBST (Thermo) and 5% milk, membranes were incubated with primary antibodies, including GAPDH (1:2000, Proteintech, 60,004–1-Lg), anti-GPX4 (1:1000, Santa, Sc-166570), anti-FTH1 (1:1000, Santa, Sc-376594) overnight at 4 °C. Next, secondary Goat Anti-Mouse IgG H&L (1:1000, abcam, ab205719) was incubated for 2 h. Protein bands were identified with a luminescent detection reagent, and the expression levels were measured using Image J software.

Fe²⁺ and malondialdehyde (MDA) detection

According to the manufacturer’s instructions, Iron Colorimetric Assay Kit (Applygen, #E1042, China) and MDA detection kit (Beyotime, S0131, China) were used to detect Fe²⁺ and MDA levels in hippocampus. In simple terms, the cells are lysed with phosphate-buffered saline (PBS). Prepare the standard working liquid, and set up the blank control, standard quality control and sample tube. Then add detection agent, mix and heat for incubation. Finally, supernatant was added to the 96-well plate, and the absorbance was measured at 550 nm and 532 nm by an enzyme labeler (Infinite M1000, TECAN).

Immunofluorescence (IF) staining

IF staining of mice hippocampus was performed as described previously (Zheng et al. 2018). Slice and against NeuN (1:300, ab177487, Abcam, America), GFAP (1:5000, ab7260, Abcam, America) and Iba-l (1:500, ab153696, Abcam, America) of primary antibodies in 3 ℃ incubation for the night. Goat anti-mouse IgG-FITC (1:500, ab97264, Abcam, America) was used as secondary antibody and incubated for 1 h before stained with DAPI. Captured images were examined utilizing a fluorescence microscope (Olympus DP80, Japan).

Small RNA sequencing (RNA-seq) and data analysis

Mice hippocampal RNA was purified employing the RNeasy Mini Kit (Qiagen, Hilden, Germany). Independent small RNA libraries were constructed according to manufacturer’s protocol. Prior to the start of library construction, we pre-processed the total RNA to remove internal modifications of the RNA utilizing the T4PNK (#M0201L, New England Biolabs, China) and AlkB (#R0639S, Beyotime Biotechnology, China). After passing quality control tests, libraries were established using Illumina’s Multiplex Small RNA Library (NEB, MA, USA). The specimens from the library were subjected to paired sequencing utilizing the Illumina Hiseq2500 platform (Illumina, San Diego, California).

We use the Illumina chastity filter to filter the raw data. FastQC (v0.11.7) was used to detect the sequencing quality. Next, cutadapt (1.17) was used to cut the 5', 3'-adaptor bases. The trimmed sequence was aligned to the mature tRNA sequence, and the unmapped sequence was then aligned to the precursor tRNA sequence using Bowtie software (V1.2.2). TsRNA expression levels were quantified from the aligned and normalized tRNA read counts. Utilizing the R package DEseq2, we identified differentially expressed tsRNA. |log2 (fold change)| > 1 and p < 0.05 was considered statistically significant.

Bioinformatics prediction

We used the package clusterProfiler (3.18.1) for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes database (KEGG) pathway annotation. GO enrichment analysis was used to reveal the biologic process, cellular composition and molecular function of the target genes. KEGG enrichment analysis was used to identify the key pathways. p < 0.05 indicates significant enrichment of GO and KEGG pathways.

Quantitative real-time PCR (qPCR)

Total RNA was extracted using TRIzol reagent (Invitrogen, America), followed by cDNA synthesis with a reverse transcription kit (Takara, China). U6 served as the normalizer for gene expression analysis. The 2^−△△Ct method was employed for relative quantification of gene expression. All primer sequences used in the experiments are listed in Table S1.

