IL-38 attenuates vascular calcification by upregulating GPX3-mediated antioxidant defense via the PPAR-γ/NRF2 axis

Abstract

Vascular calcification is a pathological process commonly associated with cardiovascular diseases, diabetes, and renal insufficiency, driven by inflammation, oxidative stress, and endoplasmic reticulum stress. IL-38, a member of the IL-1 cytokine family, has shown anti-inflammatory, antioxidant, and anti-fibrotic effects in cardiovascular contexts. However, its role in vascular calcification remains unknown. This study demonstrated significantly reduced IL-38 expression in human calcified coronary tissues, patient plasma, and experimental models. IL-38 knockout mice exhibited aggravated vascular calcification, while treatment with recombinant IL-38 protein markedly suppressed calcification. RNA sequencing revealed that IL-38 alleviates oxidative stress by upregulating glutathione peroxidase 3 (GPX3). siRNA-mediated knockdown of GPX3 abolished the protective effects of IL-38, confirming its essential role as a downstream mediator. Further mechanistic studies established that IL-38 activates the PPAR-γ/NRF2 signaling axis to regulate GPX3 expression, as evidenced by pharmacological inhibition. These results identify IL-38 as a novel endogenous inhibitor of vascular calcification operating through the PPAR-γ/NRF2/GPX3 pathway, providing mechanistic insight and suggesting a potential therapeutic strategy.

Graphical abstract

IL-38 is revealed as a novel anti-vascular calcification molecule that alleviates calcification primarily by upregulating GPX3 and its mediated antioxidant effects through the PPAR-γ/NRF2 axis (Created in https://BioRender.com).

Supplementary Information

The online version contains supplementary material available at 10.1007/s00018-025-05990-5.

Introduction

Vascular calcification is defined as the ectopic mineralization of calcium phosphate within vascular tissue, commonly observed in various diseases associated with aging and metabolic dysfunction, such as atherosclerotic cardiovascular disease, chronic kidney disease, and diabetes [1, 2]. It is recognized as a significant risk factor for cardiovascular events, leading to increased morbidity and mortality in patients [3]. It also serves as a robust predictor of cardiovascular disease incidence and mortality, independent of other cardiovascular risk factors [4]. Vascular smooth muscle cells (VSMCs), the predominant cell type in the vascular wall, play a pivotal role in vascular calcification [5]. The reprogramming and transdifferentiation of VSMCs into an osteochondrogenic phenotype are key events driving vascular calcification. The osteogenic phenotypic switch of VSMCs is primarily characterized by the progressive downregulation of contractile markers (particularly α-SMA and SM22α) and the coordinated upregulation of osteochondrogenic transcription factors (Runx2, Sox9) and calcification-related proteins (BMP2, OPN, osteocalcin, ALP) [6]. Factors such as high phosphate, elevated calcium levels, oxidative stress, and reactive oxygen species (ROS) can promote the osteogenic transformation of VSMCs [7]. Despite significant advancements in recent years in understanding the molecular regulation of vascular calcification, the complexity of these mechanisms and their clinical translation have hindered the development of effective therapeutic strategies. As a result, finding effective treatments and novel intervention targets for vascular calcification is of significant importance.

Interleukin-38 (IL-38) is a newly identified member of the IL-1 family that exerts anti-inflammatory effects by competitively binding to the IL-36 receptor (IL-36R), IL-1 receptor 1 (IL-1R1), and IL-1 receptor accessory protein-like 1 (IL-1RAPL1) [8, 9]. A number of studies have demonstrated that IL-38 provides protective effects in different cardiovascular diseases. For instance, IL-38 has been found to reduce the injury of myocardial ischemia-reperfusion by inhibiting the activation of the NLRP3 inflammasome in cardiac fibroblasts [10]. Additionally, IL-38 suppresses macrophage accumulation and modulates inflammatory phenotypes, thereby inhibiting abdominal aortic aneurysm formation in mice [11]. In addition, recent studies reveal that IL-38 not only has anti-inflammatory properties but also exhibits antioxidant and anti-fibrotic effects [12]. These findings suggest that IL-38 may serve as a promising therapeutic target for cardiovascular diseases. However, the role of IL-38 in vascular calcification and its underlying mechanisms remain unexplored.

Numerous investigations confirm that oxidative stress is deeply involved in the pathogenesis of vascular calcification [13, 14]. A previous study demonstrated that increased ROS levels promote vascular calcification, whereas reducing ROS significantly inhibits osteogenic transdifferentiation of VSMCs [15]. GPX3, as a member of the glutathione peroxidase (GPXs) family that primarily functions to protect cells from oxidative damage, serves as a major ROS scavenger. GPX3 plays a crucial role in protecting the extracellular environment of tissues from oxidative injury by eliminating ROS [16]. Recent studies have shown that GPX3 alleviates renal fibrosis and renal ischemia-reperfusion injury through its antioxidant effects [17, 18]. Furthermore, GPX3 has been shown to reduce cardiac injury following myocardial infarction by upregulating Hif1α [19]. However, it has not been reported in relevant studies whether GPX3 and its antioxidant protective mechanisms also play a role in protecting against vascular calcification.

In this study, we used both in vivo and in vitro methods to investigate the beneficial effects of IL-38 in reducing vascular calcification. RNA sequencing analysis further clarified the underlying mechanisms, potentially offering new therapeutic targets for the treatment of vascular calcification.

Materials and methods

Patient and sample handling

The human samples and related data used in this study were approved by the Ethics Committee of the First Affiliated Hospital of Air Force Medical University (Ethics Approval Number: XJLL-KY-20252322). The committee is exempted from obtaining informed consent, and all procedures fully comply with the ethical standards stipulated in the Declaration of Helsinki.

