Sevoflurane preconditioning attenuates myocardial cell damage caused by hypoxia and reoxygenation via regulating the NORAD/miR-144-3p axis

Abstract

Objective

This study aimed to investigate the function and mechanism of lncRNA NORAD in Sevoflurane (Sev) protection against myocardial hypoxia-reoxygenation (H/R).

Methods

Preprocess rat cardiomyocytes H9c2 cells with Sev at concentrations of 0.5%, 1.0%, and 1.5%, and subjected them to H/R treatment. qRT-PCR was used to detect levels of NORAD and miR-144-3p. Measure concentrations of the inflammatory cytokines IL-6, TNF-α, and IL-10, as well as cardiac injury markers cTnI, CK-MB, and LDH using ELISA. Assess cell proliferation and apoptosis using CCK-8 and flow cytometry. Perform dual-luciferase reporter assay and RIP assay to validate the targeting relationship between NORAD and miR-144-3p.

Results

H/R induced inhibition of cell proliferation, increase in apoptosis, and production of IL-6, TNF-α, CK-MB, LDH, and cTnI were significantly attenuated by Sev. As hypoxic treatment time lengthened, the NORAD levels in myocardial cells showed an increase, with Sev pretreatment being able to suppress the NORAD levels elevation. The overexpression of NORAD notably weakened the cardioprotective effect of Sev. NORAD targetedly binds to miR-144-3p and negatively regulates miR-144-3p. Increased miR-144-3p levels inhibited the antagonistic effect of NORAD on the cardioprotective effects of Sev.

Conclusion

The current study confirmed that sevoflurane attenuated H/R-induced cardiomyocyte injury via the NORAD/miR-144-3p axis.

Keywords

Sevoflurane NORAD miR-144-3p hypoxia/reoxygenation myocardial ischemia-reperfusion injury

Introduction

In recent years, cardiovascular disease has become a primary reason of human mortality,¹ with incidence and mortality rates steadily rising in many developing countries. Myocardial cell injury is a common mechanism underlying various cardiovascular diseases. Ischemic preconditioning can protect the ischemic heart, but hypoxia-reoxygenation (H/R) can cause greater damage to the myocardium.² Myocardial H/R is an ideal model for studying ischemia-reperfusion injury (I/R).^3,4 The specific mechanisms of IR are still unclear, and further research into I/R can provide a theoretical basis for the prevention and treatment of cardiovascular diseases.

Many patients undergoing treatment for cardiovascular conditions often require anesthesia. Some scholars believe that anesthetic preconditioning (APC) can provide a certain level of protection to the myocardium.⁵ Sevoflurane (Sev) is a commonly used inhalational anesthetic with features such as rapid onset of anesthesia and easy adjustment of anesthesia depth.⁶ Although there are reports that Sev can cause brain injury in newborn rats, more scholars believe that Sev treatment has a cardioprotective effect in the context of myocardial I/R injury. Investigations have shown that the intraoperative use of Sev as an anesthetic can significantly reduce the incidence and mortality of myocardial infarction in patients.⁷ Current studies suggest that Sev pre-treatment may counteract I/R injury by inhibiting apoptotic pathways,⁸ reducing harmful ROS release,⁹ and through other molecular mechanisms. Sev is a widely used anesthetic agent also applicable in surgeries involving young children, further research into the specific mechanisms of myocardial protection by Sev pre-treatment can enhance the safe and widespread clinical application of Sev in anesthesia.

Long non-coding RNAs (lncRNAs) are a class of non-coding RNA molecules that are more than 200 nucleotides in length,¹⁰ participating in various physiological processes such as transcriptional regulation within cells and post-transcriptional control.^11,12 Some studies have found that certain lncRNAs exacerbate myocardial inflammation,¹³ but others have found that lncRNAs have cardioprotective effects; for example, TTN-AS1 can alleviate sepsis-induced myocardial injury by regulating miRNAs.¹⁴ More importantly, it has been reported that lncRNAs play a key role in combating cardiac I/R injury.¹⁵ LncRNA non-coding RNA activated by DNA damage (NORAD) is a newly identified lncRNA, and it has been found that NORAD was found in Human Umbilical Vein Endothelial Cells (HUVECs) and mouse myocardial infarction (MI) left ventricular tissue expression is upregulated and may be involved in hypoxia-induced angiogenesis by regulating miR-590-3p.¹⁶ Knocking out NORAD can alleviate endothelial cell damage and the development of atherosclerosis.¹⁷ Furthermore, studies have shown that NORAD lentivirus shRNA can improve heart function, reduce fibrosis, and inflammatory responses.¹⁸ However, there has not yet been a specific study on the role of NORAD in the cardioprotective effects of Sev. Given that NORAD is involved in the occurrence of myocardial inflammation and plays an important role in other cardiac physiological processes, we selected NORAD as the object of our research.

