SNHG5 Exacerbates Sepsis‐Induced Inflammatory Injury in Coronary Artery Endothelial Cells by Regulating METAP2‐Mediated IL‐8 Secretion

Abstract

Background

Long noncoding RNAs (lncRNAs) are involved in regulating inflammatory responses in sepsis. This study aimed to investigate the potential roles of lncRNA SNHG5 in the pathogenesis of sepsis‐induced coronary artery injury.

Methods

Cecal ligation and puncture were used to establish a sepsis mouse model. Serum and coronary artery tissues were collected to assess inflammatory markers and lncRNA expression using enzyme‐linked immunosorbent assay (ELISA) and real‐time quantitative polymerase chain reaction (qPCR). In vitro, mouse aortic endothelial cells were exposed to septic serum. The SNHG5/miR‐377‐3p/methionyl aminopeptidase 2 (METAP2) axis was investigated using dual‐luciferase reporter gene experiments. Loss‐of‐function, gain‐of‐function, and rescue assays were performed to elucidate the functions of these genes in sepsis.

Results

Septic mice exhibited elevated systemic tumor necrosis factor alpha and interleukin (IL)‐6 levels, as well as dysregulated endothelial markers (eNOS downregulation and ET‐1 upregulation). Among the inflammation‐related lncRNAs, SNHG5 was the most significantly upregulated in septic coronary arteries and serum. Bioinformatic and experimental analyses revealed that SNHG5 acts as a competitive endogenous RNA (ceRNA) for miR‐377‐3p, thereby upregulating (METAP2). Silencing SNHG5 or METAP2 reduced IL‐8 secretion and endothelial apoptosis; these effects were reversed by miR‐377‐3p inhibition. Conversely, METAP2 overexpression increased IL‐8 secretion and apoptosis, which were attenuated by miR‐377‐3p mimics.

Conclusion

We identified the SNHG5/miR‐377‐3p/METAP2 axis as a novel regulatory pathway in sepsis‐induced endothelial inflammation and apoptosis. SNHG5 promotes METAP2 expression by sponging miR‐377‐3p, leading to increased IL‐8 secretion and endothelial dysfunction. These findings provide new insights into lncRNA‐mediated mechanisms in sepsis.

1. Introduction

Sepsis, a life‐threatening condition resulting from the body’s response to infection, poses a significant challenge to modern healthcare. The increasing prevalence of sepsis in recent years can be attributed to multiple factors, including an aging population [1], the rise in antibiotic‐resistant infections [2], and the increasing complexity of medical interventions. Sepsis pathophysiology involves a dysregulated interplay between the host immune system and invading pathogens. This interaction triggers a cascade of biological responses that can lead to organ dysfunction and, ultimately, death if not treated promptly and effectively [3].

The cardiovascular system is vulnerable in sepsis, with endothelial dysfunction serving as a central driver of subsequent organ injury [4]. Sepsis‐induced cardiovascular injury is characterized by a complex interplay of systemic inflammation, hemodynamic instability, and direct cellular damage, often culminating in myocardial depression, vascular hyporeactivity, and microcirculatory dysfunction [5]. Recent research on sepsis‐induced cardiovascular injury has progressed significantly. For instance, Duignan et al. [4] reported that cardiovascular dysfunction is common in neonatal sepsis. Moreover, sepsis can lead to long‐term cardiovascular dysfunction [6]. Dolmatova et al. [7] reported that, at the cellular level, sepsis exerts multiple effects on endothelial cells, including increased immune activation, dysregulated coagulation, and oxidative stress, all contributing to endothelial barrier disruption and organ hypoperfusion. Despite these insights, the precise mechanism through which sepsis‐induced inflammatory signaling converges on endothelial pathways to induce cardiovascular injury remains poorly understood, underscoring the need to elucidate specific regulatory mechanisms.

Long noncoding RNAs (lncRNAs) are RNA molecules exceeding 200 nucleotides that lack protein‐coding potential. They act as versatile regulators of gene expression, with functions spanning chromatin modification, transcription, and posttranscriptional processing [8]. Substantial evidence has revealed that lncRNAs play specific roles in the pathogenesis of sepsis, influencing immune responses, cell survival, and organ injury [9, 10]. Zhang et al. [11] discovered that lncRNA LUADT1 was downregulated in patients with sepsis, and that LUADT1 overexpression prevents lipopolysaccharide (LPS)‐induced cardiac endothelial cell apoptosis. lncRNAs are involved in the regulation of inflammatory responses and oxidative stress in coronary artery endothelial cells. Li et al. [12] discovered that oxidized low‐density lipoprotein increased lncRNA COLCA1 expression and induced oxidative stress, thereby promoting sustained inflammatory signaling. However, the roles of many inflammation‐related lncRNAs in sepsis‐induced coronary artery endothelial injury remain largely unexplored, highlighting a significant gap in our understanding of the regulatory networks governing endothelial dysfunction in sepsis.