Generation of neurons in primary culture

Neuronal cell culture was performed with reference to the relevant literature (Wei et al. 2020). Pregnant mice were anesthetized and sacrificed on day 16 of gestation, chunks of brain tissue containing the cortex and hippocampus were dissected from the embryos. After trypsinization, the single-cell suspension, and in the basal medium (Gibco, 21,103,049) in the heavy suspension, including 2% of B27 supplements (Gibco, 17,504–044), 1% of penicillin streptomycin (HyClone, SV30010) and 1% of L—glutamic acid (Gibco, 35,050–038). Cells were cultured in 6-well plates precoated with poly-D-lysine (50 μg/ml) at 37 °C in 5% CO₂. The culture medium was replaced at intervals of 3 days. Following 12-day cultivation, neurons underwent treatment with NC or tsRNA-5006c mimics.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 9.0 (La Jolla, CA, America). Two-tailed t test was used for comparison between the two groups, and one-way ANOVA was used for comparison between the three groups. All continuous variables are shown as mean ± standard deviation (SD). p < 0.05 was considered statistically significant.

Results

Postoperative cognitive decline in PND mice

Initially, a PND mouse model was established, followed by a series of behavioral assessments conducted for four consecutive days. The results of OFT showed that the immobility time of the PND mice was notably increased compared with the sham group on days 5–8 (Fig. 2A). This indicated that the total number and time of open field activities were reduced in mice after surgery, meaning anxiety and exploratory behaviors were decreased. The Y-maze test assessed the short-term working memory ability of mice, with the spontaneous alternation correct rate being quantified (Amoah et al. 2023; Kim et al. 2023). The spontaneous alternation correct rate was 46.8% ± 14.5% in sham group and 25.8% ± 12.1% in PND group. Compared to the sham group, the spontaneous alternation correct rate of PND mice were notably decreased on days 5–8 (Fig. 2B). S100β and IL-6 have been extensively validated as biomarkers in models of neurocognitive disorders, and it has potential application value in the diagnosis and prognosis evaluation of Alzheimer's disease, Parkinson's disease, brain injury and other diseases (Lopez-Rodriguez et al. 2021; MacMahon Copas et al. 2024; Rodríguez et al. 2023; Zhang et al. 2024). Furthermore, we utilized ELISA and WB to assess cognitive impairment markers, specifically measuring the levels of S100β and IL-6 in mice serum (Huang et al. 2022, Lin et al. 2024, Schaefer et al. 2019). The results indicated the concentrations of S100β and IL-6 elevated in PND mice compared to the sham group (Fig. 2C, D). Thus, these results suggest a degree of cognitive deficits in PND mice.

Fig. 2.

Behavioral tests showed decreased memory and learning in PND mice. A Images of open field test results of mice in sham group and PND group from day 5–8 and statistical analysis of immobility time were performed. B Images of Y-Maze test results of mice in sham group and PND group from day 5–8 and statistical analysis of correct rate of spontaneous alternation. C The levels of S100β and IL-6 in serum of sham and PND mice were detected by enzyme-linked immunosorbent assay (ELISA). D The levels of S100β and IL-6 in serum of sham and PND mice were detected by western blot (WB). *p < 0.05, **p < 0.01, n = 3 or 5

PND mice hippocampal neurons are damaged and accompanied by ferroptosis

Subsequently, we examined hippocampal tissue neurons from PND mice on day 9. The results of Nissl staining showed that the hippocampal cells of the mice were lightly stained, the cells were loosely arranged and broken, and the average number of neurons was significantly reduced after surgery, indicating that the neuronal cells were dead (Fig. 3A). To further explore whether ferroptosis occurs in the hippocampus of PND mice, we detected the expression of ferroptosis related parameters in the hippocampus of mice. We detected the ferroptosis markers GPX4 and FTH1 in the hippocampus, as well as Fe²⁺ and MDA levels. Ferroptosis of astrocytes was detected by immunofluorescence double staining of GFAP and GPX4, and ferroptosis of neurons was detected by immunofluorescence double staining of NeuN and GPX4. GPX4 and FTH1 expression were notably reduced in the hippocampus of PND mice on day 3 and day 9 compared with the sham group (Fig. 3B), while Fe²⁺ and MDA levels were increased in the hippocampus of PND mice on day 9 (Fig. 3C), indicating ferroptosis in hippocampal neurons of PND mice. In addition, the co-localization and expression of GPX4 with NeuN, GFAP were detected by IF double staining. The results showed that the amount of neuronal marker NeuN and astrocyte marker GFAP were notably reduced in PND mice compared with sham group (Fig. 3D, E). GPX4 main positioning with neurons NeuN occured at the same time, such a total positioning was weakened in postoperative mice (Fig. 3D, E). Due to the occurrence of ferroptosis in neuronal and astrocyte in PND, the detectable signals of GFAP and NeuN were weak. These findings indicate that neuronal damage in PND mice is accompanied by ferroptosis.

