Diacerein improves liver fibrosis, steatosis, and atherosclerosis in ApoE knockout mice
Jie-Eun Lee, Isom Jin, Jung-Jae Lee, Jisu Jung, Jee-In Lee, Bo-Rahm Kim, Tae Jung Oh, Yun Kyung Lee, Sung Hee Choi
TL;DR
Diacerein reduces liver damage and atherosclerosis in mice with a genetic predisposition to these conditions.
Contribution
This study demonstrates diacerein's dual anti-inflammatory and anti-fibrotic effects in a mouse model of metabolic disorders.
Findings
Diacerein reduced liver fat accumulation and collagen fibers in a dose-dependent manner.
Atherosclerotic plaque burden decreased with diacerein treatment, along with reduced pro-inflammatory cytokine expression.
The drug improved liver fibrosis and steatosis in high-fat diet-fed apoE knockout mice.
Abstract
Non-alcoholic fatty liver disease and atherosclerosis may share a common pathogenesis involving chronic IL-1β-induced inflammation. We aimed to evaluate the efficacy of diacerein, an IL-1 pathway inhibitor, in improving liver fibrosis, steatosis, and atherosclerosis in apolipoprotein E-knockout (apoE k/o) mice. ApoE k/o mice fed a high-fat diet (HFD) were divided into three groups based on diacerein dosage. Liver fat accumulation and fibrosis severity were compared across groups, along with changes in the expression of genes related to lipid metabolism and fibrosis. Atherosclerotic burden in the aorta was evaluated via en face analysis, and the related signaling pathway was verified in vitro. Diacerein treatment reduced the amount of collagen fibers and fat accumulation in the liver in a dose-dependent manner as well as fibrosis-related gene expression. Atherosclerotic plaque burden in…
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Figure 8- —National Research Foundation of Korea
- —Seoul National University
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Taxonomy
TopicsLiver Disease Diagnosis and Treatment · Liver physiology and pathology · Adipokines, Inflammation, and Metabolic Diseases
Introduction
Metabolic dysfunction–associated steatotic liver disease (MASLD), the most common chronic liver disease globally, includes conditions ranging from simple steatosis to metabolic dysfunction–associated steatohepatitis (MASH) and liver fibrosis. Recent meta-analyses estimate the global prevalence of MASLD at approximately 32% [1] with projections rising to over 55.4% by 2024 [2] due to associations with obesity and aging. A proportion of patients with fatty liver may develop progressive fibrosis and, eventually, cirrhosis, a marker of poor prognosis and increased mortality [3]. Due to the increasing morbidity associated with fatty liver and the high mortality rate of liver cirrhosis, prevention and early treatment of MASLD are necessary [4].
Recent studies suggest that IL-1 family cytokines are primary contributors to the inflammatory response in non-alcoholic fatty liver, and their activation alters the signaling pathways involved in insulin and lipid metabolism [5]. IL-1β, a pivotal cytokine in chronic inflammation, is involved in hepatic triglyceride (TAG) accumulation by activating hepatocyte steatosis pathways [6] and increasing the expression of other inflammatory cytokines, IL-6 and TNF-α [7], leading to localized liver inflammation as well as a chronic inflammatory state, eventually causing liver fibrosis through the activation of hepatostellate cells (HSCs) [8].
Patients with MASLD have a markedly higher risk of cardiovascular disease (CVD), with a higher incidence of risk factors such as visceral obesity, insulin resistance, type 2 diabetes (T2DM), dyslipidemia, and hypertension than the general population [9] Atherosclerosis is a chronic low-grade inflammatory disease of the arterial wall [10–12], and the IL-1 pathway contributes to its pathogenesis. Several anti-atherosclerotic drugs, including those that inhibit inflammatory pathways such as the IL-1 pathway, have been investigated; however, only a few drugs are currently available in clinical practice [13].
According to the results of the Canakinumab Anti-inflammatory Thrombosis Outcome Study, directly reducing inflammation with a monoclonal antibody against IL-1β (canakinumab) reduces cardiovascular events independent of LDL-C [14]. Anakinra, an IL-1 receptor antagonist, reduced atherosclerotic burden in apolipoprotein E [15], improved endothelial function and coronary flow reserve, and reduced the levels of inflammatory biomarkers in a small clinical trial involving rheumatoid arthritis patients [16]. A two-week treatment with anakinra in STEMI patients showed improvement in acute inflammation and potential prevention of new onset heart failure [17]. However, a pooled analysis of these clinical trials showed no significant differences in death, recurrent MI, or stroke between the control and therapy groups [17].
IL-1 inhibitors have also been evaluated for MASLD treatment; however, none are approved for clinical use. Clinical trials of anakinra (NCT04072822) [18] and canakinumab (NCT03775109) in alcoholic hepatitis are ongoing, but no positive results have yet been reported.
Diacerein, another IL-1β pathway inhibitor, has been used clinically for over 20 years to treat osteoarthritis, making it a promising candidate for the treatment of liver steatosis, fibrosis, and atherosclerosis. In the present study, we investigated the efficacy and underlying mechanism of diacerein in improving liver steatosis, fibrosis, and atherosclerosis using ApoE-knockout mice.