The above studies collectively underscore the significant roles of Sev and NORAD in the onset and progression of cardiovascular diseases. However, there remains a lack of research both domestically and internationally on whether Sev can mitigate myocardial cell damage induced by H/R through the modulation of NORAD. Therefore, the present study aimed to investigate the effects of Sev and NORAD on myocardial I/R injury.

Materials and methods

Cell culture and H/R model establishment

Based on previous similar studies, we chose the H9c2 rat myocardial cell line as the experimental material.¹⁹ H9c2 cells were cultured in high glucose DMEM medium with 10% fetal bovine serum and 1% penicillin/streptomycin. Once the cells reached the logarithmic growth phase, the normal culture medium of H9c2 cells was replaced with a hypoxic culture medium and transferred to a hypoxic incubator containing 95% N₂ and 5% CO₂ for treatment durations of 1 h, 6 h, and 24 h under hypoxia. After hypoxia treatment, the culture medium was replaced with a reoxygenation medium and the cells were placed in a culture chamber with 95% O₂ and 5% CO₂ for 3 h. The control group cells were continuously cultured in a chamber with 95% O₂ and 5% CO₂.²⁰

Sev pre-treatment

H9c2 cells were divided into a control group, a H/R group, and a Sev group. The treatments were as follows: Control group, cells were not subjected to any treatment and were cultured under normal conditions; H/R group, cells underwent 6 h of hypoxia followed by 3 h of reoxygenation; Sev group, H9c2 cells were treated with Sev at concentrations of 0.5%, 1.0%, and 1.5% for 20 min, followed by 6 h of hypoxia and 3 h of reoxygenation.²¹

Cell transfection

H9c2 cells were seeded in 6-well plates and transfected with pcDNA3.1, pcDNA3.1-NORAD, mimic NC, miR-144-3p mimic, miR-144-3p inhibitor and inhibitor NC, with Lipofectamine 2000, once they reached the logarithmic phase.

RNA extraction and qRT-PCR

Trizol reagent was added to H9c2 cells to extract total RNA, followed by reverse transcription to cDNA. The expression levels of miR-144-3p were detected using the miR-X miRNA qRT-PCR SYBR Kit on the Applied Biosystems 7300 real-time PCR system, with U6 as the internal reference gene analyzed using the 2^−ΔΔCt method. Expression of NORAD was analyzed by qRT-PCR on DNA Engine Optican 2 system using QuantiTect SYBR Green RT-PCR kit. The qRT-PCR was performed with an initial cycle at 95°C for 15 min, followed by 40 cycles at 95°C for 10 s each, and then at 60°C for 20 s. The primers used are shown in Table 1.

Table 1.

The sequences of primer pairs used in qRT-PCR.

NORAD, forward	5′-CCTGGAAGGTGAGCGAAGT-3′
NORAD, reverse	5′-AGAGGGTGGTGGGCATTT-3′
miR-144-3p, forward	5′-GCTG GGATATCATCATATACTG-3′
miR-144-3p, reverse	5′-CGGACTAGTACATCATCTATACTG-3′
U6, forward	5′-CTTCGGCAGCACATATAC-3′
U6, reverse	5′-GAACGCTTCACGAATTTGC-3′

Cell proliferation

Transfected H9c2 cells were seeded in 96-well plates at a density of 4 × 10⁴ cells/well and continuously cultured for 3 days. Every 24 h, CCK-8 reagent was added to each well, and incubated for 1 h, and the OD values were measured using a microplate reader.

Cell apoptosis

Cell apoptosis was detected using the Annexin V-FITC/PI Kit and flow cytometry. Cells from different groups were collected, washed with cold PBS buffer, stained with Annexin V-FITC and PI, and apoptosis rates were analyzed using flow cytometry.

