Simvastatin Alleviates ConA-Induced Autoimmune Hepatitis by Inhibiting CD4⁺ T Cell Activation via Calcium-NFATC3 Pathway

Abstract

Although simvastatin plays a crucial role in lipid management, tumor therapy and acute liver injury, its potential effects in autoimmune hepatitis (AIH) has received limited investigative attention. Our study demonstrated that in the ConA-induced AIH model, HMG-CoA reductase (HMGCR), the pharmacological target of simvastatin (SIM), was significantly upregulated in T cells, particularly in CD4⁺ T cells. Furthermore, our results showed that simvastatin treatment in ConA-induced AIH model reduced the level of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and alleviated liver injury. Flow cytometric analysis revealed that simvastatin treatment promoted CD4⁺ T cell apoptosis while significantly reducing the secretion of crucial inflammatory cytokines in vivo and vitro, including IL-17A, IL-6, IFN-γ, and TNF-α. To explore the underlying mechanisms, we performed transcriptome sequencing on the CD4⁺ T cells from mice treated with or without simvastatin. RNA-sequencing analysis revealed the involvement of the calcium signaling pathway and transcription factor NFATC3 in the regulation of CD4⁺ T cells. qPCR and flow cytometry analyses further confirmed that simvastatin exerted its therapeutic effects by suppressing the calcium signaling pathway and downregulating the expression of nuclear factor of activated T cells 3 (NFATC3). Collectively, our study demonstrates that simvastatin alleviates CD4⁺ T cell inflammatory responses in AIH through calcium-dependent signaling pathway.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10753-026-02462-1.

Introduction

Autoimmune hepatitis (AIH) represents a chronic hepatic inflammatory condition predominantly orchestrated by T lymphocytes, which is histologically marked by dense hepatic infiltration of lymphocytes, macrophages, and plasma cells [1, 2]. Although autoimmune hepatitis (AIH) is a rare disease worldwide, its incidence and prevalence are rising, and without timely treatment, it can progress to cirrhosis and liver failure [3, 4]. Published research suggests that the disruption of equilibrium between pro-inflammatory and anti-inflammatory mechanisms plays a pivotal role in the pathogenesis of AIH [5]. Concanavalin A (ConA)-induced liver injury has been a well-established experimental model for AIH. The ConA-induced murine model triggers CD4⁺ T cell activation in liver and spleen compartments, inducing IFN-γ and TNF-α emission that mediates subsequent hepatocellular damage [6]. Consequently, elucidating how distinct immune cell subsets, particularly CD4⁺ T cells, contribute to AIH pathogenesis may inform future diagnostic and therapeutic approaches.

The mevalonate pathway, governed by the rate-limiting enzyme HMG-CoA reductase (HMGCR), is essential for cholesterol biosynthesis and has emerged as a key nexus linking metabolism and immunity. Beyond sterol synthesis, pharmacological inhibition of this pathway by statins confers immunomodulatory effects, largely through suppression of protein geranylgeranylation and farnesylation. Augmentation of the mevalonate pathway promotes RAC1 post-translational modification, driving macrophage and fibroblast signaling and exacerbating pulmonary fibrosis [7]. Conversely, statins such as lovastatin can dampen T cell responses by reducing proliferation, calcium influx, and IL-2 secretion [8]. In addition, the intermediate metabolite mevalonate has been shown to be indispensable for the induction of trained innate immunity, acting via the IGF1-R–mTOR axis to promote histone modifications in inflammatory pathways [9].

By competitively inhibiting HMG-CoA reductase, statins reduce mevalonate production and cholesterol biosynthesis, and are widely used in managing hyperlipidemia and coronary atherosclerotic heart disease [10–13]. Beyond lipid-lowering, statins suppress T cell inflammatory activity via a KLF2-dependent mechanism and, by mitigating inflammation and oxidative stress, inhibit hepatic stellate cell (HSC) activation, slowing fatty liver–associated fibrosis [14]. Simvastatin has also shown efficacy in acute liver injury models, including acetaminophen (APAP)-induced injury and hepatic ischemia-reperfusion injury (HIRI) [15–17]. However, its therapeutic potential in AIH remains unclear.

In our study, we demonstrated that simvastatin ameliorated ConA-induced liver immune injury by suppressing CD4⁺ T cell survival and cytokine secretion through inhibition of calcium influx and downstream transcription factor NFATC3 expression. These results confirm that simvastatin alleviates ConA-induced AIH through suppressing the survival and function of CD4⁺ T cells via calcium signaling-NFATC3 pathway.