Materials and methods
Reagents
Diacerein (Artrodar 50 mg) was purchased from Myungmoon Pharm. Co., Ltd. (Seoul, Korea). TNF-α was acquired from Prospec-Tany Technogene (Rehovot, Israel). Antibodies for phosphorylated p65, phosphorylated p38, p38, phosphorylated ERK1/2, ERK, phosphorylated JNK, and JNK were sourced from Cell Signaling Technology (MA, USA). Antibodies for α-SMA and β-actin were obtained from Sigma-Aldrich (MO, USA), anti-CD68 from Abcam (Cambridge, UK), and anti-p65 from Santa Cruz Biotechnology (TX, USA). Lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA) were from Sigma-Aldrich (MO, USA).
Animal experiments
All animal care and experimental procedures were approved by the Seoul National University Bundang Hospital Institutional Animal Care and Use Committee (BA1511-188/070–01). All animal experiments complied with the Guide for Experimental Animal Research of the Laboratory for Experimental Animal Research, Clinical Research Institute, Seoul National University Bundang Hospital, Republic of Korea.
To ensure reproducibility and enhance statistical power, two independent experimental cohorts were conducted under identical environmental and dietary conditions. Male ApoE knockout mice on a C57BL/6 J background (Jackson Laboratory, ME, USA) were housed with free access to food and water under a 12-h light/dark cycle. At 8 weeks old, male mice were randomly divided into three groups: (1) control (normal saline, 154 mmol/l NaCl), (2) diacerein 50 mg/kg, and (3) diacerein 80 mg/kg. Randomization of ApoE knockout mice into treatment groups was performed using a computerized random number generator.
Data from the two cohorts were pooled for final analysis after confirming that baseline metabolic and molecular profiles were consistent between batches. The final sample sizes for the pooled analysis were n = 9 for the control, n = 11 for the 50 mg/kg group, n = 11 for the 80 mg/kg group (total N = 31).
They were fed a standard chow diet pretreated with diacerein for 4 weeks, followed by an atherogenic diet containing 40 kcal% fat, 1.25% cholesterol, and 0.5% sodium cholic acid (D12109; Research Diets Inc., NJ, USA) for 12 weeks with diacerein treatment at 50 mg/kg or 80 mg/kg. Normal saline (control) and diacerein were administered daily via oral gavage. Body weight and food intake were monitored weekly. All mice were sacrificed under sodium pentobarbital anesthesia. The aorta, heart, liver, and white adipose tissue were harvested and snap frozen in liquid nitrogen for further analysis.
Histological analysis of lipid accumulation
To measure aortic atherosclerosis lesions, the aortic root was dissected longitudinally and subjected to the en face method with Oil Red O staining. Additionally, to evaluate hepatic steatosis, fresh-frozen liver tissues were embedded in OCT compound and sectioned at a thickness of 7–10 μm. These sections were stained with Oil Red O (Sigma-Aldrich, MO, USA) to visualize and quantify neutral lipid accumulation in the liver. Section images were analyzed using an Olympus BX51 imaging system (Olympus, Tokyo, Japan). All lipid-stained areas (aortic plaques and hepatic lipid droplets) were quantified using ImageJ software (NIH, Bethesda, MD, USA) to ensure precise and consistent measurements. Atherosclerotic plaque area was expressed as a percentage of the entire area of the aorta, while hepatic lipid accumulation was expressed as a percentage of the total liver section area. The protocol for plaque fibrotic staining was performed as described previously [19].
Immunohistochemistry (IHC)
For histological analysis, white adipose tissue, heart, and liver tissues were fixed in 10% neutral formaldehyde for paraffin embedding (H&E, Masson’s trichome, and Sirius Red staining) or snap-frozen for frozen section (Oil Red O staining). Paraffin sections were prepared and stained with H&E. Macrophages in white adipose tissue sections were detected using an anti-CD68 antibody (1:500). Liver collagen content and collagen fibers were determined using Masson Trichrome and Sirius Red histochemistry as previously described [20]. Immune-stained section images were observed with an Axiophot microscope (Carl Zeiss AG, Feldbach, Switzerland) and captured using an Axiocam color camera (Zeiss, Feldbach, Switzerland). Total triglycerides were measured with the Infinity triglycerides reagent (Thermofisher Scientific, Waltham, USA) following the manufacturer’s instructions.
Enzyme-linked immunosorbent assay (ELISA)
Liver samples were collected and homogenized in protein extraction buffer with protease inhibitors. The samples were then centrifuged for 10 min at 4 °C. IL-1β and cytokine levels were measured using an Elisa Duo Set Kit (R&D System, Minneapolis, MN, USA).
In vitro cell culture
Human umbilical vein endothelial cells (HUVEC) and Rat Aortic Smooth Muscle Cells (RAoSMC) were obtained from Lonza (Basel, Switzerland) and Bio-bud (Seoul, South Korea), respectively. HUVECs were cultured in Endothelial cell Growth Medium 2 (EGM2) (Lonza, Basel, Switzerland) and Rao SMC in high glucose Dulbecco's Modified Eagle's Medium (DMEM) (Gibco, NH, USA) containing 10% FBS (Invitrogen, CA, USA) and 1% penicillin/streptomycin. Mouse 3T3-L1 cells, rat hepatoma cell McA-RH7777, and human hepatic stellate cell line LX-2 were obtained from ATCC (VA, USA) and cultured in high glucose DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. All assays were performed in 6-well cell culture plates (SPL Life Science, Gyeonggi-do, South Korea). Once cells reached 80% confluence, conditioned media with or without diacerein were applied to the cells for 24 h.