Dual-luciferase reporter (DLR) assay system

ENCORI, LncRNASNP2, DIANA, LncACTdb3.0 and miRDB databases predicted miR-144-3p as a target miRNA for NORAD. To validate this prediction, NORAD fragments were cloned into the luciferase reporter gene vector pcDNA3.1 to generate wild-type NORAD-WT and mutant recombinant plasmid NORAD-MUT. H9c2 cells were co-transfected with miR-144-3p inhibitor or miR-144-3p mimic and NORAD-WT or NORAD-MUT, and after 48 h of incubation, luciferase activity was measured using a luciferase activity detection kit, with Renilla luciferase as an internal control.

Enzyme-linked immunosorbent assay (ELISA)

Biochemical markers cTnI, CK-MB, LDH, and inflammatory factors IL-6, TNF-αand IL-10 in H9c2 cells were measured using ELISA kits according to the manufacturer’s instructions.

RNA immunoprecipitation (RIP) assay

Following the manufacturer’s instructions, RIP was performed using the EZ-Magna RNA Immunoprecipitation Kit. In short, H9c2 cells were lysed in RIP lysis buffer. Subsequently, the whole-cell extract was incubated with magnetic beads conjugated with anti-AGO2 or control IgG in RIP buffer. Finally, the immunoprecipitated RNA was isolated and subjected to qRT-PCR analysis.

Nuclear and cytoplasmic RNA fraction isolations

Using the NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit according to the manufacturer’s instructions, cytoplasmic and nuclear RNA were separated and collected. Subsequently, qRT-PCR was used to detect the expression levels of NORAD in the cytoplasmic and nuclear fractions. U6 and GAPDH served as the nuclear control transcript and cytoplasmic control transcript.

Statistical analysis

Data were presented as mean ± standard deviation (SD). GraphPad Prism 9.0 and SPSS 22.0 were performed for data analysis. Student’s t-test was used to compare two independent groups, and differences between ≥3 groups were compared using the one-way ANOVA combined with Tukey’s multiple comparisons post hoc test. The normality of the data distribution was assessed using the D’Agostino-Pearson omnibus normality test. p < .05 was considered a significant difference.²²

Results

Protective effect of Sev on H/R-induced myocardial injury

As shown in Figure 1A, with the prolongation of hypoxia treatment time, cell proliferation gradually decreased (p < .05). However, as depicted in Figure 1B, when H9c2 cells were treated with Sev, cell proliferation was significantly enhanced (p < .01), while the apoptosis rate in the H/R group was also significantly higher than that in the control group, and the apoptosis rate was significantly reduced after Sev treatment (p < .001, Figure 1C). Furthermore, compared to the control group, the concentrations of cardiac cell damage markers cTnI, CK-MB, and LDH were significantly elevated in the H/R group (p < .01), but this elevation was significantly attenuated by Sev (p < .01, Figure 1D–F), indicating that Sev can resist H/R-induced myocardial injury. The study also found a significant increase in the pro-inflammatory factors IL-6 and TNF-α concentration in H/R cells, while the concentration of the anti-inflammatory factor IL-10 decreased (p < .001, Figure 1G). However, they were both significantly reversed by Sev (p < .001, Figure 1G).

Figure 1.

Sev attenuation of H/R-induced myocardial injury. (A) Impact of different hypoxia durations on cell proliferation. (B) Effect of varying Sev concentrations on cell proliferation. (C) Impact of hypoxia and Sev on cell apoptosis. Effect of hypoxia and Sev on cardiac injury marker CK-MB (D) and cTnI (E) and LDH (F) as well as inflammatory factors (G). **p < .01, ***p < .001 versus 0 h of hypoxic treatment time; *p < .05, ***p < .001 versus control group; ##p < .01, ###p < .001 versus HR group.

Effects of H/R and Sev on NORAD levels

As shown in Figure 2A, the levels of NORAD in H9c2 cells gradually increased with the prolongation of hypoxia treatment time compared with the 0 h (p < .01), based on previous studies, 6 h of hypoxia was chosen for follow-up study). Furthermore, both 1.0% and 1.5% Sev notably reversed the promotion of H/R on NORAD levels (p < .001, Figure 2B). 1.0% Sev was adopted for the follow-up study based on the previous studies.

Figure 2.

Sev reduces H/R promotion of NOARD levels in rat cardiomyocytes. (A) Influence of hypoxia duration on NORAD levels. (B) Effect of different Sev concentrations on NORAD levels. **p < .01, ***p < .001 versus 0 h of hypoxic treatment time; *p < .05, ***p < .001 versus Control group; ##p < .01, ###p < .001 versus HR group.

