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-AKI³. Therefore, SA-AKI has become a major challenge in intensive care, contributing significantly to morbidity and mortality⁴. At present, the mechanism of SA-AKI remains incompletely understood. Although preclinical studies have involved microcirculatory dysfunction, alterations in cellular metabolism, and imbalances within the renin-angiotensin-aldosterone system (RAAS), mitochondrial dysfunction, and inflammatory dysregulation, the pathophysiological mechanism of SA-AKI remains elusive, and there is currently a lack of optimal measures to support early diagnosis and ongoing management^5,6,7,8. SA-AKI features heightened apoptosis, excessive oxidative stress, and inflammation⁹. Within this research framework, lipopolysaccharide (LPS) was employed in an in vitro system to mimic the manifestations of sepsis-associated AKI in human umbilical vein endothelial cells (HUVECs) for preliminary target exploration⁵.

In recent years, circular RNAs (circRNAs), a recently identified category of non-coding RNA molecules, have gained increasing attention for their role in various diseases¹⁰. CircRNAs possess a stable circular structure and participate in cellular physiological and pathological processes by serving as molecular sponges for microRNAs (miRNAs), regulating gene transcription, or engaging in protein interactions¹¹. Investigations have indicated that circRNAs are critically involved in modulating inflammation, oxidative stress, and apoptotic pathways, thereby generating increased research focus on their possible implications in sepsis and related conditions, including acute kidney injury¹². For instance, hsa_circ_0072463 has been identified as a promising diagnostic marker and therapeutic target for SA-AKI¹³. Circ_001653 was reported to alleviate SA-AKI through the recruitment of BUD13¹⁴. However, the functional significance of circRNAs in SA-AKI remains largely unexplored.

This study was designed to examine the influence of circZBTB46 on endothelial cell function, oxidative stress, and inflammatory responses using an in vitro SA-AKI system. By constructing an LPS-induced HUVEC model, we investigated endothelial injury under endotoxin stimulation. It should be noted that LPS-challenged HUVECs represent a reductionist endotoxin/TLR4-driven endothelial stress model that is relevant to, but not equivalent to, the multifactorial pathophysiology of sepsis-associated acute kidney injury. This model primarily captures TLR4-mediated inflammatory activation, oxidative stress, and apoptosis-related endothelial responses, while omitting key in vivo components such as hemodynamic alterations, complement and coagulation cascades, damage-associated molecular patterns, and heterogeneous microbial stimuli. Within this experimental framework, we investigated whether circZBTB46 alleviates LPS-induced oxidative stress and apoptosis by modulating the ERBB2 signaling pathway, thereby protecting endothelial cell function. To our knowledge, this study provides the first evidence linking circZBTB46 to endothelial stress responses in the context of sepsis-associated acute kidney injury. By elucidating the molecular mechanisms underlying circZBTB46-mediated endothelial protection, this research may deepen the comprehension of SA-AKI's pathogenic mechanisms and pave the way for innovative therapeutic approaches centered on circRNA.

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^15,16. HUVECs were cultured in DMEM supplemented with 10% (v/v) fetal bovine serum and antibiotic-antimycotic cocktail at 1% concentration. All cell cultures were maintained under standard physiological conditions in a humidified chamber (37 °C, 5% CO₂), which promoted sustained cellular proliferation while preserving >95% viability as confirmed by trypan blue exclusion assay. Although endothelial-specific media (e.g., EGM-2 or M199 supplemented with endothelial growth supplements) are commonly recommended for long-term HUVEC maintenance, DMEM was used in this study to ensure consistency across experimental treatments and compatibility with LPS stimulation and pharmacological inhibitor assays. For the AG-825 inhibitor assays, incubate HUVECs with fresh DMEM containing 10 µM AG-825 for 1 h. Then, add the LPS solution to the wells to achieve a final concentration of 10 µg/mL, and continue the culture for 24 h. To minimize potential phenotypic drift, HUVECs were used at low passage numbers and exposed to DMEM-based experimental conditions only for short-term assays. All functional experiments were performed within defined treatment windows rather than during prolonged culture.

Cell transfection
To elucidate the biological role of circZBTB46, overexpression plasmids (oe-circZBTB46) and matched negative control constructs (oe-NC) were synthesized and validated by GenScript. HUVECs were seeded into 6-well plates at a density of 1 x 10⁵ cells/well and cultured overnight to reach ~70%-80% confluence at the time of transfection. For each well, 2.5 µg plasmid DNA (oe-circZBTB46 or oe-NC) was diluted in 125 µL of reduced serum medium and mixed with 2.5 µL of P3000 reagent (Tube A). In parallel, 3.75 µL of Lipofectamine 3000 was diluted in 125 µL of reduced serum medium (Tube B). Tube A and Tube B were then combined, gently mixed, and incubated at room temperature for 15-20 min to allow complex formation (final complex volume: 250 µL/well). The complexes were added dropwise to the cells in antibiotic-free complete medium. After 6 h of incubation at 37 °C, 5% CO₂, the transfection medium was replaced with fresh complete growth medium. Cells were cultured for 48 h prior to subsequent assays. All procedures followed the manufacturer's instructions.

