Orally Administered Self-Microemulsifying Celastrol Alleviates Rheumatoid Arthritis by Modulating the Expression of TNF-α

Abstract

Objective: This study aimed to develop an oral celastrol-loaded self-microemulsifying drug delivery system (Cel-SMEDDS) to enhance the therapeutic efficacy against rheumatoid arthritis and reduce toxicity.

Methods: The optimal Cel-SMEDDS formulation, identified through solubility screening, excipient compatibility assays, and pseudo-ternary phase diagram analysis, was characterized by particle size, PDI, zeta potential, in vitro release, and stability. In vitro anti-inflammatory activity was evaluated in LPS-induced RAW264.7 macrophages, while in vivo anti-RA efficacy was assessed in CIA mice via paw swelling, clinical scoring, serum TNF-α, and joint histopathology. Preliminary safety was examined by hematological, serum biochemical, and histopathological analyses in mice.

Results: The optimal Cel-SMEDDS formulation consisted of LABRAFIL M 1944 CS–Kolliphor RH40–CAPRYOL 90 (0.2:0.48:0.32, w/w/w) with a drug loading of 1.5% (w/w). It spontaneously formed uniform microemulsions with a mean particle size of 26.70 nm, PDI of 0.067, and zeta potential of −2.87 mV. In vitro, Cel-SMEDDS showed enhanced cytotoxicity against M1-type macrophages (IC₅₀ = 0.1753 μg/mL vs. 0.2684 μg/mL for free Cel), significantly suppressed pro-inflammatory TNF-α and IL-1β expression, and upregulated anti-inflammatory IL-10. In CIA mice, oral Cel-SMEDDS reduced paw swelling by 37.42% (vs. 22.79% for free Cel), markedly decreased serum and intra-articular TNF-α levels, and alleviated articular cartilage damage. Preliminary safety evaluation demonstrated no significant abnormalities in hematological parameters, liver/kidney function, or major organ histology.

Conclusions: The optimized oral Cel-SMEDDS effectively inhibits the expression of pro-inflammatory cytokine TNF-α both in vitro and in vivo, exhibits superior anti-RA activity compared to free Cel, and possesses favorable safety. This formulation addresses the key limitations of celastrol and shows promising potential for clinical translation in RA treatment.

Introduction

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease characterized by joint inflammation, autoantibody production, and cartilage and bone destruction [1,2]. Its early clinical manifestations include joint redness, swelling, warmth, pain, and functional impairment, while advanced stages may present with joint stiffness and deformity, accompanied by skeletal muscle atrophy, potentially leading to disability in severe cases [3]. The pathogenesis of RA involves the complex regulation of various immune cells, immune factors, and signaling pathways [4]. Multiple innate immune cells, including monocyte-macrophages, natural killer (NK) cells, and mast cells, have been detected in the synovium of RA patients, collectively participating in joint inflammation and bone erosion. Among these, M1-type macrophages derived from monocyte differentiation are the primary drivers of local inflammation [5]. During the progression of RA, monocytes in the blood migrate into the synovium and differentiate into pro-inflammatory M1 macrophages, which release cytokines and chemokines such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β), thereby promoting and amplifying local inflammation and causing tissue damage [6,7]. Chronic inflammation leads to the persistent accumulation of inflammatory mediators, resulting in synovial cell hyperplasia, synovial thickening, and ultimately cartilage and bone destruction as well as joint dysfunction [8]. Therefore, the main therapeutic strategy for RA is to alleviate inflammation and mitigate joint damage through various approaches [9].

Tripterygium wilfordii is a traditional Chinese medicine widely used in the treatment of rheumatic diseases, with effects including dispelling wind and removing dampness, activating blood circulation and unblocking collaterals, and reducing swelling and alleviating pain [10]. Celastrol (Cel), a pentacyclic triterpenoid compound, is one of its major active components responsible for the anti-RA effects [11]. This compound inhibits RA by suppressing neutrophil-mediated inflammatory responses and reducing the secretion of TNF-α and IL-6. Additionally, it modulates the NF-κB signaling pathway and inhibits the polarization of macrophages toward the pro-inflammatory M1 phenotype, thereby reducing the secretion of inflammatory cytokines [12,13,14]. Other studies have shown that Cel can alleviate bone erosion and destruction in RA mice by inducing apoptosis of osteoclast precursor cells and inhibiting inflammatory cell infiltration [15,16]. However, despite its outstanding medicinal value, the drawbacks of Cel cannot be overlooked. Cel often exhibits significant toxic and side effects, particularly when used in excess, producing notable hepatotoxicity [17] and nephrotoxicity [18], as well as cardiotoxicity [19] and reproductive toxicity [20]. Moreover, Cel suffers from poor solubility, low oral bioavailability, and a narrow therapeutic window, which severely limit its clinical application [21].

A microemulsion is a homogeneous dispersion system that spontaneously forms from an oil phase, an aqueous phase, an emulsifier, and a co-emulsifier at appropriate ratios. It appears transparent or nearly transparent, is thermodynamically stable, and typically exhibits a uniform droplet size distribution in the range of 10~100 nm, which can effectively improve the solubility and oral absorption of poorly soluble drugs [22]. With in-depth research into the various properties of microemulsions, the self-microemulsifying drug delivery system (SMEDDS) has emerged. Compared with nanoformulations such as liposomes and micelles, SMEDDS offers a simpler preparation process and superior physical stability. After oral administration, it can spontaneously form stable oil-in-water (O/W) microemulsions in the gastrointestinal tract [23]. This stability is primarily attributed to the synergistic action of the emulsifier and co-emulsifier, which together maintain the system’s surface tension below the critical value required for microemulsion formation. In recent years, SMEDDS has been widely used to improve the oral bioavailability of poorly water-soluble drugs, including curcumin [24], resveratrol [25], puerarin [26], andrgrapholide [27], and berberine hydrochloride [28].