Atherosclerosis in vitro model
To induce inflammation in vitro, conditioned media were prepared by differentiating monocytes into macrophages. The human monocytic leukemia cell line THP-1 was cultured in Roswell Park Memorial Institute (RPMI) 1640 (Gibco, NH, USA) containing 10% FBS and 1% penicillin/streptomycin at 37 °C with 5% CO_2_ atmosphere (5.5 × 10^6^/well). THP-1 cells were differentiated by PMA treatment for 16 h, then incubated for another 24 h in RPMI 1650 without PMA. Differentiated THP-1 cells (macrophages) were stimulated with LPS and TNFα for 6 h, after which culture media were collected as conditioned media. To examine the anti-inflammatory effect of diacerein in an in vitro model of atherosclerosis, HUVECs, RAo-SMCs, and 3T3-L1 cells were used. These cells were incubated in growth media (containing 20% of conditioned media) with or without diacerein for 24 h, then harvested for further analysis.
Metabolic dysfunction-associated streatotic liver disease (MASLD)/liver fibrosis in vitro model
LX-2, an immortalized human cell line, was used for in vitro experiments. We evaluated the effect of diacerein on LX-2 cells cultured in a low-amino acid medium to create an environment for LX-2 cells similar to that of hepatic stellate cells (HSCs) in a cirrhotic liver. After incubating for 24 h, LX-2 cells were transferred to a low-amino acid medium and starved for an additional 24 h. Cells were then treated with diacerein for 1 and 6 h before adding TGF-β1 (5 ng/ml). After overnight incubation with TGF-β1, cells were harvested for further analysis.
RNA preparation and RT-qPCR
Total RNA was extracted from frozen tissue samples or cells using TRIzol (Ambion, CA, USA). Reverse transcription was performed using the High Capacity cDNA Reverse Transcription kit (Thermo Scientific, CA, USA). SYBR Green reactions were set up with the SYBR Green PCR Master mix (Enzynomics, Korea) and 10 pM primers, following the manufacturer’s instructions, and run on a ViiA7 applied biosystems system(Thermo Scientific, CA, USA). Relative mRNA levels were calculated using the comparative CT method and normalized to β-Actin or GAPDH. Expression levels of mRNA were displayed relative to controls, as specifically detailed for each experiment. Real-time PCR results are represented as the mean ± SD from at least three independent experiments, each performed in duplicate or triplicate.
Western blot analysis
Total proteins were extracted using cell lysis buffer (Cell signaling, MA, USA), and protein concentration was determined with the BCA protein assay reagent (Thermo, Waltham, MA, USA). Proteins were separated on 8 to 10% SDS-PAGE and transferred to nitrocellulose membranes (Millipore, Burlington, MA, USA). Membranes were blocked in 5% bovine serum albumin (Millipore, Burlington, MA, USA) at room temperature for one hour before adding primary antibodies. The membranes were incubated with primary antibodies at 4 ℃ overnight and with secondary antibodies at room temperature for 2 h. Protein bands were visualized using an enhanced chemiluminescence solution (Enzynomics, South Korea), and band densities were standardized to loading controls using Image J software (NIH, Bethesda, MD, USA).
Human gene-disease association
Differentially expressed genes identified following Diacerein administration were analyzed for disease associations using the DisGeNET database (https://disgenet.com/) through the Enrichr platform (https://maayanlab.cloud/Enrichr/). Diseases were filtered based on an adjusted p-value below 0.05. From the top 20 associated diseases, those related to liver and metabolic disorders were selected for further analysis.
For comparative gene expression analysis, RNA-sequencing (RNA-seq) data from healthy controls were obtained from the Genotype-Tissue Expression (GTEx) project (GTEx Analysis V8, liver tissue; https://gtexportal.org/home/downloads/adult-gtex/bulk_tissue_expression). Additionally, gene expression data from 94 non-alcoholic fatty liver disease (MASLD) patients were sourced from the public dataset GSE174478. Both gene expression profiles were normalized to transcripts per kilobase million (nTPM). Subsequently, genes downregulated by Diacerein administration were analyzed to assess their expression patterns in MASLD patients compared to healthy controls.
To evaluate the potential of these downregulated genes as blood-based diagnostic markers, protein expression levels in serum were investigated using the Serum Protein Risk Stratification Score database (GSE251855). This analysis examined the expression of the identified proteins in blood based on the severity of metabolic liver diseases, including Metabolic Associated Fatty Liver Disease (MAFLD) and Metabolic Associated Steatohepatitis (MASH).
Statistical analysis
Data represent at least three independent experiments and are expressed as mean ± standard error of the mean. Statistical significance was determined using one‐way analysis of variance (ANOVA) with Tukey’s post-hoc analysis for multiple group comparisons. Values of two-sided P < 0.05 were considered statistically significant. Statistical analyses were conducted using SPSS Statistics for Windows, version 24.0 (IBM, NY, USA).
