Circular RNA ZBTB46 Attenuates Apoptosis and Oxidative Stress in Lipopolysaccharide-Injured Human Endothelial Cells by Modulating ERBB2-AKT Signaling

Summary

Sepsis-associated acute kidney injury (SA-AKI) is associated with considerable mortality rates. This study finds circZBTB46 downregulated in LPS-induced HUVECs (SA-AKI in vitro). Its overexpression boosts HUVEC viability, reduces apoptosis, inflammation, and oxidative stress in association with ERBB2/AKT signaling, suggesting a potential therapeutic target relevant to SA-AKI.

Abstract

Circular RNAs are emerging regulators of stress responses, yet their roles in endothelial injury that leads to sepsis-related acute kidney injury remain incompletely defined. We hypothesized that the circular RNA ZBTB46 (circZBTB46) confers endothelial protection by engaging ERBB2-AKT signaling. Using a cell-based model in which human umbilical vein endothelial cells were challenged with lipopolysaccharide, we quantified circZBTB46 expression and tested the effects of its forced expression on survival, apoptosis, inflammatory mediators, and redox homeostasis. Cell viability assays and flow cytometry assessed survival and apoptosis. Enzyme-linked immunosorbent assays measured interleukin-6, tumor necrosis factor-α, and interleukin-1β, while reactive oxygen species, malondialdehyde, superoxide dismutase, and catalase were evaluated as indices of oxidative injury and antioxidant capacity. To define the mechanism, we performed transcriptome profiling with gene set enrichment analysis, confirmed pathway proteins by Western blotting, and assessed the necessity using the ERBB2 inhibitor AG-825. Lipopolysaccharide suppressed circZBTB46. CircZBTB46 overexpression increased viability, lowered apoptosis, reduced pro-inflammatory cytokines and reactive oxygen species, decreased malondialdehyde, and raised superoxide dismutase and catalase activities. Transcriptomic and protein analyses supported activation of the ERBB2-AKT axis, and pharmacologic ERBB2 blockade blunted cytoprotection and reversed gains in redox balance. These findings identify circZBTB46 as an endogenous brake on lipopolysaccharide-induced endothelial damage through ERBB2-AKT signaling and nominate circZBTB46 as a mechanistic node and potential therapeutic target for sepsis-related acute kidney injury.

Introduction

Sepsis, a life-threatening systemic inflammatory cascade initiated by pathogenic invasion, frequently culminates in multi-organ failure¹. Within this critical context, SA-AKI emerges as a predominant and clinically significant complication in intensive care units². Characterized by abrupt deterioration of renal filtration capacity concurrent with systemic infection, this condition demonstrates mortality rates exceeding those of non-septic AKI by 38%-42% in clinical cohorts². Approximately 60% of individuals diagnosed with sepsis or septic shock are anticipated to develop SA-AKI^3<....

Protocol

This protocol describes the experimental procedures used to investigate the effects of circZBTB46 modulation on inflammatory signaling, oxidative stress, and ERBB2-AKT pathway activation in HUVECs.

Cell culture
The HUVECs cells used in this study were obtained from a commercial source. HUVECs were used as a representative endothelial cell model because of their wide availability, reproducible culture conditions, and high transfection efficiency, which facilitate genetic manipulation and mechanistic investigation of endothelial signaling pathways under inflammatory stress^15,<....

Results

qRT-PCR analysis of circZBTB46 expression in an in vitro SA-AKI model
This set of experiments was conducted to test the hypothesis that circZBTB46 expression is altered under LPS stimulation and that modulation of circZBTB46 affects endothelial cell viability and apoptosis in an in vitro SA-AKI model. Initially, circZBTB46 expression was examined under control conditions and in an.......

Discussion

During sepsis, pathogen-associated molecular patterns (PAMPs), such as LPS released by pathogens, activate Toll-like receptors (e.g., TLR4), initiating a systemic inflammatory response²⁶. This process triggers the secretion of multiple pro-inflammatory mediators, such as TNF-α, IL-6, and IL-1β, resulting in a cytokine storm²⁷. Simultaneously, LPS directly damages vascular endothelial cells, resulting in microcirculatory disturbances, increased vascular permeabilit.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
AG-825 (ErbB2 Inhibitor)	MedChemExpress	HY-10197	10 μM for pathway blockade
Agilent 2100 Bioanalyzer	Agilent	G2939BA	RNA integrity (RIN≥7)
AKT Antibody	Proteintech	60203-2-Ig	WB (1:15000)
Annexin V-APC/PI Apoptosis Kit	Elabscience	E-CK-A327	Apoptosis detection
Antibiotic-Antimycotic	Gibco	15240062	1% concentration
BCA Protein Quantification Kit	Beyotime	P0012	Protein concentration
CAT ELISA Kit	MEIMIAN	MM-0897H2	Antioxidant enzyme
CCK-8 Kit	Dojindo	CK04	Cell viability assay
CircRNA seq	Fuzhou Huarong Kehui Biotechnology Co., Ltd.	PE150	CircRNA sequencing and all associated bioinformatic analyses
CytoFLEX Flow Cytometer	Beckman Coulter	B53000	Apoptosis analysis
DMEM	Gibco (Invitrogen)	11965092	Cell culture medium
ECL Substrate Kit	Millipore	WBKLS0100	Protein detection
ERBB2 Antibody	Sino Biological	100271-T32	WB (1:10000)
Fetal Bovine Serum (FBS)	Biological Industries	04-001-1ACS	10% (v/v) supplement
GAPDH Antibody	Proteintech	60004-1-Ig	WB (1:50000)
HUVECs	Procell system	CP-H082	Source of cell line
IL-1β ELISA Kit	MEIMIAN	MM-0181H2	Inflammatory factor
IL-6 ELISA Kit	MEIMIAN	MM-0049H2	Inflammatory factor
Illumina NovaSeq 6000	Illumina	20012850	RNA sequencing
Lipofectamine 3000	Invitrogen	L3000008	Transfection reagent
LPS (E. coli O111:B4)	Sigma-Aldrich	L2630	10 μg/mL for SA-AKI model
MDA ELISA Kit	MEIMIAN	MM-2037H2	Oxidative stress marker
NanoDrop 2000	Thermo Fisher	ND-2000	RNA quantification
NEBNext Ultra II RNA Library Kit	NEB	E7770S	RNA-seq library prep
oe-circZBTB46 plasmid	GenScript	Custom synthesis	circZBTB46 overexpression vector
oe-NC plasmid	GenScript	Custom synthesis	Negative control vector
p-AKT (Ser473) Antibody	Proteintech	66444-1-Ig	WB (1:1200)
PrimeScript RT Reagent Kit	Takara	RR047A	cDNA synthesis
PVDF Membranes	Millipore	IPVH00010	Western blot transfer
QuantStudio 5 System	Thermo Fisher	A28137	Real-time PCR
Ribonuclease R	Lucigen	RNR07250	rRNA removal
RIPA Lysis Buffer	Beyotime	P0013B	Protein extraction
RNAiso Plus	Takara	9109	Total RNA extraction
SOD ELISA Kit	MEIMIAN	MM-25469H2	Antioxidant enzyme
TB Green Premix Ex Taq	Takara	RR420A	qPCR reagent
TNF-α ELISA Kit	MEIMIAN	MM-0122H2	Inflammatory factor