Overexpression of NORAD attenuated the cardioprotective effect of Sev

Intended to explore the role of NOARD in Sev exerting myocardial protection, pcDNA3.1-NORAD plasmid was transfected into H9c2 cells, which significantly increased the levels of NOARD (p < .001, Figure 3A). Meanwhile, qRT-PCR showed that Sev greatly attenuated the promotion of NORAD levels by H/R, while NORAD levels were rebounded after transfection with pcDNA3.1-NOARD (p < .001, Figure 3B). Furthermore, Sev markedly reduced the inhibition of H/R group on the cell proliferation, while high NOARD expression dampened the inhibition of Sev (p < .001, Figure 3C). H/R induced apoptosis more than the control, but Sev reversed this effect, although the reversal was weakened by higher levels of NORAD (p < .001, Figure 3D). The detection results of cardiac cell damage markers in H9c2 cells subjected to different treatments were shown in Figure 3E–G, after H/R treatment, the concentrations of cTnI, CK-MB, and LDH significantly increased, while Sev treatment alleviated the release of myocardial injury markers. However, after transfecting NORAD, the concentrations of cTnI, CK-MB, and LDH increased again (p < .01). Moreover, Sev was able to inhibit the release of pro-inflammatory factors IL-6 and TNF-α induced by H/R, and the decrease of the anti-inflammatory factor IL-10, but the high expression of NORAD could partially reverse the effects of Sev (p < .001, Figure 3H).

Figure 3.

Overexpression of NORAD weakens the cardioprotective effect of Sev. (A) NORAD levels in H9c2 cells after transfection with NORAD overexpression plasmid. Effects of overexpression of NORAD on NORAD levels (B) and cell proliferation (C) and apoptosis (D) and CK-MB (E) and cTnI (F) and LDH (G) as well as inflammatory factors (H). **p < .01 versus pcDNA3.1; ***p < .001 versus control group; ###p < .001 versus H/R group; && p < .01, &&& p < .001 versus pcDNA3.1 + Sev + H/R group.

NORAD targeted miR-144-3p

There was evidence that lncRNAs in the cytoplasm mainly act as competitive endogenous RNAs (ceRNAs), degrading target genes by adsorbing specific miRNAs. NORAD was detected to be predominantly present in the cytoplasm (Figure 4A). Using ENCORI, LncRNASNP2, DIANA, LncACTdb3.0, and miRDB databases to predict which miRNAs may target NORAD, we identified two potential miRNA targets of NORAD, namely miR-144-3p and miR-155-5p (Figure 4B). The implication of miR-144-3p in myocardial injury piqued our interest. The binding site between NORAD and miR-144-3p was shown in Figure 4C. Dual-luciferase reporter assays showed that after transfecting H9c2 cells with miR-144-3p mimic, the luciferase activity of the NORAD-WT group significantly decreased, while the NORAD-MUT group was not significantly affected (p < .001, Figure 4D). RIP experiments also verified that NORAD can target bind miR-144-3p (Figure 4E).

Figure 4.

The target relationship between NORAD and miRNA-144-3p. (A) Subcellular localization analysis of LncRNA. (B) Venn diagram analyzing common target miRNAs from ENCORI, LncRNASNP2, DIANA, LncACTdb3.0, and miRDB databases. (C) The binding site of NORAD with miR-144-3p. (D) Dual-luciferase reporter assay. (E) RIP experiment. (F) Impact of hypoxia duration on miR-144-3p levels. (G) Restoration of miR-144-3p levels by Sev. (H) Overexpression of NORAD reduced the level of miR-144-3p. **p < .01, ***p < .001 versus control group; ##p < .01, ###p < .001 versus H/R group; &&&p < .001 versus pcDNA3.1 + Sev + H/R group.

Moreover, the level of miR-144-3p in H9c2 cells treated with hypoxia was notably lower than that in the control group, and it decreased further with prolonged hypoxia treatment (p < .01, Figure 4F). However, the expression level of miR-144-3p in H/R cells treated with Sev showed an increase (p < .01, Figure 4G), while overexpression of NORAD inhibited miR-144-3p expression (p < .001, Figure 4H).