In this study, six inflammation‐related lncRNAs were selected, and their expression levels in the coronary arteries and serum of septic mice were determined. Among them, SNHG5 exhibited the most significant upregulation in septic mice compared with sham controls. Although SNHG5 acts as a molecular sponge for miR‐374a‐3 p and modulates the TLR4/NF‐κB pathway in renal epithelial cells during septic acute kidney injury [13], its role in sepsis‐induced coronary artery injury remains unclear. Bioinformatic analyses (binding sites and expression correlation) revealed that SNHG5 might function as a molecular sponge for miR‐377‐3p, thereby regulating methionyl aminopeptidase 2 (METAP2) expression. Given the documented involvement of miR‐377‐3p and METAP2 in inflammatory processes, we hypothesized that the SNHG5/miR‐377‐3p/METAP2 axis might contribute to endothelial inflammatory injury in sepsis. To evaluate this, we performed in vitro functional assays to validate the regulatory interactions among these molecules and to elucidate their downstream mechanisms in sepsis‐like endothelial injury. Our results reveal a previously unrecognized signaling pathway that exacerbates coronary artery endothelial damage during sepsis, offering new insights into the molecular basis of septic cardiovascular pathology.

2. Materials and Methods

2.1. Animal Experiments

A total of 10 male C57B1/6 mice were acquired from Charles River (Beijing, China). The mice were raised in a 12 h day/night alternating environment and provided with sufficient food and water. The rats were randomly divided into two groups: the sham group (n = 6) and the sepsis group (n = 6). Cecal ligation and puncture were used to establish a sepsis model. The mice were anesthetized using an intraperitoneal injection of pentobarbital sodium (30 mg/kg), and the abdominal skin was prepared. The abdominal cavity was cut open along the midline, and the cecum was separated. Then, a part of the cecum was ligated with a suture, and the ligated area was punctured with a needle to ensure that a small amount of intestinal contents spilled out. The processed cecum was retracted into the abdominal cavity, and the incision was sutured. The mice were euthanized after 24 h by excessive anesthesia (intraperitoneal injection of pentobarbital sodium [250 mg/kg]). Finally, the serum and hearts were collected for testing. The experiment was approved by the Ethics Review Committee for Animal Welfare of Hunan Provincial People’s Hospital (Number 20230516).

2.2. Cell Culture and Treatment

Mouse arterial endothelial cells (mAECs) were obtained from Wuhan Pricella Biotechnology Co., Ltd. (Wuhan, China). The cells were cultured in M199 medium containing 20% fetal bovine serum and 2 mmol/L of L‐glutamine (Sangon Biotech, Shanghai, China). To induce injury in coronary artery endothelial cells in the sepsis model, mAECs were treated with >30% serum collected from sham or sepsis mice.

2.3. Cell Transfection and Dual Fluorescence Detection

To validate the function of SNHG5/METAP2 in mAECs, short interfering RNA specifically targeting SNHG5/METAP2 was synthesized and cotransfected into mAECs with miR‐377‐3p inhibitors (GenePharma, Shanghai, China). To demonstrate the targeted regulation of METAP2 by miR‐377‐3p, the sequence of METAP2 3^′‐untranslated region (3^′‐UTR) was cloned and recombined into the psiCHECK‐2 plasmid vector. Additionally, the bases of the miR‐377‐3p binding site on the 3^′‐UTR were mutated and recombined into the plasmid. The recombinant psiCHECK‐2 plasmid vector was cotransfected into mAECs using miR‐377‐3p mimics (GenePharma). After adding fluorescent substrates, Renilla and firefly luciferases were detected using a fluorescence reader (Thermo Fisher Scientific, Waltham, MA, USA).

2.4. Real‐Time Quantitative Polymerase Chain Reaction (qPCR)

Total RNA was extracted from cells or tissues using TRIzol (Invitrogen, Carlsbad, CA, USA), followed by reverse transcription into cDNA. To detect miR‐377‐3p, primers designed using the stem–loop method were used in reverse transcription. Specific primers were designed for the target sequences of SNHG5, miR‐377‐3p, and METAP2. GAPDH and U6 were used as internal reference genes. Then, PCR amplification was performed using SybrGreen qPCR Master Mix (Sangon Biotech). PCR reaction conditions were as follows: 2 min at 94°C, followed by 40 cycles of 20 s at 94°C, 20 s at 58°C, and 20 s at 72°C. The relative expression levels of genes were determined using the 2^–ΔΔCt method. The primer sequences are listed in Table S1.