Method and Materials

Murine and Experimental Animal Models

Male wild-type (WT) C57BL/6 mice (6–8 weeks, 20–22 g) were purchased from Beijing Vital River Laboratory (Beijing, China) and maintained under specific pathogen-free conditions with a 12-h light/dark cycle and free access to food and water at Capital Medical University. A total of 58 mice were used in this study and randomly assigned to different experimental groups, with 4–5 mice per group. ConA-induced liver injury was established by tail vein injection of 15 mg/kg ConA (L7647, Sigma, USA) dissolved in PBS. All procedures were approved by the Institutional Animal Care and Use Committee of Capital Medical University (AEEI-2023-282).

Simvastatin Administration

Experimental mice were randomly assigned to three groups: normal control, ConA, and ConA plus simvastatin. Mice in the treatment group received simvastatin (HY-17502, MCE, USA) at 30 mg/kg via oral gavage once daily for 5 days prior to ConA injection, 1 h before injection, and 24 h post-injection, based on previous studies and preliminary optimization [18–21]. ConA group mice received vehicle at the same time points. All mice were euthanized 48 h after ConA injection for analysis [22, 23].

Isolation and Flow Cytometry Analysis of Intrahepatic Immune Cells

Intrahepatic immune cells were isolated by enzymatic digestion as previously described [24]. Surface marker expression was analyzed by flow cytometry using fluorochrome-conjugated antibodies. For intracellular cytokine staining, cells were fixed with Cyto-Fast™ Fix/Perm buffer and stained with antibodies such as anti-granzyme B or anti-TNF-α. Intranuclear antigens were detected using the True-Nuclear™ Transcription Factor Buffer Set followed by staining with antibodies like anti-Ki67. Annexin V staining was performed using Annexin V Binding Buffer (Supplementary Table S1). Samples were analyzed on a FACS Aria II cytometer, and data were processed with FlowJo software.

Intracellular Calcium Detection

Intracellular calcium concentration in CD4⁺ T cells was evaluated using the Fluo-4 AM fluorescent probe (HY-101896, MCE, USA). Briefly, the cells were resuspended in PBS at a density of 2 × 10⁶/ml and incubated with 1 µM Fluo-4 AM (prepared from a 1 mM stock solution) for 1 h at 37 ℃. After three washes with PBS, fluorescence intensity was analyzed by flow cytometry.

Histological and Immunohistochemical Staining, and Serum Aminotransferase Measurement

Liver samples from the largest lateral lobe were fixed, dehydrated, embedded, and sectioned for H&E staining (BA4027, Baso Diagnostics, China). Tissue sections were scanned using a PANNORAMIC Diagnostic Scanner (3DHISTECH, Switzerland). Five randomly selected fields per section were analyzed for necrotic areas. Quantification was performed using ImageJ (NIH, USA), and the average necrotic area per section was determined.

For immunohistochemical (IHC) analysis, 5-µm-thick liver sections were stained with an anti-mouse CD4 antibody (1:200, GB15064, Servicebio, China). CD4⁺ T cells were quantified using ImageJ software and expressed as cells/mm².

Blood samples were centrifuged at 800 g for 10 min to collect serum, and ALT (C009-2) and AST (C010-2-1) levels were measured with commercial assay kits (Nanjing Jiancheng Bioengineering Institute, China).

Stimulation of Murine Splenocytes and CD4⁺ T Cells in Vitro

Splenocytes from C57BL/6 mice were subjected to erythrocyte lysis, and CD4⁺ T cells were isolated using the MojoSort™ Mouse CD4⁺ T Cell Isolation Kit (480033, Biolegend, USA). Sorted CD4⁺ T cells or splenocytes (5 × 10⁵ cells/well) were seeded in 96-well U-bottom plates and treated for 72 h under different conditions: untreated control, 5 µg/mL ConA, 5 µg/mL ConA plus 20 µmol simvastatin, 5 µg/mL ConA plus 1 µM ionomycin (56092-82-1, Sigma), or 5 µg/mL ConA with 20 µmol simvastatin and 1 µM ionomycin. For experiments involving ionomycin, cells were first stimulated with ionomycin for 1 h, followed by the addition of ConA and simvastatin. Cells were cultured for a total of 72 h. After treatment, cells from each group were enumerated, and equal numbers of cells were harvested for subsequent flow cytometric analysis. For proliferation analysis, splenocytes were stained with CellTrace™ Violet (C34571, Thermo Fisher, USA) following the manufacturer’s protocol before treatment.

Western Blot Analysis

Jurkat T cells were stimulated for 72 h with either ConA alone or ConA in combination with simvastatin. Nuclear proteins were then extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents (78833, Thermo Fisher), and NFATC3 expression was analyzed by Western blotting. Equal amounts of protein (30 µg) were separated by 12% SDS-PAGE and transferred onto PVDF membranes. The following primary antibodies were used: NFAT4 Antibody (1:1000, AF6420, Affinity), Lamin B1 Antibody(1:1000, AF5161, Affinity), GAPDH antibody (1:10000, 10494-1-AP, Proteintech, USA), and DYKDDDDK tag monoclonal antibody (1:5000, 80801-2-RR, Proteintech), followed by appropriate HRP-conjugated secondary antibodies. Relative protein expression was quantified using ImageJ software.

