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. 2026 Jan 9;16:4186. doi: 10.1038/s41598-025-34240-8

Morin improves postoperative cognitive dysfunction by mediating the miR-138-5p/SIRT1 axis to regulate ferroptosis

Yang Yang 1,2,#, Wen Hu 2,#, Zhen Wu 1,2, Mi Chen 1,2, Hui Li 1,2, Jiatian Cui 2, Chaoying Wang 2, Yushan Luo 2, Bailong Hu 1,2,✉, Xiaohua Zou 1,2,✉
PMCID: PMC12858968  PMID: 41513685

Abstract

This study aimed to explore whether Morin could improve postoperative cognitive dysfunction (POCD) by inhibiting hippocampal ferroptosis through miR-138-5p/SIRT1. Morin improves the cognitive function of POCD mice by inhibiting the expression of miR-138-5p and promoting the expression of SIRT1 protein. When detecting ferroptosis-related indicators (such as GSH, MDA, and iron ion levels), it was found that miR-138-5p agomir blocked the regulatory effect of Morin on these indicators. Protein detection showed that Morin regulated the ferroptosis process by controlling the miR-138-5p/SIRT1 axis to affect the expression of p53, SLC7A11, and GPX4. In vitro cell experiments showed that after Erastin induction, the GSH level in HT22 cells decreased, the MDA and Fe2+ contents increased, and the ROS fluorescence intensity increased; after Morin treatment, these oxidative stress indicators were significantly improved, the expression of SIRT1, SLC7A11 and GPX4 increased, and the expression of p53 decreased. The miR-138-5p mimic aggravated the upregulation of oxidative stress and the regulation of ferroptosis-related genes, while the miR-138-5p inhibitor had the opposite effect. Morin improved cognitive function and neuronal morphology in POCD mice by inhibiting miR-138-5p and upregulating SIRT1. It also inhibited ferroptosis through the miR-138-5p/SIRT1 pathway, further confirming its protective role in POCD.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-34240-8.

Keywords: miR-138-5p, SIRT1, Morin, Ferroptosis, POCD

Subject terms: Cognitive neuroscience, Learning and memory

Introduction

Postoperative cognitive dysfunction (POCD) is a common central nervous system complication that occurs one week or even months after surgical anesthesia. In a few patients, it may last longer. Patients mainly show cognitive function changes such as postoperative memory loss, inattention, and decreased executive ability. In severe cases, personality changes and decreased social skills may occur1,2. The incidence of POCD in patients over 65 years of age can be as high as 40% in major and medium-sized non-cardiac surgery and up to 60% in cardiac surgery3,4. POCD negatively affects patients’ cognitive domains such as memory and attention. It not only prolongs hospitalization and increases healthcare costs, but can also seriously affect patients’ postoperative quality of life and postoperative rehabilitation, and even increase the mortality rate, among other things, and aggravate the burdens of the family and society5,6. Therefore, POCD has also become an urgent medical problem and research hotspot in today’s society. Thus, there is an urgent need for drugs that can effectively improve POCD and elucidate its mechanism, which is of great clinical significance and social value for the prevention and treatment of POCD and for improving patient’s prognosis.

Current studies suggest that the pathogenesis of POCD may involve several aspects, including amplification of the inflammatory response of the central nervous system, oxidative stress, reduced function of the central cholinergic system, changes in cerebral blood flow, deposition of β-amyloid, hyperphosphorylation of tau proteins, and impaired synaptic function, but to date, the pathogenesis of POCD has not been fully elucidated7,8. In recent years, the role of ferroptosis in the pathophysiological mechanisms of POCD due to various triggers has received increasing attention9,10. Studies on animal models of POCD have found that an increase in brain iron is associated with a decline in cognitive function after surgery, suggesting a potential link between iron deposition and POCD9,10. Mice with a knockout of glutathione peroxidase 4 (GPX4) in specific cerebral cortex and hippocampal neurons showed significant cognitive dysfunction and degeneration of hippocampal neurons11. Administration of vitamin E or the ferroptosis inhibitor Ferrostatin-1 reduced the extent of neurodegenerative lesions12. These studies indicate that neuronal ferroptosis is closely related to the occurrence and development of POCD.

MicroRNAs (miRNAs) are a class of non-coding RNAs approximately 22 bp in length that exert their effects primarily by binding to specific sequences of target genes to silence their expression13. miRNAs are highly expressed in the nervous system and are widely involved in various life processes, including neural development, tissue differentiation, and synapse formation13,14. Numerous studies have shown that cognitive impairment disorders are often associated with abnormal expression of cognitive-related proteins in the brain15. Furthermore, regulatory genes involved in protein expression also contribute to the development and progression of cognitive impairment. Studies report that miR-138-5p drives neuronal cell death16,17. Therefore, the relationship between miRNAs and the nervous system, and the mechanisms by which they influence cognitive impairment disorder (POCD), warrant further investigation. miRNAs hold promise as potential targets in the clinical diagnosis and treatment of cognitive impairment disorders.

Morin is a bioflavonoid extracted from Moraceae plants such as yellow mulberry wood and orange tree and many Chinese herbal medicines. It has obvious antioxidant, anti-apoptotic, anti-inflammatory, and neuroprotective properties18–21 and is widely found in fruits, vegetables, green tea, and herbs22. Previous studies have found that Morin treatment can downregulate the expression of serum IL-6, MCP-1, TNF-α and IL-10 in septic mice, reduce microglial activation, reduce tau protein phosphorylation, reduce Aβ deposition, and protect synaptic integrity, indicating that Morin has anti-inflammatory and anti-neurodegeneration effects and can reverse cognitive dysfunction in septic mice23. However, there are still few studies on how Morin can improve POCD by inhibiting ferroptosis.