Results
Interleukin 1β inhibitor and fatty liver fibrosis
IL-1β inhibitor attenuated liver inflammation and fibrosis in in vitro models of liver fibrosis
TGF-β1 treatment activates the IL-1β pathway in LX-2 cells. Treatment of LX-2 cells with TGF-β1 resulted in a flattened cell morphology (Fig. 1A). Diacerein treatment reduced the mRNA expression of caspase-3, a key mediator of the IL-1β pathway, whereas the expression levels of SMAD2/4 were not significantly affected. IL-6 expression showed a decreasing trend following diacerein treatment, although the change did not reach statistical significance (Fig. 1B). Next, we compared the mRNA expression of genes associated with liver fibrosis and found that αSMA and fibronectin (FN1) were significantly upregulated by TGF-β1 treatment, whereas pretreatment with diacerein inhibited this upregulation (Fig. 1C). Another gene family linked to fibrosis, Col1A1 and Col3A1, showed similar results, with their expression reduced by diacerein treatment (Fig. 1C). Thus, inhibiting the IL-1β pathway with diacerein can reduce the expression of genes associated with liver fibrosis.Fig. 1IL-1β inhibitor attenuated liver inflammation and fibrosis in in vitro models of liver fibrosis. a Morphological changes of LX-2 cells were observed under light microscope images (100 × magnification). mRNA levels of b liver inflammation related genes and c fibrosis related genes in LX-2 cells after pretreatment with diacerein 20 μM for 1 h and 6 h then 5 ng/ml TGF-β1 overnight. All data represent the mean ± S.E. from 3 independent experiments **, p < 0.01; ***, p < 0.005; ****, p < 0.001 versus without pretreatment of diacerein group, as determined with a one-way ANOVA. All qRT-PCR data were normalized to the mRNA levels of GAPDH
IL-1β inhibitor reduced hepatic lipid accumulation
To evaluate the effects of diacerein on fatty liver, ApoE-knockout mice were fed an atherogenic diet. H&E staining showed decreased lipid droplet size in the liver with diacerein administration at 50 mg/kg and 80 mg/kg compared to that in the controls (Fig. 2A). For a quantitative comparison, total TAG levels in liver tissue were measured, revealing a significant reduction in TAG and IL-1β levels after diacerein administration (Fig. 2B).Fig. 2. Effect of IL-1β inhibitor on liver steatosis. a Representative H&E staining in the liver of atherogenic diet-fed ApoE^−/−^ mice (n = 4–7), and representative Oil red O staining and quantification for lipid accumulation in the liver of atherogenic diet-fed ApoE^−/−^ mice (n = 8, 6, 5). b Liver triglyceride and IL-1β quantification in atherogenic diet-fed ApoE^−/−^ mice (n = 9, 10, 11 and n = 9, 9, 9). c mRNA expression of lipolysis-related genes in the livers of atherogenic diet-fed ApoE^−/−^ mice (n = 3–7). d Protein expression of carboxyesterase (CES) 1 and PPARγ coactivator (PGC) 1α in the liver of atherogenic diet-fed ApoE^−/−^ mice (n = 4–6). e Expression of enzymes related to fat metabolism in the liver. e-1) lipolysis, e-2) fatty acid oxidation, e-3) adipogenesis, e-4) lipogenesis. All data represent the mean ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.005; ****, p < 0.001 versus the Control group, by Student’s t test. All qRT-PCR data were normalized to the mRNA levels of GAPDH
To explore the mechanism underlying TAG reduction in liver tissue, we examined changes in the expression of enzymes involved in TAG metabolism. Adipose TAG lipase, responsible for TAG hydrolysis, showed a modest increase in the 50 mg/kg diacerein-treated group compared with the control, which was not statistically significant (Fig. 2C). In the 80 mg/kg group, lipid accumulation was significantly reduced, accompanied by a significant increase in adipose TAG lipase expression (Fig. 2C). We also examined the mRNA expression of long-chain fatty acyl-CoA dehydrogenase, involved in fatty acid oxidation, which was increased after diacerein treatment (Fig. 2C). This increase, however, was not significant relative to the reduction in TAG levels. Furthermore, the mRNA and protein levels of carboxylesterase 1, involved in ester hydrolysis, and those of PGC1α, associated with lipid metabolism in the liver, showed significant changes (Fig. 2D). No changes were observed in the expression of enzymes involved in lipid metabolism in