Lentiviral Transduction Assay

Jurkat T cells were transduced with either an empty vector (Lv.Con467) or an NFATC3-overexpressing lentivirus (Lv.NFATC3), and overexpression was confirmed by Western blot and qPCR. Lv.Con467 and Lv.NFATC3 cells were then stimulated for 72 h with ConA alone or in combination with simvastatin. Apoptosis and cytokine production were subsequently assessed by flow cytometry.

Quantitative Real-Time PCR (qPCR) Analysis

Total RNA was extracted with FreeZol Reagent (R711, Vazyme) and were reverse transcribed to cDNA with PrimeScript™ RT Master Mix (RR036A, TAKARA, Japan). Real-time qPCR was conducted by the AB QuantStudio™ 1 Plus system (Applied Biosystems, USA) with SYBR Green Master Mix (Applied Biosystems). Amplicon expression in each sample was calculated using the 2^{^−ΔΔCt} method and normalized to Gapdh expression. The primers used in this study are listed in Supplementary Table S2.

RNA Sequencing Analysis

Mice pretreated with simvastatin via oral gavage were injected with ConA through the tail vein. After 48 h, they were euthanized, and liver tissue was enzymatically digested to isolate immune cells [24]. Cells were stained with fluorochrome-conjugated antibodies (CD45, 7-AAD, CD3, CD4, CD8a), and hepatic CD4⁺ T cells were purified by fluorescence-activated cell sorting (FACS). Transcriptome sequencing was performed by Annoroad Gene Technology, and the data were deposited in the NCBI GEO under accession number GSE301394.

Single-Cell RNA Sequencing Analysis

We analyzed the single-cell dataset GSE201006 from GEO. Raw scRNA-seq data were processed in R (v4.3.0) using the Seurat V5 package. After quality control (QC), dimensionality reduction was performed with PCA, and UMAP was applied for visualization. CD4⁺ T cells were identified by the co-expression of Cd3d, Cd3e, Cd3g, and Cd4, and were subsequently subdivided into Th1, Th2, Th17, and Treg subsets. In addition, CD4⁺ T cells were further stratified into Nfatc3⁺ and Nfatc3⁻ populations. Differentially expressed genes (DEGs) between the two subsets were determined using the Wilcoxon rank-sum test, followed by GO and KEGG enrichment analyses with the clusterProfiler (v3.18.1) package.

Quantification and Statistical Analysis

Prism 9.0 software and SPSS Statistics were performed to statistical analyses. The normality of dataset was assessed with the Shapiro-Wilk test. For normally distributed variables, a two-tailed t-test was applied for comparisons between two groups and one-way ANOVA was applied for multiple group comparisons. While non-normal data were assessed with Kruskal-Wallis tests. Values are presented as the mean ± SD and differences were considered significant at P < 0.05.

Results

HMGCR Expression is Significantly Upregulated in CD4⁺ T Cells During ConA-Induced Hepatic Injury

To systematically evaluate HMGCR expression across distinct immune cell subpopulations in ConA-induced AIH, we analyzed single-cell RNA sequencing (scRNA-seq) data from an open-access dataset (GSE201006) on immune cells infiltrating the liver during ConA-induced hepatitis. Following quality control, dimensionality reduction and clustering analysis, samples were segregated into 8 distinct clusters (Fig. 1A). As shown in Fig. 1B, the expression of Hmgcr significantly upregulated in intrahepatic T cells compared with other immune cells including NK/NKT cells and macrophages. Further subdivision of T cells into CD4⁺ and CD8⁺ subsets, we found that the expression of Hmgcr upregulated in both populations following ConA treatment, with more pronounced upregulation in CD4⁺ T cells (Fig. 1C).

Fig. 1.