This study will use the myocardial ischemia reperfusion injury to construct a cardiac surgery animal model to simulate POCD. First, differential miRNAs and target genes will be screened through sequencing. Then, animal experiments and cell experiments will be conducted to explore the role and mechanism of Morin in mediating ferroptosis in POCD through miRNA and target genes. Through this in-depth study, we hope to inject new vitality into the treatment of POCD and bring a better quality of life to patients.

Results

Transcriptome sequencing analysis of differential miRNAs and potential mechanisms of POCD

After sequencing miRNAs in the sham and OP groups, 28 upregulated miRNAs and 9 downregulated miRNAs were found (Fig. 1A). The 37 differential miRNAs were displayed in a heat map, and we focused on miR-138-5p, which is closely related to neurological diseases24,25. Compared with the sham group, miR-138-5p was highly expressed in the OP group (Fig. 1B). Next, we performed GO annotations analysis on miR-138-5p target genes using miRTarBase (Fig. 1C). The predicted GO functional pathways are datalytic activity, binding, organelle, cell part, biological regulation, and cellular process. In neurology, we pay more attention to synapse parts and synapse. Figure 1D shows the enriched pathways of KEGG annotation analysis of miR-138-5p target genes, and Neuro degenerative disease and Nervous system are worth noting. The regulatory network of miRNA-138-5p and its downstream target gene is shown in Fig. 1E, in which SIRT1 is enriched in GO and KEGG entries related to nerves (Fig. 1E).

Fig. 1.

Fig. 1

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Transcriptome analysis of miRNA expression profiles and potential mechanisms of POCD. (A) Volcano plot comparing Sham and OP groups. (B) Heat map of 37 differential miRNAs. (C) GO annotations analysis of target genes of miR-138-5p. (D) KEGG annotations analysis of target genes of miR-138-5p26–28 (KEGG Copyright Permission has been obtained from Kanehisa Laboratories, Ref: 254078). (E) Regulatory network of miR-138-5p.

Targeting validation of miR-138-5p and SIRT1

As indicated in Fig. 2A, the perfect base pairing was observed between the 3’UTR (wt) of SIRT1 mRNA and the seed sequence of miR-138-5p. However, SIRT1-3’UTR (mu) did not have a complementary pairing with miR-138-5p. Dual-luciferase reporter assay shows that mmu-miR-138-5p mimic has a targeted inhibitory effect on m-sirt1-3UTR-wt, but has no impact on m-sirt1-3’UTR-mu. In addition, NC mimics did not affect m-sirt1-3’UTR-wt and m-sirt1-3’UTR-mu (Fig. 2B). In vivo, the detection of miR-138-5p and SIRT1 showed that the expression of miR-138-5p was increased and the mRNA expression level of SIRT1 was decreased in the OP group compared with the sham group (Fig. 2C and D).

Fig. 2.

Fig. 2

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Expression and targeting relationship of miR-138-5p and SIRT1 in the hippocampus of POCD mice. (A) Cloned into a luciferase reporter vector to generate the WT or Mut SIRT1-3’UTR reporter plasmid. (B) Relative luciferase activity of 293T cells transfected with NC mimic or mmu-miR-138-5p mimic, and WT or Mut SIRT1 3’UTR reporter plasmid. (C) Expression of miR-138-5p in the hippocampus of sham-operated and OP-operated mice. (D) Expression of SIRT1 in the hippocampus of sham-operated and OP-operated mice. *Compared with the sham group, *** p < 0.001.

Protective effect of Morin on POCD mice

After identifying the potential mechanism of POCD, we selected Morin (Fig. 3A), an active natural compound with neuroprotective and cognitive dysfunction-improving properties23,29, to validate the involvement of the miR-138-5p/SIRT1 axis in POCD. As shown in Fig. 3B, compared with the sham group, the expression of miR-138-5p in the OP group mice increased, and the expression of miR-138-5p decreased after Morin treatment. By contrast, compared with the sham group, the expression of SIRT1 protein decreased in the OP group, and the expression of SIRT1 increased after treatment (Fig. 3C and D). In Y Maze, the spontaneous alternation rate in the OP group decreased compared with that in the sham group. However, the spontaneous alternation rate increased after Morin treatment compared to the OP group (Fig. 3E). Regarding Novel location recognition and Novel object recognition, compared with the sham group, the time of both in the OP group mice was reduced, and after Morin treatment, the time of both was increased (Fig. 3F and G). The results of TEM showed that compared with the sham group, the morphological structure of neurons in the OP group was abnormal. The cell volume was reduced, the nucleus was shrunken, the chromatin was aggregated, and the cytoplasmic electron density was increased; the mitochondria became smaller and rounder, and the cristae were broken; the rough endoplasmic reticulum expanded, and the Golgi apparatus expanded. Compared with the OP group, these ultrastructural alterations were restored to a certain extent in the Morin-treated group (Fig. 3H).

Fig. 3.

Fig. 3

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Protective effect of Morin on cognitive dysfunction in POCD mice. (A) Chemical structure of Morin. (B) Effect of Morin on miR-138-5p. (C and D) Effect of Morin on SIRT1 protein. Effect of Morin on spontaneous alternation rate in Y Maze (E), novel location recognition (F), and novel object recognition (G) in POCD mice. (H) Effect of Morin on the ultrastructure of hippocampal tissue in mice observed by TEM. *P < 0.05, **P < 0.01, and ***P < 0.001.

