The Role Of HMOX1 In Propofol-induced Ferroptosis Of Developmental Hippocampal Neurons And Long-term Cognitive Impairment

Research Background:Propofol is one of the most commonly used intravenous anesthetics in clinical practice today.Its potential effects on the developing brain are not only a matter of concern in the field of anesthesiology but also a hot topic of social attention.The perinatal and infancy periods are crucial for neural development,during which neurons are more vulnerable to external disruptions leading to damage.Animal studies have shown that early postnatal exposure to propofol increases neuronal apoptosis and results in long-term cognitive impairment.While human epidemiological studies have not reached a consensus,it is widely believed that increased anesthesia time and frequency elevate the risk of long-term learning,memory deficits,and behavioral disorders.Heme oxygenase-1（HMOX1/HO-1）is the rate-limiting enzyme in heme degradation.Although HMOX1 is considered a protective cellular factor in various models,excessive activation of HMOX1 can augment heme breakdown,releasing free iron,thus leading to an imbalance in iron ion homeostasis.Ferroptosis is closely related to brain development,yet research on whether it is involved in propofol-induced neurotoxicity during development is limited.This study explores the mechanism of propofol-induced developmental neurotoxicity at the transcriptional level of genes using high-throughput sequencing technology.It employs a mouse hippocampal neuron HT22 cell model and a propofol-induced developmental neurotoxicity mouse model to investigate whether propofol induces developmental neurotoxicity by regulating the expression of HMOX1 and subsequently triggering cellular.Through the investigation of the mechanism of propofol-induced developmental neurotoxicity,we aim to discover its pathogenesis and potential therapeutic targets,providing insights for the prevention of propofol-induced developmental neurotoxicity and rational clinical drug use.Objective:To investigate whether propofol causes neurotoxicity during development and long-term cognitive impairment,and to determine if its neurotoxicity mechanism is related to the upregulation of HMOX1 expression leading to cellular ferroptosis.Methods:Part I:We selected 7-day-old male C57BL/J mice from the same litter as our research subjects.Six mice were randomly divided into a Control group（Con group）and a Propofol group（Pro group）.The Con group received intraperitoneal injection of lipid emulsion for 6 hours,while the Pro group underwent anesthesia induction with 100mg/kg of propofol via intraperitoneal injection,followed by a maintenance dose of 50mg/kg/h,totaling 6 hours of anesthesia.After the drug administration,the mice were euthanized,and their brain tissues were collected for transcriptome sequencing.Differential genes were identified through bioinformatics analysis,followed by enrichment analysis of differential expression,construction of protein-protein interaction networks,and selection of key genes.Finally,the binding ability of the key target HMOX1 was verified by molecular docking.Part II:We chose mouse hippocampal neuron HT22 cells as our research subject and first investigated the effects of propofol at different concentrations and exposure times on these cells.Cell viability was measured using the CCK-8 assay,apoptosis was assessed by flow cytometry,and the expression of HMOX1 and ferroptosis-related genes GPX4,ACSL4,SLC7A11 was evaluated using q RT-PCR and Western blot.Next,to explore whether the increased expression of HMOX1 led to ferroptosis and subsequent cellular damage,we used si RNA transfection technology to silence HMOX1.The experiment was divided into four groups:Con group,Pro group,Pro+si-NC group,and Pro+si-HMOX1 group.After si RNA transfection,the cells were treated with 100μM propofol for 24 hours,while the control group received0.1%DMSO in the culture medium as a control.Cell viability was assessed using the CCK-8 assay,apoptosis was measured by flow cytometry,and the expression of HMOX1 and ferroptosis-related genes GPX4,ACSL4,SLC7A11 was analyzed using q RT-PCR and Western blot.Additionally,the levels of malondialdehyde（MDA）,iron(Fe²⁺),glutathione（GSH）,and reactive oxygen species（ROS）were determined using assay kits to assess their intracellular levels.Part III:We selected 7-day-old C57BL/J mice as the research subjects.Initially,we investigated the effects of different durations of propofol exposure on HMOX1expression and ferroptosis in mouse hippocampal tissue.A total of 72 seven-day-old C57BL/J mice were selected and were subjected to propofol anesthesia for 0,2,4,and6 hours.We assessed the expression of HMOX1 and ferroptosis-related genes GPX4,ACSL4,SLC7A11 using q RT-PCR,Western blot,and immunofluorescence staining.Hippocampal tissue levels of MDA,Fe²⁺,GSH,and ROS were measured using assay kits,and changes in hippocampal tissue structure were examined through HE staining and Nissl staining.To