HMGCR expression is significantly upregulated in intrahepatic CD4⁺ T cells following ConA stimulation. (A). Murine single-cell RNA sequencing data (GSE201006) from liver mononuclear cells of control and ConA were analyzed to confirm the expression of Hmgcr in distinct cell subpopulation. UMAP plots show 8 clusters: B cells, T cells, NK/NKT cells, macrophages, dendritic cells (DC), endothelial cell (EC), neutrophils and others. (B). Bubble diagram showing the expression of Hmgcr in distinct cell subpopulations. (C). Violin plots and statistical analysis showed the expression of Hmgcr in CD4⁺ T cells and CD8⁺ T cells. (D). Relative expressions of HMGCR in Kupffer cells, neutrophils, monocytes, CD3⁺ T, NK, and NKT cell were measured by flow cytometry in control and ConA groups. (E). Representative flow cytometry image (left) and statistical analysis (right) of the expression of HMGCR in CD4⁺ T cells and CD8⁺ T cells. n = 5 per group. *P < 0.05, **P < 0.01

To verify these findings, we employed the flow cytometry gating strategy presented in Fig. S1A to quantify HMGCR expression levels in distinct intrahepatic immune cell populations following ConA challenge in mice. Consistent with prior findings, our data demonstrated that HMGCR was highly expressed in T cells compared with Kupffer cells, neutrophils and monocytes following ConA treatment (Fig. 1D). Further analysis revealed that this alteration was predominantly in CD4⁺ T cells, whereas CD8⁺ T cells showed no significant alteration (Fig. 1E).

Simvastatin Treatment Ameliorates ConA-Induced Immune Hepatitis

Considering that simvastatin is an effective inhibitor for HMGCR, we investigated its therapeutic effects on ConA-induced liver injury. Specifically, mice were administered simvastatin (30 mg/kg/day) via oral gavage for 5 consecutive days prior to ConA challenge, with additional doses administered 1 h before and 24 h after ConA injection (Fig. 2A). We evaluated the degree of liver injury by measuring serum ALT and AST levels, along with histopathological examination of liver tissue. As shown in Fig. 2B, the levels of serum ALT and AST were significantly decreased in the ConA model following simvastatin treatment compared with those without simvastatin treatment. H&E staining also showed that simvastatin treatment attenuated hepatic inflammatory infiltration and necrotic areas in mice (Fig. 2C). We subsequently conducted flow cytometric analysis to measure immune cell subsets in liver. The results showed an increase in the proportion of intrahepatic CD4⁺ T cells and CD8⁺ T cells in the ConA model, while simvastatin treatment primarily reduced CD4⁺ T cells (Fig. 2D). Furthermore, immunohistochemical analysis confirmed that simvastatin treatment significantly reduced CD4⁺ T cell infiltration in the liver (Fig. 2E). Consistent with these observations, quantitative analysis of hepatic chemokine ligands showed markedly increased mRNA levels of Cxcl9, Cxcl10, and Cxcl11 in ConA-induced liver injury, which were substantially suppressed following simvastatin administration (Fig. 2F). Together, these results suggest that increased hepatic accumulation of CD4⁺ T cells in ConA-induced liver injury may, at least in part, be linked to elevated chemokine expression.

Fig. 2.

Simvastatin (SIM) suppressed hepatic CD4⁺ T cells both the survival and proinflammatory cytokine secretion in ConA model. (A). The strategy of simvastatin administration in the ConA-induced AIH model (The schematic diagrams were created using BioRender). (B). The levels of serum ALT and AST in mice of normal control, ConA (15 mg/kg) and ConA with simvastatin (SIM) administration. (C). Representation and quantification of hematoxylin and eosin (H&E) staining in liver paraffin sections of each group. Scale bars: 100 μm and 400 μm. (D). The proportions of intrahepatic CD4⁺ T, CD8⁺ T, NK and NKT cells among CD45⁺ cells in vivo were measured by flow cytometry. (E). Representative images and statistical analysis of CD4 staining in liver paraffin sections of each group. Scale bars: 100 μm and 400 μm. (F). Relative mRNA levels of chemokine ligands in liver tissues of each group. (G). The levels of apoptosis and proliferation in liver CD4⁺ T cells were detected by flow cytometry. (H). Bubble diagram showing Hmgcr expression across Th1, Th2, Th17, and Treg cell subsets. (I). Relative mRNA expression levels of cytokine genes in liver tissues. (J and K). The proportions of IFN-γ⁺, TNF-α⁺, IL-4⁺, IL-13⁺, IL-17 A⁺, RORγt⁺, and FOXP3⁺ CD4⁺ T cells were analyzed by flow cytometry. n = 4–5 per group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Flow cytometry showed that simvastatin increased apoptosis and reduced proliferation of CD4⁺ T cells (Fig. 2G). Single-cell RNA-seq data (GSE201006) revealed that HMGCR expression was upregulated in Th1 cells during ConA-induced liver injury (Fig. 2H). In mice, simvastatin suppressed Th1 and Th17 responses, as reflected by reduced IFN-γ, TNF-α, IL-2, IL-17 A, and RORγt, while enhancing Treg-associated markers IL-10 and FOXP3. Th2 cytokines (IL-4, IL-13) were not significantly affected (Fig. 2I–K). Collectively, these results indicate that simvastatin alleviates ConA-induced liver injury by inhibiting the survival and pro-inflammatory function of CD4⁺ T cells and by reshaping the balance of CD4⁺ T cell subsets.