Morin promotes cognitive recovery in POCD by inhibiting miR-138-5p

Morin has been previously identified to inhibit miR-138-5p in the mouse hippocampus, and the miR-138-5p agomir was introduced to block the efficacy of Morin. As shown in Fig. 4A, in Y Maze, agomir NC did not affect the effect of Morin. However, the spontaneous alternation rate of Morin-treated POCD mice was reduced after the action of miR-138-5p agomir. Compared with the OP + Morin group, the novel location recognition and novel object recognition were reduced after the miR-138-5p agomir treatment (Fig. 4B and C). When we shifted our focus to the ferroptosis, compared with the sham group, GSH decreased in the OP group, while MDA and Iron increased. Compared with the OP group, Morin increased GSH, while MDA and Iron decreased. Compared with OP + Morin, miR-138-5p agomir inhibited GSH and restored the levels of MDA and Iron (Fig. 4D, E, and F). TEM results showed that the morphological structure of hippocampal neurons in the OP + morin group was relatively intact. Compared with the OP + morin group, the morphological structure of hippocampal neurons in the OP + morin + miR-138-5p agomir group was abnormal. The neuronal cell bodies were larger, the mitochondria in the cytoplasm became smaller and rounder, the outer membrane ruptured, the cristae broke and disappeared, and vacuolated (Fig. 4G).

Fig. 4.

Fig. 4

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Effect of Morin on cognitive function in POCD via miR-138-5p. Effect of Morin inhibiting miR-138-5p on spontaneous alternation rate in Y Maze (A), novel location recognition (B), and novel object recognition (C) in POCD mice. Levels of GSH (D), MDA (E), and Iron (F) in hippocampal tissue of POCD mice. (G) TEM observation of hippocampal ultrastructure. *P < 0.05, **P < 0.01, and ***P < 0.001.

Mechanism of Morin mediating ferroptosis through miR-138-5p/SIRT1

Based on the fact that Morin regulates miR-138-5p to affect iron ions and mitochondrial ultrastructure, we then studied Morin’s regulation of p53, SLC7A11, and GPX4 through miR-138-5p/SIRT1. At the mRNA level, compared with the sham group, the expression of SIRT1, SLC7A11, and GPX4 in the OP group was reduced, and the expression of p53 was increased. Compared with the OP group, the expression of SIRT1, SLC7A11, and GPX4 increased after Morin treatment, and the expression of p53 decreased. In POCD mice, miR-138-5p agomir was added based on Morin treatment. After miR-138-5p agomir intervention, the expression of SIRT1, SLC7A11, and GPX4 decreased, and the expression of p53 increased (Fig. 5A). Protein detection showed consistency with mRNA levels. Compared with the sham group, SIRT1, SLC7A11, and GPX4 decreased in the OP group, and p53 expression increased. However, after Morin treatment, SIRT1, SLC7A11, and GPX4 protein expression increased, and p53 expression decreased. In addition, the effect of Morin on these proteins was weakened after miR-138-5p agomir intervention (Fig. 5B and C).

Fig. 5.

Fig. 5

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Morin regulates p53, SLC7A11, and GPX4 through miR-138-5p/SIRT1. (A) SIRT1, p53, SLC7A11, and GPX4 mRNA levels. (B and C) Changes in SIRT1, p53, SLC7A11, and GPX4 protein levels and relative expression. *P ˂ 0.05, **P < 0.01, and ***P < 0.001.

In vitro cell experiments verified the effect of Morin on ferroptosis through miR-138-5p/SIRT1

In vitro experiments continued to study the mechanism of Morin on ferroptosis through miR-138-5p/SIRT1. As shown in Fig. 6A, the expression of miR-138-5p in the ferroptosis cell model induced by Erastin increased, and the expression of miR-138-5p decreased after Morin incubation. Compared with the Erastin and Morin co-incubation group, the expression of miR-138-5p increased after the action of miR-138-5p mimic. In addition, compared with the control group, after Erastin established the ferroptosis model, the GSH of HT22 cells decreased, and the MDA and Fe2+ contents increased. After the continued addition of Morin, the GSH increased, and the MDA and Fe2+ contents decreased. Compared with the Erastin + Morin group, the GSH decreased, and the MDA and Fe2+ contents increased after the miR-138-5p mimic. On the contrary, the GSH increased, and the MDA and Fe2+ level decreased after the miR-138-5p inhibitor (Fig. 6B, C, and D). Regarding ROS, Erastin-induced HT22 hippocampal cells had the strongest ROS fluorescence, and Morin weakened the intensity of ROS fluorescence. Based on Erastin and Morin, miR-138-5p mimic can induce ROS, while miR-138-5p inhibitor can inhibit the intensity of ROS (Fig. 6E and F). Furthermore, the mRNA levels of SIRT1 and ferroptosis-related genes p53, SLC7A11, and GPX4 were analyzed. Compared with the control group, the expression of SIRT1, SLC7A11, and GPX4 decreased and the expression of p53 increased after Erastin induction. Compared with Erastin, the expression of SIRT1, SLC7A11, and GPX4 increased and the expression of p53 decreased after Morin. Compared with the Erastin + Morin group, the expression of SIRT1, SLC7A11, and GPX4 decreased and the expression of p53 increased after miR-138-5p mimic. The miR-138-5p inhibitor had the opposite effect to the miR-138-5p mimic (Fig. 7A). At the protein level, Erastin and miR-138-5p mimic can regulate the expression of SIRT1, SLC7A11, and GPX4 to decrease, and the expression of p53 to increase. Morin and miR-138-5p inhibitor affect the expression of SIRT1, SLC7A11, and GPX4 to increase, and the expression of p53 to decrease (Fig. 7B and C).

