Research Paper Volume 15, Issue 10 pp 4288—4303
DNMT1 regulates polarization of macrophage-induced intervertebral disc degeneration by modulating SIRT6 expression and promoting pyroptosis in vivo
- 1 Department of Orthopaedic Surgery, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China
Received: February 22, 2023 Accepted: April 24, 2023 Published: May 17, 2023
https://doi.org/10.18632/aging.204729How to Cite
Copyright: © 2023 Hou et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Abstract
Background: Intervertebral disc degeneration (IDD) is a complex phenomenon and a multifactorial degenerative disease that creates a heavy economic burden on health systems globally. Currently, there is no specific treatment proven to be effective in reversing and delaying the progression of IDD.
Method: This study consisted of animal and cell culture experiments. The role of DNA methyltransferase 1 (DNMT1) on regulating the M1/M2 macrophages polarization and pyroptosis, as well as its effect on Sirtuin 6 (SIRT6) expression in an IDD rat model and in tert-butyl hydroperoxide (TBHP)-treated nucleus pulposus cells (NPCs) were explored. Rat models were constructed, followed by transfection with lentiviral vector to inhibit DNMT1 or overexpress SIRT6. The NPCs were treated with THP-1-cells conditioned medium, and their pyroptosis, apoptosis, and viability were evaluated. Western blot, histological and immunohistochemistry staining, ELISA, PCR, and flow cytometry were all used to evaluate the role of DNMT1/ SIRT6 on macrophage polarization.
Results: Silencing DNMT1 inhibited apoptosis, the expression of related inflammatory mediators (e.g., iNOS) and inflammatory cytokines (e.g., IL6 and TNF-α). Moreover, silencing DNMT1 significantly inhibited the expression of pyroptosis markers IL- 1β, IL-6, and IL-18 and decreased the NLRP3, ASC, and caspase-1 expression. On the other hand, M2 macrophage specific markers CD163, Arg-1, and MR were overexpressed upon silencing DNMT1 or SIRT6 overexpression. At the same time, silencing DNMT1 exerted a regulatory effect on increasing the SIRT6 expression.
Conclusions: DNMT1 may be a promising potential target for IDD treatment due to its ability to ameliorate the progression of the disease.
Abbreviations
IDD: Intervertebral disc degeneration; TBHP: Tert-butyl hydroperoxide; NPCs: Nucleus pulposus cells; DNMT1: DNA methyltransferase 1; SD: Sprague-Dawley; PBS: Phosphate-buffered saline; H&E: Hematoxylin and eosin; BSA: Bovine serum albumin; THP-1: Human monocyte cell line; PMA: Phorbol 12-myristate 13-acetate; CCK-8: Cell Counting kit-8; TUNEL: Terminal deoxynucleotidyl transferase dUTP nick-end labeling; Co-IP: Co-immunoprecipitation; PVDF: Polyvinylidene difluoride; ELISA: Enzyme-linked immunosorbent assay; BER: Base excision repair.