Simvastatin Inhibits the Survival and Cytokine Secretion of CD4⁺ T Cells in Vitro

We designed experiments to further confirm the role of simvastatin on CD4⁺ T cell survival and function in vitro. Splenocytes were stimulated with ConA in vitro for 72 h, and HMGCR expression was assessed in CD4⁺ T cells, revealing significant upregulation upon stimulation (Fig. 3A). The cells were then treated with ConA in the presence or absence of simvastatin, followed by evaluation of CD4⁺ T cell apoptosis, proliferation, and cytokine secretion via flow cytometry. Consistent with our earlier observations in vivo, CD4⁺ T cell apoptosis was reduced following ConA stimulation, whereas simvastatin treatment promoted it (Fig. 3B). To evaluate CD4⁺ T cell proliferation, splenocytes were stained with Cell Trace Violet (CTV) and the percentage of CTV⁻ cells was assessed on day 3, 5 and 7. As expected, CD4⁺ T cells presented significantly proliferation following ConA challenge, which was markedly suppressed with simvastatin treatment (Fig. 3C and D). CD69, an early activation marker of lymphocytes, was markedly upregulated following ConA stimulation. However, CD4⁺ T cell activation was significantly attenuated in the simvastatin-treated group compared to the ConA-only group (Fig. 3E).

Fig. 3.

Simvastatinsignificantly suppressed ConA-mediated CD4⁺ T cell activation and proinflammatory cytokine secretion in vitro. (A). Splenocytes were stimulated with ConA (5 µg/mL) for 72 h in vitro. HMGCR expression in CD4⁺ T cells was analyzed by flow cytometry. Representative plots (left) and quantification (right) are shown. (B). Splenocytes were stimulated with ConA (5 ug/ml) and simvastatin (20 µmol) for 72 h in vitro. The proportions of Annexin V positive and caspase-3 positive CD4⁺ T cells were analyzed by flow cytometry. (C and D). After Cell Trace Violet (CTV) labeling, splenocytes were stimulated with either ConA alone or ConA plus simvastatin, and CD4⁺ T cell proliferation was assessed by flow cytometry on days 3, 5, and 7 after stimulation. (E). Representative flow cytometry plots (left) and statistical analysis (right) of CD69 in CD4⁺ T cells in vitro. (F and G). TNF-α, IFN-γ, IL-17A, IL-10, IL-6, and IL-2 positive CD4⁺ T cells were analyzed after in vitro stimulation with ConA or ConA plus simvastatin. n = 5 per group. *P < 0.05, **P < 0.01, ****P < 0.0001

Moreover, ConA treatment increased the levels of proinflammatory cytokines, including TNF-α, IFN-γ, IL-17A, IL-6, and IL-2, which were significantly suppressed with simvastatin stimulation. In contrast, the anti-inflammatory cytokine IL-10 exhibited an opposite trend, with levels increased following simvastatin treatment (Fig. 3F-G). Our findings indicate that ConA induces CD4⁺ T cell activation, as well as survival and proinflammatory cytokine secretion increase, while simvastatin potently inhibits these effects.

RNA-Sequencing Analysis of CD4⁺ T Cells with or Without Simvastatin Stimulation in Vitro

To mechanistically dissect how simvastatin regulates CD4⁺ T cells, we sorted liver CD4⁺ T cells from ConA model with or without simvastatin and conducted transcriptomic sequencing (Fig. 4A). Compared with the control group, the simvastatin-stimulated group displayed 1829 differentially expressed genes (DEGs) totally, including 1003 upregulated and 826 downregulated genes (Fig. 4B). To investigate the functional enrichment and annotation of DEGs, we performed GO and KEGG analysis. As shown in Fig. 4C, the DEGs were predominantly enriched in pathways related to calcium ion, inflammatory response, apoptotic process and activated T cell proliferation. Correspondingly, KEGG pathway enrichment analysis highlighted T cell receptor signaling pathway and interferon signaling. GSEA analysis showed that apoptotic signaling pathway was upregulated in simvastatin-treat mice derived CD4⁺ T cells compared with the ConA model, while inflammatory response, calcium dependent protein kinase activity and oxidative stress response were downregulated (Fig. 4D). Heatmap analysis revealed that simvastatin stimulation significantly increased the expression of pro-apoptotic genes, while genes associated with proliferation, inflammation and calcium ion pathway were downregulated (Fig. 4E). In summary, our results imply that simvastatin could inhibit the survival, inflammatory and calcium ion pathway of CD4⁺ T cells.

Fig. 4.

