4-IPP Alleviates Dexamethasone-Induced Muscle Atrophy by Targeting the MIF/TXNIP/NLRP3 Axis

4-IPP Alleviates Dexamethasone-Induced Muscle Atrophy by Targeting the MIF/TXNIP/NLRP3 Axis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article 4-IPP Alleviates Dexamethasone-Induced Muscle Atrophy by Targeting the MIF/TXNIP/NLRP3 Axis Weiqing Li, Yu Bai, Yahong Lu, Lin Ye, Haiwei Ma, Zhiguo Zhou, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8979207/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 9 You are reading this latest preprint version Abstract Sarcopenia, an age-related degenerative skeletal muscle disorder, is strongly associated with adverse clinical outcomes including recurrent falls, functional disability, frailty, and increased mortality. Emerging evidence suggests that systemic chronic inflammation plays a central role in muscle wasting. Although macrophage migration inhibitory factor (MIF) is a known pro-inflammatory cytokine in various inflammatory diseases, its role in sarcopenia development remains unclear. We found significantly elevated plasma levels of MIF, TNF-α, IL-6, and IL-8 in sarcopenia patients, which were inversely correlated with muscle strength. In vitro , we demonstrated that MIF exposure directly induced atrophy in C2C12 myotubes. Furthermore, our study showed that MIF induces excessive ROS production, activating the TXNIP/NLRP3 inflammasome pathway and subsequent pro-inflammatory responses. Consistent with these findings, in vivo administration of 4-IPP significantly alleviated muscle mass loss and functional decline in a Dexamethasone (DEX)-induced murine sarcopenia model. Our findings demonstrate that MIF is a key regulator of inflammatory muscle atrophy, supporting its potential as a therapeutic target for sarcopenia. Sarcopenia MIF ROS TXNIP/NLRP3 4-IPP Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Sarcopenia is a common, progressive musculoskeletal disorder marked by age-related declines in skeletal muscle mass, strength, and function. It is associated with an increased risk of adverse outcomes, including falls, functional impairment, frailty, and mortality, thereby substantially diminishing quality of life and imposing a significant socioeconomic burden [ 1 , 2 ]. Epidemiological research indicates that sarcopenia affects about 5–13% of people aged 60–70, with prevalence rising to 11–50% in individuals aged 80 and above. With the ongoing trend of global population aging, the economic burden associated with sarcopenia is expected to escalate further [ 3 ]. However, despite its clinical significance, effective therapeutic strategies for sarcopenia remain limited. The core pathological feature of sarcopenia is an imbalance between protein synthesis and proteolysis. Under physiological conditions, skeletal muscle maintains mass and function through balanced protein synthesis and degradation [ 4 ]. The ubiquitin-proteasome system (UPS) is essential in proteolytic processes, with E3 ubiquitin ligases Atrogin-1 and MuRF-1 serving as key regulators of muscle atrophy [ 5 ]. Bodine et al. Overexpression of Atrogin-1 causes atrophy in cultured myotubes, whereas genetic deficiency in either ligase provides resistance to atrophy in mice [ 6 ]. Research indicates that Atrogin-1 and MuRF1 expression is significantly increased by various catabolic stimuli, such as oxidative stress, inflammatory cytokines, and glucocorticoids, which are associated with enhanced muscle degradation [ 7 ]. Atrogin-1 and MuRF1 are involved in muscle wasting associated with various common human diseases, including cancer, diabetes, chronic kidney disease (CKD), chronic obstructive pulmonary disease (COPD), and age-related sarcopenia [ 8 ]. The pathogenesis of sarcopenia involves complex physiological processes, with systemic chronic inflammation considered a critical contributing factor [ 9 , 10 ]. Research indicates that inflammatory factors such as TNF-α, IL-6, IL-1β, and IFN-γ are notably increased in sarcopenia patients and closely linked to reduced muscle strength. Chronic inflammation leads to the substantial release of pro-inflammatory cytokines into the bloodstream, which directly affect the immune system and facilitate muscle protein degradation through metabolic pathway modulation. TNF-α induces atrophy by activating the IKKβ/NF-κB signaling pathway, which upregulates the expression of the ubiquitin carrier protein UbcH2 and thereby activates the UPS. Alternatively, it can directly induce the expression of the E3 ubiquitin ligase MuRF1, facilitating the ubiquitin-mediated degradation of myosin heavy chain (MyHC) [ 11 ]. IL-6 activates NF-κB via the gp130/JAK/STAT3 pathway, leading to increased expression of E3 ubiquitin ligases and subsequent muscle wasting. Concomitantly, skeletal muscle-specific IL-6 knockout effectively attenuates this pathological process [ 12 – 14 ]. Furthermore, IL-1β activates p38 MAPK and NF-κB signaling through mechanisms independent of oxidative stress and the AKT/Foxo pathway. It specifically upregulates the Atrogin-1 and MuRF1, thereby accelerating myofibrillar protein degradation and contributing to muscle atrophy [ 15 ]. Inflammatory mechanisms are pivotal in sarcopenia pathogenesis, making chronic inflammation a potential therapeutic target. Macrophage migration inhibitory factor (MIF) is a widely distributed pleiotropic proinflammatory cytokine produced by various cell types, playing a role in inflammatory responses through multiple signaling pathways. MIF is associated with the development and advancement of inflammation-related diseases, such as glomerulonephritis, inflammatory bowel disease, and pancreatitis, as well as immune-mediated disorders like rheumatoid arthritis, systemic lupus erythematosus, and spondyloarthritis [ 16 ]. MIF, upon binding to its receptor CD74 and coreceptors, activates signaling pathways such as NF-κB, MAPK, and PI3K/Akt. These pathways trigger the release of pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6, which enhance localized and systemic inflammation [ 17 – 19 ]. Previous research indicates that MIF stimulates reactive oxygen species (ROS) production and autophagy in hepatocellular carcinoma cell lines [ 20 ]. Moreover, MIF-mediated activation of ROS signaling has been implicated, at least in part, in promoting breast cancer metastasis [ 21 ]. Current research on MIF in the muscular system has primarily focused on its cardioprotective effects in myocardial ischemia and its involvement in anti-senescence mechanisms [ 22 – 24 ]. However, its specific role and underlying mechanisms in sarcopenia remain largely unexplored. Investigating MIF's role in inflammatory