2.5. Western Blotting

Cells or tissues were lysed using RIPA lysis buffer (Invitrogen) to extract proteins. The proteins were then separated based on their molecular weight using polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane. After blocking with 5% bovine serum albumin, specific primary antibodies targeting eNOS (ab300071, 1:1000; Abcam, Cambridge, MA, USA), ET‐1 (ab2786, 1:1000), METAP2 (ab124953, 1:2000), caspase‐3 (ab184787, 1:2000), BCL‐2 (ab182858, 1:2000), Bax (ab32503, 1:2000), and β‐actin (ab13772, 1:5000) were incubated with the membrane for 12 h at 4°C. Following incubation with an IgG H&L secondary antibody (ab6728, 1:5000), the proteins were visualized using enhanced chemiluminescence (Invitrogen). The grayscale values of the bands were analyzed and quantified using the Image‐Pro Plus software (Media Cybernetics, Silver Spring, MD, USA).

2.6. Enzyme‐Linked Immunosorbent Assay (ELISA)

Serum TNF‐α, interleukin (IL)‐1α, IL‐1β, IL‐6, IL‐8, and IL‐10 in supernatant were measured using an ELISA Kit (Solaibio, Beijing, China) according to the instructions. After diluting five times, the serum (100 μL) was added to a precoated 96‐well plate. After incubation for 1 h, primary and secondary antibody solutions were added separately. The absorbance was measured using a microplate reader (Thermo Fisher Scientific).

2.7. Cell Viability Testing

A Cell Counting Kit‐8 (CCK8; Beyotime Biotechnology) assay was used to determine cell viability. The transfected and serum‐treated mAECs (1 × 10⁴/100 μL) were seeded into a 96‐well plate. Then, CCK8 solution (100 μL) was added to each well at 12, 24, 36, and 48 h, respectively. After incubation for 1 h, the absorbance at 450 nm was measured using a microplate reader (Thermo Fisher Scientific).

2.8. Apoptosis Detection via Flow Cytometry

After serum treatment, cell apoptosis was determined using flow cytometry with a PI/Annexin V Test Kit (Beyotime Biotechnology). The mAECs were fixed using formaldehyde, resuspended in 100 μL of 1 × annexin V binding buffer, and incubated with 5 μL annexin V‐Ab Flour TM 488 solution and 2 µL PI solution in the dark for 15 min. After staining the cells with 400 μL 1 × annexin V binding buffer for 30 min, the samples were analyzed using flow cytometry (FACSCalibur, BD, Franklin Lakes, NJ, USA).

2.9. Co‐Immunoprecipitation (Co‐IP)

Cells with/without shMETAP2 were treated with LPS. The treated cells were washed and lysed using NP‐40 lysis buffer (Beyotime). The supernatant was collected, and a small aliquot (5%) was used as the “Input” control. The remaining lysate was precleared by incubation with 20 µL of Protein A/G Magnetic Beads (Beyotime) for 1 h at 4°C. The precleared lysates were incubated overnight with 2 µg of either rabbit anti‐METAP2 antibody (Abcam) or anti‐IL‐8 antibody (Abcam). Subsequently, each sample was incubated with 40 µL of protein A/G magnetic beads for 4 h with gentle rotation. Bound proteins were eluted by boiling the beads in 40 µL of 2 × loading buffer at 95°C for 10 min. The eluted proteins and the saved input samples were analyzed using western blotting.

2.10. Statistical Analysis

All experiments were independently repeated three times. The data are presented as average ± standard deviation. The Student t‐test was used for comparison between two groups, while one‐way analysis of variance with Tukey’s post hoc test was used for comparison between multiple groups. A p < 0.05 indicated a statistically significant difference.