Animal models

The experiments of animal were conducted in accordance with the ethical guidelines approved by the Animal Welfare and Ethics Committee of Air Force Medical University (Ethical Approval Number: 20240026). This study utilized 6 to 8-week-old male C57BL/6 mice from Air Force Medical University in Shaanxi, China, as well as IL-38 knockout (IL-38 KO) mice provided by Cyagen Biotechnology Co., Ltd. By using specific primers (F1: 5 ‘-CTCAAGATGATATTCTCCACTATG-3’); the tail DNA of (R1: 5 ‘-TCTCTCACACCTTTCTGAATTGCT-3’) was amplified by PCR for genotype identification, and verified by agarose gel electrophoresis and DNA sequencing. All animals are raised in specific pathogen-free (SPF) facilities at the Animal Experiment Center of Air Force Medical University. All mice completed an environmental adaptation period prior to the study, during which temperature was maintained at 25 °C and lighting followed a 12-hour circadian rhythm. They can freely obtain commercial rodent feed sterilized by cobalt-60 and drinking water sterilized by high pressure. Mice from the same group were randomly assigned to the experimental group, with 6 mice in each cage. A final sample size of n = 6 mice per experimental group was used for all subsequent analyses. This research plan has been approved by the Institutional Animal Care and Use Committee of the Air Force Medical University and adheres to the standards stipulated in the “Guidelines for the Care and Use of Laboratory Animals” of the National Research Council of China.

Vitamin D3-induced vascular calcification model

In the experimental protocol, both IL-38-deficient male mice (6–8 weeks old) and WT littermate controls were treated with either vitamin D3 (VitD3) (600,000 IU/kg, Sigma-Aldrich) dissolved in olive oil or control solution through subcutaneous injections for three consecutive days. Following a 7-day acclimation period on standard chow, animals were humanely sacrificed, and blood specimens were obtained. The aortic tissue was rapidly frozen in liquid nitrogen or fixed in formalin for further analysis. To evaluate the role of IL-38 in vascular calcification in vivo, another group of mice received recombinant IL-38 (rIL-38, MCE (HY-P72570)) supplementation. These mice were intraperitoneally injected with rIL-38 on the first day of treatment, once every other day until euthanasia.

Cell culture

Primary human aortic smooth muscle cells (hASMCs) were obtained from ETHEPHON and preserved in smooth muscle cell culture medium (Sciencell, 1101). All experiments were conducted using cells that had been passaged between 3 and 8 times. Calcification was induced in hASMCs by culture in high-glucose DMEM mineralization medium with 2% FBS supplementation, 10 mM β-glycerophosphate (β-GP; Sigma, G9422), and 4 mM CaCl2 (Sigma, C5670). To investigate the role of IL-38 in this process, rIL-38 was added to the calcified medium. The PPAR-γ inhibitor Oleuropein (HY-N0292) and the NRF2 inhibitor ML385 (HY-100523), used for mechanistic studies, were purchased from MCE. Throughout the experiment, the culture medium was refreshed every 48 h.

Western blot (WB) analysis

Tissue and cell lysates were prepared by adding protease and phosphatase inhibitors to RIPA lysis buffer. Nuclear protein extraction was performed according to the instructions of the commercial kit (Beyotime, P0027). After centrifugation, the supernatant was harvested for protein quantification using the BCA method. Protein samples of equal concentration were electrophoresed through 12% SDS-PAGE gels and transferred to 0.45 μm PVDF membranes. To reduce non-specific binding, the membrane was blocked with 5% skimmed milk at room temperature for 1 h. Subsequently, the primary antibody was incubated overnight at 4 °C, and then the enzyme-labeled secondary antibody was incubated at room temperature for 2 h. Protein bands were detected by enhanced chemiluminescence technology and images were obtained by chemiluminescence imaging system. Density analysis was conducted using ImageJ software to quantitatively analyze the spectral band intensity.

RNA extraction and quantitative real-time PCR (RT-qPCR)

Total RNA was isolated from the treated cells using Trizol reagent (plate number 1, Tiangen, China), following the manufacturer’s instructions. Next, first-strand cDNA synthesis was conducted using the 5× StarLighter Script RT All-in-One Mix (FOREVERSTAR, Beijing, China). Finally, quantitative PCR analysis was performed with the 2× StarLighter HP SYBR Green qPCR Mix (FOREVERSTAR, Beijing, China) on a real-time PCR system. All primers were synthesized by Tsingke Biotech (Beijing, China). PCR amplification was conducted using the following protocol: 3 min at 95 °C for initial denaturation, followed by 40 cycles of 10 s at 95 °C for denaturation and 30 s at 60 °C for annealing/extension. All primer sequences are detailed in the resource table. mRNA expression was determined by the ΔΔCT method using GAPDH as endogenous control.

Alizarin red S staining

Cells cultured in 12-well plates were washed twice with PBS and fixed at room temperature for 15 min with 4% paraformaldehyde (PFA). After washing twice with distilled water, stain with 2% Alizarin red S solution at room temperature for 30 min. After two more washes with distilled water, the calcified cells showed a red staining. The tissue specimens were fixed with 4% PFA, embedded in paraffin, and sectioned into thin slices 5 μm thick. After dewaxing and rehydration, the sections were stained with 2% Alizarin red S solution for 15 min. Then the stained sections were washed twice with distilled water and imaged using the Olympus VS2000 sliding scanner (Olympus, Tokyo, Japan).

Von kossa staining

Aortic specimens were fixed in 4% paraformaldehyde (PFA), paraffin-embedded, and sectioned at 5 μm thickness. Tissue sections underwent silver nitrate staining (5% solution, 30 min UV exposure) followed by treatment with 5% sodium thiosulfate (5 min). Counterstaining was performed using hematoxylin and eosin (H&E) for histological evaluation.

Alkaline phosphatase (ALP) activity assay

ALP activity was quantified using a commercial assay kit (Beyotime, P0321S) following the manufacturer’s instructions. Briefly, samples were homogenized in lysis buffer (Beyotime, P0013J) and centrifuged. The resulting supernatant was combined with the reaction working solution and incubated at 37 °C for 30 min. Reactions were terminated with stop solution, and absorbance was measured at 450 nm. Values were normalized to total protein concentration.

Calcium content measurement

According to the manufacturer’s instructions, the calcium content in tissues and cells was determined using the colorimetric assay kit (Beyotime, S1063S). The sample was lysed in the lysis buffer and centrifuged to obtain the supernatant. Incubate the supernatant with the reaction working solution at room temperature in the dark for 10 min. Read the absorbance at 450 nm using a microplate. For data normalization, the total protein concentration was determined by the BCA method, and the calcium content was expressed relative to the protein concentration.