RNA sequencing analysis of CD4⁺ T cells with or without simvastatin treatment in vivo. (A). Hepatic immune cells were prepared from mice with or without simvastatin treatment. Following surface antibody staining, CD4⁺ T cells were sorted by flow cytometry and subjected to transcriptome sequencing (The schematic diagrams were created using BioRender). (B). Volcano plot showed all DEGs identified in CD4⁺ T cells stimulated with or without simvastatin (absolute fold change ≥ 1.2 and p value < 0.05). Significantly differentially upregulated genes and downregulated genes were respectively represented by red dots and blue dots. (C). GO and KEGG pathway analysis were performed on the significantly upregulated and downregulated genes in CD4⁺ T cells. (D). GSEA analysis was performed in hepatic CD4⁺ T cells from ConA with or without simvastatin treatment mice. (E). Heatmap showed genes related to apoptosis, proliferation, inflammation, and calcium ion pathway in hepatic CD4⁺ T cells from mice treated with ConA alone or with a combination of ConA and simvastatin. n = 3 per group.

Simvastatin Affects the Survival and Function of CD4⁺ T Cells via Calcium-NFATC3 Pathway

Our sequencing data showed that simvastatin treatment effectively influenced calcium ion pathway in CD4⁺ T cells (Fig. 4C-E). Calcium ion signals, especially intracellular Ca²⁺ concentration modulates the activation of T cells, subsequently affecting T cell cytokine secretion and adaptive immunity [25–27]. Based on these findings, we hypothesized that simvastatin may suppress the calcium ion influx to impact CD4⁺ T cell survival and function. First, we utilized calcium probe to detect intracellular Ca²⁺ concentration in CD4⁺ T cells. ConA treatment significantly increased intracellular calcium levels in CD4⁺ T cells both in vivo and in vitro, while simvastatin treatment markedly attenuated this elevation (Fig. 5A and B). Then, we treated CD4⁺ T cells with ionomycin, a calcium ionophore that effectively elevated intracellular Ca²⁺ concentration, and measured the activation and cytokine secretion to assess the influence of calcium signaling on CD4⁺ T cells. We observed that compared with both ConA/ionomycin treatment, simvastatin supplementation did not significantly alter the CD69 expression in CD4⁺ T cells (Fig. 5C) and cytokine secretion (Fig. 5D). Taken together, these findings establish that simvastatin suppresses ConA-induced CD4⁺ T cell activation via calcium signaling. Accumulated evidence indicates that calcium signaling govern the expression of nuclear factor of activated T cells (NFAT) transcription factors [28, 29]. Heatmap analysis showed that simvastatin treatment significantly downregulated the expression of NFATC1 and NFATC3 in CD4⁺ T cells. qPCR analysis showed that Nfatc3, but not Nfatc1, was significantly upregulated by ConA challenge and suppressed by simvastatin treatment (Figs. 5E and S2A), which was consistently observed in CD4⁺ T cells in vivo (Fig. 5F). NFATC3 activation depends on its dephosphorylation and nuclear translocation. Accordingly, nuclear extracts were analyzed by Western blot to assess NFATC3 activation. ConA stimulation markedly increased nuclear NFATC3 levels, whereas simvastatin treatment significantly reduced its nuclear accumulation, indicating effective inhibition of NFATC3 activation (Fig. 5G). Meanwhile, flow cytometry results displayed that simvastatin significantly suppressed the expression of NFATC3 rather than NFATC1 in CD4⁺ T cells. This inhibitory effect of simvastatin on NFATC3 was abolished by co-treatment with the calcium ionophore ionomycin, indicating that simvastatin inhibits NFATC3 activation via suppression of calcium influx (Figs. 5H and S2B).

Fig. 5.

Simvastatin regulates CD4⁺ T cell survival and cytokine secretion via calcium signaling and downstream transcription factor NFATC3. (A). Representative flow cytometry image (left) and statistical analysis (right) of intracellular calcium concentration in hepatic CD4⁺ T cells in vivo. (B). Representative flow cytometry image (left) and statistical analysis (right) of intracellular calcium concentration in CD4⁺ T cells following ConA with or without simvastatin treatment in vitro. (C). Representative flow cytometry image (left) and statistical analysis (right) of the expression of CD69 in CD4⁺ T cells of four groups in vitro: (i) ConA (5 µg/mL), (ii) ConA + simvastatin (20 µmol), (iii) ConA + ionomycin (1 µmol), and (iv) ConA + simvastatin + ionomycin. (D). The proportions of TNF-α, IFN-γ, IL-17A, IL-10, and IL-2 positive CD4⁺ T cells were statistically analyzed by flow cytometry in vitro. (E). The mRNA expression levels of Nfatc3 in CD4⁺ T cells after in vitro stimulation with ConA or ConA plus simvastatin were determined by real-time qPCR. (F). The NFATC3 expression in hepatic CD4⁺ T cells from the control, ConA, and ConA + simvastatin groups were detected by flow cytometry in vivo. (G). After 72 h of in vitro treatment with ConA or ConA plus simvastatin, nuclear proteins were extracted and NFATC3 expression was analyzed by Western blot relative to Lamin B1. (H). NFATC3 expression was measured by flow cytometry in CD4⁺ T cells of four groups in vitro. (I). Enrichment pathway analysis was conducted on the DEGs in Nfatc3 positive group compared with Nfatc3 negative groups in the single-cell RNA sequencing data (GSE201006). (J). Jurkat T cells were transduced with either empty vector (Lv.Con467) or NFATC3 overexpression (Lv.NFATC3) constructs. After 72 h of in vitro stimulation with ConA (5 µg/mL) or ConA plus simvastatin (20 µmol), Annexin V positivity and cytokine production were analyzed. (K). Mechanism by which simvastatin regulates CD4⁺ T cells via the calcium signaling pathway (The schematic diagrams were created using BioRender). n = 3–5 per group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