3. Results

3.1. Abnormally High Expression of SNHG5 in the Coronary Arteries of Septic Mice

Following the induction of sepsis via surgical modeling, blood samples and coronary artery tissues were obtained from the mice. Blood samples were analyzed for inflammatory factors, including TNF‐α, IL‐1α, IL‐1β, IL‐6, IL‐8, and IL‐10. The results revealed that the levels of these six inflammatory factors were significantly elevated in the sera of septic mice (Figure 1a), indicating a systemic inflammatory response. To investigate the effect of septic inflammatory storm on the function of coronary endothelial cells, we assessed the protein expression of endothelial dysfunction markers in coronary artery tissues. The results demonstrated a significant downregulation of eNOS and a marked upregulation of ET‐1 in the sepsis group (Figure 1b), indicating impaired endothelial homeostasis. Additionally, the expression levels of inflammation‐related lncRNAs were measured using qPCR. The results revealed that lncRNAs MYCNOS, SNHG5, and SNHG14 were significantly elevated in the coronary artery tissue of septic mice, while MEG3 was significantly downregulated, and lncRNAs TUG1 and GAS5 were unchanged (Figure 1c). Furthermore, MYCNOS, SNHG5, and SNHG14 levels were significantly elevated in the serum of septic mice (Figure 1d). Considering the maximum fold change in SNHG5, it was selected as the target for future research. It is worth investigating whether the abnormally high expression of SNHG5 caused by a septic inflammatory storm is related to coronary artery endothelial injury (Figure 1e).

Figure 1.

Abnormal expression of lncRNA in the coronary arteries of septic mice. (a) Serum levels of TNF‐α, IL‐1α, IL‐1β,IL‐6, IL‐8, and IL‐10 were measured using ELISA. (b) Protein concentrations of eNOS and ET‐1 in coronary artery tissue. (c, d) Expression levels of MYCNOS, TUG1, GAS5, MEG3, and SNHG14 in coronary artery tissue and serum. (e) Sepsis‐induced SNHG5 may be related to the function of coronary artery endothelial cells. n = 6 each group;  ^∗ p < 0.05,  ^∗∗∗ p < 0.001.

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3.2. SNHG5 May Act as a Competitive Endogenous RNA (ceRNA) for miR‐377‐3p to Regulate METAP2 Expression

Previous studies indicate that SNHG5 may function as a ceRNA in inflammatory environments [14]. To systematically identify its potential miRNA–mRNA regulatory axes, the starBase (Version 2.0) database (https://rnasysu.com/encori/ceRNA.php?source=lncRNA) [15] was used to predict SNHG5‐associated miRNAs and their target genes. Among these, the miR‐377‐3p–METAP2 axis was selected for further validation based on three criteria: First, METAP2 has been implicated in the regulation of vascular endothelial cell proliferation [16], indicating its potential relevance in sepsis‐induced endothelial cell dysfunction. Second, the binding sites among SNHG5, miR‐377‐3p, and METAP2 demonstrated high predictive scores and conservation (Figure 2a). Third, the expression level of miR‐377‐3p, which was downregulated in the septic coronary artery, was negatively correlated with the expression levels of SNHG5 and METAP2, while METAP2 expression, which was upregulated in the septic coronary artery, demonstrated a strong positive correlation with SNHG5 expression (Figure 2b, c). A dual‐luciferase reporter assay was performed to validate the predicted binding interactions. The wild‐type or mutant 3^′‐UTR of METAP2 was cloned into the psiCHECK‐2 vector and cotransfected with miR‐377‐3p mimics into mAECs (Figure 2d). Luciferase activity was significantly inhibited by miR‐377‐3p mimics exclusively for the wild‐type 3^′‐UTR of METAP2 and SNHG5, with no effect on their mutant counterparts (Figure 2e, f). These findings illustrate the specific binding of miR‐377‐3p to both transcripts, thereby providing mechanistic support for the ceRNA regulatory axis involving SNHG5, miR‐377‐3p, and METAP2 in sepsis‐induced coronary injury.

Figure 2.

SNHG5 may regulate the expression levels of miR‐377‐3p and METAP2. (a) Target genes that may be regulated by SNHG5 in the starBase (Version 2.0) database (https://rnasysu.com/encori/ceRNA.php?source=lncRNA). (b) Expression levels of miR‐377‐3p and METAP2 in the coronary artery tissue of septic mice; n = 6. (c) Correlation of expression levels of SNHG5/miR‐377‐3p, SNHG5/METAP2, and miR‐377‐3p/METAP2. (d) Potential binding site sequences for SNHG5/miR‐377‐3p and miR‐377‐3p/METAP2 3^′‐UTR. (e) Dual‐luciferase reporter gene experiment to verify miR‐377‐3p targeting METAP2. (f) Dual‐luciferase reporter gene experiment to validate miR‐377‐3p binding to SNHG5; n = 3 replicate wells per sample.  ^∗∗∗ p < 0.001; ns, no significant difference.