signaling and chronic diseases may reveal new therapeutic targets for age-related muscle atrophy by clarifying its specific function in muscle wasting. This study investigates the role of MIF in muscle wasting and assesses the therapeutic potential of 4-IPP, a specific MIF inhibitor, through the use of in vitro and in vivo muscle atrophy models. The research improves our understanding of inflammation-induced sarcopenia by clarifying MIF-mediated pathological pathways, providing new insights for clinical intervention. Materials and methods Reagents Fetal bovine serum (FBS), horse serum, DMEM, and streptomycin-penicillin (SP) solution were sourced from Thermo Fisher Scientific (Waltham, MA, USA). Recombinant Mouse MIF was obtained from R&D Systems (Minneapolis, MN, USA). Dexamethasone (DEX) was acquired from Sigma-Aldrich (St. Louis, MO, USA), and 4-iodo-6-phenylpyrimidine (4-IPP) was procured from MedChemExpress (Monmouth Junction, NJ, USA). Primary antibodies against MuRF1, Atrogin-1, NLRP3, Caspase-1, and IL-1β were procured from Proteintech (Rosemont, IL, USA). Antibodies against TXNIP and GAPDH were obtained from Cell Signaling Technology (Danvers, MA, USA) and Abcam (Cambridge, UK), respectively. Species-matched secondary antibodies (anti-rabbit and anti-mouse) were acquired from Cell Signaling Technology. Human subjects The Ethics Committee of Lishui Central Hospital, Zhejiang Province, approved this retrospective study ( approval number 2020-17). Sarcopenia was classified according to the Asian Working Group on Sarcopenia 2019 (AWGS 2019) criteria, which include low grip strength (male < 28 kg, female < 18 kg), low calf circumference (male < 34 cm, female < 33 cm), and low skeletal muscle index (SMI; male < 7. 0 kg/m², female < 5. 4 kg/m²) as measured by dual-energy X-ray absorptiometry (DXA). Plasma samples were collected from the antecubital vein between 6:00 and 8:00 a. m., then centrifuged and stored at -80°C. Written informed consent was obtained from all participants. Patients were excluded from the study if they had chronic diseases (such as COPD, CHF, diabetes mellitus, or cancer), autoimmune disorders (including rheumatoid arthritis, psoriasis, multiple sclerosis, systemic sclerosis, or inflammatory bowel disease), a recent infection (within the past week), or had used corticosteroids in the past month to eliminate the impact of these factors on MIF levels. ELISA (Enzyme-linked immunosorbent assay) Venous blood samples from patients were collected into anticoagulant tubes between 6:00–8:00 AM and processed immediately. Plasma was obtained by centrifugation at 3, 000×g for 10 min at 4°C. Cell culture supernatants were centrifuged at 1, 000×g for 5 minutes to eliminate cellular debris, then aliquoted and stored consistently. MIF, TNF-α, IL-6, and IL-1β concentrations were quantified using ELISA kits from Proteintech (Group, Inc., Rose, USA.). Cytotoxicity assay The cytotoxic effect of 4-IPP on C2C12 cells was assessed using the CCK-8 cell proliferation and cytotoxicity assay kit (Dalian, Liaoning, China). Cells were initially seeded at a density of 5×10^3 per well in a 96-well plate. After 24 hours of culture to allow cell adhesion, the cells were further cultured until reaching 80% confluence. The growth medium was subsequently substituted with a differentiation medium containing 2% horse serum to promote myotube formation over a period of 6 days. Post-differentiation, cells were exposed to 4-IPP at concentrations of 0 to 640 µM, alongside DEX, for 48 hours. Each concentration was tested in five replicate wells. In the last hour of incubation, 10 µL of CCK-8 reagent was added to each well with 100 µL of DMEM and incubated at 37°C. Absorbance at 450 nm was recorded with a TECAN Infinite M200 microplate reader (TECAN, Männedorf, Switzerland). Extraction of RNA and quantitative real-time PCR (qPCR) Cells were plated in a 12-well dish to facilitate adhesion. Cells were subjected to total RNA extraction utilizing the RNA Rapid Extraction Kit from Yishan Biotech, Shanghai, China. cDNA was synthesized via reverse transcription using the Strand cDNA Synthesis SuperMix for qPCR kit (Yishan Biotech, Shanghai, China). qPCR was used to evaluate relative mRNA expression levels. Detection was performed using qPCR SYBR Green Master Mix (Yishan Biotech, Shanghai, China) and an ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The cycling protocol involved initial denaturation at 95℃, followed by 40 amplification cycles at 60℃. The 2 − ΔΔCT method was employed to calculate the results. The figure below presents the primer sequences. Mouse MuRF1 primers: Forward 5′-TGGAGGTCGTTTCCGTTGC-3′, Reverse 5′-CTGAGGTTCTGTCTGCGGTAG-3′. Mouse Atrogin-1 primers: Forward 5′-CAGCTTCGTGAGCGACCTC-3′, Reverse 5′-GGCAGTCGAGAAGTCCAGTC-3′. Mouse GAPDH primers: Forward 5′-TAGGGCCTCTCTTGCTCAGT-3′, Reverse 5′-TGTCAAGCTCATTTCCTGGT-3′. Western blotting Total protein was extracted by lysing cells using RIPA lysis buffer (Solarbio Science & Technology Co., Ltd., Beijing, China) with added protease inhibitors. Protein levels were measured with a BCA Protein Assay Kit from Beyotime Biotechnology (Shanghai, China). For the Western blot experiment, 0 µg of protein samples were separated using SDS‒PAGE and then transferred onto a PVDF membrane (Bio-Rad Laboratories, Hercules, CA, USA). Membranes were incubated in 5% non-fat milk in TBST at room temperature for 1–2 hours to block them. After blocking, membranes were briefly rinsed with TBST and cut into strips according to the molecular weight markers to isolate the regions containing the proteins of interest. The membrane strips were incubated with primary antibodies at 4°C for 12–16 hours overnight. The following day, membranes underwent three 5-minute washes with TBST and were then incubated with species-specific secondary antibodies (anti-mouse or anti-rabbit IgG, 1:5, 000 dilution) for 1–2 hours at room temperature. After three additional 5-minute TBST washes, protein bands were detected using an Enhanced ECL Ultra-Sensitive Chemiluminescence Kit (Yeasen Biotech, Shanghai, China) and captured with an Invitrogen iBright 1500 Imaging System (Thermo Fisher Scientific, Waltham, MA, USA). Band intensity was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The protein bands in the figure represent typical outcomes from three independent experiments, with quantitative data presented as mean ± SD. Giemsa Staining Cells were seeded into 6-well plates and differentiated into multinucleated myotubes. After treatment for 48h, removing the culture medium, cells were gently washed twice with PBS. Fixation was performed by incubating cells with 