LPS-induced model
To generate an in vitro SA-AKI model, HUVECs (5 x 10⁵ cells/well) transfected with oe-circZBTB46 or NC were seeded in 96-well plates and maintained for 24 h until 80% confluency was achieved. Following this, these cells underwent treatment with LPS at 10 µg/mL for 24 h. HUVECs without LPS treatment served as the control group.

qRT-PCR
Total RNA extraction from HUVECs utilized a commercial reagent. Reverse transcription for cDNA synthesis was performed with the commercial reagent kit. Quantitative PCR (qPCR) was executed in triplicate using a Real-Time PCR System alongside a master mix. The expression quantities of target genes were calibrated against U6, with analysis conducted via the 2^−ΔΔCT method^17,18. For circRNA expression analysis, U6 small nuclear RNA was used as the internal reference for normalization, as it is commonly employed for quantification of small non-coding RNAs and has been reported to exhibit relatively stable expression across short-term in vitro experimental conditions. Primer sequences applied in this study are listed as follows:

circZBTB46-Forward: 5'-CGGCGCTCATGAGTAAGAAC-3'

circZBTB46-Reverse: 5'-CGCCTCTTCTACAGACTGGG-3'

U6-Forward: 5'-TGCTATCACTTCAGCAGCA-3'

U6-Reverse: 5'-GAGGTCATGCTAATCTTCTCTG-3'.

CCK-8 assay
HUVECs' viability was assessed using CCK-8 purchased commercially. In brief, cells were seeded into a 96-well plate, and the cells underwent adhesion culture for 24 h. After this, 10 µL of CCK-8 solution was added to each well, and the cells were further incubated at 37 °C for 2 h. A microplate reader was employed to measure the optical density (OD) at a wavelength of 450 nm. Preliminary observations demonstrated that 10 µg/mL of LPS significantly decreased the viability of HUVECs, leading to the selection of this concentration for subsequent experimental processes.

Flow cytometry
Initially, HUVECs were detached with EDTA-free trypsin according to standard procedures. In accordance with the manufacturer's guidelines for the Annexin V-APC/PI Apoptosis Assay Kit, cell suspensions were centrifuged at 1,000 x g for 5 min at 4 °C, after enzymatic dissociation. The pelleted cells were then washed 2x with ice-cold PBS under identical conditions and resuspended in 100 µL of 1x binding buffer. Each sample was treated with 5 µL of Annexin V-FITC conjugate and 5 µL of propidium iodide (PI) staining solution, followed by a 10 min incubation in darkness at room temperature (20-25 °C). Finally, 400 µL of 1x binding buffer was added to all samples, and apoptosis was analyzed within 1 h using a flow cytometer. Events were first gated by FSC/SSC to exclude debris. For apoptosis analysis, quadrant gates were then applied on the Annexin V-FITC vs PI plot using appropriate controls (unstained and single-stained/compensation controls) to define live (Annexin V^-/PI^-), early apoptotic (Annexin V⁺/PI^-), late apoptotic (Annexin V⁺/PI⁺), and necrotic (Annexin V^-/PI⁺) populations. The same gating template and thresholds were applied across all samples.

ELISA assay
The concentrations of inflammatory factors-tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β)-in the supernatant of cultured HUVECs were measured using commercially available ELISA kits. Specifically, the Human TNF-α ELISA Kit, Human IL-6 ELISA Kit, and Human IL-1β ELISA Kit were used according to the manufacturers' instructions. For each assay, 50 µL of undiluted cell culture supernatant was loaded per well. Standard curves were generated using the provided serial dilutions (TNF-α: 0-1000 pg/mL; IL-6: 0-500 pg/mL; IL-1β: 0-200 pg/mL). After sample and standard addition, plates were incubated at 37 °C for 90 min, followed by five washes with wash buffer. Biotin-conjugated detection antibody was then added and incubated at 37 °C for 60 min. After another washing step, streptavidin-HRP was added and incubated at 37 °C for 30 min. Following a final wash, 3,3',5,5'-tetramethylbenzidine (TMB) substrate was added and incubated at 37 °C in the dark for 15 min. The reaction was terminated with stop solution, and absorbance was immediately measured at 450 nm with a reference wavelength of 630 nm. Each sample and standard was assayed in duplicate, and all experiments included three independent biological replicates. The levels of oxidative stress markers-malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT)-in the supernatant were evaluated using the MDA ELISA Kit, SOD ELISA Kit, and CAT ELISA Kit, respectively, following the provided protocols^19,20,21. For MDA and SOD assays, 40 µL of supernatant was used per well without dilution; for CAT, samples were diluted 1:10 with assay buffer prior to loading. Standard curves were prepared according to kit specifications (MDA: 0-60 nmol/mL; SOD: 0-100 U/mL; CAT: 0-100 U/mL). Incubation steps were performed as follows: MDA - 60 min at 37 °C; SOD - 60 min at 37 °C; CAT - 30 min at 37 °C. Plates were washed 4x between key incubation steps. Color development was achieved by adding the respective substrates and incubating at 37 °C (MDA: 15 min; SOD: 20 min; CAT: 15 min). Absorbance was read at 450 nm for SOD and CAT, and at 532 nm for MDA. All measurements were performed in duplicate wells and repeated across three independent biological replicates.