Fig. 6.

Fig. 6

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Effects of Morin on oxidative stress in hippocampal cells via miR-138-5p. (A) Expression of miR-138-5p in HT22 hippocampal cells. (B) GSH content in HT22 cells. (C) MDA content in HT22 cells. (D) Fe2+ level. (E) Representative flow cytometry images of ROS. (F) ROS statistical results. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 7.

Fig. 7

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Effects of Morin on ferroptosis of hippocampal cells via miR-138-5p/SIRT1. (A) SIRT1, p53, SLC7A11, and GPX4 mRNA levels in HT22 cells. (B and C) Changes of SIRT1, p53, SLC7A11, and GPX4 protein levels and relative expression in HT22 cells. *P < 0.05, **P < 0.01, and ***P < 0.001.

Discussion

Postoperative cognitive dysfunction (POCD) often occurs after surgery, especially in the elderly. It is characterized by a significant decline in mental performance, including memory, attention, coordination, verbal fluency, and executive function1. The decrease in cognitive ability leads to prolonged hospital stays and increased mortality1. In particular, the prevalence of POCD can reach 40% within 1 week after cardiovascular surgery and remains as high as 17% 3 months after surgery2. In addition, POCD increases the long-term risk of Alzheimer’s disease (AD)30. Coronary artery disease is a highly fatal disease worldwide, and its morbidity and mortality have recently increased. Coronary artery bypass grafting is widely considered to be the most effective treatment for coronary artery disease31. And, it is associated with an increased risk of POCD, especially after coronary artery bypass grafting and cardiopulmonary bypass (CPB)32. In contemporary research, the construction of POCD models has reached a mature stage, and cognitive function assessment involves tests such as the Y maze test, novel location recognition, and novel object recognition experiment33. In this study, we chose to ligate the left anterior descending branch of the mouse coronary artery to establish a cardiac surgery animal model. After surgery, the spontaneous alternation rate in Y Maze, novel location recognition, and novel object recognition of the mice were reduced, indicating that the POCD model was successfully established.

The changes in the hippocampus in cognitive impairment and its potential therapeutic mechanisms have always been a hot topic of research. The hippocampus is an important structure in the brain that is responsible for regulating cognitive functions such as memory and learning34. POCD is usually associated with neuronal damage and insufficient neuronal regeneration35. As an important memory center, damage to the hippocampus may have a profound impact on cognitive function36. Studies have shown that the hippocampus plays an important role in forming spatial memory, but its structure and function are susceptible to negative effects from external surgical/anesthesia stimulation37. Non-coding RNA in hippocampal tissue is also a hot topic in the current field of neuroscience research. Non-coding RNA plays an important role in regulating gene expression and nervous system function38. The hippocampus is a structure in the brain that is closely related to learning and memory. The expression and function of its non-coding RNA may play a key role in the maintenance and abnormality of cognitive function39. miRNA is a type of non-coding RNA that is believed to play an important regulatory role in a variety of biological processes, including cell proliferation, differentiation, and apoptosis40. In this study, transcriptome sequencing analysis confirmed that miR-138-5p was highly expressed in POCD. Further, GO and KEGG annotation analysis showed that the target genes of miR-138-5p were involved in neurodegenerative diseases and key pathways of the nervous system. Dual luciferase reporter gene experiments verified the direct interaction between miR-138-5p and the 3’UTR region of SIRT1, and in the POCD group, the expression of miR-138-5p increased while the mRNA level of SIRT1 decreased. These findings reveal the potential role of miR-138-5p in POCD and its regulatory mechanism.

Morin, a natural active product with neuroprotective properties41, showed favorable inhibition of miR-138-5p. miRNA genes are transcribed by RNA polymerase II or III to produce primary transcripts (pri-miRNA) of approximately several thousand base pairs in length42. Subsequently, under the action of the protein complex Drosha-DGCR8, these are further processed into precursor miRNAs (pre-miRNA) with stem-loop structures43. Pre-miRNA is transported from the nucleus to the cytoplasm with the assistance of the Ran-GTP-Exportin-5 transporter. In the cytoplasm, pre-miRNA is recognized by Dicer-TRBP, and through cleavage and modification of the stem-loop structure, miRNA dimers are formed44. One strand rapidly degrades, while the other strand is transferred into the AGO2 protein, forming a RISC (RNA-induced silencing complex), ultimately generating mature, functional single-stranded miRNA. Morin’s inhibition of miR-138-5p may be involved in the maturation process of the above miRNAs. Morin significantly improved cognitive and spatial memory abilities in healthy adult mice by increasing BDNF and synapse-associated protein expression, as well as promoting hippocampal dendrite growth45. Long-term feeding of Morin significantly improved the spatial learning and memory abilities of APPswe/PS1dE9 mice by promoting Aβ degradation and inhibiting the CDK5 signaling pathway to reduce tau protein phosphorylation29. We found that Morin improved cognitive functions in POCD mice by mediating the miR-138-5p/SIRT1 Axis, including an increase in spontaneous alternation rate and a prolonged time for novel location and object recognition, as well as repairing ultrastructural damage to neurons such as cell volume, nuclear morphology, and restoration of mitochondria and endoplasmic reticulum.