70% ethanol at room temperature for 10 minutes. Fixed cells were then stained with the freshly prepared modified Giemsa working solution (1×) (Beyotime Biotechnology, Shanghai, China) After staining for 45 minutes. Unbound dye was removed by thorough rinsing with distilled water until the effluent became clear. Plates were air-dried, and myotube morphology images were captured using an inverted light microscope with a digital camera. Myotube diameters were quantified from ten random fields per well using ImageJ software. Animal model and treatment Eight-week-old C57BL/6J mice were obtained from HangSi Biotech in Hangzhou, China. Animal experiments received approval from the Ethics Committee of Lishui Central Hospital (Research Ethics Committee Number 2024YD0161) and were conducted following the Guidelines for Animal Treatment and the NIH Guide for the Care and Use of Experimental Animals. An 8-week-old mouse model for DEX-induced muscle atrophy was developed using 24 male C57BL/6J mice, divided into four groups for daily intraperitoneal injections over 15 days. The Control group received 30% PEG400 in 0. 9% saline (10 mL/kg body weight). The DEX group was administered DEX (25 mg/kg) in 30% PEG400 in 0. 9% saline. The DEX + 4-IPP (Low-dose) group received DEX with 4-IPP (1 mg/kg) in 10% DMSO in corn oil. The DEX + 4-IPP (High-dose) group received DEX with 4-IPP (5 mg/kg) in 10% DMSO in corn oil. Throughout the treatment period, grip strength and body weight were monitored. Twenty-four hours post-final injection, mice were euthanized, and their gastrocnemius muscles, tibiae, and organs (heart, liver, spleen, lungs, and kidneys) were collected and fixed in 4% paraformaldehyde (PFA) for histological analysis. Four-Limb Grip Strength Test A digital grip strength meter (model ZX-ZL; Zhixiang, Shanghai Yuyan Scientific Instruments Co., Ltd., China) was used to evaluate four-limb grip strength of mice. During testing, the mouse was placed on the grip strength meter, ensuring all four limbs firmly grasped the horizontal bar. The tail was gently pulled horizontally until the mouse released its grip, and the device's maximum force value was recorded. Each mouse underwent five consecutive trials with 10-second rest intervals between measurements. After excluding the highest and lowest values, the average maximal grip strength was determined by calculating the mean of the remaining three measurements. Haematoxylin–eosin (H&E) staining Tissue sections embedded in paraffin were deparaffinized using xylene and then rehydrated with a series of graded ethanol. Following this, sections were stained with haematoxylin and eosin and subsequently dehydrated through an ascending ethanol gradient. Sections were ultimately cleared in xylene and mounted using neutral balsam. Images were captured with a Leica DMi8 confocal microscope (Leica, Wetzlar, Germany). ImageJ software was utilized to quantify muscle fiber cross-sectional area (CSA). Immunohistochemical staining Gastrocnemius muscles were fixed in 4% PFA, embedded in paraffin, and sectioned to a thickness of 4 µm. Following deparaffinization in xylene and a graded ethanol series, antigen retrieval was conducted using heat in 10 mM citrate buffer (pH 6. 0) at 95°C for 20 minutes. Endogenous peroxidase activity was quenched with 3% H₂O₂ in methanol for 30 min at room temperature. Sections were blocked with 5% normal goat serum for 1 hour and incubated overnight at 4°C with primary antibodies: anti-TXNIP (1:200), anti-NLRP3 (1:300), anti-caspase-1 (1:200), and anti-IL-1β (1:100). Following PBS washes, a 1:500 dilution of HRP-conjugated secondary antibody was applied for 1 hour at room temperature. Signal detection used 3, 3'-diaminobenzidine (DAB) chromogen with hematoxylin counterstaining. Images were captured with a Leica DMi8 confocal microscope. An investigator, blinded to group allocation, quantified the positive staining area (%) from five random fields per section at 200× magnification using ImageJ. Statistical analysis Experiments were conducted in triplicate, yielding consistent results across independent replicates. Data are expressed as mean ± standard deviation (SD) or shown as representative images from a minimum of three independent experiments. GraphPad Prism 10 (GraphPad Software, La Jolla, CA, USA) was used for statistical analyses. Group differences were evaluated using one-way ANOVA with post-hoc tests or Student's t-test for pairwise comparisons. A P-value less than 0. 05 was deemed statistically significant. Results 1. Elevated MIF and cytokines correlate with muscle weakness in sarcopenic patients To assess inflammatory levels and differences in MIF between groups, participants were stratified into two categories according to AWGS2019: Sarcopenia and Healthy controls (Figure 1A). Demographic parameters showed no significant differences between the two groups (Figure 1B). However, the sarcopenia group exhibited significantly lower calf circumference (Male: 35.89±1.21 cm vs 30.79±1.07 cm, p<0.001；Female: 34.41±1.04 cm vs 27.83±2.33 cm, p<0.001; Figure 1C), grip strength(Male: 32.39±3.42 kg vs 15.86±1.98 kg , p<0.001；Female: 22.52±2.45 kg vs 13.01±3.72 kg, p<0.001; Figure 1D), and skeletal muscle parameters (Male: 7.43±0.35 kg/m² vs 6.72±0.18 kg/m², p<0.01；Female: 6.08±0.60 kg/m² vs 4.91±0.37 kg/m², p<0.001; Figure 1E) compared to the healthy control group. In contrast , plasma levels of MIF (1.47±0.41 pg/ml vs 2.11±0.42 pg/ml, p<0.001), TNF-α (133.2±33.47 pg/ml vs 233.4±61.67 pg/ml, p<0.001), IL-6 (159.0±34.27 pg/ml vs 291.6±65.72 pg/ml, p<0.001), and IL-8 (259.1±70.35 pg/ml vs 546.1±127.60 pg/ml, p<0.001) were notably higher in the sarcopenia group than in the healthy controls (Figure 1F). Correlation analyses revealed significant negative correlations between plasma MIF concentrations and both skeletal muscle index (p=0.0038, Figure 1G) and muscle strength (p=0.0016, Figure 1H). These results suggest that elevated MIF levels are associated with reduced muscle function in sarcopenia patients. 2. Exogenous MIF induces myotube atrophy and upregulates MuRF1 and Atrogin-1 expression The role of MIF in C2C12 myotubes was further investigated. C2C12 myoblasts were differentiated into myotubes by treating them with 2% horse serum for 6 days. Differentiated myotubes were then exposed to increasing concentrations of murine recombinant MIF protein (0, 6.25, 12.5, 25, 50, 100 ng/ml) for 48 hours to evaluate its effects. qPCR analysis showed that MIF treatment significantly upregulated the expression of atrophy-related markers, MuRF1 and Atrogin-1 , in a dose-dependent manner, with the effect becoming more pronounced at higher concentrations (Figure 2A). Consistent with these results, western blot analysis also confirmed the dose-dependent increase in marker expression (Figure 2B-C). Morphologically, Giemsa staining demonstrated dose-dependent myotube thinning with increasing MIF concentrations (Figures 2D-E). Taken together, these data demonstrate that exogenous MIF induces myotube atrophy through the activation of proteolytic pathways. 