the liver (Fig. 2E); additionally, the expression of Plin 1, HSL (involved in lipolysis) (Fig. 2E-1); PPARα, PGC1α (involved in fatty acid oxidation) (Fig. 2E-2); PPARγ, SREBP1c (involved in adipo/lipogenesis) (Fig. 2E-3); and adiponectin aP2 (involved in lipogenic target genes) (Fig. 2E-4) remained unaltered.Fig. 3IL-1β inhibitor attenuated liver fibrosis and downregulated the expression of related genes. a IL-1β reduced the deposition of collagen fibers in the liver tissue, as confirmed by Masson’s trichrome and Sirius red staining from atherogenic diet-fed ApoE^−/−^ mice (n = 4–7). b Hepatic mRNA expression involved in liver fibrosis from atherogenic diet-fed ApoE^−/−^ mice (n = 4–6). All data represent mean ± S.E. *, p < 0.05; ** p < 0.01; *** p < 0.005 versus Control group by One-way ANOVA. All qRT-PCR data were normalized to the mRNA levels of GAPDHFig. 4IL-1β inhibitor attenuated NLRP3 inflammasome activation in in vitro models of atherosclerosis. a The mRNA expression of NLRP3 and related genes, IL-1β, IL-6, and MMP9I, in HUVECs after culture with Control (NA), conditioned media (CM), diacerein 10, 25, 50, and 100 μM. b mRNA levels of IL-1β, ICAM1 and phosphorylated p65 in RAo-SMCs after culture with Control(NA), conditioned media (CM), diacerein 10, 25, 50, and 100 μM. c Gene expression levels of IL-1β, ICAM1, phosphorylated p65 and TNFα in 3T3-L1 cells after culture with Control (NA), conditioned media (CM), diacerein 10, 25, 50, and 100 μM. d-e NLRP3 pathway related protein expression of phosphorylated p65, phosphorylated p38, and pJNK in HUVECs and RAo-SMCs after culture with Control (NA), conditioned media (CM), diacerein 10, 25, 50, and 100 μM. f Protein expression of phosphorylated p65 in 3T3-L1 cells after culture with Control (NA), conditioned media (CM), diacerein ` 10, 25, 50, and 100 μM. All data represent the mean ± S.E. of values from 2–5 independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.005; ****, p < 0.001 versus CM group, by a one-way ANOVA. All qRT-PCR data were normalized to the mRNA levels of GAPDH. All western blot data were normalized to the band intensity of β actin, determined using ImageJ (NIH)
IL-1β inhibitor attenuated liver fibrosis and downregulated the related genes
Masson’s trichrome and Sirius red staining revealed decreased collagen fibers in the diacerein-treated group compared with those in the control (Fig. 3A). Figure 3B shows that the mRNA expression of TIMP1, αSMA, Col1a1, and Col3a1 was significantly decreased after diacerein treatment compared with that in the control group. The mRNA expression of MMP-2 was also significantly reduced in the diacerein-treated group, along with the expression of Cxcl10, another pro-fibrotic factor (Fig. 3B).
Interleukin 1ß inhibitor and atherosclerosis
IL-1ß inhibitor attenuated NLRP3 inflammasome activation in in vitro models of atherosclerosis
HUVECs were incubated with conditioned media (CM) pretreated with LPS and TNFα from THP-1 cells. The levels of IL-1β and IL-6 were significantly increased compared with those in the controls without CM (Fig. 4A). Additionally, MMP9 was significantly induced by CM compared with the control without CM. Furthermore, IL-1β, IL-6, and MMP9 levels were significantly decreased in a dose-dependent manner after treatment with diacerein and CM (Fig. 4A). In RAoSMCs treated similarly to HUVECs, the mRNA levels of IL-1β, ICAM, and phosphorylated NK-κB (p65) were significantly increased compared with those in the control without CM (Fig. 4B). Moreover, the gene expression of IL-1β, ICAM, and phosphorylated NK-κB (p65) was significantly reduced in a dose-dependent manner by diacerein CM (Fig. 4B). The gene expression of IL-1β, ICAM, NF-κB (p65), and TNFα also significantly increased with CM and was subsequently reduced in a dose-dependent manner in 3T3-L1 cells (Fig. 4C). Moreover, phosphorylated NK-κB (p65-Ser536), p38 MAPK, and pJNK protein levels were also increased by CM; nevertheless, this change was reversed upon diacerein treatment, showing a dose-dependent response in HUVECs and RAoSMCs (Fig. 4D and 4E). In 3T3-L1 cells, phosphorylated NK-κB (p65-Ser536) and IL-1β levels were significantly reduced by diacerein treatment, and the downstream signaling ICAM as well as TNFα showed a decreasing trend (Fig. 4F).