MiR-138 originates from two primary transcripts, pri-miR-138-1 and pri-miR-138-2. These two primary transcripts are located on chromosome 3 (Ch3p21.32) and chromosome 16 (Ch16q13), respectively, and after a series of reactions, mature miR-138 is formed46. In addition to regulating tumor malignant proliferation and cardiovascular diseases, miR-138 has also been found to be involved in the occurrence and development of neurological diseases such as Parkinson’s disease and depression. MiR-138 enriched in the brain can regulate the morphogenesis of dendritic spines in rat hippocampal neurons47. miR-138 is differentially expressed in a variety of nervous system diseases and is involved in regulating the growth activities of neurons, glial cells, and neural stem cells, as well as the proliferation, invasion, migration, apoptosis, angiogenesis, and other biological behaviors of nervous system tumor cells24,48. The miR-138-5p agomir weakened the improvement of Morin on cognitive function in POCD mice, including the reduction of spontaneous alternation rate and novel location and object recognition time, and reversed the protective effect of Morin on oxidative stress indicators (GSH, MDA, Iron) and neuronal ultrastructure, as well as inhibited the regulatory of Morin on p53, SLC7A11, and GPX4 through the miR-138-5p/SIRT1 axis.

In a mouse model of depression, miR-138 negatively regulates hippocampal SIRT1 (silent information regulator of transcription 1, SIRT1) gene expression, thereby promoting depressive symptoms in mice49. SIRT1 is involved in the regulation of a variety of cellular and physiological processes, including neurodevelopment, cerebral ischemia, neuropathic pain, mood disorders, aging, etc.50. SIRT1 has also been shown to be highly expressed in neurons of the hippocampus, which is a learning and memory key structure, essential for normal cognitive function51. Recent studies have shown that reduced SIRT1 function is associated with cognitive impairment and dementia52. It has also been suggested that SIRT1 affects spatial memory and synaptic plasticity in the mouse hippocampus53. Morin inhibited the onset of ferroptosis by inhibiting miR-138-5p and upregulating SIRT1 expression. In contrast, miR-138-5p mimic promoted the onset of ferroptosis by down-regulating the expression of SIRT1. miR-138-5p inhibitor suppressed the onset of ferroptosis by up-regulating the expression of SIRT1. These results suggest that miR-138-5p/SIRT1 plays a key role in Morin-mediated regulation of ferroptosis.

Recent studies have shown that activation of SIRT1 alleviates ferroptosis in early brain injury after subarachnoid hemorrhage52–54. SIRT1 directly regulates ferroptosis mainly through related signaling pathways such as p53 and Nrf2 and has been widely used in cardiovascular, respiratory, and neurological diseases55,56. In lung tissue, overexpression of SIRT1 inhibited lipid peroxidation in lung epithelial cells, reduced MDA levels, and reversed acute lung injury in mice, involving reversed downregulation of SLC7A11 and GPX4, regulation acetylated p53, and inhibition of ferroptosis57. In addition, p53 can regulate ferroptosis by directly inhibiting GPX4 or inhibiting SLC7A11 and enhancing LOX function through ROS, while SIRT1 can inhibit ferroptosis through p53 deacetylation58,59. We found that in the mouse hippocampal regulatory network, Morin can inhibit the expression of miR-138-5p, promote the activity of SIRT1, and then inhibit p53 and upregulate the expression levels of SLC7A11 and GPX4. Through the Erastin-induced ferroptosis hippocampal cell model, Morin and miR-138-5p overexpression or inhibition determined that these genes and proteins play a key role in the ferroptosis process, forming a complex regulatory network.

Ferroptosis is a regulated cell death characterized by lethal lipid peroxidation and iron overload60. Among them, GSH is an important antioxidant that serves as a substrate for GPX4 in cells to reduce lipid peroxides and prevent the further development of lipid peroxidation61. MDA is a product of lipid peroxidation and an important indicator of lipid peroxidation levels62. In ferroptosis, the increase in ROS is closely related to the aggravation of lipid peroxidation, and high levels of ROS are an important factor in inducing ferroptosis63. Previous studies have shown that Morin can improve dopaminergic, glutamatergic, serotonergic, and cholinergic neurotransmission, reduce the increase in the levels of MDA and nitrite and increase the concentration of reduced GSH in the brain, reduce the activity of superoxide dismutase and catalase, and prevent and reverse cognitive impairment in mice19,64. This study found that Morin affects ferroptosis by regulating the miR-138-5p/SIRT1 axis, which is manifested by increased GSH levels and corresponding decreases in MDA and iron ion concentrations and ROS levels.

Conclusion

In conclusion, the present findings identify that Morin inhibits the expression of miR-138-5p, thereby upregulating the expression of SIRT1, downregulating the expression of p53, and upregulating the expression of SLC7A11. This, in turn, upregulates the expression of GSH, thereby upregulating the expression of GPX4 and then downregulating ROS, ultimately improving POCD caused by ferroptosis. However, unlike animal experiments, the causes of POCD in humans are more complex and require more comprehensive studies.

Methods and materials

Animals and grouping

8 weeks old C57BL/6J mice without specific pathogens (SPF) were purchased from the Experimental Animal Center of Guizhou Medical University (SPF (Beijing) biotechnology co., Ltd., Beijing, China), body weight 18–22 g, and animal license number: SCXK (jing) 2019-0010. The animals were housed in an SPF-grade laboratory at the Experimental Animal Center of Guizhou Medical University and fed and watered ad libitum. All experimental protocols of the present study were approved by the Animal Ethics Committee of Guizhou Medical University (license number: SCXK (qian) 2018-0001) (IRB number: 2100557). All authors complied with the ARRIVE guidelines. All treatments and experimental procedures were performed in accordance with the National Institutes of Health guidelines and were approved by the Animal Ethics Committee of Guizhou Medical University (license number: SCXK (qian) 2018-0001) (IRB number: 2100557).