3. 4-IPP reverses DEX-induced myotube atrophy, reduces MuRF1 and Atrogin-1 expression To confirm the role of MIF in muscle atrophy and assess the therapeutic potential of inhibiting MIF in sarcopenia, we used the specific inhibitor 4-IPP for further experiments. C2C12 myotubes were exposed to 20 μM DEX to establish an in vitro model of muscle atrophy. To determine the optimal 4-IPP concentration, C2C12 myotubes were co-treated with DEX and increasing 4-IPP doses (0–640 μM) for 48 hours. CCK-8 assay results indicated that cell viability remained unaffected at concentrations ≤80 μM but significantly decreased in a dose-dependent manner at higher doses (>80 μM) (Figure 3A). qPCR analysis revealed that DEX significantly increased the expression of MuRF1 and Atrogin-1, serving as evidence of the atrophy model. This upregulation progressively decreased as the concentration of 4-IPP increased (Figure 3B). Western blot analysis confirmed these results, further validating the inhibitory effect of 4-IPP on DEX-induced upregulation (Figure 3C). Consistently, Giemsa staining confirmed the morphological rescue effects of 4-IPP in a concentration-dependent manner (Figure 3D-E). These results collectively confirm the pathogenic role of MIF in sarcopenia and provide preliminary evidence for the therapeutic potential of 4-IPP in alleviating muscle wasting. 4. MIF drives ROS production and activates TXNIP/NLRP3 inflammasome Inflammation and oxidative stress are closely interconnected, and their vicious cycle serves as a critical driver of sarcopenia pathogenesis. To explore the role of MIF in this context, We treated C2C12 myotubes with increasing concentrations of murine recombinant MIF protein (0, 6.25, 12.5, 25, 50, 100 ng/ml) and evaluated intracellular ROS generation in myotubes. DCFH-DA fluorescence shows MIF exposure markedly elevated ROS production in a concentration-dependent manner (Figures 4A-B). Subsequently, we performed Western blot analysis to examine the key components of the TXNIP/NLRP3 inflammasome pathway. MIF treatment increased the expression of TXNIP, NLRP3, Caspase-1, and mature IL-1β (Figures 4C-D). ELISA analysis of culture supernatants revealed that TNF-α, IL-1β secretion increased significantly and in a concentration-dependent manner following MIF treatment (Figure 4E). Collectively, these findings demonstrate that MIF mediates myotube degeneration through ROS-dependent activation of the TXNIP/NLRP3 inflammasome pathway, thereby triggering inflammatory responses characterized by intracellular inflammasome assembly and extracellular cytokine release. 5. 4-IPP blocks ROS/NLRP3 axis and reduces IL-1β secretion To further investigate the mechanistic role of MIF in muscle atrophy, we established the in vitro atrophy model as previously described by treating C2C12 myotubes with 20 μM DEX. Concomitantly, myotubes were co-treated with low-dose (10 μM) or high-dose (40 μM) 4-IPP. DCFH-DA fluorescence showed that DEX treatment induced excessive ROS production in myotubes, while co-treatment with 4-IPP effectively attenuated this ROS accumulation (Figures 5A-B). Western blot analysis confirmed that DEX increased the expression of TXNIP, NLRP3, Caspase-1, leading to elevated intracellular expression of mature IL-1β. Conversely, high-dose 4-IPP (40 μM) significantly suppressed the expression of TXNIP, NLRP3, Caspase-1 and reduced IL-1β production (Figures 5C-D). ELISA analysis indicated that DEX significantly increased the level of MIF, TNF-α, and IL-1β in culture supernatants, while high-dose 4-IPP substantially decreased these inflammatory mediators (Figure 5E). These findings substantiate the pathogenic role of MIF in the development of sarcopeniaand further suggest a mechanistic cascade wherein DEX induces MIF secretion, leading to intracellular ROS elevation and upregulating TXNIP/NLRP3 inflammasome expression, ultimately driving muscle atrophy. 6. 4-IPP Mitigates DEX-Induced muscle atrophy and functional decline in vivo We further validated 4-IPP efficacy in vivo . Eight-week-old male C57BL/6J mice were used to establish the DEX-induced muscle atrophy model and were divided into four groups for daily intraperitoneal injections over 15 days: (1) Control group receiving PEG400 vehicle (10 mL/kg), (2) DEX group receiving 25 mg/kg DEX in PEG400, (3) DEX+4-IPP Low-dose group receiving DEX with 1 mg/kg 4-IPP in 10% DMSO/90% corn oil, and (4) DEX+4-IPP High-dose group receiving DEX with 5 mg/kg 4-IPP. Body weight and grip strength were monitored daily throughout the treatment period. Following euthanasia on day 15, gastrocnemius muscles and tibiae were dissected for morphometric analysis (Figure 6A). Longitudinal monitoring revealed distinct treatment effects on body weight and muscle function. DEX administration induced progressive body weight loss, which was attenuated by 4-IPP co-treatment (Figure 6B). Concurrently, DEX-treated mice exhibited significantly impaired grip strength, which was substantially rescued by 4-IPP intervention (Figure 6C). Terminal measurements on day 15 demonstrated significantly reduced body weight in the DEX group compared to controls. While statistical significance was not reached, both low-dose and high-dose 4-IPP groups showed higher body weights than DEX-treated animals (Figure 6D). Conversely, grip strength analysis confirmed marked impairment in DEX-treated mice versus controls，with both 4-IPP doses significantly restoring muscle function (Figure 6D). Additionally, morphological assessment of gastrocnemius muscles showed visible atrophy in DEX-treated mice, while the 4-IPP cohorts maintained relatively preserved muscle volume (Figure 6E). Quantitative analysis demonstrated significantly lower gastrocnemius weights in the DEX group compared to controls, with 4-IPP treatment restoring muscle mass to near-normal levels (Figure 6F). Furthermore, H&E staining confirmed a significant reduction in muscle cross-sectional area in DEX-treated mice, an atrophic phenotype that was notably attenuated by 4-IPP co-treatment (Figures 6G-H). These in vivo results demonstrate that systemic administration of 4-IPP effectively mitigates dexamethasone-induced muscle atrophy in mice. 7. 