Therefore, the present study was designed to develop an oral celastrol-loaded self-microemulsifying drug delivery system (Cel-SMEDDS) to address the limitations of poor aqueous solubility, low oral absorption, and potential toxicity of Cel, with the goal of improving therapeutic efficacy while reducing toxicity. Although Qi et al. developed a solid self-microemulsifying dispersible tablet of Cel that improved oral bioavailability by 2.3-fold, they did not evaluate its anti-arthritic efficacy or long-term stability [29]. Another study by Onyeabor et al. prepared celastrol-loaded silk fibroin nanoparticles for oral delivery, but focused solely on pharmacokinetic parameters without investigating anti-inflammatory mechanisms or systemic toxicity [30]. Notably, no prior Cel-SMEDDS formulation has been comprehensively evaluated for rheumatoid arthritis treatment, including both in vitro macrophage polarization modulation and in vivo CIA model validation. Furthermore, the majority of these prior works constructed pseudo-ternary phase diagrams directly, omitting excipient compatibility pre-screening and thereby potentially compromising formulation stability and batch-to-batch reproducibility. To overcome these limitations, we systematically developed Cel-SMEDDS by integrating compatibility testing and phase diagram optimization. The novelty of the present study includes the comprehensive evaluation of long-term stability under diverse storage conditions, elucidation of the anti-inflammatory mechanisms involving macrophage polarization and TNF-α modulation, rigorous in vivo validation of anti-arthritic efficacy in collagen-induced arthritis (CIA) mice, and a preliminary safety assessment at a high therapeutic dose. Collectively, this study advances the mechanistic understanding of SMEDDS-based Cel delivery and provides a promising oral formulation candidate for the treatment of rheumatoid arthritis.

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Materials

Celastrol (batch No. P2430964, purity 98%) and sulforhodamine B (batch No.: P2409495, purity 70%) were purchased from Shanghai Titan Scientific Co., Ltd. (Shanghai, China). LABRAFIL M 1944 CS (batch No. 162835), PLUROL OLEIQUE CC 497 (batch No. 162324), and CAPRYOL 90 (batch No. 172122) were obtained from Gattefossé (Saint-Genis-Laval, France). Kolliphor RH 40 (batch No. 28653868E0) was purchased from Beijing Fengli Jingqiu Pharmaceutical Technology Co., Ltd. (Beijing, China). Ethyl oleate (batch No. 20220209) was obtained from Jiangxi Alpha Hi-Tech Pharmaceutical Co., Ltd. (Pingxiang, China). Tween 20 (batch No. 20240924) and 1,2-propylene glycol (batch No. 20180921) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Anhydrous ethanol (batch No. 01101143) was obtained from Nanjing Chemical Reagent Co., Ltd. (Nanjing, China). CMC-NA (batch No. 0552117505) was purchased from Shenzhen Youpuhui Pharmaceutical Technology Co., Ltd. (Shenzhen, China). DMEM medium (batch No. 6125016) was obtained from Gibco (Grand Island, NY, USA). Fetal bovine serum (batch No. SA240119) was purchased from Wuhan Pricella Life Science & Technology Co., Ltd. (Wuhan, China).

The magnetic bead-based tissue/cell/blood total RNA extraction kit (batch No. A0925A) and RNase-free/DNase I (batch No. B0721B) were obtained from Tiangen Biotech (Beijing) Co., Ltd. (Beijing, China). Hifair® III 1st Strand cDNA Synthesis Supermix for qPCR (gDNA digester plus) (batch No. H9405020), Hieff UNICON® Universal Blue qPCR SYBR Green Master Mix (batch No. H74272080), and DiR Iodide (DiIC 18 (7)) (batch No. D2322051) were purchased from Yeasen Biotechnology (Shanghai) Co., Ltd. (Shanghai, China). Primers for GAPDH, TNF-α, IL-1β, and IL-10 (batch No. 2414247) were obtained from Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). Bovine type II collagen, Freund’s incomplete adjuvant, and Freund’s complete adjuvant (batch No. 240009) were purchased from Chondrex, Inc. (Redmond, WA, USA). Mouse TNF-α ELISA kit (batch No. A28240735) was obtained from Lianke Bio (Hangzhou) Co., Ltd. (Hangzhou, China). TNF Alpha/TNFA antibody (batch No. 24BP9019J2554H21) was purchased from Boster Biological Technology Co., Ltd. (Pleasanton, CA, USA). Assay kits for AST (batch No. 140124020), ALT (batch No. 140223007), CREA (batch No. 141124042), and UREA (batch No. 141325015) were obtained from Shenzhen Mindray Bio-Medical Electronics Co., Ltd. (Shenzhen, China).

Ma, B.; Li, Y.; Zhang, J.; Fu, Y.; Cao, H. Orally Administered Self-Microemulsifying Celastrol Alleviates Rheumatoid Arthritis by Modulating the Expression of TNF-α. Pharmaceutics 2026, 18, 695. https://doi.org/10.3390/pharmaceutics18060695