Transfection of miR-144-3p mimic hampered the weakening effect of NORAD on Sev cardioprotection

As shown in Figure 5A, after transfection of H9c2 cells with miR-144-3p mimic, the level of miR-144-3p in H9c2cells significantly increased (p < .001), indicating successful transfection of miR-144-3p mimic into the cells. Figure 5B showed that the level of miR-144-3p in the H/R + Sev + pcDNA3.1-NORAD + miR-144-3p mimic group was significantly higher than that in the H/R + Sev + pcDNA3.1-NORAD + mimic NC group (p < .01). Cell proliferation experiments also showed that the cell proliferation in the H/R + Sev + pcDNA3.1-NORAD + miR-144-3p mimic group was significantly higher than that in the H/R + Sev + pcDNA3.1-NORAD + mimic NC group (p < .01, Figure 5C). Additionally, cell apoptosis detection revealed that the apoptosis rate in the H/R + Sev + pcDNA3.1-NORAD + miR-144-3p mimic group was significantly lower than that in the H/R + Sev + pcDNA3.1-NORAD + mimic NC group (p < .001, Figure 5D).

Figure 5.

Inhibition of NORAD’s weakening effect on Sev cardioprotection by miR-144-3p mimic transfection. (A) Expression levels of miR-144-3p post-transfection of miR-144-3p mimic. . Effect of modulating NORAD and miR-144-3p on on miR-144-3p levels (B) and cell proliferation (C) and apoptosis (D) and CK-MB (E) and cTnI (F) and LDH (G) as well as inflammatory factors (H). ***p < .001 versus mimic NC group; ###p < .001 versus H/R group; &&p < .01, &&&p < .001 versus pcDNA3.1 + Sev + H/R group; $p < .05, $$p < .01, $$$p < .001 versus H/R + Sev + pcDNA3.1 + mimic NC.

The detection results of myocardial cell damage markers in H9c2 cells treated with different interventions were shown in Figure 5E–G. The concentrations of cTnI, CK-MB, and LDH in the H/R + Sev + pcDNA3.1-NORAD + miR-144-3p mimic group were significantly lower than those in the H/R + Sev + pcDNA3.1-NORAD + mimic NC group (p < .05). Furthermore, it was found that the concentrations of IL-6 and TNF-α in the H/R + Sev + pcDNA3.1-NORAD + miR-144-3p mimic group were significantly lower than those in the H/R + Sev + pcDNA3.1-NORAD + mimic NC group (p < .05), while the concentration of IL-10 significantly increased (p < .001, Figure 5H).

Discussion

Although myocardial I/R can alleviate acute ischemic heart disease, the re-perfusion process itself may lead to further myocardial damage, resulting in more severe consequences.²³ Sev, well-known as a widely used anesthetic, exhibits certain myocardial protective effects. Sev treatment can be classified based on the timing of administration into Sev preconditioning and Sev postconditioning.^24,25 Sev reconditioning plays a role similar to ischemic preconditioning (IPC) in reducing reperfusion-induced injury.²⁶ Our study revealed a decrease in cell proliferation and an increase in apoptosis rate in H9c2 cardiomyocytes under H/R conditions. However, after Sev preconditioning, cell proliferation improved, apoptosis rate decreased, and cardiac injury markers concentrations decreased.

Numerous studies have highlighted inflammation as a key factor in myocardial I/R injury, with the release of pro-inflammatory cytokines like TNF-α and IL-6 exacerbating myocardial damage and leading to cell apoptosis.²⁷ Following Sev preconditioning under H/R conditions, the release of pro-inflammatory cytokines IL-6 and TNF-α decreased, while anti-inflammatory cytokine IL-10 increased. These results demonstrate that Sev can attenuate H/R-induced cardiomyocyte apoptosis and inhibit cell inflammatory responses, further confirming the protective effect of Sev on the myocardium, in line with previous research findings.^28,29

Previous studies have shown upregulation of the NORAD in various cardiovascular diseases, implicating NORAD in regulating apoptosis inflammatory responses. NORAD is also involved in the development and progression of cardiovascular diseases such as coronary artery disease.³⁰ Xiong et al. found that NORAD promotes fibrosis and cell apoptosis through the miR-577/COBLL axis, exacerbating the condition of acute myocardial infarction.³¹ However, some studies have shown that the knockout of NORAD can exacerbate atherosclerosis by stimulating cellular inflammation and oxidative stress.³² This may be due to the differential expression of NORAD in different locations. Our study found that H/R induced an increase in NORAD levels. Moreover, overexpression of NORAD resulted in decreased cell proliferation, significantly increased apoptosis rate, elevated concentrations of cTnI, CK-MB, and LDH, increased pro-inflammatory cytokines, and decreased anti-inflammatory cytokines, indicating that NORAD induced cell inflammation, accelerated cell apoptosis, and impaired myocardial health, displaying an ability to counteract the cardioprotective effect of Sev.