4-IPP suppresses inflammasome activation in skeletal muscle Next, we explored the mechanism of 4-IPP in vitro . Our study shows that DEX-induced muscle atrophy in mice significantly activates the TXNIP/NLRP3 inflammasome pathway, as evidenced by increased expression of TXNIP, NLRP3, caspase-1, and IL-1β in gastrocnemius muscle tissues (Figure 7A-D). Immunohistochemical analysis revealed a significant increase in the positive staining area (%) of the TXNIP/NLRP3 inflammasome pathway in the DEX group compared to controls. Notably, 4-IPP treatment inhibited pathway activation in a dose-dependent manner, with the high-dose group (5 mg/kg) showing nearly complete suppression of TXNIP, NLRP3, and caspase-1 expression, alongside reduced IL-1β levels. These results suggest that pharmacological inhibition of MIF may offer a novel therapeutic strategy to alleviate sarcopenia-associated inflammation and muscle wasting. Further research is required to evaluate the clinical potential of targeting MIF. Discussion Sarcopenia is a clinical syndrome characterized by a reduction in muscle mass and strength, typically associated with aging but also observed in other pathological conditions. With the global aging population, sarcopenia has become a significant public health issue. This study specifically focused on the inflammatory mediator MIF to elucidate its mechanistic role in sarcopenia. Ultimately, we used a MIF inhibitor in in vivo experiments to treat sarcopenia. Inflammation plays a crucial role in the development of sarcopenia. It disrupts muscle metabolic homeostasis by inhibiting protein synthesis, activating the ubiquitin-proteasome system and autophagic pathways for protein degradation, impairing muscle stem cell function, and inducing insulin resistance [ 25 ]. Numerous studies have demonstrated elevated levels of various inflammatory cytokines in sarcopenia patients, indicative of a chronic inflammatory state. Previous research investigating MIF levels in patients with sarcopenia comorbid with COPD or CHF revealed significantly increased MIF concentrations in sarcopenic individuals, which correlated significantly with reduced muscle strength [ 26 , 27 ]. However, these studies did not account for the potential confounding effects of COPD and CHF on MIF levels. Consequently, during clinical plasma sample collection for the present study, sarcopenia patients with comorbid COPD or CHF were excluded. Our results align with prior findings: MIF levels were significantly elevated in sarcopenia patients, and associated inflammatory cytokines were also markedly increased. The results indicate MIF's involvement in sarcopenia development. MIF is a highly conserved, pleiotropic pro-inflammatory molecule implicated in numerous infectious, inflammatory, and autoimmune diseases. MIF influences the release of various cytokines, such as TNF-α, IFN-γ, IL-2, IL-6, and IL-8, through both direct and indirect pathways, playing a role in the development of inflammatory disorders [ 16 , 17 ]. This study found that adding exogenous MIF to C2C12 myoblast cell lines increased TNF-α and IL-1β levels in the cell culture supernatant, suggesting that MIF triggers extracellular inflammatory responses. In a DEX-induced muscle atrophy model, MIF and its related inflammatory factors, TNF-α and IL-1β, showed significant increases. The specific MIF inhibitor 4-IPP significantly decreased inflammatory cytokine levels in the supernatant, reinforcing MIF's role in promoting their release. Exposure of myoblast cell lines to MIF significantly enhanced the gene and protein expression of MuRF1 and Atrogin-1, essential E3 ubiquitin ligases in the UPS. These enzymes are established core mediators of sarcopenia and are upregulated in multiple muscle atrophy models [ 8 ]. The upregulation of MuRF1 and Atrogin-1 expression induced by MIF was inhibited by 4-IPP, indicating that MIF promotes muscle atrophy through inflammation and activation of the UPS pathway. Oxidative stress refers to an imbalance between pro-oxidant production and antioxidant defenses, resulting in the accumulation of oxidatively damaged molecules and the generation of ROS and their derivatives [ 28 ]. In humans, the mitochondrial respiratory chain is the primary source of ROS; as a high-energy-demand organ, skeletal muscle is rich in mitochondria [ 5 ]. Oxidative stress occurs when ROS production within the muscle exceeds its clearance capacity. TXNIP naturally inhibits thioredoxin (Trx), a protein that scavenges reactive oxygen species (ROS). Excessive ROS generation causes TXNIP to detach from the TXNIP-Trx complex, subsequently binding to NLRP3 and recruiting ASC and procaspase-1 to assemble the NLRP3 inflammasome. This process initiates inflammatory responses and pyroptosis [ 29 ]. Previous studies have reported that in tumor cells, MIF can bind to TXNIP, thereby alleviating TXNIP-mediated suppression of the NF-κB pathway and consequently promoting inflammation [ 30 ]. In this study, we demonstrated in muscle cells that MIF significantly elevates intracellular ROS levels. The increase in elevation leads to TXNIP protein expression, facilitating NLRP3 inflammasome assembly and boosting IL-1β expression and release. Furthermore, in a subsequent DEX-induced muscle atrophy model, we validated that inhibiting MIF effectively reduces ROS levels in myotubes and suppresses the activation of the TXNIP/NLRP3 pathway. The direct binding of MIF to TXNIP in muscle cells and its role in mediating the inflammatory response is yet to be clarified. In this study, we employed the classic glucocorticoid dexamethasone to establish both cellular and murine models of muscle atrophy. MIF and glucocorticoids exhibit a bell-shaped dose-response relationship, where high glucocorticoid levels inhibit MIF secretion, while low physiological levels promote its release from monocytes, macrophages, and T cells [ 31 , 32 ]. In vitro, MIF counteracts the suppressive impact of glucocorticoids on the secretion of pro-inflammatory cytokines, such as TNF, IL-1, IL-6, and IL-8, by activated macrophages. In vivo, within a lethal endotoxemia inflammation model, MIF completely counteracts the protective effects of glucocorticoids. Although glucocorticoids are generally known to suppress MIF expression at high doses, our experiments revealed that treatment of C2C12 myoblasts with 20 µM DEX markedly increased MIF concentration in the cell culture supernatant. Studies further confirm that DEX administration significantly upregulates MIF protein expression in tissues sensitive to glucocorticoid-induced growth inhibition, including immune/endocrine organs, skin, and skeletal muscle [ 32 , 33 ]. This paradoxical upregulation suggests a potential tissue-specific regulatory mechanism in skeletal muscle and indicates that MIF may serve as a key mediator in DEX-induced muscle atrophy. 