Fig. 3.

Ferroptosis occurs in neurons in the hippocampus of PND mice. A Nissl staining of hippocampal tissue neurons from sham and PND mice on day 9. Scale = 50 μm. B WB was used to detect the expression of GPX4 and FTH1. C Fe²⁺ and malondialdehyde (MDA) were detected by kit. D IF detection of GPX4 and GFAP colocalization. Scale = 50 μm. E Co-localization of GPX4 and NeuN detected by IF. Scale = 50 μm. *p < 0.05, **p < 0.01, n = 3 or 5

Inhibition of ferroptosis can ameliorate cognitive impairment in PND mice

Given the above results showing neuronal ferroptosis in PND, it prompted us to wonder whether pharmacologic inhibition of neuronal ferroptosis is a new strategy for the treatment of PND. In the experimental setup, PND mice were administered either a vehicle or Fer-1 treatment. Similarly, we used the OFT and Y-maze test to examine mice behavior. In comparison with the PND + vehicle group, the immobility time of the PND + Fer-1 mice was significantly decreased on days 5–8 (Fig. 4A). This indicated that PND mice treated with Fer-1 exhibited increasing anxious and exploratory behaviors. The spontaneous alternation correct rate was 29.9% ± 6.7% in PND + vehicle group and 45.3% ± 9.4% in PND + Fer-1 group. Compared to the PND + vehicle group, the spontaneous alternation correct rate of PND + Fer-1 mice was significantly increased on days 5–8 (Fig. 4B). Concentrations of S100β and IL-6 in PND + Fer-1 mice were notably reduced in PND + vehicle group (Fig. 4C, D). Consequently, Fer-1 ameliorated cognitive impairment in PND mice. In additional, Fer-1 effectively reduced the occurrence of the neuronal ferroptosis (Fig. 4E) and increased in the number of neurons (Fig. 4F). These results indicate that inhibition of ferroptosis can reduce the pathologic damage of hippocampus induced by surgery in mice.

Fig. 4.

Inhibit ferroptosis improve cognitive impairment of mice. A Images of open field test results of mice in PND + vehicle group and PND + Fer-1 group from day 5–8 and statistical analysis of immobility time were performed. B Images of Y-Maze test results of mice in PND + vehicle group and PND + Fer-1 group from day 5–8 and statistical analysis of correct rate of spontaneous alternation. C The levels of S100β and IL-6 in serum of mice were detected by ELISA. D The levels of S100β and IL-6 were detected by WB. E Fe²⁺ and MDA levels were detected by kit. F Nissl staining of hippocampal tissue neurons from PND + vehicle and PND + Fer-1 mice on day 9. Scale = 50 μm. *p < 0.05, **p < 0.01, n = 3 or 5

The tsRNA expression profile of PND mice is disordered

To further explore the regulatory mechanism of ferroptosis in neurons, the hippocampus from PND and sham groups were subjected for small RNA-seq. Relative to the sham group, the hippocampal tsRNA expression landscape in PND-affected mice exhibited significant dysregulation, containing three up-regulated tsRNAs and eight down-regulated tsRNAs (Fig. 5A). These abnormal tsRNAs enrichment in biology functions associated with neuronal development, such as neural development, neural regulation of synaptic plasticity (Fig. 5B). Simultaneously, KEGG analysis revealed that the identified tsRNAs were involved in Wnt signaling pathway, FoxO signaling pathway and MAPK signaling pathway (Fig. 5C), all of which were implicated in ferroptosis (Chen et al. 2023; Li et al. 2024; Wang et al. 2023). Consequently, our findings propose a link between the dysregulation of these tsRNAs and the modulation of cognitive impairment in PND, potentially through the influence on neuronal and ferroptotic processes (Fig. 5D).