Immunofluorescence (IF) staining

Tissue specimens were fixed with 4% PFA, embedded in paraffin, and 5 μm sections were prepared. These sections were subjected to standard dewaxing and hydration, and antigen-repaired in 95 °C EDTA buffer (pH 9.0) for 20 min. For cell samples, the cultures on confocal dishes were fixed with 4% PFA (15 min, RT) and washed with PBS. Then, block the tissue and cell preparations with QuickBlock™ buffer (Beyotime, P0260) at room temperature for 30 min, and incubate overnight with the primary antibody at 4 °C. After washing three times with PBS, incubate with a fluorescin-bound secondary antibody for 2 h (photoprotected), then stain with DAPI (1 µg/mL, 5 min), and seal with an anti-fluorescence quenching mounting medium. Fluorescence imaging was performed using a Leica STELLARIS 5 confocal microscope.

Enzyme-linked immunosorbent assay (ELISA)

The concentration of plasma IL-38 in clinical and experimental samples was determined using ELISA kits. Human plasma samples from patients with coronary artery calcification were tested using the Cusabio kit (EL011615HU). In contrast, plasma samples from VitD3 overloaded mice were tested using the Mlbio kit (mlC50322-1), following the manufacturer’s protocol. Absorbance is measured at a wavelength of 450 nm, and the sample concentration is calculated through the corresponding standard curve.

Detection of intracellular reactive oxygen species (ROS) levels

For reactive oxygen species (ROS) detection, cells were seeded in confocal dishes at a density of 5 × 10⁴ cells per well. Following PBS washing, the cells were treated with 10 µM DCFH-DA (1 mL per well) and incubated at 37 °C in the dark for 30 min. After extensive PBS washing, fluorescence images were acquired using a Leica STELLARIS 5 confocal laser scanning microscope.

Quantification of intracellular malondialdehyde (MDA) levels

For MDA quantification, cells were seeded in 12-well plates at 5 × 10⁴ cells/well. Following experimental treatments, cells were lysed, and the homogenate was centrifuged to isolate the supernatant. For the assay, 100 µL of supernatant was combined with 200 µL of commercial MDA detection reagent (Solarbio, BC0025), followed by incubation in a 95–100 °C water bath for 90 min. After cooling to ambient temperature, samples were centrifuged at 10,000×g for 10 min, and the optical density of the resulting supernatant was determined spectrophotometrically at 532 nm.

Quantification of superoxide dismutase (SOD) activity

According to the instructions of the SOD Activity Assay Kit (Beyotime, S0101S), after lysing the cells, the supernatant was collected by centrifugation. The supernatant was then mixed with the working solution and incubated at 37 °C for 30 min. The absorbance was measured at 450 nm, and the SOD activity was calculated accordingly. For data normalization, the total protein concentration was determined by the BCA method, and the SOD activity was expressed relative to the protein concentration.

RNA sequencing (RNA-seq) and analysis

Total RNA was extracted from the aortic tissue of mice, and mRNA was enriched using Oligo(dT)-attached magnetic beads. RNA sequencing was performed by Bepro Biotechnology Co., Ltd. on the Illumina sequencing platform. The raw reads obtained from sequencing were quality-controlled and aligned to the reference genome using HISAT2 software. Gene expression quantification was performed using HTSeq, which generated raw read counts mapped to each gene. To normalize for gene length and sequencing depth, we calculated FPKM (Fragments Per Kilobase of transcript per Million fragments mapped) values. FPKM represents the number of fragments per kilobase of transcript length per million mapped fragments, providing a standardized measure of gene expression levels.

Differential expression analysis between experimental groups was conducted using the DESeq2 algorithm. Differentially expressed genes (DEGs) were identified using stringent criteria requiring an absolute log2-fold change (|log2FC|) ≥ 1 and a Benjamini-Hochberg adjusted p-value (FDR) < 0.05. Genes meeting these criteria were classified as either up-regulated or down-regulated relative to the control group.

Functional annotation of the DEGs was performed through Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. These analyses aimed to identify significantly overrepresented biological processes, molecular functions, cellular components, and metabolic pathways among the DEGs. Cluster analysis and visualization of the results were conducted using the Pheatmap package in R software.

Small interfering RNA (siRNA) and transfection

The siRNA targeting GPX3 (hs-GPX3) and IL-38 (hs-IL-38) were custom-synthesized by Shanghai Hanao Biotechnology Co., Ltd. In the RNA interference experiment, we mixed 5 µL of 20 µM siRNA duplex with 200 µL of Opti-MEM medium to prepare the transfection mixture. Add 4.5 µL of RNAFit transfection reagent, vortex mix for 10 s, and incubate at room temperature for 10 min. Then, the obtained transfection complex was introduced into the cells and suspended in a complete medium without antibiotics. Six hours after transfection, replace the culture medium with fresh growth medium containing standard serum and antibiotics. The transfection efficiency was evaluated 48 h after transfection.

Statistical analysis

The data are expressed as mean ± SD. All the analyses were conducted without the researchers’ knowledge of the experimental conditions. The Shapiro-Wilk test was used to evaluate the normality of the data. For the comparison between the two groups, the parametric data were analyzed using the two-tailed Student’s t-test, and the non-parametric data were evaluated using the Mann-Whitney U test. For multiple group comparisons, normally distributed datasets were subjected to one-way or two-way ANOVA, followed by Bonferroni correction for post-hoc analysis. Non-parametric data were evaluated using Dunn’s multiple comparison test. Statistical significance was defined as p < 0.05. All analyses were conducted using GraphPad Prism 10.5 (GraphPad Software, CA) and SPSS 23.0 (IBM, IL).

Results

IL-38 level is decreased in coronary artery tissues and plasma of coronary artery calcification (CAC) patients

To investigate changes in IL-38 expression levels in the coronary artery tissues of patients with coronary artery calcification (CAC), we compared tissue samples from CAC and non-CAC groups. Based on coronary CT imaging criteria, patients with calcified plaques (CT value >130 HU and area >1 mm²) were assigned to the CAC group [20], while those without detectable calcification served as non-CAC controls. Our results showed that IL-38 protein expression was significantly decreased in the CAC group. Concurrently, the expression of the vascular smooth muscle contractile marker Calponin1 was also downregulated, whereas osteogenic-related markers such as BMP2, Runx2, and OPN were markedly upregulated (Fig. 1A and B). Consistent results were observed at the mRNA level (Fig. 1C). Furthermore, plasma IL-38 quantification demonstrated lower circulating IL-38 levels in CAC patients versus non-CAC subjects (Fig. 1D). Pearson correlation analysis confirmed a negative correlation between plasma IL-38 concentrations and Agatston scores in the CAC cohort (Fig. 1E).