4-IPP is a suicide substrate that irreversibly inhibits MIF's biological activity via covalent binding. Current studies demonstrate that 4-IPP has application value in tumor treatment and inflammation regulation by inhibiting MIF activity [ 34 – 36 ]. Zheng et al. Research indicates that 4-IPP inhibits MIF expression in HOS/143B cells [ 37 ]. In this study, treatment with a relatively high concentration of 4-IPP (40 µM) effectively counteracted DEX-induced upregulation of MIF protein levels, which is consistent with previous research findings. Through concentration-gradient experiments conducted in DEX-induced C2C12 myotube atrophy models, it was further verified that 4-IPP can effectively alleviate intracellular oxidative stress in myotubes and downregulate the activation level of the oxidative stress-associated TXNIP/NLRP3 pathway. In DEX-induced murine muscle atrophy models, 4-IPP significantly improved muscle mass and contractile strength in mice, with these results aligning with the cellular experimental conclusions. Although no direct reports exist on interactions between 4-IPP and DEX, whether synergistic or antagonistic effects exist between them requires further investigation. Conclusion This study elucidates the mechanistic role of MIF in sarcopenia pathogenesis through integrated in vitro and in vivo investigations. We demonstrate that MIF directly induces excessive ROS generation, thereby activating the TXNIP/NLRP3 inflammasome pathway and amplifying inflammatory cascades, ultimately driving muscle atrophy in vitro. Pharmacological inhibition of MIF using the specific inhibitor 4-IPP significantly improves muscle mass and strength in a murine sarcopenia model. These findings establish MIF as a pivotal mediator of inflammatory muscle wasting and validate its therapeutic targeting for sarcopenia intervention. Declarations Funding This research was funded by the Lishui Key Research and Development Program (Grant No. 2022ZDYF10) and the Zhejiang Province Medical and Health Science and Technology Projects (Grant No. 2024KY565). Data Availability Statement The original contributions presented in the study are included in the article and supplementary material, further inquiries can be directed to the corresponding authors. Authors’ Contributions The research was designed by Weiqing Li, Zhenzhong Chen, and Dengwei He. Weiqing Li, Yahong Lu and Yu Bai performed the experiments and acquired the data. Weiqing Li, Ye lin, Haiwei Ma, Zhiguo Zhou and Chendi Wang analyzed the data. Dengwei He and Hehuan Lai supervised the project. All authors participated in the creation of the article and consented to its submission. Disclosures The authors have stated that there are no conflicts of interest related to this article. References Cruz-Jentoft, A. J. et al. 2019. Sarcopenia: revised European consensus on definition and diagnosis Age Ageing 48(1):16–31. Fielding, R. A. et al. 2011. Sarcopenia: an undiagnosed condition in older adults. Current consensus definition: prevalence, etiology, and consequences. International working group on sarcopenia. Journal Of The American Medical Directors Association 12(4):249–256. Liu, D. et al. 2024. Frontiers in sarcopenia: Advancements in diagnostics, molecular mechanisms, and therapeutic strategies. Molecular Aspects Of Medicine 97:101270. Sandri, M. 2013. Protein breakdown in muscle wasting: role of autophagy-lysosome and ubiquitin-proteasome. International Journal Of Biochemistry & Cell Biology 45(10):2121–2129. Attaix, D. et al. 2005., The ubiquitin-proteasome system and skeletal muscle wasting. Essays Biochem, 41: pp. 173 – 86. Bodine, S. C. et al. 2001. Identification of ubiquitin ligases required for skeletal muscle atrophy Science 294(5547):1704–1708. Glass, D. J. 2005. Skeletal muscle hypertrophy and atrophy signaling pathways. Int. J Biochem Cell Biol 37(10):1974–1984. Rom, O., and A. Z. Reznick. 2016. The role of E3 ubiquitin-ligases MuRF-1 and MAFbx in loss of skeletal muscle mass. Free Radical Biology And Medicine 98:218–230. Chang, K. V. et al. 2023., Enhanced serum levels of tumor necrosis factor-alpha, interleukin-1beta, and – 6 in sarcopenia: alleviation through exercise and nutrition intervention. Aging (Albany NY), 15(22): pp. 13471–13485. Franceschi, C., and J. Campisi. 2014. Chronic inflammation (inflammaging) and its potential contribution to age-associated diseases. Journals Of Gerontology. Series A, Biological Sciences And Medical Sciences 69(Suppl 1):S4–9. Cai, D. et al. 2004., IKKbeta/NF-kappaB activation causes severe muscle wasting in mice. Cell, 119(2): pp. 285–298. Bonetto, A. et al. 2012., JAK/STAT3 pathway inhibition blocks skeletal muscle wasting downstream of IL-6 and in experimental cancer cachexia. Am J Physiol Endocrinol Metab, 303(3): pp. E410-21. Bonetto, A. et al. 2011. STAT3 activation in skeletal muscle links muscle wasting and the acute phase response in cancer cachexia. PLoS One 6(7):e22538. Zanders, L. et al. 2022., Sepsis induces interleukin 6, gp130/JAK2/STAT3, and muscle wasting. J Cachexia Sarcopenia Muscle, 13(1): pp. 713–727. Li, W. et al. 2009. Interleukin-1 stimulates catabolism in C2C12 myotubes. American Journal Of Physiology. Cell Physiology 297(3):C706–C714. Harris, J. et al. 2019. Rediscovering MIF: New Tricks for an Old Cytokine. Trends In Immunology 40(5):447–462. Calandra, T., and T. Roger. 2003. Macrophage migration inhibitory factor: a regulator of innate immunity. Nature Reviews Immunology 3(10):791–800. Lue, H. et al. 2002. Macrophage migration inhibitory factor (MIF): mechanisms of action and role in disease. Microbes And Infection 4(4):449–460. Osipyan, A., D. Chen, and F. J. Dekker. 