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3.3. SNHG5 Promotes METAP2 Expression in mAECs

To validate the regulatory relationship between SNHG5, miR‐377‐3p, and METAP2, mouse serum from sepsis models was used to stimulate mAECs transfected with SNHG5 siRNA or miR‐377‐3p inhibitors (Figure 3a). The qPCR results revealed that stimulation with sepsis mouse serum significantly upregulated the expression of SNHG5 in mAECs more than the normal serum group (Figure 3b). Meanwhile, stimulation with serum from septic mice significantly upregulated METAP2 expression and downregulated miR‐377‐3p expression. SNHG5 knockdown upregulated miR‐377‐3p expression and inhibited the expression of METAP2, and these effects were reversed by miR‐377‐3p inhibitors. Additionally, western blotting results revealed that the level of METAP2 protein was consistent with its RNA expression level (Figure 3c, d). Immunofluorescence staining was used to confirm METAP2 expression patterns and subcellular localization (Figure 3e). These results demonstrated a direct regulatory relationship between SNHG5, miR‐377, and METAP2 in mAECs.

Figure 3.

SNHG5 knockdown inhibited METAP2 expression. (a) Mouse arterial endothelial cells were stimulated with 30% serum collected from sham or sepsis mice and transfected with SNHG5 siRNA or miR‐377‐3p inhibitors. (b) Expression levels of SNHG5, miR‐377‐3p, and METAP2 were determined using qPCR. (c, d) Protein levels of METAP2 were measured using western blotting. (e) Immunofluorescence revealed the expression distribution of METAP2. DAPI: 4^′,6‐diamidino‐2‐phenylindole. n = 3 replicate wells per sample;  ^∗ p < 0.05,  ^∗∗ p < 0.01,  ^∗∗∗ p < 0.001.

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3.4. SNHG5 Knockdown Improves Sepsis‐Induced Endothelial Cell Apoptosis

To investigate the functional role of SNHG5 in sepsis‐induced vascular endothelial injury, we first evaluated the activity of mAECs after serum stimulation. The results revealed that the treatment with sepsis mouse serum significantly decreased the cell activity of mAECs, while SNHG5 knockdown increased the cell activity (Figure 4a). The miR‐377‐3p inhibitors completely reversed the effects of SNHG5 knockdown on cell activity. Additionally, SNHG5 knockdown reduced the sepsis mouse serum‐induced apoptosis of mAECs, while miR‐377‐3p inhibitors promoted it (Figure 4b). Furthermore, stimulation with sepsis mouse serum decreased the protein level of eNOS and increased that of ET‐1 (Figure 4c). SNHG5 knockdown increased the protein level of eNOS and decreased that of ET‐1, while miR‐377‐3p inhibitors produced the opposite results. Proapoptotic proteins, caspase‐3 and Bax, changed like the ET‐1 protein, while the BCL‐2 protein changed like the eNOS protein (Figure 4d). Moreover, we discovered that IL‐8 was significantly upregulated by stimulation with sepsis mouse serum, while SNHG5 knockdown reduced IL‐8 secretion, an effect reversed by miR‐377‐3p inhibition (Figure 4e–g). The results revealed that SNHG5 knockdown improved sepsis‐induced endothelial cell apoptosis, possibly by regulating miR‐377‐3p/METAP2.

Figure 4.

SNHG5 promotes sepsis‐induced inflammatory apoptosis of coronary artery endothelial cells. (a) Cell activity detected using CCK8 assay. (b) Cell apoptosis was detected using flow cytometry. (c) Protein levels of eNOS and ET‐1. (d) Protein levels of caspase‐3, BCL‐2, and Bax were determined using western blotting. (e) The expression level of IL‐8 was detected using qPCR. (f) Protein levels of IL‐8 in cells. (g) IL‐8 levels in the culture supernatant. ns: no significant difference. n = 3 replicate wells per sample;  ^∗ p < 0.05,  ^∗∗ p < 0.01,  ^∗∗∗ p < 0.001.

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3.5. miR‐377–3p Inhibits METAP2‐Promoted Endothelial Cell Apoptosis in Sepsis

To investigate the functional role of METAP2 in sepsis‐induced vascular endothelial injury, we knocked down METAP2 expression in mAECs using siRNA, followed by stimulation with serum from septic mice. miR‐377‐3p mimics inhibited METAP2 expression (Figure 5a, e). Furthermore, despite unchanged IL‐8 mRNA levels, METAP2 downregulation reduced IL‐8 secretion and increased intracellular IL‐8 levels, indicating posttranscriptional regulation (Figure 5a–c). Co‐IP assay revealed that METAP2 interacted with IL‐8 under septic conditions (Figure 5d). METAP2 knockdown and miR‐377‐3p mimic treatment significantly decreased the rate of apoptosis (Figure 5f). Meanwhile, caspase‐3 and Bax levels decreased upon METAP2 suppression, while eNOS and BCL‐2 levels were upregulated (Figure 5g, h).