Fig. 1.

IL-38 Expression is Decreased in Coronary Artery Tissues and Plasma of CAC Patients. (A) Representative Western blot results of IL-38, Calponin1 and osteogenic markers (BMP2, Runx2 and OPN) from coronary artery tissues. (B) Quantification of immunoblots (A) (n = 3). (C) Relative mRNA expression of IL-38 and osteogenic markers from coronary artery tissues (n = 3). (D) Plasma IL-38 levels in patients with control or CAC (n = 40). (E) Correlation between plasma IL-38 levels and Agatston score of CAC patients was analyzed using Pearson correlation coefficient analysis (n = 40). Statistical significance was evaluated via two-tailed unpaired Student’s t-test (B, BMP2, OPN, Calponin1; C, D), Mann-Whitney U test (B, Runx2, IL-38). All data were presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001

IL-38 expression is decreased in both In vivo and In vitro models of vascular calcification

To further validate the changes in IL-38 levels during calcification, we established a vascular calcification model in mice with VitD3 overload, immunofluorescence staining was used to examine mouse aortic tissue sections, we observed that compared to the control group, the expression levels of IL-38 and Calponin1 were significantly downregulated in the aortic tissues of VitD3-overloaded mice, while Runx2 expression was markedly upregulated (Fig. 2A-C). We then measured the protein and mRNA expression levels in the aortic tissues of both groups and obtained consistent results: IL-38 and the contractile marker Calponin1 were downregulated, whereas osteogenic-related markers BMP2, Runx2, and OPN were significantly upregulated (Fig. 2D-F). Additionally, we found that VitD3-overloaded mice had lower plasma IL-38 levels than the control group (Fig. 2G). In addition, we established an in vitro calcification model using human aortic smooth muscle Cells (hASMCs). Immunofluorescence analysis revealed that hASMCs cultured in calcification medium exhibited downregulated expression of IL-38 and Calponin1, while Runx2 expression was upregulated. Consistent with the in vivo findings, protein and mRNA expression analyses demonstrated the same trends (Fig. 2H-M).

Fig. 2.

IL-38 Expression is Decreased in both In vivo and In vitro Models of Vascular Calcification. (A) Representative images of Alizarin red staining, Von Kossa staining and immunofluorescence staining of IL-38 (Magenta), Runx2 (Red), Calponin1 (Green) and DAPI (Blue) in aortic sections, scale bar = 100 μm. (B) Quantification of Alizarin red staining and Von Kossa staining (A) (n = 6). (C) Quantification of immunofluorescence staining (A) (n = 4). (D) Relative mRNA expression of IL-38 and osteogenic markers from aortas (n = 6). (E) Representative Western blot results of IL-38, Calponin1 and osteogenic markers (BMP2, Runx2 and OPN) from aortas. (F) Quantification of immunoblots (E) (n = 6). (G) Plasma IL-38 levels in mice with control or overloaded VitD3 (n = 12). (H) Representative images of Alizarin red staining and immunofluorescence staining of IL-38 (Magenta), Runx2 (Yellow), Calponin1 (Green) and DAPI (Blue) in hASMCs with control or β-glycerophosphate (β-GP), scale bar = 50 μm. (I) Quantification of Alizarin red staining (H) (n = 6). (J) Quantification of immunofluorescence staining (H) (n = 4). (K) Relative mRNA expression of IL-38 and osteogenic markers from hASMCs (n = 6). (L) Representative Western blot results of IL-38, Calponin1 and osteogenic markers from hASMCs. (M) Quantification of immunoblots (L) (n = 6). Statistical significance was evaluated via two-tailed, unpaired Student’s t-test (B, C, D, BMP2, Runx2; F, G, I, J, K, M), Mann-Whitney U test (D, OPN, IL-38). All data were presented as mean ± SD. **p < 0.01, ***p < 0.001

IL-38 deficiency aggravates vascular calcification

To elucidate the role of IL-38 in vascular calcification, we generated IL-38 knockout (IL-38 KO) (Fig. S2A and B) mice and induced vascular calcification through subcutaneous injection of VitD3 (Fig. 3A). Histological sections of aortic tissues were stained with Alizarin red and Von Kossa, revealing that IL-38 deficiency alone did not cause spontaneous vascular calcification. However, compared to wild-type (WT) mice, IL-38 KO mice exhibited significantly aggravated calcification and enhanced calcium deposition following VitD3 administration (Fig. 3B and C). Subsequent quantitative analyses revealed that IL-38 knockout significantly increased alkaline phosphatase (ALP) activity and calcium content in calcified aortas (Fig. 3D and E). Protein expression further demonstrated that IL-38 deficiency upregulated the expression of BMP2, Runx2, and OPN in calcified aortic tissues (Fig. 3F and G).

Fig. 3.

IL-38 Deficiency Aggravates Vascular Calcification. (A) Schematic diagram of the in vivo experimental procedure. WT mice and IL-38 KO mice were subcutaneously injected with VitD3 for 3 consecutive days, followed by 7 days of normal diet feeding before sacrifice. (B) Representative Alizarin red staining and Von Kossa staining of aortas from WT and IL-38 KO mice with control or overloaded VitD3, scale bar = 100 μm. (C) Quantification of Alizarin red staining and Von Kossa staining (A) (n = 6). (D) Quantification of ALP activity of aortas (n = 6). (E) Quantification of calcium content in aortas (n = 6). (F) Representative Western blot results of osteogenic markers (BMP2, Runx2 and OPN) from aortas. (G) Quantification of immunoblots (F) (n = 6). Statistical significance was evaluated via two-way ANOVA followed by the Bonferroni multiple-comparison test (C, D, E, G). All data were presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001

IL-38 ameliorates vascular calcification

We administered recombinant IL-38 (rIL-38) to VitD3-overloaded mice to further confirm the role of IL-38 in vascular calcification. Based on the protocol established by Han et al. [21], we intraperitoneally injected rIL-38 (1 mg/kg) every other day concurrently with subcutaneous VitD3 administration until sacrifice (Fig. 4A). Histological analysis using Alizarin red and Von Kossa staining demonstrated that exogenous rIL-38 supplementation significantly ameliorated vascular calcification and reduced calcium deposition in aortic tissues (Fig. 4B and C). Furthermore, rIL-38 administration reduced both ALP activity and calcium content in arterial tissues (Fig. 4D and E). Protein expression analysis showed consistent results, with rIL-38 significantly downregulating osteogenic markers BMP2, Runx2, and OPN in aortic tissues of VitD3-overloaded mice (Fig. 4F and G).