2021. Epigenetic regulation in macrophage migration inhibitory factor (MIF)-mediated signaling in cancer and inflammation. Drug Discov Today, 26(7): pp. 1728–1734. Chuang, Y. C. et al. 2012. Macrophage migration inhibitory factor induces autophagy via reactive oxygen species generation. PLoS One 7(5):e37613. Lv, W. et al. 2016., Macrophage migration inhibitory factor promotes breast cancer metastasis via activation of HMGB1/TLR4/NF kappa B axis. Cancer Lett, 375(2): pp. 245–255. Zhuang, L. et al. 2020., Exosomal LncRNA-NEAT1 derived from MIF-treated mesenchymal stem cells protected against doxorubicin-induced cardiac senescence through sponging miR-221-3p. J Nanobiotechnology, 18(1): p. 157. Wang, H. et al. 2024., A small molecule macrophage migration inhibitory factor agonist ameliorates age-related myocardial intolerance to ischemia-reperfusion insults via metabolic regulation. Metabolism, 153: p. 155792. Zhu, W. et al. 2021., Macrophage migration inhibitory factor facilitates the therapeutic efficacy of mesenchymal stem cells derived exosomes in acute myocardial infarction through upregulating miR-133a-3p. J Nanobiotechnology, 19(1): p. 61. Li, Y. P. et al. 2003. TNF-alpha increases ubiquitin-conjugating activity in skeletal muscle by up-regulating UbcH2/E220k. The Faseb Journal 17(9):1048–1057. Kwak, J. Y. et al. 2018. Prediction of sarcopenia using a combination of multiple serum biomarkers. Scientific Reports 8(1):8574. Qaisar, R. et al. 2021. Prediction of sarcopenia using a battery of circulating biomarkers Sci Rep 11(1):8632. Xu, H. et al. 2021. Muscle mitochondrial catalase expression prevents neuromuscular junction disruption, atrophy, and weakness in a mouse model of accelerated sarcopenia. J Cachexia Sarcopenia Muscle 12(6):1582–1596. Tschopp, J., and K. Schroder. 2010. NLRP3 inflammasome activation: The convergence of multiple signalling pathways on ROS production? Nature Reviews Immunology 10(3):210–215. Kim, M. J. et al. 2017., Macrophage migration inhibitory factor interacts with thioredoxin-interacting protein and induces NF-kappaB activity. Cell Signal, 34: pp. 110–120. Leng, L. et al. 2009., Glucocorticoid-induced MIF expression by human CEM T cells. Cytokine, 48(3): pp. 177–185. Calandra, T. et al. 1995. MIF as a glucocorticoid-induced modulator of cytokine production. Nature 377(6544):68–71. Fingerle-Rowson, G. et al. 2003. Regulation of macrophage migration inhibitory factor expression by glucocorticoids in vivo. American Journal Of Pathology 162(1):47–56. Zheng, Y. et al. 2016., Role of Myeloma-Derived MIF in Myeloma Cell Adhesion to Bone Marrow and Chemotherapy Response . Journal Of The National Cancer Institute , 108(11). Winner, M. et al. 2008. A novel, macrophage migration inhibitory factor suicide substrate inhibits motility and growth of lung cancer cells. Cancer Research 68(18):7253–7257. Bozzi, F. et al. 2017., MIF/CD74 axis is a target for novel therapies in colon carcinomatosis. J Exp Clin Cancer Res, 36(1): p. 16. Zheng, L. et al. 2022. Destabilization of macrophage migration inhibitory factor by 4-IPP reduces NF-kappaB/P-TEFb complex-mediated c-Myb transcription to suppress osteosarcoma tumourigenesis. Clin Transl Med 12(1):e652. Additional Declarations No competing interests reported. Supplementary Files supplementaryfile.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 08 Apr, 2026 Reviews received at journal 07 Apr, 2026 Reviewers agreed at journal 02 Apr, 2026 Reviews received at journal 22 Mar, 2026 Reviewers agreed at journal 12 Mar, 2026 Reviewers invited by journal 09 Mar, 2026 Editor assigned by journal 02 Mar, 2026 Submission checks completed at journal 02 Mar, 2026 First submitted to journal 26 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8979207","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":604822734,"identity":"de580b8d-d766-452e-abd8-043090a6efe7","order_by":0,"name":"Weiqing Li","email":"","orcid":"","institution":"Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Weiqing","middleName":"","lastName":"Li","suffix":""},{"id":604822735,"identity":"0d9f6bd5-1070-465a-b91e-31a97feac9e5","order_by":1,"name":"Yu Bai","email":"","orcid":"","institution":"Lishui Central Hospital and Fifth Affiliated Hospital of Wenzhou Medical College","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Bai","suffix":""},{"id":604822737,"identity":"22c0b536-86f3-4723-9b35-b675ffa66fe9","order_by":2,"name":"Yahong Lu","email":"","orcid":"","institution":"Lishui Central Hospital and Fifth Affiliated Hospital of Wenzhou Medical College","correspondingAuthor":false,"prefix":"","firstName":"Yahong","middleName":"","lastName":"Lu","suffix":""},{"id":604822738,"identity":"f22acd74-6640-4118-975e-e61e1c63ff18","order_by":3,"name":"Lin Ye","email":"","orcid":"","institution":"Lishui Central Hospital and Fifth Affiliated Hospital of Wenzhou Medical College","correspondingAuthor":false,"prefix":"","firstName":"Lin","middleName":"","lastName":"Ye","suffix":""},{"id":604822739,"identity":"9475dd73-8482-40f9-8115-eb3f7172f658","order_by":4,"name":"Haiwei Ma","email":"","orcid":"","institution":"Lishui Central Hospital and Fifth Affiliated Hospital of Wenzhou Medical College","correspondingAuthor":false,"prefix":"","firstName":"Haiwei","middleName":"","lastName":"Ma","suffix":""},{"id":604822741,"identity":"7711f959-572b-408b-81cb-071aade0a5c2","order_by":5,"name":"Zhiguo Zhou","email":"","orcid":"","institution":"Lishui Central Hospital and Fifth Affiliated Hospital of Wenzhou Medical College","correspondingAuthor":false,"prefix":"","firstName":"Zhiguo","middleName":"","lastName":"Zhou","suffix":""},{"id":604822745,"identity":"3c3cf3f5-1f2c-4a82-b9fc-40160d3e34c4","order_by":6,"name":"Chendi Wang","email":"","orcid":"","institution":"Lishui Central Hospital and Fifth Affiliated Hospital of Wenzhou Medical College","correspondingAuthor":false,"prefix":"","firstName":"Chendi","middleName":"","lastName":"Wang","suffix":""},{"id":604822747,"identity":"7a2ca10f-23c0-4ab6-915d-b79f16f7b0c9","order_by":7,"name":"Hehuan Lai","email":"","orcid":"","institution":"Lishui Central Hospital and Fifth Affiliated Hospital of Wenzhou Medical College","correspondingAuthor":false,"prefix":"","firstName":"Hehuan","middleName":"","lastName":"Lai","suffix":""},{"id":604822749,"identity":"b900d609-30a3-4e07-a57e-7e4b57e0f975","order_by":8,"name":"Zhenzhong Chen","email":"","orcid":"","institution":"Lishui Central Hospital and Fifth Affiliated Hospital of Wenzhou Medical College","correspondingAuthor":false,"prefix":"","firstName":"Zhenzhong","middleName":"","lastName":"Chen","suffix":""},{"id":604822752,"identity":"4f1462a2-27ca-495b-862c-d4931ca5aa65","order_by":9,"name":"Dengwei He","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYBACAzBiYOBhYGA+cODDD9K0sCUenNlDghaQLuPDHGxEaDFnb94mwfDnjow5e8+Hw0DL5PnFDuDXYtlzrEyCse0Zj2XP2Q2HCywYDGfOTiDgsBs5ZjcYGw7zGNzI3XB4Bg9DgsFtQlruvzG7wfAHqOX+mweHediI0XKDB6iFDWQLDwORWs6klf8A+QXIMAAGsgQRfjl+eLMBMMTsgYzHHz78sJHnlyagBQSY/zAcgLElCCuHggMEVYyCUTAKRsEIBgCuJUl8tfdJWAAAAABJRU5ErkJggg==","orcid":"","institution":"Zhejiang University School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Dengwei","middleName":"","lastName":"He","suffix":""}],"badges":[],"createdAt":"2026-02-26 14:54:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8979207/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8979207/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104583472,"identity":"bb1b158a-f450-4965-8888-1fb13d99576f","added_by":"auto","created_at":"2026-03-13 15:21:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1004907,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePatients with sarcopenia exhibit elevated systemic inflammatory levels \u003c/strong\u003e(A) Schematic diagram of plasma sample collection and processing from study participants (B) Demographic characteristics (height, weight, sex, age, BMI) of each group. (C-E) calf circumference(C) grip strength(D) and SMI(E) in each group. (F) Plasma levels of MIF, TNF-α, IL-6, and IL-8 in each group. (G) Correlation analyses between plasma MIF concentrations and skeletal muscle index. (H) Correlation analyses between plasma MIF and muscle strength. Data are expressed as mean ± standard deviation. *P \u0026lt;. 