Currently, the regulation of non-coding RNAs has become a very promising method for disease treatment, with lncRNAs being one of the hot topics. Exploring the mechanisms of lncRNAs in Sev-induced myocardial protection can provide new directions for the diagnosis and treatment of myocardial injury, and lay a foundation for the safe clinical use of Sev. Prior research has established that Sev exerts cardioprotective effects against H/R injury in cardiomyocytes, mediated in part by LINC01133.³³ Notably, augmented levels of the lncRNA NEAT1 were shown to dampen the favorable effects of Sev on cardiac performance, infarct size reduction, and apoptosis mitigation in mouse models.³⁴ Consistent with the results of previous studies, we found that Sev could resist myocardial injury in H/R and exert cardioprotective effects by modulating lncRNA levels. Unlike previous studies, our study represented the first to uncover a significant upregulation of NORAD in the context of H/R-induced myocardial injury. Furthermore, we demonstrated that Sev significantly alleviates H/R-mediated myocardial damage, cardiomyocyte apoptosis, and inflammatory responses, accomplished through the inhibition of NORAD expression. This novel finding expanded our understanding of the complex regulatory networks governing cardioprotection and highlighted the potential therapeutic implications of targeting NORAD.

LncRNAs usually interacted with miRNAs.³⁵ In this study, we used bioinformatics methods to identify miR-144-3p and miR-155-5p as targets of NORAD. However, the role of miR-155-5p in myocardial injury is somewhat controversial, so we chose to conduct an in-depth study on miR-144-3p in this article. The role of miR-155-5p in myocardial protection in Sev will be explored in future research. Dual luciferase reporter assay and RIP assay validated the targeting relationship between NORAD and miR-144-3p. Previous reports have suggested that upregulation of miR-144-3p can alleviate myocardial ischemia-reperfusion injury.³⁶ Our results showed that miR-144-3p expression was higher in Sev-treated H/R H9c2 cells compared to H/R group, while overexpression of NORAD inhibited the expression of miR-144-3p. This indicated that NORAD not only targeted miR-144-3p but also exerted a negative regulatory effect on miR-144-3p. The study further investigated the effects of transfecting miR-144-3p mimic on the antagonistic effects of NORAD on Sev-induced cardioprotection. The results demonstrated that elevated miR-144-3p levels could inhibit a series of adverse effects caused by NORAD overexpression in H9c2 cells, including decreased cell proliferation, increased apoptosis rate, and elevated pro-inflammatory cytokine concentrations, which may have similarities with the process of miR-144-3p inhibiting LPS-induced cardiomyocyte damage.³⁷ In brief, previous studies have found that NORAD and miR-144-3p are involved in myocardial injury, respectively, while our research confirms the regulatory role of the NORAD/miR-144-3p axis. Furthermore, we discovered that the NORAD/miR-144-3p axis is closely associated with the myocardial protective effects of Sev. In H/R-induced myocardial injury, Sev may exert cardioprotective effects by inhibiting the NORAD/miR-144-3p axis. Further suppression of this axis in clinical studies holds promise to further mitigate the detrimental consequences of H/R-induced myocardial injury. Although research on lncRNAs is still in its infancy and has not yet reached the level of clinical application,³⁸ in-depth research can provide new perspectives for their application.

This study inevitably encounters several limitations that warrant further exploration. Primarily, while we have analyzed and validated in rat cardiomyocytes the involvement of NORAD in mediating the cardioprotective mechanisms of Sevoflurane through the modulation of miR-144-3p, it is acknowledged that the utilization of human myocardial cardiomyocytes would offer a more disease-responsive model, which constitutes a critical aspect to be addressed in subsequent investigations. Furthermore, the absence of animal studies represents another significant limitation, underscoring the need for future endeavors to focus on this dimension, thereby enhancing the translational potential and clinical relevance of our findings. Furthermore, the downstream genes and signaling pathways of the NORAD/miR-144-3p axis were addressed in subsequent studies.

Conclusion

In conclusion, our research further confirmed that Sev could counteract myocardial I/R injury and discovered that NORAD antagonized the cardioprotective effects of Sev. Additionally, NORAD was a target gene of miR-144-3p; the influence of NORAD on the cardioprotective effect of Sev was achieved through the negative regulation of miR-144-3p. These findings provided new insights into the role of the NORAD/miR-144-3p axis in the cardioprotective effects of Sev.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Long Wang

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