Fig. 4.

IL-38 Ameliorates Vascular Calcification both in vivo and In vitro. (A) Schematic diagram of the in vivo experimental procedure. WT mice were subcutaneously injected with VitD3 for 3 consecutive days. Meanwhile, the experimental group received rIL-38 injections every other day (EOD) until sacrifice. All mice were maintained on a normal diet for 7 days before euthanasia. (B) Representative Alizarin red staining and Von Kossa staining of aortas from control mice or VitD3-overloaded mice with or without rIL-38 treatment, scale bar = 100 μm. (C) Quantification of Alizarin red staining and Von Kossa staining (A) (n = 6). (D) Quantification of ALP activity of aortas (n = 6). (E) Quantification of calcium content in aortas (n = 6). (F) Representative Western blot results of osteogenic markers (BMP2, Runx2 and OPN) from aortas. (G) Quantification of immunoblots (F) (n = 6). (H) Schematic diagram of the in vitro experimental procedure. HASMCs were cultured in medium containing β-glycerophosphate (β-GP), with the experimental group supplemented with rIL-38. (I) Representative Alizarin red staining of β-GP-induced calcification in hASMCs with or without rIL-38 treatment. (J) Representative images of immunofluorescence staining of Runx2 (Magenta), Calponin1 (Green) and DAPI (Blue) in hASMCs, scale bar = 50 μm. (K) Quantification of ALP activity of hASMCs (n = 6). (L) Quantification of calcium content in hASMCs (n = 6). (M) Representative Western blot results of osteogenic markers (BMP2, Runx2 and OPN) from hASMCs. Statistical significance was evaluated via two-way ANOVA followed by the Bonferroni multiple-comparison test (C, D, E, G, K, L). All data were presented as mean ± SD. ***p < 0.001

To further elucidate the role of IL-38 in vascular calcification through in vitro experiments, we added rIL-38 to the culture medium simultaneously with the switch to calcification-inducing medium. Based on previous research [21], rIL-38 was administered at a concentration of 100 ng/mL, with medium replacement and rIL-38 supplementation performed every two days until the endpoint (Fig. 4H). Alizarin red staining of hASMCs demonstrated that rIL-38 treatment significantly reduced calcium deposition (Fig. 4I and Fig. S3A). Immunofluorescence staining revealed that rIL-38 downregulated Runx2 expression while upregulating Calponin1 levels (Fig. 4J and Fig. S3B). Furthermore, rIL-38 markedly decreased both ALP activity and calcium content in hASMCs (Fig. 4K and L). Protein expression analysis yielded results consistent with in vivo findings (Fig. 4M and Fig. S3C). Collectively, these in vitro and in vivo results confirm the protective role of IL-38 in vascular calcification.

IL-38 suppresses oxidative stress in vascular calcification

Previous studies have found that IL-38 can reduce oxidative stress levels in skeletal muscle cells [12], however, whether IL-38 exerts an anti-vascular calcification effect by alleviating oxidative stress in vascular calcification has not yet been reported. Therefore, to explore the specific mechanism by which IL-38 alleviates vascular calcification, we performed RNA transcriptome sequencing (RNA-seq) on aortic tissues from WT and IL-38 KO mice with a VitD3 overload model. The sequencing results showed 585 genes were downregulated and 45 genes were upregulated (Fig. S4A and B). Interestingly, in the molecular function (MF) of GO enrichment analysis, we found that IL-38 KO significantly downregulated the antioxidant activity pathway (Fig. 5A and B). To further verify whether IL-38 reduces oxidative stress levels in vascular calcification, we used the DCFH-DA fluorescent probe to detect ROS levels in hASMCs cultured in calcification medium. The results showed that supplementation with rIL-38 significantly reduced ROS levels in hASMCs (Fig. 5D and E). Additionally, we measured the intracellular content of malondialdehyde (MDA), assayed the activity of superoxide dismutase (SOD), and detected the expression level of 4-hydroxynonenal (4-HNE) by immunofluorescence staining. Consistent results showed that rIL-38 significantly reduced MDA content and 4-HNE expression levels, while concurrently upregulating SOD activity (Fig. 5F-I).

Fig. 5.

IL-38 Suppresses Oxidative Stress in Vascular Calcification. (A) Gene ontology (GO) analysis showing major related molecular function signaling pathways in aortas from WT mice and IL-38 KO mice with VitD3 treatment. (B) Gene set enrichment analysis (GSEA) showing enrichment of genes involved in antioxidant activity. (C) Heatmap of down-regulated genes in antioxidant activity signaling pathway (n = 4). (D) Representative images of DCFH-DA staining of intracellular ROS in hASMCs. (E) Quantification of DCFH-DA staining of intracellular ROS in hASMCs (n = 4). (F) Quantification of malondialdehyde (MDA) of hASMCs (n = 6). (G) Representative images of immunofluorescence staining of 4-HNE (Red), α-SMA (Green) and DAPI (Blue) in hASMCs, scale bar = 50 μm. (H) Quantification of immunofluorescence staining (G) (n = 4). (I) Quantification of superoxide dismutase (SOD) activity of hASMCs (n = 6). (J) Relative mRNA expression of GPX3 from hASMCs (n = 6). (K) Representative Western blot results of GPX3 from hASMCs. (L) Quantification of immunoblots (K) (n = 6). Statistical significance was evaluated via two-way ANOVA followed by the Bonferroni multiple-comparison test (E, F, H, I, J, L). All data were presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001

After confirming that IL-38 reduces oxidative stress levels in vascular calcification, we further investigated the molecular mechanisms by which IL-38 alleviates oxidative stress during this pathological process. Among the antioxidant activity pathway significantly downregulated in IL-38 KO mice tissue, a total of nine genes were downregulated (Fig. 5C and Fig. S4C). We examined the expression levels of these nine genes both in vivo and in vitro and found that only the mRNA level of GPX3 was significantly upregulated after supplementation with rIL-38, while the other genes were not (Fig. 5J, Fig. S4D and E, Fig. S5A-C). Subsequently, we detected the protein expression level of GPX3, and the results showed that supplementation with rIL-38 upregulated the protein expression level of GPX3 (Fig. 5K and L).