The mice were randomly divided into the sham group, OP group, OP + morin group, OP + morin + agomir NC group, and OP + morin + miR-138-5p agomir group, with 6 mice in each group. OP (POCD) group: The cardiac surgery animal model in the OP group was established by continuous ligation of the left anterior descending coronary artery (LAD) for 45 min and then removing the ligature for blood reperfusion. The mice were maintained under general anesthesia by continuous inhalation of a mixture of 1–2% sevoflurane and 0.5 L/min oxygen. A skin incision and purse-string suture were made on the left anterior chest of C57 mice. The pectoralis major and pectoralis minor muscles were separated with hemostatic forceps and the 3rd to 4th ribs were spread to expose the heart. The left anterior descending coronary artery was quickly ligated, and then the heart was repositioned, sutured, and a purse-string suture was made. After the operation, the mice were placed on a warming blanket, and waited for them to wake up. After 45 min, the slipknot was loosened for myocardial reperfusion to complete the model-making process. Sham group: 8.0 suture was only passed through the back of LAD, no slipknot or ligation was done, and the rest of the operation was the same as that of the OP group. OP + morin group: before the surgical modeling of mice, 200 mg/kg/day morin was intragastrically administered once a day for 14 consecutive days. OP + morin + agomir NC group: morin 200 mg/kg/day was given by gavage for 14 days before surgery, agomir NC (miR4N0000001-4-5, RiboBio) was injected intrathecally at 5 µl (60 µM) once a day for 3 consecutive days starting the day after surgical modeling. OP + morin + miR-138-5p agomir group: morin 200 mg/kg/day was given by gavage for 14 days before surgery, miR-138-5p agomir (miR4N0000150, RiboBio) was injected intrathecally at 5 µl (60 µM) once a day for 3 consecutive days starting the day after surgical modeling. After surgery and drug administration, behavioral experiments were performed. At the end of the experiment, all mice were euthanized by cervical dislocation, and samples were collected for molecular biological testing.

Y maze

The Y maze apparatus is a spatial recognition memory test consisting of three identical arms (45 × 13 cm, with 45 cm high walls) that were intersected at 120° around a central triangle. On the third day after surgery, the mice were placed in the Y maze experiment room for 1 h of adaptation. On the fourth day after surgery, the mice were placed at the end of arm A and allowed to freely explore arms A, B, and C for 5 min. The order and total number of arms entered were recorded. The criterion for mice to enter each arm was that all four limbs entered. Successful alternation was defined as entering consecutive arms (such as ABC, ACB, BCA, BAC, CAB, and CBA) in sequence, and the number of alternations was counted. The alternation rate was calculated using the following formula: alternation rate = [number of successful alternations/(total number of entries into the arm-2)] × 100% 65. After each trial, the maze was treated with 75% alcohol before the next trial.

Novel location recognition (NLR) and novel object recognition (NOR) assay

The experiment utilized the Panlab SMART 3.0 system for image acquisition and video analysis. A square open-field box (40 × 40 × 40 cm) was selected as the testing arena. On postoperative day 4, mice were acclimated to the testing room for 1 h and then placed into the empty box to habituate for 10 min. The box and objects were cleaned with 75% ethanol after each session to eliminate any residual odor. On the day of testing, mice (n = 6 per group) were placed in an arena containing two identical objects (A and B) placed in symmetrically opposite corners for a 10-minute acquisition trial. The time spent exploring each object (A and B) was recorded. Afterward, the box and objects were cleaned with 75% ethanol.

For NLR task, 1 h after training, object A was moved to a new location (A’). Mice were reintroduced to the box and allowed to explore for 10 min. The time spent exploring the object at the new location (A’) and the object at the old location (B) was recorded. The box and objects were cleaned with 75% ethanol after testing.

For NOR task, 1 h after the NLR, object B was replaced with a novel object (C). Mice were tested for 10 min, and the time spent exploring the novel object (C) and the familiar object at the location (A’) was recorded66.

The percentage of exploration time is used to quantitatively represent the animal’s interest in a novel location or object during the test. It is calculated as follows:

Inline graphic​

T (Novel): Time spent exploring the novel location or novel object.

T (Total): Total exploration time, calculated as: T (Total) = T (Novel) + T (Familiar).

T (Familiar): Time spent exploring the familiar location or familiar object.

Transcriptome sequencing of miRNAs

The Sham and OP groups each had 3 mice. All mice in the OP group were confirmed to have POCD by cognitive assessment tests before sequencing. The extraction, quality check, and library construction of total RNA from mouse hippocampal tissue were done by Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China). Total RNA concentration and purity were measured using an Agilent 5300 Bioanalyzer. A small RNA library was constructed using QIAseq miRNA Library Kit (96) (331505, Qiagen). Next, the library was enriched by PCR amplification with the addition of sequenced connectors and Index sections. Single-stranded libraries were used as templates for bridge PCR amplification, sequencing primer annealing, and sequencing while synthesizing. This project focused on the characterization and expressions of miRNAs. After completing the statistical analysis, clustering analysis and target gene prediction (miRTarBase) were performed on the differential miRNAs, and the predicted target genes were further enriched for GO and KEGG analysis. As for mRNAs, After the samples were RNA extracted, purified, and library built, these libraries were sequenced using Next-Generation Sequencing (NGS) technology, based on the NovaSeq 5000/6000 S2 Reagent Kit (20012860, illumina), for double-end (Paired-end, PE) sequencing.