To investigate the impact of NFATC3 on CD4⁺ T cells, we analyzed scRNA-seq data (GSE201006) of liver mononuclear cells from ConA-induced liver injury. CD4⁺ T cells were identified based on co-expression of Cd3d, Cd3e, Cd3g, and Cd4, then stratified into Nfatc3 positive and Nfatc3 negative subsets. The analysis of DEGs was performed between two groups, followed by functional enrichment pathway analysis. Pathway enrichment analysis displayed that DEGs were predominantly enriched in pathways associated with T cell survival, activation, and inflammatory responses between two groups (Fig. 5I). We also observed that Nfatc3 positive group displayed significantly increased secretion of proinflammatory cytokines along with reduced apoptosis compared with Nfatc3 negative group (Fig. S3A). Finally, we generated Jurkat T cells overexpressing NFATC3, and overexpression was confirmed by Western blot and qPCR (Figs. S3B and S3C). Cells were stimulated for 72 h with ConA alone or ConA in combination with simvastatin. In control cells (Lv.Con467), simvastatin significantly reduced cell viability and cytokine secretion, whereas these inhibitory effects were abolished by NFATC3 overexpression. Moreover, compared with Lv.Con467 cells, NFATC3 overexpression (Lv.NFATC3) enhanced T-cell activation and cytokine production under simvastatin treatment (Fig. 5J). Collectively, these results indicate that simvastatin suppresses T-cell activity through NFATC3. Collectively, as evidenced above, simvastatin attenuates the proinflammatory response of hepatic CD4⁺ T cells in ConA-induced liver injury through reducing intracellular calcium concentration and subsequently suppressing NFATC3 expression.

Discussion

The liver, an immunologically complex and immune-privileged organ, harbors multiple immune cell populations that maintain microenvironmental homeostasis. Among them, CD4⁺ T cells act as the primary effectors in the ConA model. In our study, liver injury in male mice was induced via tail vein injection of ConA, establishing a model that partially recapitulates human AIH-like hepatic damage. Notably, AIH shows higher incidence in females, mirrored in mouse models, where female mice are more susceptible to ConA-induced liver injury, with elevated serum ALT/AST and more severe inflammation and necrosis. However, the ConA model in females exhibits greater inter-individual variability and higher mortality than males [30, 31]. Therefore, most studies, including ours, use male mice to investigate ConA-induced hepatitis [32]. Using this model, we observed that ConA stimulation induced CD4⁺ T cell hyperactivation and HMGCR upregulation, mediating calcium influx and downstream transcription factor expression.

Simvastatin inhibits cholesterol biosynthesis, promoting clearance of harmful cholesterol and protecting vascular endothelium [33], supporting its clinical use in hypercholesterolemia, coronary heart disease, hypertension, and cerebrovascular diseases [10–13, 34, 35]. Beyond lipid regulation, the mevalonate pathway is essential for immune homeostasis and tolerance, with its rate-limiting enzyme HMGCR implicated in T cell immunoregulation. As a potent HMGCR inhibitor, simvastatin suppresses T cell proliferation and cytotoxicity [36, 37]. HMGCR also plays a key role in liver injury pathogenesis [38]. Simvastatin has shown hepatoprotective effects in acute liver injury models, including APAP-induced hepatotoxicity and HIRI, by reducing oxidative stress, inflammatory infiltration, and cytokine production while enhancing protective mediators [15–17].