IL-38 alleviates vascular calcification via GPX3-mediated antioxidant defenses

To determine whether IL-38 alleviates vascular calcification through GPX3-mediated antioxidant effects, we transfected hASMCs with GPX3-specific siRNA (Fig. 6A and Fig. S6A-C). Alizarin red staining revealed that GPX3 knockdown exacerbated calcification in hASMCs, increased calcium deposition, and antagonized IL-38’s inhibitory effect on calcification (Fig. 6B and Fig. S6D). Immunofluorescence staining demonstrated that GPX3 knockdown upregulated Runx2 expression and abolished IL-38’s protective effects (Fig. 6C and D). Both ALP activity and calcium content in hASMCs were significantly increased after siRNA transfection, and supplementation with rIL-38 failed to rescue this phenotype (Fig. 6E and F). Western blot analysis confirmed these findings, showing that GPX3 knockdown significantly upregulated osteogenic markers (Fig. 6G and Fig. S6E). Subsequent evaluation of oxidative stress indicators in hASMCs showed that GPX3 knockdown markedly increased intracellular ROS levels, 4-HNE expression, and MDA content, while eliminating IL-38’s ability to mitigate oxidative stress (Fig. 6H-L). These results show that GPX3 and its mediated antioxidant defense constitute a crucial downstream mechanism through which IL-38 alleviates vascular calcification.

Fig. 6.

IL-38 Alleviates Vascular Calcification via GPX3-mediated Antioxidant Defenses. (A) Schematic diagram of the In vitro experimental procedure. HASMCs with siGPX3 or siNC treatment were cultured in medium containing β-GP, with the experimental group supplemented with rIL-38. (B) Representative Alizarin red staining of β-GP-induced calcification in hASMCs. (C) Representative images of immunofluorescence staining of Runx2 (Magenta), Calponin1 (Green) and DAPI (Blue) in hASMCs, scale bar = 50 μm. (D) Quantification of immunofluorescence staining (C) (n = 4). (E) Quantification of ALP activity of hASMCs (n = 6). (F) Quantification of calcium content in hASMCs (n = 6). (G) Representative Western blot results of osteogenic markers (BMP2, Runx2 and OPN) from hASMCs. (H) Representative images of DCFH-DA staining of intracellular ROS in hASMCs. (I) Quantification of DCFH-DA staining of intracellular ROS in hASMCs (n = 4). (J) Quantification of MDA of hASMCs (n = 6). (K) Representative images of immunofluorescence staining of 4-HNE (Red), α-SMA (Green) and DAPI (Blue) in hASMCs, scale bar = 50 μm. (L) Quantification of immunofluorescence staining (K) (n = 4). Statistical significance was evaluated via two-way ANOVA followed by the Bonferroni multiple-comparison test (D, E, F, I, J, L). All data were presented as mean ± SD. **p < 0.01, ***p < 0.001

IL-38 alleviates vascular calcification by activating the PPAR-γ/NRF2 axis to enhance GPX3 expression

To further investigate how IL-38 regulates GPX3, we found that the PPAR signaling pathway was significantly enriched in KEGG enrichment analysis of RNA-seq data (Fig. 7A and Fig. S7A). We examined the expression levels of PPARs, including PPAR-α, PPAR-δ, and PPAR-γ. The results showed that PPAR-γ was significantly upregulated by rIL-38 in calcified hASMCs, while no notable changes were observed in PPAR-α or PPAR-δ (Fig. 7B and Fig. S7B). To verify whether PPAR-γ is the key mediator through which IL-38 regulates GPX3, we used the PPAR-γ inhibitor Oleuropein. According to previous study, Oleuropein (100 µM) [22] was added to the hASMCs culture medium simultaneously with rIL-38 (Fig. 7C). Oleuropein significantly inhibited the upregulation of GPX3 by rIL-38 and increased the protein expression levels of osteogenic markers (Fig. 7D and Fig. S7C). Alizarin red staining, ALP activity, and calcium content assays all showed that Oleuropein suppressed the anti-calcific effects of IL-38 (Fig. 7E–G and Fig. S7D). Furthermore, Oleuropein also aggravated oxidative stress in hASMCs, as indicated by significantly increased levels of ROS, MDA, and A-HNE (Fig. 7H–K and Fig. S7E).

Fig. 7.

IL-38 Alleviates Vascular Calcification by Activating the PPAR-γ/NRF2 Axis to Enhance GPX3 Expression. (A) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed downregulated signaling pathways. (B) Representative Western blot results of PPARs (PPAR-α, PPAR-δ and PPAR-γ) from hASMCs. (C) Schematic diagram of the in vitro experimental procedure. HASMCs with Oleuropein or DMSO treatment were cultured in medium containing β-GP, with the experimental group supplemented with rIL-38. (D) Representative Western blot results of GPX3 and osteogenic markers (BMP2, Runx2 and OPN) from hASMCs. (E) Representative Alizarin red staining of β-GP-induced calcification in hASMCs. (F) Quantification of ALP activity of hASMCs (n = 6). (G) Quantification of calcium content in hASMCs (n = 6). (H) Representative images of DCFH-DA staining of intracellular ROS in hASMCs. (I) Quantification of DCFH-DA staining of intracellular ROS in hASMCs (n = 4). (J) Quantification of MDA of hASMCs (n = 6). (K) Representative images of immunofluorescence staining of 4-HNE (Red), α-SMA (Green) and DAPI (Blue) in hASMCs, scale bar = 50 μm. (L) Representative images of immunofluorescence staining of NRF2 (Red), α-SMA (Green) and DAPI (Blue) in hASMCs, scale bar = 20 μm. (M) Representative Western blot results of total NRF2, nuclear NRF2 and cytoplasm NRF2 from hASMCs. Statistical significance was evaluated via two-way ANOVA followed by the Bonferroni multiple-comparison test (F, G, I, J). All data were presented as mean ± SD. ***p < 0.001