Ultrastructure of hippocampal nerve cells under transmission electron microscope (TEM)

The hippocampal tissue was placed in 3% glutaraldehyde fixative, dehydrated with gradient ethanol, embedded (epoxy resin), and ultrathin sections were prepared. The ultrastructure changes of hippocampal nerve cells were observed (under TEM, JEOL, JEM-1400FLASH).

Plasmid construction and dual-luciferase assay

WT SIRT1 3’-UTR (wild type) containing miR-138-5p binding site plasmid and Mut SIRT1 3’-UTR (mutant) without miR-138-5p binding site were constructed into pSI-Check2 vector (Hanbio Biotechnology, Shanghai, China) to obtain a fluorescent reporter gene plasmid. LipoFiterTM Liposomal Transfection Reagent (HB-TRLF-1000, Hanbio Biotechnology, Shanghai, China) was used to transfect NC-mimic and miR-138-5p for 48 h. The Dual-Luciferase Reporter Assay Kit (Hanbio Biotechnology, Shanghai, China) was used to detect the fluorescence intensity of each group to determine the targeting of miR-138-5p and SIRT1. All plasmids were constructed in Hanbio Biotechnology Co., Ltd. (Shanghai, China).

HT22 cell culture, drug administration and transfection assay

Neuronal HT22 cells were provided by Procell Life Science&Technology Co.,Ltd. (CL-0697, Wuhan, China) and cultured in DMEM (100 U/ml penicillin-streptomycin) supplemented with 10 g/dl fetal bovine serum (C04001, Viva Cell, Shanghai, China) in a 37 °C incubator. The culture medium was replaced every 48 h and the cells were passaged when the cell growth density reached more than 70%. The logarithmic growth phase cells were inoculated with the appropriate number of cells in a 6-well plate (Nest, China), and when their density grew to about 70%, miR-138-5p mimic (50 nM, RiboBio), mimic NC (50 nM, RiboBio), miR-138-5p inhibitor (50 nM, RiboBio), inhibitor NC (50 nM, RiboBio) were transient transfection according to the instructions of ribo FECT™CP Transfection (RiboBio, Guangzhou, China). Cells were first transfected with miR-138-5p mimic, mimic NC, miR-138-5p inhibitor, and inhibitor NC for 24 h to 48 h, respectively, and then 200 µM Morin (M813301, Macklin) was added. 1 h later, 320 nM Erastin (B1524, APExBIO) was added, and relevant tests were performed 24 h later.

RT-qPCR assay

Fresh hippocampal tissues/hippocampal neural cell lines (HT22) of mice in each group were added into centrifuge tubes. The total RNA of tissues/cells was extracted using the TRIzol method (R1100, Solarbio®). Target cDNA was obtained by reverse transcription kit (RR047A, TaKaRa). Then the reaction system was configured according to the fluorescence quantitative PCR kit (RR820A, TaKaRa): 1 µL cDNA, forward and reverse primers 0.5 µL each, TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) 10.0 µL, RNase-free H2O 8.0 µL were added into reaction centrifuge tube. Next, the cDNA and primers were added, and amplify the target gene by real-time fluorescence quantitative PCR. The amplification conditions were: 50 °C pre-denaturation for 15–30 min, 95 °C denaturation for 2 min, 95 °C annealing for 10 s, 60 °C extensions for 30 s, and 40 cycles. After the reaction, the Ct value was obtained, and the relative expression of these mRNAs was calculated using the 2−ΔΔCt method with normal tissue as the control group and GAPDH as the internal reference. The relative expression of miR-138-5p was measured using U6 as a reference. The primer sequences are shown in Table 1.

Table 1.

Primers used in this study.

Gene Primer (5′ to 3′)
P53_F 5′-TGAACCGCCGACCTATCCTTAC-3′
P53_R 5′-GCACAAACACGAACCTCAAAGC-3′
sirt1_F 5′-GCCAGAGTCCAAGTTTAGAAGAACC-3′
sirt1_R 5′-TCCAGATCCTCCAGCACATTCG-3′
GPX4_F 5′-CCCGATATGCTGAGTGTGGTTTAC-3′
GPX4_R 5′-TTTCTTGATTACTTCCTGGCTCCTG-3′
SLC7A11_F 5′-GTTCGCTGTCTCCAGGTTATTCTAC-3′
SLC7A11_R 5′-AGAGCATCACCATCGTCAGAGG-3′
GAPDH_F 5′-GGTTGTCTCCTGCGACTTCA-3′
GAPDH_R 5′-TGGTCCAGGGTTTCTTACTCC-3′
mmu-miR-138-5p Stem-loop RT Primer

5′-GTCGTATCCAGTGCAGGGTCC

GAGGTATTCGCACTGGATACGACCGGCCT-3′
M-miR-138-5p-F 5′-GGGAGCTGGTGTTGTGAATC-3′
M-miR-138-5p-R 5′-AGTGCAGGGTCCGAGGTATT-3′
U6-F 5′-GGAACGATACAGAGAAGATTAGC-3′
U6-R 5′-TGGAACGCTTCACGAATTTGCG-3′
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Western blot assay