Figure 5.

METAP2 knockdown or miR‐377‐3p overexpression inhibits endothelial cell damage and inflammation. (a) Expression levels of METAP2 and miR‐377‐3p were determined using qPCR. (b) IL‐8 levels in the culture supernatant. (c) Protein levels of METAP2 and IL‐8 in cells. (d) Co‐IP confirmed a direct interaction between METAP2 and IL‐8. (e) Immunofluorescence was used to elucidate the expression distribution of METAP2. (f) Cell apoptosis was determined using flow cytometry. (g) Protein levels of eNOS and ET‐1. (h) Protein levels of caspase‐3, BCL‐2, and Bax were determined using western blotting. ns, no significant difference. n = 3 replicate wells per sample;  ^∗∗∗ p < 0.001.

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Furthermore, METAP2 was overexpressed using a vector containing its CDS and 3^′‐UTR. Overexpression was confirmed via qPCR, and this effect was attenuated by miR‐377‐3p mimics (Figure 6a, c). Under septic serum stimulation, METAP2 overexpression increased IL‐8 secretion, which was reversed by miR‐377‐3p. In contrast, intracellular IL‐8 levels demonstrated an opposite trend (Figure 6b, d). Consistent with the secretion data, METAP2 overexpression elevated the expression levels of ET‐1, caspase‐3, and Bax, which were rescued by miR‐377‐3p. Meanwhile, eNOS and BCL‐2 expression levels demonstrated an opposite trend (Figure 6e, f). METAP2 overexpression increased endothelial cell apoptosis, and this effect was significantly suppressed by miR‐377‐3p mimics (Figure 6g). These findings demonstrate that METAP2, which is downregulated by miR‐377‐3p, promotes endothelial cell apoptosis and IL‐8 secretion during sepsis.

Figure 6.

METAP2 overexpression promotes IL‐8 release from endothelial cells. The METAP2 coding sequence carrying a 3^′‐UTR was co‐overexpressed with miR‐377‐3p mimics in endothelial cells. (a) Expression levels of METAP2 and miR‐377‐3p were determined using qPCR. (b) IL‐8 levels in the culture supernatant. (c) Immunofluorescence was used to elucidate the expression distribution of METAP2. (d) Protein levels of METAP2 and IL‐8 in cells. (e) Protein levels of eNOS and ET‐1. (f) Protein levels of caspase‐3, BCL‐2, and Bax were determined using western blotting. ns: no significant difference. (g) Cell apoptosis was determined using flow cytometry. ns: no significant difference. n = 3 replicate wells per sample;  ^∗ p < 0.05,  ^∗∗∗ p < 0.001.

4. Discussion

The endothelium serves as a vital interface between the bloodstream and vascular tissues, playing indispensable roles in cardiovascular homeostasis. It acts not only as a semipermeable barrier but also as a dynamic regulator of vascular tone through the balanced release of vasodilators (for instance, nitric oxide) and vasoconstrictors. Furthermore, it maintains thromboresistance by expressing anticoagulant factors and modulates inflammatory responses by controlling leukocyte adhesion and transmigration, as well as influences vascular growth and repair. Endothelial dysfunction is a hallmark of cardiovascular injury. As a type of nonconventional immune cell, endothelial cells play a major role in the systemic response during septic shock [17]. The excessive generation of reactive oxygen/nitrogen species in endothelial cells during septic shock constitutes a key contributor to endothelial dysfunction [18]. Some biomarkers of endothelial dysfunction related to endothelial activation/injury (vWF, sE‐selectin, and sVCAM‐1), vascular barrier function/permeability (ANGPT1, ANGPT2, and VE‐cadherin), and dysregulation of coagulation and anticoagulation (thrombomodulin) are dysregulated in sepsis and may be prognostic markers for sepsis [19, 20]. In our experimental model, sepsis caused a significant reduction in coronary eNOS protein and an elevation in ET‐1. Given that eNOS promotes vasodilation and vascular homeostasis via nitric oxide production, while ET‐1 is a key endothelial vasoconstrictor, their dysregulated expression indicates impaired endothelial function. This interpretation is reinforced by our in vitro findings that septic serum significantly increases endothelial cell apoptosis [21, 22].