further investigate the role of HMOX1 in propofol-induced developmental neurotoxicity and ferroptosis,we used HMOX1-specific inhibitor Znpp and inducer Hemin.A total of 112 mice were randomly divided into four groups:Con group,Pro group,Pro+Znpp group,and Pro+Hemin group.Among them,72mice were immediately assessed for the expression of HMOX1 and ferroptosis-related genes GPX4,ACSL4,SLC7A11 after drug administration,and hippocampal tissue MDA,Fe²⁺,GSH,ROS levels,as well as hippocampal tissue structural changes were measured.The remaining 40 mice,with 10 in each group,were continued to be raised for 30 days to undergo behavioral tests,aimed at validating the long-term cognitive effects of propofol on mice.Results:Part I:Transcriptome sequencing was performed on mouse hippocampal tissue,and differential genes were selected using a criterion of P-Value<0.05,resulting in 1,962differentially expressed genes.GO enrichment analysis and KEGG pathway analysis were conducted on these differential genes,revealing that among the top 10significantly enriched pathways,ferroptosis is closely related to neural development.Protein-protein interaction network analysis of differential genes and the selection of key genes identified Hmox1as a crucial gene playing a role in the ferroptosis pathway.The application of molecular docking again demonstrated that propofol had a strong binding affinity with HMOX1.Part II:Propofol induced damage to mouse hippocampal neuron HT22 cells,with a decrease in cell viability as concentration and exposure time increased.Apoptosis increased,HMOX1 expression increased,and expression of ferroptosis-related protein ACSL4 increased,while GPX4 and SLC7A11 expression decreased.Intracellular MDA levels increased,Fe²⁺levels rose,GSH levels decreased,and ROS accumulated,leading to cellular ferroptosis.Utilizing si RNA to silence the Hmox1 gene effectively inhibited the occurrence of ferroptosis.Compared to the Pro group,cell viability increased,apoptosis decreased,ferroptosis-related protein ACSL4 was downregulated,and GPX4 and SLC7A11 were upregulated.MDA levels decreased,Fe²⁺levels decreased,GSH levels increased,and ROS levels decreased.Part III:As the exposure time to propofol increased,q RT-PCR,Western blot,and immunofluorescence staining confirmed an increase in HMOX1 expression.The expression of ferroptosis-related gene ACSL4 increased,while GPX4 and SLC7A11expression decreased.MDA levels increased,Fe²⁺levels rose,GSH levels decreased,and ROS accumulated in brain tissue.HE staining and Nissl staining confirmed significant structural changes in the hippocampal tissue after 6 hours of propofol anesthesia,with the neurotoxic effects of propofol during developmental stages showing dose-dependent increases.Following the application of the HMOX1inhibitor Znpp and the inducer Hemin,the Pro+Znpp group showed a decrease in HMOX1 expression compared to the Pro group in q RT-PCR,Western blot,and immunofluorescence staining results.The expression of ferroptosis-related genes GPX4 and SLC7A11 increased,while ACSL4 expression decreased.Brain tissue MDA levels decreased,Fe²⁺levels decreased,GSH levels increased,and ROS levels decreased.HE staining and Nissl staining indicated reduced hippocampal tissue damage.In the Pro+Hemin group,HMOX1 expression increased,ferroptosis-related gene ACSL4 expression increased,GPX4 and SLC7A11 expression decreased.Brain tissue MDA levels increased,Fe²⁺levels rose,GSH levels decreased,and ROS accumulated.HE staining and Nissl staining exacerbated hippocampal tissue damage.In behavioral experiments,the open field test showed no significant differences among the four groups.However,the novel object recognition test and the Y-maze test revealed that the mice in the Pro group exhibited a decline in learning and memory abilities.In contrast,the Pro+Znpp group showed some mitigation of this damage.On the other hand,the Pro+Hemin group exhibited an exacerbation of damage in the novel object recognition test but did not show significant changes in the Y-maze test.The behavioral results suggest that propofol does not induce anxiety or motor abnormalities but does impair cognitive functions such as learning and memory in young mice.The HMOX1 inhibitor Znpp appears to alleviate this damage,while the inducer Hemin exacerbates it.Conclusion:（1）.Transcriptome sequencing suggests that differential genes in the developmental neurotoxicity model induced by propofol are significantly enriched in the ferroptosis pathway,with Hmox1 being a key gene and playing a role in the ferroptosis pathway.HMOX1 is closely related to the occurrence of ferroptosis and neurotoxicity.（2）.Exposure to propofol leads to damage to the developing hippocampal neurons and long-term behavioral changes,which worsen with increasing time and dose.（3）.Both cellular and animal experiments have confirmed that propofol induces ferroptosis in developing hippocampal neurons.（4）.Propofol causes ferroptosis by upregulating HMOX1,leading to developmental neurotoxicity.