IL-1β inhibitor alleviated atherosclerosis in ApoE knockout mice
Aortic lipid accumulation was evaluated via oil red O staining of the aortic root; quantification of lipid accumulation showed a decreasing trend with diacerein (Fig. 5A). To investigate the underlying molecular mechanisms alongside the atherosclerotic burden evaluated in the aortic root, heart tissue was utilized as a surrogate for analysis. Since isolating sufficient aortic tissue for multiple simultaneous mRNA and protein assays in a mouse model is technically challenging, molecular changes in the myocardium were analyzed to reflect the drug’s broader cardiovascular impact and the systemic nature of IL-1β-mediated inflammation. Accordingly, the gene expression of IL-1β, TNFα, phosphorylated NK-κB (p65-Ser536), and ICAM1 was reduced in heart tissue after diacerein administration (Fig. 5B). The protein expression of phosphorylated NK-κB (p65-ser536) and phosphorylated p44/42 (ERK1/2) was also reduced (Fig. 5C). Immunohistochemical analysis of CD68 in adipose tissue from atherogenic diet-fed ApoE-knockout mice showed decreased macrophage infiltration with diacerein treatment (Fig. 5D). The mRNA expression of IL-6, IL-8, and TNFα was also reduced in adipose tissue (Fig. 5E). The gene expression of IL-1β, TNFα, phosphorylated NK-κB (p65-Ser536), and ICAM1 was reduced in heart tissue after diacerein administration (Fig. 5B). The protein expression of phosphorylated NF-κB (p65-Ser536) and phosphorylated p44/42 (ERK1/2) was also reduced (Fig. 5C). Immunohistochemical analysis of CD68 in adipose tissue from atherogenic diet-fed ApoE-knockout mice showed decreased macrophage infiltration with diacerein treatment (Fig. 5D). The mRNA expression of IL-6, IL-8, and TNFα was also reduced in adipose tissue (Fig. 5E).Fig. 5IL-1β inhibitor alleviated atherosclerosis in atherogenic diet-fed ApoE^−/−^ mice. a Representative images of Oil Red O staining of aortas en face from atherogenic diet-fed ApoE^−/−^ mice and quantification of plaque lesion area on surface of the aortic root (n = 3–4). b mRNA expression of IL-1β, TNF-α, phosphorylated p65, and ICAM in heart tissue (n = 3–5) c Protein expression of phosphorylated NF-kB and MAPK in heart tissue (n = 4–6). d Representative images of Hematoxylin–eosin (H&E) staining and immunohistochemical staining of CD68 in adipose tissue from ApoE^−/−^ mice (n = 4–6). e mRNA expression of IL-6, IL-8, and TNF-α in adipose tissue (n = 3–6). All data represent mean ± S.E. *, p < 0.05 versus the Control group, by One-way ANOVA. All qRT-PCR data were normalized to the mRNA levels of cyclophilin
Predicting response in human clinical trials using human gene-disease association
As shown in Table 1, seven randomized controlled trials have been conducted to date on the metabolic effects of diacerein, with most studies reporting a reduction in HbA1c and/or FPG levels compared with that in the control group (Table 1). In addition to its glucose-lowering effects, it has been reported to increase insulin secretion [21], reduce microalbuminuria in patients with CKD [22], and improve liver fibrosis in those with MASLD [23]. Table 1. Summary of randomized controlled trials of diacereinFirst author, yearTarget-diseaseDiacerein dosesPatientsAge (yrs)DurationOutcomesResultsNCT numberRamos-Zavala et al., 2011 [21]T2DM50 mg/day40 drug-naïve adult patients with type 2 diabetes47.82mo1) Metabolic profile (IL-1β, TNF-α, IL-6, and fasting insulin levels2) Insulin secretion and insulin sensitivity (hyperglycemic-hyperinsulinemic clamp technique)Metabolic profile improvedInsulin secretion increased01298882Leite et al. 2011 [23]T2DM with MASLD100 mg/day69 Patients with type 2 diabetes and non-alcoholic fatty liver disease64.524moDifference in mean liver stiffness and steatosisReduced liver stiffness in contrast to placebo by 1.6 kPa (Fibroscan)02242149Piovesan et al., 2017 [22]T2DMwith CKD100 mg/day(50 mg bid)72 participants with CKD with T2DM62.590 daysReduction > 15% in ACRMarked reduction in ACR was detected in both treatment groups without significant differenceCardoso et al., 2017 [46]T2DM100 mg/day84 patients with HbA1c between 7.5 and 9.5%64.848wksDifference in mean HbA1c changesDifference in HbA1c 0.35%02242149Villar et al., 2017 [47]T2DM50 mg/day12 patients with HbA1c ≥ 7% with metformin as monotherapy (≥ 1500 mg per day)41.390daysDifferences in FG,PPG and HbA1C concentrationsSignificantly decreasing HbA1C, FG and PPG concentrationsTres et al., 2018 [48]T2DM50 mg/day72 participants5912wks1) Change in HbA1c2) Proportion of patients achieving metabolic control (HbA1c ≤ 7 0%)and change in inflammatory mediatorsThe difference in HbA1c level −1.3% in those with < 14 years of diabetes durationJangsiripornpakorn et al., 2022 [49]T2DM50 mg/day25 T2DM patients with poor glycaemic control, despite being treated with at least three glucose-lowering agents5212wksChanges in HbA1c at the 4th and 12th weeksSignificant reduction in HbA1c levels from baselineT2DM, type 2 diabetes mellitus; MASLD, metabolic dysfunction–associated steatotic liver disease; HbA1c, glycated hemoglobin; CKD, chronic kidney disease; IL, interleukin; TNF, tumor necrosis factor; FG, fasting plasma glucose, PPG, postprandial plasma glucose; ACR albumin/creatinine ratio; NCT number, national clinical trial number
The disease associations of differentially expressed genes resulting from IL-1β inhibition by diacerein were investigated using the DisGeNET human gene-disease association database. The analysis revealed strong associations between these genes and conditions such as liver cirrhosis, myocardial infarction, liver fibrosis, and MASLD (Fig. 6A). Consequently, the expression of these genes was further examined. For comparative gene expression analysis, data from 221 healthy donors were obtained from the GTEx project and data from 94 patients with MASLD were sourced from the public dataset GSE174478. The genes were significantly upregulated in MASLD patients (Fig. 6B).Fig. 6. Predicting the Clinical Implications of Diacerein on MASLD. a Gene-Disease Association Analysis of Differentially Expressed Genes. The genes differentially expressed following diacerein administration were analyzed using the DisGeNET database. Associated diseases were visualized as a bubble plot, where the color of each bubble represents the significance level (−log₁₀(p-value)) and the size indicates the odds ratio. The grey dotted line denotes the significance threshold (p-value = 0.05). b Comparative Gene Expression Analysis in MASLD Patients. Gene expression levels were visualized using a dot-bar plot. **** indicates a p-value < 0.0001. c Serum Risk Stratification Score of Genes Inhibited by Diacerein Administration. *** indicates a p-value < 0.001
To evaluate the potential of these genes as blood-based biomarkers for predicting the severity of hepatic metabolic disorders, serum protein levels were compared using the public dataset GSE251855. Specifically, the concentrations of secreted proteins TIMP1, MMP2, and CXCL10, whose expression is inhibited by diacerein, were assessed. TIMP1 levels exhibited an increasing trend, correlating with the progression of metabolic disease severity from metabolic associated fatty liver disease to metabolic associated steatohepatitis (Fig. 6C).