Fig. 5.

tsRNA expressed in the bioinformatics analysis of sham vs PND. A Radar plot showing tsRNA expression profile. B Top 20 of GO enrichment based on the target genes of differentially expressed tsRNAs. C Top 20 of pathway enrichment based on the target genes of differentially expressed tsRNAs. D tsRNA-KEGG-GO network diagram

tsRNA-5006c inhibits neuronal ferroptosis

To screen the key tsRNAs, we analyzed the expression profiles of 11 differentially expressed tsRNAs, and finally selected 7 tsRNAs with high expression abundance, large fold change and small P value as candidate tsRNAs. Then q-PCR and conservation analysis revealed that the expression trend of tsRNA-5006c was not only consistent with the sequencing results but also the best conserved (Fig. 6A, B), so tsRNA-5006c was selected for subsequent studies. According to MINTbase v2.0 (http://cm.jefferson.edu/MINTbase/), the tsRNA-5006c (5’-GCCCGGCTAGCTCAGTCGGTAGAGCATGGGAC-3’) also termed as tRF-32-PSQP4PW3FJIK1 and belonged to 5’-half tsRNA (Holmes et al. 2023; Xia et al. 2022). The tsRNA-5006c was derived from the mature tRNA cleavage at the site of Lys-CTT. Subsequently, we isolated and cultured primary neuronal cells and treated them with NC or tsRNA-5006c mimics. Compared with NC, tsRNA-5006c mimics not only promoted tsRNA-5006c expression (Fig. 6C) but also promoted GPX4 and FTH1 expression in neurons (Fig. 6D), and reduced the Fe²⁺ and MDA (Fig. 6E). Taken together, tsRNA-5006c has an inhibitory effect on neuronal ferroptosis.

Fig. 6.

tsRNA-5006c inhibits neuronal cell ferroptosis. A tsRNA expression by small RNA-seq. B q-PCR verify differentially expressed tsRNA. C The transfection efficiency of tsRNA-5006c mimics was detected by q-PCR. D The expression of GPX4 and FTH1 in NC and tsRNA-5006c mimics was detected by WB. E Fe²⁺ and MDA levels in NC and tsRNA-5006c mimics were detected by kits. *p < 0.05, **p < 0.01, n = 3

Discussion

Ferroptosis occurs in a variety of neurologic diseases with cognitive impairment (Weiland et al. 2019). Studying the involvement of tsRNA in the regulation of ferroptosis in PND may provide a new understanding of the pathogenesis of PND. As mentioned earlier, advanced age is considered an important risk factor for PND. Therefore, we selected 18-month-old mice to mimic the physiology and pathology of elderly patients. To avoid variability thought to be caused by the oestrous cycle, we constructed a PND model using aged male mice (Singh et al. 2024). Our data clearly showed that PND mice exhibited cognitive dysfunction and hippocampal neuronal ferroptosis, and Fer-1 treatment could significantly ameliorate cognitive impairment, reduce neuronal damage and the occurrence of ferroptosis in PND mice. Similar to our results, studies have shown that the use of iron chelator deferoxamine can prevent neuroinflammation-induced learning and memory impairment by preventing iron accumulation and reducing oxidative stress (Ramakrishna et al. 2024; Zhang et al. 2015). Furthermore, certain studies have documented the neuroprotective capabilities of ferroptosis inhibitors, such as liprostatin-1 and Fer-1, which have been observed to alleviate cognitive impairments in animal models simulating neurodegenerative conditions (Alim et al. 2019; Bao et al. 2021).

