NEAT1/miR-181a-5p/HMGB1 Axis Regulates Macrophage Polarization and Inflammation in Sepsis Models

Summary

This protocol integrates clinical samples, cultured macrophages, and a cecal ligation and puncture (CLP) mouse model to investigate how the NEAT1/miR-181a-5p/HMGB1 axis regulates macrophage polarization and inflammation in sepsis, providing step-by-step guidance that can be adapted to other lncRNA-mediated immune pathways.

Abstract

Sepsis is characterized by a dysregulated host immune response and remains a leading cause of mortality worldwide. Long non-coding RNA NEAT1 has been implicated in inflammatory diseases, but its specific role in macrophage polarization during sepsis has not been fully defined. Here, we systematically examine the NEAT1/miR-181a-5p/HMGB1 axis across clinical samples, cultured macrophages, and a CLP mouse model. Quantitative PCR, western blotting, dual-luciferase reporter assays, and RNA pull-down experiments are used to confirm the competitive endogenous RNA (ceRNA) interaction among NEAT1, miR-181a-5p, and HMGB1. Functional assays, including immunofluorescence, transwell migration, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, are applied to assess macrophage polarization, migration, and apoptosis. In vivo, the CLP model combined with ELISA and histopathology validates the impact of NEAT1 knockdown on cytokine profiles and organ injury. NEAT1 and HMGB1 are upregulated, whereas miR-181a-5p is downregulated, in patients with sepsis and in lipopolysaccharide-stimulated macrophages. Silencing NEAT1 promotes M2 macrophage polarization, reduces pro-inflammatory cytokines, impairs macrophage migration, and alleviates tissue damage in septic mice via the miR-181a-5p/HMGB1 axis. To our knowledge, this is the first integrated protocol to characterize the lncRNA-microRNA-HMGB1 regulatory circuit in sepsis using harmonized clinical, in vitro, and in vivo approaches. It provides a methodological framework for targeting NEAT1-related ceRNA networks as potential therapeutic strategies.

Introduction

Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection¹. Despite advances in clinical management, its mortality remains high, and the molecular mechanisms underlying immune imbalance are not fully understood^2,3. Among immune regulators, long non-coding RNAs (lncRNAs) have emerged as important modulators of inflammatory signaling and macrophage function^4,5. NEAT1, a nuclear-enriched abundant transcript, is a structural component of paraspeckles and has been associated with immu....

Protocol

All procedures involving human participants were approved by the Ethics Review Board of the Affiliated Hospital of Hebei University (Approval No. HDFY-LL-2021-232). Written informed consent was obtained from all participants or, when applicable, from their legal surrogates. All animal experiments were conducted in accordance with institutional guidelines and national regulations, and were approved by the same ethics committee. The approved protocol specified details of the cecal ligation and puncture (CLP) procedure, adenoviral administration, anesthetic regimen, and humane endpoints.

Patient sample collection

Results

Detection of NEAT1/miR-181a-5p/HMGB1 expression and interactions in sepsis

In serum from patients with sepsis, NEAT1 and HMGB1 levels were significantly higher than in healthy controls, whereas miR-181a-5p was markedly reduced. Similar expression patterns were observed in PBMCs and in RAW264.7 macrophages stimulated with lipopolysaccharide, indicating that this axis is consistently altered in sepsis and sepsis-like inflammatory conditions (Figure 1A

Discussion

Sepsis is characterized by profound and often long-lasting immune dysregulation, in which phases of hyperinflammation and immunosuppression coexist and jointly contribute to organ failure and poor outcomes^21,22. Accumulating evidence indicates that long non-coding RNAs (lncRNAs) participate in these processes by modulating inflammatory signaling and macrophage polarization^23,24. For example, lncRNA-MAAMT .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Adenoviral Vector (shNEAT1)	10692-V5	Vigene Biosciences	Adenovirus used for knocking down NEAT1 expression in vivo and in vitro.
BCA Protein Assay Kit	23227	Thermo Fisher	Kit used to quantify protein concentration in cell lysates.
Dual-Luciferase Reporter Assay Kit	E1910	Promega	Kit used for dual-luciferase reporter assays to measure miR-181a-5p interaction.
ELISA Kits (Cytokines)	N/A	R&D Systems	Kits used for measuring cytokine levels in serum and tissue samples.
Fetal Bovine Serum (FBS)	16000-044	Gibco	Supplement used in culture media for cell growth.
GAPDH Antibody	2118S	Cell Signaling Technology	Primary antibody against GAPDH used as loading control in western blot.
Hematoxylin and Eosin Staining Kit	N/A	Thermo Fisher	Kit used for histological staining of tissue sections for pathological analysis.
Immunofluorescence Antibodies (CD206)	14693-1-AP	Proteintech	Antibody used for detecting M2 macrophage marker CD206 in tissue sections.
Lipopolysaccharide (LPS)	L2630	Sigma-Aldrich	LPS used to induce inflammatory macrophage polarization in RAW264.7 cells.
miR-181a-5p Mimic	N/A	N/A	Synthetic miR-181a-5p mimic used to modulate miR-181a-5p levels.
NEAT1 Expression Plasmid	N/A	N/A	Plasmid used for overexpression of NEAT1 in RAW264.7 macrophages.
RAW264.7 Macrophage Cells	TIB-71	ATCC	Murine macrophage cell line used for in vitro experiments.
RIPA Lysis Buffer	89901	Thermo Fisher	Buffer used for lysing cells to extract total protein.
RPMI-1640 Medium	11875-093	Gibco	Culture medium for RAW264.7 macrophages.
SYBR Green Master Mix	4385612	Thermo Fisher	Reagent used for quantitative real-time PCR analysis.
Transwell Migration Assay Chambers	3422	Corning	Chambers used for transwell migration assays to assess macrophage migration.
TUNEL Apoptosis Assay Kit	ab66110	Abcam	Kit used for detecting apoptosis in RAW264.7 cells by TUNEL staining.
Western Blotting Antibodies (HMGB1)	ab18256	Abcam	Primary antibody against HMGB1 used in western blotting.