RNA sequencing
Total RNA was extracted from HUVECs using commercial reagents according to the manufacturer's instructions. RNA purity and concentration were assessed using a spectrophotometer by measuring absorbance at 260 nm and 280 nm (A260/A280), and RNA integrity was evaluated with a Bioanalyzer. Only RNA samples with an RNA Integrity Number (RIN) ≥ 7 were used for subsequent library preparation. Ribosomal RNA was removed during library preparation using a standard rRNA depletion procedure done by a commercial company. Where indicated, RNase R treatment was applied prior to library construction to selectively digest linear RNAs and enrich circular RNA species. RNA fragmentation was then performed under elevated temperature conditions following the standard protocol of the RNA Library Preparation Kit manual to generate appropriately sized RNA fragments. Sequencing libraries were prepared using the RNA Library Preparation Kit according to the manufacturer's instructions and sequenced on an Illumina platform using a paired-end sequencing strategy with a read length of 150 bp (PE150). Sequencing depth was sufficient to support transcriptome-wide analysis. Raw sequencing reads were processed using standard quality control procedures to remove adaptor sequences and low-quality reads. Clean reads were aligned to the human reference genome, and gene expression levels were quantified accordingly^22,23. Differentially expressed genes (DEGs) between circZBTB46-overexpressing HUVECs and NC-treated HUVECs under LPS stimulation were identified using established statistical methods. Gene set enrichment analysis (GSEA) was performed to identify signaling pathways associated with circZBTB46 overexpression. Genes were ranked based on differential expression between experimental groups. Curated gene sets from the Molecular Signatures Database (MSigDB) were used for enrichment analysis. Enrichment significance was evaluated based on normalized enrichment score (NES) and false discovery rate (FDR), with FDR < 0.25 considered statistically significant. CircRNA sequencing and all associated bioinformatic analyses were outsourced.

Identification of DEGs
To screen DEGs, the expression levels between two distinct groups were contrasted via the DESeq (2012) R software package. The thresholds for statistical significance were defined as p < 0.05 with a |log2FC| value exceeding 0.58. The heatmap was generated by the pheatmap R package, and the volcano plot used the Goplot2 R package for drawing.

Functional enrichment analysis
GO and KEGG enrichment analyses derived from linear transcripts were conducted using the online platform EnrichR, and the Bubble Chart was drawn based on the GOplot R package. The GSEA software was utilized, based on the expression matrix and gene set files provided by the MSigDB database, with analysis parameters set (e.g., number of permutations and statistical methods). The enrichment of gene sets was evaluated using the normalized enrichment score (NES), P-value, and FDR value, providing an in-depth exploration of the functions and potential biological mechanisms of the differentially expressed genes.

Western blot
Total protein was extracted from HUVECs (approximately 1 x 10⁶ cells) using RIPA lysis buffer supplemented with protease and phosphatase inhibitors. Protein concentration was determined with a BCA Protein Quantification Kit. Equal amounts of protein (20 µg per lane) were resolved by electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) at 80 V for 30 min, followed by 120 V for 60 min. Subsequently, proteins were transferred to polyvinylidene fluoride (PVDF) membranes using a wet-transfer system at 100 V for 90 min at 4 °C. Membranes were blocked with 5% non-fat milk in Tris-buffered saline with 0.1% Tween-20 (TBST) for 1 h at room temperature, followed by incubation with primary antibodies diluted in the same blocking buffer (typically 1:1000 for target proteins and 1:5000 for GAPDH) at 4 °C overnight. The primary antibodies used were as follows: ERBB2, p-AKT, AKT, and GAPDH. After incubation, membranes were washed 3x (10 min per wash) with TBST and then incubated with a horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody (1:5000 in blocking buffer) for 1 h at room temperature. Following three additional TBST washes (10 min each), protein bands were visualized using an Enhanced Chemiluminescence (ECL) Western Blotting Substrate Kit and imaged with an Imaging System. Band intensities were quantified using software.

Statistical analysis
Statistical evaluations were performed using GraphPad Prism 10. Data are displayed as means ± standard error of the means (SEMs). A p-value < 0.05 was defined to indicate statistical significance.

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