Protective Effect of MiR-223-3p against Hypoxia/Reoxygenation Injury in H9c2 Cardiomyocytes via Targeting the TLR4/NF-κB Pathway

Q: What are the key findings of this study?

Inhibition of miR-223-3p in H/R-treated H9c2 cardiomyocytes reduced cell viability and markedly exacerbated TLR4, p-p65, TNF-α, IL-6, and LDH levels.

Q: What is the clinical evidence from this study?

Population: Hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury (myocardial ischemia/reperfusion injury model). Intervention: miR-223-3p inhibitor transfection prior to hypoxia/reoxygenation vs. Control (normoxic conditions), H/R treatment only, and H/R + Negative Control (NC) transfection. Primary outcome: Cell viability (CCK-8 assay), TLR4 and phosphorylated NF-κB (p-p65) protein expression (Western blot), and TNF-α, IL-6, and LDH levels (ELISA) (p=<0.05).

Q: What does this research mean for the field?

MiR-223-3p protects cardiomyocytes from hypoxia/reoxygenation injury by negatively regulating the TLR4/NF-κB inflammatory signaling pathway. Novelty: ClaimNovelty.INCREMENTAL. Consensus alignment: ConsensusAlignment.NEUTRAL.

Q: What question did this study set out to answer?

To investigate the role and mechanism of miR-223-3p in hypoxia/reoxygenation-induced injury in rat H9c2 cardiomyocytes.

Lingyu Li<sup>2</sup></p> <p>Fuqiang Kan<sup>1</sup>

doi:10.25236/ijfm.2026.080216

January 1, 2026International Journal of Frontiers in MedicineOpen Access

Protective Effect of MiR-223-3p against Hypoxia/Reoxygenation Injury in H9c2 Cardiomyocytes via Targeting the TLR4/NF-κB Pathway

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Key result

Inhibiting miR-223-3p worsens hypoxia-reoxygenation injury and exacerbates myocardial inflammation in H9c2 cells.

P<0.05

Why the study?

The specific role and underlying mechanism of miR-223-3p in hypoxia/reoxygenation-induced injury in H9c2 cardiomyocytes required investigation.

Does miR-223-3p inhibition exacerbate cellular injury and TLR4/NF-κB pathway activation in H9c2 cardiomyocytes subjected to hypoxia/reoxygenation?

Key Points

To investigate the role and mechanism of miR-223-3p in hypoxia/reoxygenation-induced injury in rat H9c2 cardiomyocytes.
In vitro study dividing H9c2 rat cardiac myoblasts into Control, H/R (6 h hypoxia in glucose-free DMEM followed by 16 h reoxygenation), H/R + NC, and H/R + miR-223-3p inhibitor groups.
Cell viability was measured by CCK-8 assay, miR-223-3p expression by RT-qPCR, TLR4 and p-p65 protein levels by Western blot, and TNF-α, IL-6, and LDH levels in culture supernatants by ELISA.
Data were derived from at least three independent experiments and evaluated across multiple groups using one-way ANOVA.
Hypoxia/reoxygenation treatment significantly reduced cell viability, upregulated miR-223-3p expression, and increased protein levels of TLR4, p-p65, TNF-α, IL-6, and LDH compared with the Control group.
No significant difference was observed between the H/R + NC and H/R groups across tested markers.
Transfection with the miR-223-3p inhibitor prior to H/R further decreased cell viability and markedly exacerbated the upregulation of TLR4, p-p65, TNF-α, IL-6, and LDH.

Structured PICO

Does miR-223-3p inhibition exacerbate cellular injury and TLR4/NF-κB pathway activation in H9c2 cardiomyocytes subjected to hypoxia/reoxygenation?

P

Population

In vitro preclinical study using H9c2 rat cardiac myoblast cells (passages 3 to 5) subjected to 6 hours of hypoxia and 16 hours of reoxygenation to model myocardial ischemia/reperfusion injury.

I

Intervention

Transfection with miR-223-3p inhibitor (mixed 1:2 with Lipofectamine 2000 for 6 h, cultured for 48 h) prior to H/R treatment (6 h hypoxia / 16 h reoxygenation).