lncRNA SNHG5 is associated with inflammation in different tissues and cells. Wang et al. [13] discovered that SNHG5 expression was significantly elevated in the serum of patients with sepsis compared with healthy controls. Mechanistically, SNHG5 acts as a sponge of miR‐374a‐3 p to regulate the TLR4/NF‐κB pathway in septic acute kidney injury. Additionally, SNHG5 knockdown increased viability while reducing apoptosis and inflammatory cytokine production in renal cells. In status epilepticus, SNHG5 knockdown inhibited LPS‐induced inflammation by regulating NF‐κB signaling [23]. However, the role of SNHG5 varies in the inflammatory environments of different tissues. SNHG5 upregulation inhibited the LPS‐induced production of TNF‐α and IL‐17 in alveolar Type II epithelial cells [24]. In the chronic constriction injury mouse model, SNHG5 inhibited the inflammatory response through sponging miR‑142‑5 p and regulating the expression of CAMK2A [25]. Our results demonstrated that SNHG5 was significantly upregulated in coronary artery tissues and serum in a murine sepsis model. Extending previous reports on its role in inflammation, we provide first‐time evidence that SNHG5 acts as a ceRNA, sequestering miR‐377‐3p and promoting coronary artery endothelial apoptosis under septic conditions. Beyond SNHG5, we identified additional lncRNAs dysregulated in sepsis. Moreover, MYCNOS and SNHG14 were significantly upregulated in septic coronary arteries and serum, indicating that they may contribute to vascular inflammation and merit further investigation. Conversely, MEG3 was downregulated in coronary tissue but unchanged in serum. This indicates a tissue‐specific, localized regulatory role during sepsis. Its downregulation likely constitutes a direct cardiac response to inflammation [26]. The stable serum levels may result from insufficient release, rapid clearance, or compensatory release from other tissues, emphasizing its primary action within the vessel wall rather than as a systemic factor.

While miR‐377‐3p has been extensively studied as a tumor suppressor that inhibits proliferation in various cancers [27–29], its role in vascular and inflammatory diseases reveals a more complex functional landscape. Previous studies have demonstrated that miR‐377‐3p suppresses the proliferation, migration, and inflammatory activation of vascular smooth muscle cells in vascular diseases [30], including atherosclerosis, by targeting neuropilin‐2 [31]. Contrary to these inhibitory roles, we discovered that miR‐377‐3p expression was significantly downregulated in the coronary arteries during sepsis. In vitro, miR‐377‐3p inhibition increased apoptosis in mAECs, indicating a protective, antiapoptotic function in this inflammatory environment. This finding aligns with previous studies that miR‐377‐3p ameliorates LPS‐induced acute lung injury by targeting RPTOR [32] and attenuates oxidized‐LDL‐induced inflammation in vascular smooth muscle cells [33]. Similarly, miR‐377‐3p overexpression in osteoarthritis improves synoviocyte proliferation and reduces apoptosis under inflammatory stimulation [34]. These studies underscore the context‐dependent duality of miR‐377‐3p, which can exert growth‐suppressive or cell‐protective effects depending on the disease microenvironment. Our results highlight its specific role in mitigating endothelial apoptosis during septic inflammation, emphasizing the significance of cellular and pathological context in determining miRNA function.

Methionine aminopeptidase mediates the excision of N‐terminal methionine residues from ~40% of proteomic substrates in mammals [35]. Currently, two methionine aminopeptidases, METAP1 and METAP2, have been identified. The removal of N‐terminal methionine typically occurs during the early stages of protein folding and modification. Following excision of the initiator methionine, the neo‐N‐terminus may reveal specific functional amino acid sequences that facilitate protein–protein interactions and functional engagement, while concurrently providing structural prerequisites for subsequent modifications (e.g., N‐acetyltransferases or lipidation). This process is critical for protein maturation, structural stability, and functional competence [36]. Therefore, METAP2 is involved in diverse physiological and pathological processes, including cellular growth, angiogenesis, tumorigenesis, obesity, diabetes, and immunity. Ehlers et al. [37] discovered that small molecule inhibitors of METAP2 suppress endothelial cell proliferation, and METAP2 might be a potential target for antiangiogenesis. Lin et al. [38] discovered that METAP2 knockdown reduced VEGF secretion and lowered its mRNA and protein expression levels, leading to inhibition of angiogenesis. Additionally, abnormally high expression of METAP2 has been detected in various human tumors [39]. METAP2 is also related to inflammation. Zhang et al. [40] discovered that in a mouse model of Alzheimer’s disease, the METAP2 inhibitor BL6 suppressed the production of key proinflammatory molecules, including nitric oxide, iNOS, IL‐1β, and IL‐6. While METAP2 is established as an angiogenesis promoter in cancer, we demonstrated its pathogenic proapoptotic role in septic endothelium, where both genetic knockdown and miR‐377‐3p‐mediated inhibition significantly attenuated caspase‐3/Bax activation, restored BCL‐2/eNOS expression, and reduced IL‐8 secretion. This functional divergence may arise from context‐dependent substrate processing; while METAP2 likely supports the maturation of proteins required for rapid proliferation in cancer, it may preferentially process proapoptotic and proinflammatory proteins during sepsis. Furthermore, we discovered that METAP2 expression affected IL‐8 secretion rather than IL‐8 mRNA and intracellular IL‐8 under the stimulation of sepsis mouse serum. Given the canonical role of METAP2 in cotranslational protein processing, as well as our Co‐IP data demonstrating a physical interaction between METAP2 and IL‐8, we propose that METAP2 may facilitate IL‐8 export through aminopeptidase‐dependent pathways. This hypothesis warrants further validation through substrate‐profiling approaches, including liquid chromatography–tandem mass spectrometry.