Clinically, simvastatin has been used to mitigate liver injury. In patients with severe portal hypertension who respond suboptimally to conventional β-blockers, combining β-blockers with simvastatin further reduces portal pressure, improves endothelial function, and attenuates pro-inflammatory cytokine release [39]. In the context of liver transplantation, prolonged cold ischemia increases the risk of ischemia-reperfusion injury and primary graft dysfunction. Randomized clinical trials have shown that simvastatin administration prior to transplantation or in organ preservation solutions can extend preservation time and reduce post-reperfusion injury [40]. These findings suggest potential clinical benefits of simvastatin in liver injury.

Statins and herbal/dietary supplements (HDS) have also been linked to drug-induced autoimmune-like hepatitis (DI-ALH), a self-limiting condition resembling AIH but differing in etiology and treatment, and distinct from the ConA-induced liver injury model used here [41]. In our study, simvastatin pre-intervention attenuated liver injury, reducing serum transaminases and necrotic areas while suppressing CD4⁺ T cell survival and the secretion of proinflammatory cytokines such as TNF-α and IFN-γ, with further analysis revealing modulation of CD4⁺ T cell subsets. However, the clinical efficacy of simvastatin in established AIH requires further investigation.

Calcium influx is a tightly regulated and essential event in T cell activation, functioning both as a marker and driver of signal amplification. Following antigen-MHC engagement, the TCR/CD3 complex initiates phosphorylation cascades that release Ca²⁺ from intracellular stores and trigger extracellular influx. This Ca²⁺ release activates downstream pathways and transcription factors such as NFAT and NF-κB [26, 42]. Statins inhibit HMGCR and thereby lower intracellular calcium via the Rho–PLC–calcium axis [43]. Using the calcium indicator Fluo-4 AM, we observed that ConA markedly increased calcium levels in CD4⁺ T cells, while simvastatin attenuated this effect. However, when calcium was artificially elevated by ionomycin, simvastatin no longer significantly affected ConA-induced apoptosis, activation, or cytokine secretion. These results indicate calcium influx is a key mediator of simvastatin’s immunomodulatory effects.

Mechanistically, the Ca²⁺–calcineurin–NFAT signaling pathway is a well-established regulator of CD4⁺ T cell activation and differentiation [44, 45]. Increasing evidence indicates that calcium signaling contributes to activation and differentiation of CD4⁺ T cells, particularly toward Th1 and Th17 subsets, through selective engagement of NFAT family members and associated feedback mechanisms [46]. Consistent with this, NFATC3 has been linked to inflammatory CD4⁺ T cell responses and Th1-related transcriptional programs [47, 48]. In this study, we found that simvastatin markedly suppressed ConA-induced calcium influx and NFATC3 expression in CD4⁺ T cells. Restoration of calcium influx counteracted the suppressive effects of simvastatin, resulting in increased NFATC3 expression and enhanced production of Th1- and Th17-associated cytokines. Similarly, enforced NFATC3 expression largely reversed the inhibitory effects of simvastatin on T cell activation and cytokine production. These results suggest that simvastatin attenuates pathogenic CD4⁺ T cell responses, at least in part, by targeting the calcium–NFATC3 axis.

Overall, our work systematically elucidates the underlying mechanism by which simvastatin modulates ConA-induced CD4⁺ T cell activation and inflammatory cytokine secretion through the calcium–NFATC3 signaling pathway. We identified simvastatin directly regulates calcium signaling pathway and downstream transcription factors in CD4⁺ T cells, uncovering novel therapeutic targets for intervention in ConA-induced acute liver injury (Fig. 5K).

Conclusion

In summary, this study demonstrates that simvastatin pretreatment alleviated ConA-induced liver injury. Meanwhile, we revealed that simvastatin inhibited the inflammatory by suppressing CD4⁺ T cells. The underlying mechanism was simvastatin-mediated inhibition of calcium signaling pathway and the expression of downstream transcription factor NFATC3, thereby suppressing CD4⁺ T cell proliferation, activation and cytokine secretion. These findings not only provide new evidence for the immunomodulatory role of simvastatin but also offer novel strategy for improving immune intervention strategies in AIH.

Supplementary Information

Below is the link to the electronic supplementary material.

Author Contributions

All authors have made contributions to this study and approved the manuscript for submission. Xiyu Wang participated in performing the research, analyzing the data, and initiating the original draft of the article. Tianhang Long, Longyang Zhou, Nan Xu, Peiyang Fang, Buer Li, Haozhe Xu participated in performing the research. Guangyong Sun supervised the studies. Dong Zhang and Hua Jin established the hypotheses, designed experiments, supervised the studies, and reviewed and edited the manuscript.

Data Availability

Raw data of deidentified RNA-seq in our work have been deposited at Gene Expression Omnibus (GEO) database under accession number GSE301394 .The following secure token has been created to allow review of record GSE301394 while it remains in private status: mxabaikyxnkvjyh.

Declarations

Competing Interests

The authors declare no competing interests.