Discussion
In this study, we demonstrated that diacerein inhibited the progression of liver fibrosis and exhibited a potential to attenuate atherosclerotic development in both in vitro and in vivo models using atherogenic diet-fed ApoE-knockout mice. Diacerein treatment reduced the expression of fibrosis-related genes and inflammatory signals in hepatic stellate cells and ameliorated both fatty liver and fibrosis in a mouse model of MASLD. Furthermore, diacerein reduced the expression of pro-inflammatory signals in endothelial, smooth muscle, and adipose cells, and tended to decrease aortic atherosclerotic burden in ApoE-knockout mice. These findings suggest that diacerein may be repurposed for metabolic disorders with chronic inflammation, such as MASLD and cardiovascular disease, given its established safety profile.
Diacerein reportedly reduces the liver injury induced by various chemicals or ischemia/reperfusion, with some studies suggesting IL-1β pathway inhibition as a possible mechanism [24]. The IL-1β pathway is a key factor in the progression from MASLD to liver fibrosis. In this study, diacerein treatment significantly reduced the expression of fibrogenic genes in LX-2 cells treated with TGF-β1 and in atherogenic diet-fed ApoE-knockout mice. Elevated TGF-β expression in liver injury, due to lipid accumulation or viral infection, activates HSCs and primes the inflammasome within them, upregulating the NF-κB pathway [25]. Consequently, the levels of proinflammatory cytokines such as IL-1β and IL-18 increase, further accelerating inflammation and fibrosis [26].
Several liver diseases are reversible in their early stages [27]. Within 72 h of thioacetamide (TAA)-induced liver injury, IL-1R-deficient mice exhibited a reduction in the elevation of serum ALT levels, decreased mRNA expression of MMP-9, and significantly decreased expression of αSMA, a marker of HSC activation, compared to wild type, which demonstrated the role of IL-1 in the early stages of liver injury and fibrosis [28] atherogenic diet used in this study increased the plasma and liver lipid levels, resulting in progressive steatosis, fibrosis, inflammation, and hepatocellular ballooning over 6–24 weeks [29]. Pretreatment with diacerein may block IL-1β activity early during the metabolic changes induced by a 12-week atherogenic diet, consistent with the results of previous studies [28]. Given that the onset of liver fibrosis is crucial in the prognosis of chronic liver disease, our findings suggest that diacerein is a promising candidate to inhibit the early and reversible stages of liver fibrosis.
α-SMA and Col1A1, whose expression is increased by TGF-β1 in LX-1 cells and reversed upon the administration of diacerein, are upregulated in the pre-fibrotic stage of hepatic steatosis and steatohepatitis [30]. In the present study, the mRNA expression of TIMP1 and Col3a1 significantly decreased in liver tissue after diacerein administration. These genes are upregulated in hepatic steatohepatitis and early fibrosis [31]. Prior studies recognize that lipid accumulation, inflammation, and fibrosis in the liver are not sequential events but rather an intricate cascade of interactions [27]. In this context, if diacerein can inhibit the expression of genes activated early during the progression of MASLD, it could be a useful tool for preventing disease progression.
Our findings demonstrated that diacerein treatment reduced lipid accumulation and fibrosis in the liver. Previous studies using HFD-fed mice reported that diacerein improved glucose tolerance and fatty liver, with a reduction in pro-inflammatory signaling molecules such as IL-1β and IL-6 in adipose, liver, and muscle tissues [32]. In our ApoE⁻/⁻ model, fibrosis was primarily localized to the perivascular region, which is consistent with the early stages of hepatic fibrogenesis. Although advanced parenchymal fibrosis—a hallmark of advanced MASH—was not prominently observed in the control group, the significant reduction in perivascular collagen deposition suggests that diacerein effectively inhibits fibrosis at its onset. This is consistent with the 'multiple parallel hits' hypothesis, which suggests that steatosis, inflammation, and fibrosis do not necessarily progress in a linear, sequential manner but rather emerge through complex, simultaneous pathways [33, 34]. In this context, diacerein may intervene in the early inflammatory cascades that prime fibrogenesis, even before extensive parenchymal damage becomes histologically evident.