IL‐8 is a biomarker for detecting the early phase of neonatal sepsis. Clinical studies have demonstrated significantly elevated IL‐8 levels in patients with sepsis [41]. Abrams et al. and Alsabani et al. [42, 43] demonstrated that IL‐8 induces sepsis‐associated neutrophil extracellular traps (NETs) formation through CXCR1/2‐mediated neutrophil chemotaxis and priming, as evidenced by ex vivo NET formation assays in patient‐derived neutrophils from intensive care units. Pharmacological inhibition of IL‐8 significantly attenuated NETosis, substantiating the chemokine’s pivotal role in driving NET formation through activation of the mitogen‐activated protein kinase pathway, a dominant signaling axis identified in septic patients. However, the regulatory mechanisms governing IL‐8 expression in sepsis remain incompletely understood. In this study, we demonstrated that serum from septic mice significantly induces IL‐8 secretion, underscoring the relevance of the septic microenvironment in endothelial activation. We identified the lncRNA SNHG5 as an upstream regulator that promotes IL‐8 secretion through a ceRNA mechanism. Genetic interference of either SNHG5 or its downstream target METAP2 substantially attenuated IL‐8 secretion, whereas supplementation with miR‐377‐3p mimics effectively reversed this suppression, confirming the functional interplay within this pathway. Therefore, our findings establish the SNHG5/miR‐377‐3p/METAP2 axis as a novel and critical regulatory circuit controlling IL‐8 secretion in sepsis. Furthermore, additional inflammatory markers, including TNF‐α, IL‐1α, IL‐1β, IL‐6, and IL‐10 [44, 45], were found to be significantly increased in the serum of septic mice. Notably, the increase in IL‐10, an anti‐inflammatory cytokine, likely represents a compensatory response by the host to counter‐regulate excessive inflammation during sepsis. These findings not only improve our understanding of the molecular basis of hyperinflammation in sepsis but also highlight potential therapeutic targets for mitigating endothelial dysfunction.

5. Conclusions

In summary, the expression level of SNHG5 was elevated in the coronary arteries of septic mice. SNHG5 knockdown attenuated the inflammatory injury of arterial endothelial cells, potentially by regulating miR‐377‐3p/METAP2/ IL‐8 (Figure 7).

Figure 7.

A schematic illustration of the proposed mechanism.

Author Contributions

All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by Tingzhi Deng, Ding Li, and Lihui Liang. The first draft of the manuscript was written by Yan Zou. The manuscript was revised by Ding Li. Funding acquisition was secured by Tingzhi Deng.

Funding

This work was supported by the Natural Science Foundation of Hunan Province (Grant 2023JJ60296).

Disclosure

All authors reviewed and approved the final manuscript.

Ethics Statement

All animal experiments were carried out strictly in accordance with international ethical guidelines and the National Experimental Animal Welfare Ethics. The experiments were approved by the Ethics Review Committee for Animal Welfare of Hunan Provincial People’s Hospital (Number 2025092).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting Information

Additional supporting information can be found online in the Supporting Information section.

Supporting information

Supporting Information Table S1: Primer sequences for target detection.

Deng, Tingzhi , Li, Ding , Liang, Lihui , Zou, Yan , SNHG5 Exacerbates Sepsis‐Induced Inflammatory Injury in Coronary Artery Endothelial Cells by Regulating METAP2‐Mediated IL‐8 Secretion, Mediators of Inflammation, 2026, 2519086, 15 pages, 2026. 10.1155/mi/2519086

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.