Molecular mechanisms and translational medicine application of Oroxylin A in microRNA 155-5p targeting IRF2BP2-NFAT1 axis in sepsis induced lung injury

[Speaker] Pai-Fang Lai:1
[Co-author] Hsin-Tzu Liu:2, Ching-Feng Cheng:2,3,4, Shi-Wei Chao:5,6, Heng Lin:7,8
1:Department of Emergency Medicine, Buddhist Tzu Chi General Hospital, Hualien, Taiwan, 2:Department of Medical Research, Buddhist Tzu Chi General Hospital, Hualien, Taiwan, 3:Department of Pediatrics, Buddhist Tzu Chi General Hospital, Hualien, Taiwan, 4:Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, 5:School of Pharmacy, Taipei Medical University, Taipei, Taiwan, 6:Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei, Taiwan, 7:PhD Program in Biotechnology Research and Development, Collage of Pharmacology, Taipei Medical University, Taipei, Taiwan, 8:Department of Physiology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan

Oroxylin A (OroA), an active ingredient of the Chinese herb Scutellariae radix (Huang Qin), exerts greater inhibitory effect on the inflammation induced physiology defect but related modulation axis via microRNA is unclear now. In C57BL/6 mice model, 24 hrs after LPS (200 ng/ml) application significantly increased iNOS, NF-kB p65, NO expression and these phenomenon were reduced rapidly by OroA treatment. (fig 1) OroA (15 mg/kg, iv) administered attenuated circulating WBC degradation and expression of iNOS caused significant attenuation of LPS-induced decreased mean arterial pressure (MAP) and increased heart rate (HR), pulmonary edema formation, and thickened interalveolar septa in lung tissues after LPS challenge.
Methods and Results:
To elucidate the relationship between OroA and microRNAs (miRs), firstly, we identified, using a microRNA array screen and qPCR, that miR-155-5p and NO content both were higher in macrophages with LPS treatment than with OroA or in normal controls. In mice with sepsis also indicated the higher level of NO and miR-155-5p in macrophages or in neutrophil compared to normal groups. Using TargetScan, we found that the 3' untranslated region (3'UTR) of anti-inflammatory protein, interferon regulatory factor 2-binding protein 2 (IRF2BP2), is complementary to miR-155-5p and miR-155-5p decreased the fluorescent reporter activity by binding to the 3'UTR of IRF2BP2. Furthermore, overexpression of shRNA of miR-155-5p in macrophages significantly attenuated iNOS contents via NFAT4-IRF2BP2 axis. Meanwhile, Intraperitoneal (I.P.) inject with NFAT1 antagonist in the WT mice dramatically reduced the LPS induced MAP, HR and inflammatory effect.
Taken together, our results provided new mechanistic insights for an understanding of the role of OroA induced miR-155-5p-IRF2BP2-NFAT1 axis in sepsis and revealed shRNA-miR-155-5p may instead of OroA serve as a potential agonist or inhibition sepsis injury.

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