Mouse hippocampal tissue/mouse hippocampal neuronal cell line (HT22) was lysed by RIPA in an ice bath. The lysate was centrifuged, the supernatant was collected, and the total protein concentration was determined by BCA. The protein samples were electrophoresed on 10% to 14% sodium dodecyl sulfate-polyacrylamide gel and transferred to a polyvinylidene fluoride membrane (PVDF). Subsequently, the PVDF membrane was incubated in Tris-buffered saline Tween-20 (TBST) containing 5% skim milk at 25 °C. The membrane was rinsed with TBST and then incubated overnight with primary antibodies: Anti-SIRT1 (1: 1000, Abcam, ab189494), Anti-p53 (acetyl K370) (1: 1000, Abcam, ab183544), Anti-SLC77A11 (1:1000, zenbio, R26116), Anti-GPX4 (1:5000, Abcam, ab125066), and using GAPDH (1:10000, proteintech, 10494-1-AP) as a loading control protein. Afterward, the membrane was washed with TBST and incubated with the secondary antibody Goat Anti-Rabbit IgG H&L (HRP) (1:20000, Abcam, ab205718) for 1 h. After the PVDF membrane was washed, the expression level of the protein was detected by an enhanced chemiluminescence detection kit (MA0186-3, MeilunBio).

Flow cytometry for ROS

HT22 cells were inoculated in 6-well plates, and 1 × 106 cells were taken in each group and washed twice with PBS. The cells were loaded into the probe to make 500 µL suspension cells containing 5 mol/L DCFH-DA (S0033S, Beyotime), and the reaction was carried out at 37 °C for 30 min protected from light, and washed twice with PBS. The FITC channel was chosen to measure the variation of ROS in each group, and the change in ROS levels was quantified based on the optical density values of the cells detected by the FITC channel (CytoFLEX, Beckman Coulter Life Sciences).

GSH, MDA, and iron ion detection

The hippocampal tissue/hippocampal cells were homogenized and then centrifuged to obtain the supernatant for the following experiments. The hippocampal tissue iron ion detection (A039-2-1, Nanjing Jiancheng Bioengineering Institute, China) was carried out using a detection kit under 520 nm wavelength. The Fe2+ level in HT22 cells was determined using an Fe2+ detection kit (Solarbio®, BC5415) according to the manufacturer’s instructions under 593 nm wavelength. The reagents used in the iron detection process did not contain iron chelators. The content of MDA was determined by the thiobarbituric acid (TBA) method (A003-1-2, Nanjing Jiancheng Bioengineering Institute, China), with a maximum absorption peak at 532 nm. The 5,5′-Dithiobis (2-nitrobenzoic acid) (DTNB) reaction (A006-2-1, Nanjing Jiancheng Bioengineering Institute, China) was used to determine GSH, and the reduced GSH content was determined by colorimetric quantitative determination at 405 nm.

Statistics and analysis

SPSS 24.0 software was used for data statistics and analysis. The data were expressed as mean ± standard deviation (Inline graphic ± s), one-way ANOVA followed by LSD post hoc test was used for comparison among multiple groups, and t-test was used for comparison between two groups. P < 0.05 was considered statistically significant.    

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (66MB, pptx)

Acknowledgements

The authors would like to thank the assistance from Dr. Xiufang Wan and Dr. Wen Yang. The authors are grateful for the technical support provided by Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China).

Author contributions

The first draft of the manuscript was written by Yang Yang and Wen Hu, and all authors commented on previous versions of the manuscript. Biochemical experiments were completed by Yang Yang and Zhen Wu. Bioinformatics analysis was performed by Wen Hu. Clear research guidance and technical support were provided by Zhen Wu. Data collection and statistical analysis were completed by Mi Chen and Hui Li. Animal behavioral tests were performed by Yang Yang, Jiatian Cui, Chaoying Wang, and Yushan Luo. Financial support was provided by Xiaohua Zou and Yang Yang. The study was conceived, designed, and supervised by Xiaohua Zou and Bailong Hu. All authors have read and approved the manuscript.

Funding

This study was supported by the National Natural Science Foundation of China (Grant number 82160224), Science and Technology Fund of Guizhou Provincial Health Commission (Grant number gzwkj2024-418), Teaching reform research project of Guizhou Medical University (Grant number JG2021041), Guizhou Medical University Key Laboratory of Anesthesia and Pain Mechanism Research (Grant number [2024]fy003).

Data availability

Sequence data that support the findings of this study have been deposited in the Sequence Read Archive with the primary accession code PRJNA1227727: https://dataview.ncbi.nlm.nih.gov/object/PRJNA1227727?reviewer=2g9vogdp796tnntbtateu79v6q.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

All authors complied with the ARRIVE guidelines. All treatments and experimental procedures were performed in accordance with the National Institutes of Health guidelines. All experimental protocols of the present study were reviewed and approved by the Animal Ethics Committee of Guizhou Medical University (license number: SCXK (qian) 2018-0001) (IRB number: 2100557).

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Yang Yang and Wen Hu contributed equally to this work.

Contributor Information

Bailong Hu, Email: 375896605@qq.com.

Xiaohua Zou, Email: zouxiaohuazxh@gmc.edu.cn.

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Associated Data

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Supplementary Materials

Supplementary Material 1 (66MB, pptx)

Data Availability Statement

Sequence data that support the findings of this study have been deposited in the Sequence Read Archive with the primary accession code PRJNA1227727: https://dataview.ncbi.nlm.nih.gov/object/PRJNA1227727?reviewer=2g9vogdp796tnntbtateu79v6q.

Articles from Scientific Reports are provided here courtesy of Nature Publishing Group

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