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Nicotinamide Mononucleotide Alleviates Hyperosmolarity-Induced IL-17a Secretion and Macrophage Activation in Corneal Epithelial Cells/Macrophage Co-Culture System

Authors Meng YF, Pu Q, Dai SY, Ma Q, Li X, Zhu W

Received 18 November 2020

Accepted for publication 27 January 2021

Published 22 February 2021 Volume 2021:14 Pages 479—493

DOI https://doi.org/10.2147/JIR.S292764

Checked for plagiarism Yes

Review by Single anonymous peer review

Peer reviewer comments 3

Editor who approved publication: Professor Ning Quan


Yi-Fang Meng,1,2,* Qi Pu,1,* San-You Dai,3,* Qian Ma,1 Xinyu Li,1 Wei Zhu2

1Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People’s Republic of China; 2Department of Ophthalmology, Changshu No. 2 People’s Hospital, Changshu, People’s Republic of China; 3Department of Ophthalmology, Lixiang Eye Hospital of Soochow University, Suzhou, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Xinyu Li
Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Ave, Wuhan, People’s Republic of China
Email lixy07@126.com
Wei Zhu
Department of Ophthalmology, Changshu No. 2 People’s Hospital, Changshu, People’s Republic of China
Tel/Fax +86-027-83663456
Email shzhuwei0722@163.com

Background: Hyperosmosis stress (HS) was a key pathological factor in the development of dry eye disease (DED). Nicotinamide mononucleotide (NMN) demonstrated protective effects in the corneal damage, however, its role in the HS-induced DED remained unclear.
Methods: A NaCl based HS in-vitro model (500 mOsm) was generated and used in a co-culture system including corneal epithelial cells (CEC) and macrophage cell line RAW264.7. The effect of NMN on NAD+ metabolism and the expression of HS biomarker, tonicity-responsive element binding protein (TonEBP), was studied in the CEC. The cellular activity, including cell viability, apoptosis status and lactate dehydrogenase (LDH) release through trypan blue staining, flow cytometry and LDH assay, respectively. The mitochondrial membrane potential (MMP) assay would be conducted using the JC1 kit. The expression of IL-17a were detected using RT-PCR, ELISA and Western blot. After co-culture with the CEC in different group for 24 h, the phagocytosis ability and macrophage polarization were assessed in RAW264.7 cells co-cultured with CEC with or without HS or NMN treatment. Besides, the involvement of Notch pathway in the RAW264.7 would be analyzed. The potential involvement of Sirtuin 1 (SIRT1) and IL-17a in the crosstalk between CEC and macrophage was studied with SIRT1 inhibitor EX 527 and anti-IL-17a monoclonal antibody, respectively.
Results: NMN treatment increased NAD+ concentration and thus improved cell viability, reduced apoptotic rate and decreased the LDH release in HS-treated CEC. Besides, NMN alleviated HS-induced MMP, intracellular ROS and LDH release. Besides, it was confirmed NMN improve SIRT1 function and decreased the HS related IL-17a expression in CEC and then alleviated macrophage phagocytosis ability and M1 polarization based on a CEC-macrophage co-culture system. Moreover, NMN treatment of CEC in the CEC could moderate the subsequent macrophage activation through Notch pathway. SIRT1 activation and IL-17a inhibition was regarded as key progress in the function of NMN based on the application of EX 527 and anti-IL-17a antibody in the CEC-macrophage co-culture system.
Conclusion: The findings demonstrated that NMN could alleviated HS-induced DED status through regulating the CEC/macrophage interaction. Our data pointed to the role of SIRT1, IL-17a and Notch pathway in the function of NMN and then provided updated knowledge of potential NMN application in the management of DED.

Keywords: dry eye disease, hyperosmosis stress, nicotinamide mononucleotide, SIRT1, notch pathway, macrophage, corneal epithelial cells

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