Nuclear factor erythroid 2-related factor 2 (NRF2), a key transcription factor in the antioxidant stress response pathway, regulates the transcription and expression of various antioxidant genes. We found that supplementation with rIL-38 significantly upregulated the protein expression level of NRF2 in the nucleus of calcified hASMCs, but this effect was inhibited by Oleuropein (Fig. 7M and Fig. S7F). Consistent with this, immunofluorescence results showed that IL-38 enhanced the nuclear translocation of NRF2, which was suppressed by Oleuropein (Fig. 7L). To clarify the regulatory role of NRF2 on GPX3, we used the NRF2 inhibitor ML385 (5 µM) [13]. The results demonstrated that ML385 downregulated the protein expression of GPX3 in calcified hASMCs (Fig. S7G and H). These results indicate that IL-38 regulates GPX3 and its mediated antioxidant effects through the PPAR-γ/NRF2 axis.

Discussion

Vascular calcification has been identified as a risk factor for cardiovascular events, increasing patient morbidity and mortality [3]. In this study, we identified a novel anti-vascular calcification molecule, IL-38. Through comprehensive analyses that included human tissue specimens, in vivo mouse models, and in vitro cellular experiments, we consistently observed a significant decrease in IL-38 expression under calcified conditions. By constructing IL-38 KO mice and exogenously supplementing rIL-38, we comprehensively validated the protective role of IL-38 in the vascular calcification process through both in vivo and in vitro experiments. Based on RNA-seq results and in vitro experimental validation, we determined that IL-38 can reduce oxidative stress levels in vascular calcification. By integrating RNA-seq, siRNA interference, and pharmacological inhibition approaches, we further elucidated the mechanistic pathway through which IL-38 alleviates vascular calcification. Specifically, IL-38 activates the PPAR-γ/NRF2 axis, leading to the upregulation of GPX3 and subsequent enhancement of antioxidative effects. Our research suggests that IL-38 could be a potential therapeutic target for vascular calcification.

IL-38, officially named IL-1F10, belongs to the IL-1 family [23]. It is primarily expressed in immune organs and some non-immune tissues, such as the heart and placenta [24]. Recent studies have shown that this molecule plays a protective role in various cardiovascular diseases. It ameliorates myocardial ischemia-reperfusion injury by inhibiting the IL-1R8/SYK axis, thereby reducing NLRP3 inflammasome activation in fibroblasts [10]. It can also modulate dendritic cells (DCs) phenotypes, suppress inflammatory responses, and attenuate cardiac remodeling after myocardial infarction [25]. In vascular contexts, IL-38 has been shown to counteract abdominal aortic aneurysm formation by reducing macrophage accumulation in the aortic wall and modulating inflammatory phenotypes [11]. It also mitigates inflammation and combats obesity-related atherosclerosis via the SIRT6/HO-1 axis [26]. In addition, while Erlinda The et al. discovered that IL-38 exerts protective effects in aortic valve interstitial cells by inhibiting NLRP3 to exert anti-inflammatory actions and alleviate aortic valve calcification [27], our study reveals a distinct mechanism. We found that supplementation with rIL-38 inhibits osteogenic phenotypic switching in human aortic vascular smooth muscle cells and attenuates vascular calcification primarily through a previously unreported antioxidant mechanism. Our research confirms that IL-38 plays a crucial protective role in the pathological process of vascular calcification.

Oxidative stress, as one of the well-established drivers of vascular calcification, primarily promotes this process by altering phosphate balance [28], inducing osteogenic differentiation of vascular smooth muscle cells [29], exacerbating inflammation [30], causing DNA damage [31], and triggering pathological extracellular matrix remodeling [32]. In this study, through RNA-seq analysis and in vitro experimental validation, we found that IL-38 significantly reduces oxidative stress levels in calcified hASMCs, suggesting that IL-38 may alleviate vascular calcification by counteracting oxidative damage. Currently, research on the interaction between IL-38 and oxidative stress is limited. A recent study reported that IL-38 can mitigate insulin resistance in hyperlipidemic skeletal muscle cells by suppressing STAT3-mediated signaling and oxidative stress via the PPARδ/SIRT1 axis [12]. Our study may provide a novel therapeutic target for reducing oxidative stress in vascular calcification.

GPX3, a glycosylated selenocysteine-containing protein and member of the GPX family, possesses antioxidant properties by catalyzing the reduction of glutathione (GSH) to oxidized glutathione (GSSG), which effectively mitigates hydrogen peroxide-induced cellular damage [33]. In this study, we identified GPX3 as one of the downregulated genes in antioxidant pathways based on RNA-seq results. Previous studies have demonstrated that GPX3 exerts protective effects in particular cardiovascular diseases. It alleviates ischemic myocardial injury post-myocardial infarction by regulating the LSD1/Hif1α axis [19], while GPX3 deficiency promotes a prothrombotic state and vascular dysfunction, thereby enhancing platelet-dependent arterial thrombosis [33]. Additionally, a clinical study revealed that GPX3 is associated with the incidence of hypertension and coronary artery disease, as well as the severity of coronary atherosclerosis [34]. In vitro experiments demonstrated that supplementation with rIL-38 significantly upregulated GPX3 levels. Furthermore, upon transfection with GPX3-specific siRNA, both the antioxidative stress and anti-vascular calcification effects of IL-38 were abolished. These findings confirm that GPX3 is a crucial mechanistic molecule mediating the antioxidative stress effects of IL-38 in vascular calcification. Additionally, GPX3 and its associated antioxidative damage mechanisms likely represent the specific molecular pathway through which IL-38 mitigates vascular calcification.