C

Comparator

Control group (normoxic culture without H/R), H/R group (H/R treatment only), and H/R + Negative Control (NC) group (transfected with negative control before H/R).

O

Outcome

Cell viability assessed by CCK-8 assay (absorbance at 450 nm after 2 h incubation) following 6 h hypoxia and 16 h reoxygenation.surrogate

Main Result

p-value: p=<0.05

This preclinical study suggests that endogenous miR-223-3p upregulation plays a protective role against hypoxia/reoxygenation cardiomyocyte injury by negatively regulating the pro-inflammatory TLR4/NF-κB signaling pathway.

Limitations

Lack of causal validation (such as dual-luciferase reporter assays or TLR4-knockout cell lines) between miR-223-3p and TLR4
Insufficient investigation into other relevant pathways such as apoptosis and autophagy
In vitro cell model only; further in vivo animal experiments are required to validate clinical potential
Lack of direct causal validation between miR-223-3p and TLR4
Lack of in vivo animal model verification to evaluate therapeutic potential

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Implication

Suggests miR-223-3p protects against H/R cardiomyocyte injury; hypothesis-generating for its therapeutic targeting in myocardial ischemia/reperfusion.

Abstract

This study aimed to investigate the role and mechanism of miR-223-3p in hypoxia/reoxygenation (H/R)-induced injury in H9c2 cardiomyocytes. An in vitro H/R injury model was established, and cells were divided into Control, H/R, H/R + Negative Control (NC), and H/R + miR-223-3p inhibitor groups. Cell viability was assessed by CCK-8 assay, miR-223-3p expression was measured via RT-qPCR, protein levels of TLR4 and phosphorylated NF-κB, p-p65 were analyzed by Western blot, and the release of inflammatory factors and lactate dehydrogenase (LDH) was detected using ELISA. The results showed that compared with the Control group, H/R treatment significantly decreased cell viability, upregulated miR-223-3p expression, and increased the levels of TLR4, p-p65, TNF-α, IL-6, and LDH. No significant difference was observed between the H/R+NC and H/R groups. However, inhibition of miR-223-3p further reduced cell viability and markedly exacerbated the expression of TLR4 and p-p65, as well as the release of inflammatory factors. These findings suggest that miR-223-3p may alleviate H/R-induced cardiomyocyte injury by negatively regulating the TLR4/NF-κB signaling pathway, whereas its inhibition aggravates inflammatory response and cellular damage, indicating its potential protective role in myocardial ischemia/reperfusion injury.

Discussion

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Cite This Study

Lingyu Li2 Fuqiang Kan1 (2026) studied Hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury (myocardial ischemia/reperfusion injury model). miR-223-3p inhibitor transfection prior to hypoxia/reoxygenation vs. Control (normoxic conditions), H/R treatment only, and H/R + Negative Control (NC) transfection was evaluated on Cell viability (CCK-8 assay), TLR4 and phosphorylated NF-κB (p-p65) protein expression (Western blot), and TNF-α, IL-6, and LDH levels (ELISA) (p=<0.05). Inhibition of miR-223-3p in H/R-treated H9c2 cardiomyocytes reduced cell viability and markedly exacerbated TLR4, p-p65, TNF-α, IL-6, and LDH levels.

synapsesocial.com/papers/6af5cd26a40bbe8669e89b9d https://doi.org/10.25236/ijfm.2026.080216

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Protective Effect of MiR-223-3p against Hypoxia/Reoxygenation Injury in H9c2 Cardiomyocytes via Targeting the TLR4/NF-κB Pathway

Key Points

Structured PICO

Main Result

Limitations

Ask about this paper

Implication

Abstract

Discussion

Cite This Study

Protective Effect of MiR-223-3p against Hypoxia/Reoxygenation Injury in H9c2 Cardiomyocytes via Targeting the TLR4/NF-κB Pathway

Key Points

Structured PICO

Main Result

Limitations

Ask about this paper

Implication

Abstract

Discussion

Cite This Study

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