Current PND-treatment strategies include: (1) inhibiting inflammatory mediators to reduce inflammation, (2) neutralizing oxidative stress linked to inflammation, and (3) preoperatively promoting neuronal health (Grossman et al. 2023; Safavynia and Goldstein 2018). These treatments mainly reduce oxidative damage to neurons by increasing antioxidant levels in the nervous system (Srivastava et al. 2019; Tripathi et al. 2019a, b). This suggests that inhibition of oxidative stress is the main therapeutic direction to improve neuronal damage, and reducing oxidative stress and inflammatory response in the brain hippocampus can ameliorate the learning and memory function of mice (Zhang et al. 2015; Zhao et al. 2017). The cerebral cortex exhibits a heightened vulnerability to oxidative stress in comparison with other bodily tissues. Generally, oxidative stress is marked by increased levels of reactive oxygen species, which is accompanied by the occurrence of ferroptosis (Li et al. 2022; Rai et al. 2020). Consequently, the suppression of neuronal ferroptosis could potentially offer a novel therapeutic avenue for the management of PND.

Surgical trauma can cause neurologic damage, involving synaptic defects, neuronal dysfunction, and neuroinflammation (Alam et al. 2018). tsRNA serves as a crucial modulator in central nervous system development and functionality. Its dysregulation can precipitate pathologic conditions. Evidence suggests that tsRNA anomalies are linked to oxidative stress, enhancing the vulnerability of motor neurons to such stress and potentially influencing neuronal redox balance under non-pathologic conditions (Hanada et al. 2013). Mutations in CLP1 have been observed to result in tRF production, which can amplify neuronal susceptibility to p53-dependent oxidative damage, contributing to the etiology of certain familial forms of Parkinson’s disease (Schaffer et al. 2014). Furthermore, research by Magee and colleagues has indicated that tRFs derived from the prefrontal cortex, cerebrospinal fluid, and serum can differentiate Parkinson's disease patients from healthy controls, highlighting their potential as credible biomarkers for PD diagnosis (Magee et al. 2019). In our research, small RNA-seq analysis of the mice hippocampus indicated a significant enrichment of aberrant tsRNAs in functions pivotal to neurodevelopment, encompassing nervous system development and modulation of synaptic plasticity in neurons. Importantly, we identified tsRNA-5006C as the pivotal tsRNA that had an inhibitory effect on primary neuronal ferroptosis in postoperative mice, offering a potential therapeutic target for PND. Only previous studies have reported that M1-EVs carrying tsRNA-5006c regulated the osteogenic differentiation of AVIC from the perspective of mitautophagy (Xia et al. 2022). But on specific tsRNA small molecules in the study of the role of the PND is less, the study of biomarkers tsRNA as PND is still in its infancy.

There are still some challenges in tsRNA related research. Our further research will be focused on tsRNA-5006c expression patterns in the PND and the downstream targets. Before tsRNA-5006c can be clinically applied for PND, its therapeutic mechanisms must be well understood. The potential of tsRNA to improve the quality of life for surgical patients with PND is considerable, suggesting significant clinical benefits. Future research in this area could lead to better patient outcomes and enhance surgical care.

In conclusion, this study shows for the first time that the pathophysiological process of PND is accompanied by the occurrence of neuronal ferroptosis, and Fer-1 can significantly ameliorate the cognitive impairment of PND mice. Dysregulated tsRNAs in the hippocampus of PND mice were enriched in biologic functions related to neuronal development and ferroptosis. The tsRNA-5006c, identified as a pivotal player, significantly suppressed ferroptosis in primary mice neurons. In brief, tsRNA-5006c ameliorate PND by targeted inhibition of ferroptosis in hippocampal neurons. This study provides support for the future development of new treatment strategies to ameliorate cognitive function in PND.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Chuanlin Zhou: conceptualization, Data curation, writing—original draft, and writing—review and editing. Fang Lian: formal analysis, data curation, software. Hejian Li: methodology, validation, visualization. Fumou Deng: project administration, funding acquisition, methodology.

Funding

Natural Science Foundation of Jiangxi Province, 20212BAB206045.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

All animal experiments in this study were approved by the Ethics Committee of Laboratory Animal Welfare of Youshu Life Technology (Shanghai) Co., LTD. [YS-m202311002], and followed the principles of the National Institute of Health Guide for the Care and Use of Laboratory Animals.