We further elucidated the specific molecular mechanism by which IL-38 regulates GPX3, namely through the PPAR-γ/NRF2 axis. PPAR-γ, a key transcription factor within the nuclear receptor superfamily, exhibits a broad range of biological functions, including anti-inflammatory and antioxidant effects, upon ligand-mediated activation [35, 36]. Previous studies found that IL-38 could inhibit oxidative stress levels in skeletal muscle cells via the PPARδ/SIRT1 axis [12]. In contrast, we did not obtain consistent results in this study; in calcified hASMCs, supplementation with rIL-38 did not upregulate the expression level of PPARδ but upregulated the expression level of PPAR-γ, and the antioxidant effect of IL-38 could be inhibited by a PPAR-γ inhibitor. We speculate that this discrepancy may be related to differences in cell types and disease models. Additionally, one study found that PPAR-γ could alleviate oxidative stress in bronchial epithelial cells exposed to cigarette smoke extract (CSE) by upregulating GPX3 [37], which is consistent with our experimental results. NRF2 is a central regulator of the cellular antioxidant response and one of the most important defense mechanisms against oxidative damage. Under homeostatic conditions, NRF2 binds to its inhibitory protein KEAP1 (Kelch-like ECH-associated protein 1) and is continuously ubiquitinated and degraded. When cells are stimulated by ROS, NRF2 accumulates and translocates into the nucleus, where it initiates the transcription of antioxidant genes by binding to the antioxidant response element (ARE) [38]. A recent study indicated that upregulating PPAR-γ in neurons during spinal cord injury promoted the nuclear translocation of NRF2, thereby alleviating oxidative stress and lipid peroxidation [39]. Consistent with the aforementioned studies, we found in this study that IL-38 promotes the nuclear translocation of NRF2, and this effect could be counteracted by a PPAR-γ inhibitor, indicating that PPAR-γ is an intermediate molecule through which IL-38 promotes NRF2 nuclear translocation. Subsequently, by using an NRF2 inhibitor, we confirmed that IL-38 regulates GPX3 and its mediated antioxidant effects via the PPAR-γ/NRF2 axis. While the synergistic protective effects of NRF2 and PPARγ activators against oxidative injury have been established in previous studies [40], our current work identifies IL-38 as a novel endogenous regulator that coordinately activates both PPAR-γ and NRF2 pathways within the context of vascular calcification, and demonstrates that the GPX3-mediated antioxidant defense mechanism serves as its crucial downstream pathway. This discovery represents an important extension to the existing knowledge in this research field.

While this study provides in-depth mechanistic insights into how IL-38 alleviates vascular calcification, these findings also highlight several considerations for future research. The research lacks investigation into the clinical translation of these molecular targets. Currently, there are no clinically approved specific agonists targeting IL-38 or GPX3. In contrast, several agonists are available for PPAR-γ and NRF2, such as thiazolidinediones (TZDs, e.g., pioglitazone and rosiglitazone) for PPAR-γ and dimethyl fumarate (an indirect NRF2 activator). These drugs have been used in both animal and clinical studies and have demonstrated anti-vascular calcification effects [41–44]. Although no specific GPX3 agonists exist, its activity can be enhanced indirectly through selenium supplementation or NRF2 upregulation. Regarding IL-38, most research remains at the mechanistic stage without established targeted therapies. Therefore, future studies should explore the use of human recombinant IL-38 protein or the development of novel small-molecule compounds targeting IL-38.

Conclusions

In conclusion, our study has uncovered a novel anti-vascular calcification regulatory mechanism, in which IL-38 alleviates vascular calcification by activating the PPAR-γ/NRF2 axis, which leads to the upregulation of GPX3, thereby enhancing antioxidant defense and ultimately attenuating calcification. This discovery not only provides new insights into the pathogenesis of vascular calcification but also offers important theoretical foundations and a potential therapeutic target for its treatment.

Supplementary Information

Below is the link to the electronic supplementary material.

Non-standard Abbreviations and Acronyms

IL-38

Interleukin-38

GPX3

Glutathione Peroxidase 3

PPAR-γ

Peroxisome Proliferator-Activated Receptor γ

NRF2

Nuclear factor erythroid 2-related factor 2

VitD3

Vitamin D3

rIL-38

Recombinant IL-38

β-GP

β-glycerophosphate

ALP

Alkaline Phosphatase

ROS

Reactive Oxygen Species

CAC

Coronary Artery Calcification

α-SMA

Alpha Smooth Muscle Actin

BMP2

Bone Morphogenetic Protein type 2

Runx2

Runt-related Transcription Factor 2

OPN

Osteopontin

hASMCs

Human Aortic Smooth Muscle Cells

4-HNE

4-hydroxynonenal

MDA

Malondialdehyde

SOD

Superoxide Dismutase

Authors’ contributions

Lei Zhu and Jun Cui designed and conceptualized the research; Qiang Wang, Bingliang Shang, Yujie Song, Bing Zhang, Wenlong Chen, Jialin Xin and Yang Cui performed the experiments and analyzed the data; Panpan Li, and Haitao Zhou established the animal model; Lanrui Jing, Wangxia Tang and Rui Liu collected the animal samples; Lei Zhu, Qiang Wang and Bingliang Shang wrote the manuscript; Yang Sun and Wei Yi revised the manuscript. All authors have reviewed the final version of the manuscript and approved its submission.

Funding

This work was supported by the National Natural Science Foundation of China (Grant No. 82170336), the Health Research and Innovation Capacity Enhancement Program of Shaanxi Province (2024TD-05), and Key Research and Development Program of Shaanxi Province (2023-ZDLSF-39).

Data availability

RNA sequencing dataset has been deposited in the National Genomics Data Center (NGDC). The dataset is now publicly accessible under accession number GSA: CRA032337.

Declarations

Ethics committee approval and patient consent

The human samples and related data used in this study were approved by the Ethics Committee of the First Affiliated Hospital of Air Force Medical University (Ethics Approval Number: XJLL-KY-20252322). The committee is exempted from obtaining informed consent, and all procedures fully comply with the ethical standards stipulated in the Declaration of Helsinki.

The experiments of animal were conducted in accordance with the ethical guidelines approved by the Animal Welfare and Ethics Committee of Air Force Medical University (Ethical Approval Number: 20240026).

Generative AI and AI-assisted technologies in the writing process

During the preparation of this work, the authors used Deepseek in order to check the accuracy of language usage and improve the readability of the manuscript. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.

Competing interests

The authors have no relevant financial or non-financial interests to disclose.