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Updated: Jan 5, 2026
Improved Rodent Model of Myocardial Ischemia and Reperfusion Injury
Published on: March 7, 2022
Long non-coding RNA FOXD3-AS1 aggravates ischemia/reperfusion injury of cardiomyocytes through promoting autophagy
Guoxin Tong1, Yongmei Wang1, Chenke Xu2
1Department of Cardiology, Affiliated Hangzhou First People's Hospital, Zhejiang University School of Medicine No. 261 Huansha Road, Hangzhou 310006, Zhejiang Province, People's Republic of China.
Insights
Long non-coding RNA FOXD3-AS1 aggravates myocardial ischemia/reperfusion (I/R) injury by promoting autophagy via activating NF-κB/iNOS/COX2 signaling. Inhibiting autophagy with 3-methyladenine reversed these detrimental effects, highlighting FOXD3-AS1 as a potential therapeutic target.
Area of Science:
- Cardiovascular Biology
- Molecular Medicine
- Genetics and Genomics
Background:
- Myocardial ischemia/reperfusion (I/R) injury is a significant clinical challenge.
- Long non-coding RNAs (lncRNAs) are emerging as critical regulators in cardiovascular diseases.
- The specific role of lncRNA FOXD3 antisense RNA 1 (FOXD3-AS1) in myocardial I/R injury remains largely unexplored.
Purpose of the Study:
- To investigate the functional role of lncRNA FOXD3-AS1 in myocardial I/R injury.
- To elucidate the underlying molecular mechanisms involving autophagy and NF-κB/iNOS/COX2 signaling.
- To assess the therapeutic potential of targeting FOXD3-AS1 or autophagy.
Main Methods:
- Oxygen-glucose deprivation and reoxygenation (OGD/R) model in H9C2 cells.
- RT-qPCR to measure lncRNA FOXD3-AS1 expression.
- Transfection with pcDNA-lncRNA FOXD3-AS1 for overexpression studies.
- Immunofluorescence and Western blot to assess autophagy markers (LC3 II, Beclin1, ATG5, p62) and apoptosis-related proteins (Bcl-2, Bax, cleaved caspase 3).
- Evaluation of cardiac injury markers (CK, CK-MB, cTnI), inflammation factors, ROS, and NO.
- Assessment of NF-κB/iNOS/COX2 signaling pathway activation.
- Treatment with 3-methyladenine (3 M), an autophagy inhibitor.
Main Results:
- FOXD3-AS1 expression was significantly upregulated in OGD/R-treated H9C2 cells.
- FOXD3-AS1 overexpression promoted autophagy (increased LC3 II, Beclin1, ATG5; decreased p62) and exacerbated myocardial injury markers, inflammation, ROS, and NO production.
- FOXD3-AS1 overexpression enhanced apoptosis and altered apoptosis-related protein expression (decreased Bcl-2, increased Bax, cleaved caspase 3).
- FOXD3-AS1 promoted NF-κB/iNOS/COX2 signaling activation.
- Inhibition of autophagy by 3 M reversed all the detrimental effects induced by FOXD3-AS1 overexpression.
Conclusions:
- Overexpression of lncRNA FOXD3-AS1 aggravates myocardial I/R injury.
- FOXD3-AS1 exerts its detrimental effects by promoting autophagy and activating the NF-κB/iNOS/COX2 signaling pathway.
- Autophagy inhibition represents a potential therapeutic strategy to mitigate FOXD3-AS1-induced myocardial I/R injury.
Abstract:
We aimed to investigate the role of long non-coding RNA (lncRNA) FOXD3 antisense RNA 1 (FOXD3-AS1) in myocardial Ischemia/reperfusion (I/R) injury. In our study, H9C2 cells were treated with oxygen-glucose deprivation and reoxygenation (OGD/R). RT-qPCR was performed to detect the expression level of lncRNA FOXD3-AS1 in OGD/R induced H9C2 cells. Then, pcDNA-lncRNA FOXD3-AS1 was transfected into H9C2 cells. The level of LC3 II was measured by immunofluorescence assay. And the expression of autophagy related genes were detected using western blot. In addition, 3-methyladenine (3 M), an autophagy inhibitor, was recruited to treat with H9C2 cells. The contents of creatine kinase (CK), CK isoenzymes (CK-MB), cardiac troponin I (cTnI), inflammation associated factors, reactive oxygen (ROS) and NO were evaluated by kits. Moreover, cell apoptosis was measured by a flow cytometry assay and the expression levels of apoptosis associated proteins were evaluated by western blot. Furthermore, the expression of NF-κB/iNOS/COX2 signaling were measured in our study. The results indicated that FOXD3-AS1 expression was increased in OGD/R-treated H9C2 cells and overexpression of FOXD3-AS1 upregulated the expression of LC3 II, Beclin1, ATG5 accompanied by a downregulated expression of p62. In addition, FOXD3-AS1 overexpression increased the levels of CK, CK-MB, cTnI, TNF-α, IL-1β, IL-6, ROS and NO, whereas the increase of above factors were reversed following treatment with 3 M. Moreover, FOXD3-AS1 overexpression enhanced the rate of apoptosis cells coupled with a decrease of Bcl-2 expression and an increase of Bax and cleaved caspase 3 expression, which were reversed by 3 M. Furthermore, FOXD3-AS1 overexpression promoted the activation of NF-κB/iNOS/COX2 signaling, which was blocked following treatment with 3 M. These findings demonstrate that overexpression of lncRNA FOXD3-AS1 aggravates myocardial I/R injury through promoting autophagy, which was regulated by activating NF-κB/iNOS/COX2 signaling.
