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miR-330-5p inhibits NLRP3 inflammasome-mediated myocardial ischaemia–reperfusion injury by targeting TIM3

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Abstract

Background/aims

The Nod-like receptor protein-3 (NLRP3) inflammasome signalling pathway is involved in the inflammatory reaction of myocardial ischaemia–reperfusion (I/R) injury. Our previous study showed that miR-330-5p was differentially expressed in both cerebral and myocardial I/R injury, and thus might be a biomarker for I/R injury-related diseases. Another study also indicated that miR-330-5p could promote NLRP3 inflammasome activation in renal IRI. However, the role of miR-330-5p in myocardial I/R injury-induced inflammatory responses is unknown. This study aimed to investigate the role of miR-330-5p in NLRP3 inflammasome-mediated myocardial I/R injury.

Methods

Myocardial I/R injury was induced in mice by occlusion of the left anterior descending coronary artery for 45 min followed by reperfusion. For NLRP3 inflammasome stimulation in vitro, cardiomyocytes were treated with 2 h of oxygen and glucose deprivation (OGD) or LPS (100 ng/ml). Myocardial miR-330-5p expression was examined by PCR at different treatment times. A miR-330-5p antagomir and an agomir were used to regulate miR-330-5p expression. To evaluate the role of miR-330-5p in myocardial I/R injury, 2,3,5-triphenyltetrazolium chloride (TTC) staining, echocardiography, and immunoblotting were used to assess infarct volume, cardiac function, and NLRP3 inflammasome activation respectively. A luciferase binding assay was used to examine whether miR-330-5p could directly bind to the T cell immunoglobulin domain and mucin domain-containing molecule-3 (TIM3). Finally, the role of the miR-330-5p/TIM3 axis in regulating apoptosis and NLRP3 inflammasome formation was evaluated with flow cytometry assays and immunofluorescence staining.

Results

Compared to that in the model group, the inhibition of miR-330-5p significantly aggravated myocardial I/R injury, resulting in increased infarct volume and more severe cardiac dysfunction. Moreover, inhibition of miR-330-5p significantly increased the levels of NLRP3 inflammasome-related proteins, including caspase-1, IL-1β, IL-18 and TNF-α, in both in-vivo and in-vitro models. Furthermore, TIM3 was confirmed as a potential target of miR-330-5p. As predicted, suppression of TIM3 by siRNA ameliorated the anti-miR-330-5p-mediated activation of the NLRP3 inflammasome induced by OGD and LPS, thus decreasing cardiomyocyte apoptosis.

Conclusions

Our study indicated that the miR-330-5p/TIM3 axis was involved in the regulatory mechanism of NLRP3 inflammasome-mediated myocardial inflammation.

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Funding

This work was supported by funding from by National Natural Science Foundation of China Grants No. 81974183, CAMS Innovation Fund for Medical Science (CAMS-2016-I2M-1-011), Wu Jieping Medical Foundation (320.6750.2020-04-16).

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Author notes

  1. Wei Zuo and Ran Tian contributed equally to this work.

Authors and Affiliations

  1. Department of Pharmacy, Peking Union Medical College Hospital, No.1 Shuaifuyuan, Wangfujing, Dongcheng District, Beijing, 100730, China

    Wei Zuo & Bo Zhang

  2. Department of Cardiology, Peking Union Medical College Hospital, No.1 Shuaifuyuan, Wangfujing, Dongcheng District, Beijing, 100730, China

    Ran Tian, Lun Wang, Qing Gu, Yingxian Liu, Jingyi Li, Xinglin Yang & Zhenyu Liu

  3. Department of Laboratory Medicine, Peking Union Medical College Hospital, Beijing, China

    Qian Chen & Chunmei Huang

  4. State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Department of Pathophysiology, Peking Union Medical College, Beijing, China

    Hongmei Zhao

  5. Clinical Pharmacology Research Center, Peking Union Medical College Hospital, Beijing, China

    Lihong Xu

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  1. Wei Zuo
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Contributions

Animal experiment: Wei Zuo and Lun Wang; Cell experiment: Hongmei Zhao and Jingyi Li; Echocardiography: Xinglin Yang; Biochemical experiment: Wei Zuo and Lihong Xu; Data analysis: Ran Tian and Qian Chen; Project administration, Zhenyu Liu; Supervision, Bo Zhang; Writing — original draft: Wei Zuo and Zhenyu Liu; Writing — review & editing: Ran Tian and Yingxian Liu.

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Correspondence to Bo Zhang or Zhenyu Liu.

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Zuo, W., Tian, R., Chen, Q. et al. miR-330-5p inhibits NLRP3 inflammasome-mediated myocardial ischaemia–reperfusion injury by targeting TIM3. Cardiovasc Drugs Ther 35, 691–705 (2021). https://doi.org/10.1007/s10557-020-07104-8

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