Clinical evidence further supports this local, anti-fibrotic action. A randomized, placebo-controlled trial involving patients with type 2 diabetes and MASLD showed that a 2-year diacerein treatment significantly reduced liver stiffness, indicating an improvement in liver fibrosis. Notably, that study reported that diacerein did not significantly affect liver steatosis and found no significant changes in serum pro-inflammatory cytokine levels, including IL-1 and IL-6. These clinical findings align with our results, where the reduction in IL-6 expression did not reach statistical significance despite the clear anti-fibrotic effect. This suggests that diacerein may act through local inhibitory mechanisms within the liver tissue rather than by significantly altering systemic cytokine levels [23].
Overall, as the onset of liver fibrosis is a crucial determinant of prognosis, these results suggest that diacerein is a promising candidate for inhibiting the early and reversible stages of liver fibrosis. While our study highlights its potential in early-stage fibrogenesis, further research using models of advanced parenchymal fibrosis is warranted to fully evaluate its therapeutic spectrum.
Targeting IL-1 offers a novel therapeutic approach to atherosclerosis. Multiple studies over the past two decades have shown that selective IL-1β inhibition recues atherogenesis in mice [35]. However, there are few clinical trials using drugs to target IL-1β in CVD. Even the Canakinumab Anti-inflammatory Thrombosis Outcome Study did not show a significant difference in all-cause mortality compared to placebo, and a higher incidence of serious infections has been reported in a small portion of patients, which precludes clinical use [15]. Clinical trials using the IL-1 receptor antagonist anakinra and the recombinant IL-1 antagonist rilonacept are ongoing, though definitive results are pending (NCT05177822, NCT01950299, and NCT00417417).
No human clinical trials have been reported on the anti-atherogenic effect of diacerein, but the pro-inflammatory and pro-atherogenic effects of diacerein treatment in primary human coronary artery endothelial cells have been published [36]. In this study, we also confirmed anti-inflammatory action in smooth muscle cells, adipocytes, and endothelial cells, all of which are closely related to atherosclerosis pathogenesis. Furthermore, diacerein showed a trend toward reduced aortic atherogenic plaques and attenuated the inflammatory response in the heart and adipose tissue in ApoE knockout mice, suggesting potential anti-atherosclerotic effects. To the best of our knowledge, this is the first report evaluating diacerein’s impact on atherosclerosis in both cellular and animal models.
In vivo studies and experiments using animal models have shown promising results in mitigating the progression of fatty liver, liver cirrhosis, and atherosclerosis. However, clinical trials in humans have not been conducted, limiting the understanding of diacerein’s effectiveness in real-world clinical practice. In this study, genes whose expression is altered by diacerein were analyzed using human disease genomic data, revealing a strong association with metabolic liver diseases, such as fatty liver and liver cirrhosis. Additionally, some of these genes, mainly related to fibrosis, significantly increased in expression in patients with MASLD compared to healthy individuals, suggesting that diacerein treatment may inhibit the progression of metabolic liver disease by reducing the expression of these genes. Previous reports of increased expression of TIMPs, MMPs, or COL1A1 in obese patients with comorbid MASLD who underwent bariatric surgery support the findings of this analysis [37].
Furthermore, TIMP1 was identified as a protein whose expression increased in blood according to the severity of metabolic liver disease through the analysis of the Serum Protein Risk Stratification Score database (GSE251855). TIMP-1 is elevated in acute viral hepatitis, cirrhosis, and alcoholic hepatitis [38], and has also been studied as a non-invasive tool for diagnosis of liver fibrosis and is under investigation as a non-invasive tool to diagnose liver fibrosis and assess its severity [39]. Taken together, TIMP-1 is a potential biomarker that is increased in liver diseases such as liver fibrosis, and its expression may be reduced by diacerein treatment, and further studies are needed to confirm its clinical applicability.
Diacerein has been used to treat osteoarthritis and is currently being studied for other indications [40]. In terms of metabolic diseases, several human clinical trials have demonstrated improvements in glucose tolerance and insulin secretion in patients with T2DM, along with a documented reversal of liver stiffness. However, the exact mechanism of diacerein’s action is not yet fully understood; only a few possible mechanisms have been proposed [41]. Diacerein may disrupt IL-1β activation by inhibiting interleukin converting enzyme (ICE) [42], reduce IL-1β sensitivity by decreasing IL receptor levels on the cell surface [43], increase the production of IL receptor antagonist [44], or deactivate IL-1β-induced activation of NF-κB [45]. Although we observed potential modulatory effects of diacerein on atherosclerosis and significant improvement in fatty liver fibrosis in vitro and vivo, its clinical application requires further research. In our experiments, applying 50 and 80 mg/kg diacerein to mice revealed no significant toxicity. However, these doses are substantially higher than the typical doses used in humans for osteoarthritis (50–100 mg), so the potential side effects of higher doses should be carefully considered. Further studies are needed to determine the long-term efficacy and safety of diacerein in human subjects with metabolic disorders, as well as to elucidate its precise molecular mechanisms.
IL-1β inhibition is a major target to block the inflammation cascade in liver and other metabolic diseases. Although statin treatment is a key part of anti-atherosclerosis treatment strategy and preventing CVDs, various attempts to reduce chronic inflammation in various metabolic organs remain for decreasing remnant CVD risks. Here, we report that diacerin, an IL-1β inhibitor, significantly reduced the inflammation cascade, MASLD/liver fibrosis-related gene expression in vitro, and high-fat induced in vivo models and atherosclerosis-related pathways.
Supplementary Information
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