05, **P \u0026lt;. 01, ***P \u0026lt;. 001 compared to healthy controls\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8979207/v1/19af93a3f1637b85724ee14b.png"},{"id":104583469,"identity":"7de544b8-7f2a-42f1-bd3e-a74476299419","added_by":"auto","created_at":"2026-03-13 15:21:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2859084,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExogenous MIF causes atrophy in C2C12 myotubes\u003c/strong\u003e （A）qPCR analysis reveals a dose-dependent increase in MuRF1 and Atrogin-1 mRNA levels after a 48-hour treatment with varying concentrations (0, 6. 25, 12.5, 25, 50, 100 ng/ml) of recombinant MIF protein. (B-C) Western blot analysis demonstrating dose-dependent increase in MuRF1 and Atrogin-1 protein levels in myotubes treated with MIF (concentrations as in A) for 48 hours. (D-E) Giemsa staining and quantification of myotubes diameter revealing dose-dependent myotubes atrophy in myotubes exposed to MIF (concentrations as in A) for 48 hours; scale bar:200μm. Data are expressed as mean ± standard deviation. *P \u0026lt; 0. 05, **P \u0026lt; 0. 01, ***P \u0026lt; 0. 001 compared to 0 ng/ml\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8979207/v1/39d26ddf38839976ba76c487.png"},{"id":104583467,"identity":"b3044f13-9f61-4841-827d-6063aa8d51fb","added_by":"auto","created_at":"2026-03-13 15:21:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2696899,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e4-IPP attenuates DEX-induced C2C12 myotubes atrophy \u003c/strong\u003e(A) CCK-8 assay showing no significant cytotoxicity of 4-IPP (0–80 μM) in C2C12 myotubes co-treated with DEX for 48 hours. (B) qPCR analysis shows that 4-IPP (10, 40 μM) dose-dependently suppresses MuRF1 and Atrogin-1 mRNA expression in DEX-induced atrophic myotubes. (C) Western blot analysis demonstrated that 4-IPP (10–80 μM) reduced MuRF1 and Atrogin-1 protein levels in a dose-dependent manner in DEX-treated myotubes. (D) Giemsa staining and myotube diameter quantification revealing rescue of DEX-induced atrophy by 4-IPP (10, 40 μM); scale bar:200μm. Data are expressed as mean ± standard deviation. Significance levels are indicated as follows: *P \u0026lt; 0. 05, **P \u0026lt; 0. 01, ***P \u0026lt; 0. 001 when compared to the DEX-treated group.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8979207/v1/1cef95b180d0ba2830b51a20.png"},{"id":104583477,"identity":"c59fc27f-5283-447b-8b99-6bf21889f6ca","added_by":"auto","created_at":"2026-03-13 15:21:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2293340,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMIF increases intracellular ROS production and activates the TXNIP/NLRP3 pathway in C2C12 myotubes\u003c/strong\u003e (A-B)Dose-dependent increase in intracellular ROS levels in C2C12 myotubes treated for 48 hours with recombinant MIF (0–100 ng/ml), \u0026nbsp;measured by DCFH-DA fluorescence intensity; scale bar:200μm. (C-D) Western blot analysis demonstrates that MIF treatment induces a dose-dependent increase in TXNIP, NLRP3, and Caspase-1 protein levels in myotubes, \u0026nbsp;consistent with the concentrations used in A. (E) TNF-α and IL-1β levels were significantly increased in the supernatants of C2C12 myotubes treated with MIF for 48 hours, as measured by ELISA . Data are expressed as mean ± standard deviation. *P \u0026lt; 0. 05, **P \u0026lt; 0. 01, ***P \u0026lt; 0. 001 compared to 0 ng/ml\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8979207/v1/b66a0045fff0c7e049c0d976.png"},{"id":104583470,"identity":"290bbae6-c75a-46fb-b8fb-d29867716b99","added_by":"auto","created_at":"2026-03-13 15:21:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1010412,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e4-IPP rescues DEX-induced oxidative stress and TXNIP/NLRP3 pathway activation in C2C12 myotubes\u003c/strong\u003e (A-B) Suppression of DEX-induced intracellular ROS overproduction by 4-IPP (10, 40 μM), measured by DCFH-DA fluorescence intensity; scale bar: 200μm. (C-D) Western blot analysis confirming dose-dependent inhibition of DEX-induced TXNIP, NLRP3, Caspase-1 and IL-1β protein expression by 4-IPP (10, 40 μM). (E) ELISA analysis demonstrates that DEX-induced C2C12 myotubes exhibit increased levels of secreted MIF, TNF-α, and IL-1β, which are subsequently reduced following 48-hour treatment with 4-IPP at concentrations of 10 and 40 μM. Data are expressed as mean ± standard deviation. *P \u0026lt; 0. 05, **P \u0026lt; 0. 01, ***P \u0026lt; 0. 001 compared to the DEX group\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8979207/v1/1010356e49ece6979d8015c9.png"},{"id":104583473,"identity":"53be6236-e0f6-4876-9d54-8282fce12132","added_by":"auto","created_at":"2026-03-13 15:21:14","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2732948,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e4-IPP mitigates muscle atrophy caused by DEX in mice. \u003c/strong\u003e(A) Diagram illustrating the DEX-induced atrophy model and 15-day 4-IPP treatment in 8-week-old mice. n = 6 per group (B-C) Progress of body weight loss and grip strength in each group. (D) Terminal body weight and grip strength measurements at day 15. (E) Representative muscle morphology from each group. (F) Quantitative measurements of muscle weight and muscle weight/tibia length ratio. (G-H) H\u0026amp;E staining of gastrocnemius muscles and statistical analysis of muscle cross-sectional area using ImageJ software; scale bar: =100μm. Control: PEG400 vehicle (10 mL/kg/day, i. p. ); DEX: 25 mg/kg/day in PEG400 (i. p. ); Low dose: DEX + 1 mg/kg 4-IPP (in 10% DMSO/90% corn oil, i. p. ); High dose: DEX + 5 mg/kg 4-IPP (vehicle as above). Data are expressed as mean ± standard deviation. *P \u0026lt; 0. 05, **P \u0026lt; 0. 01, ***P \u0026lt; 0. 001 compared to the DEX group\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8979207/v1/a7113b36eea0d6bf62f30ec4.png"},{"id":104583476,"identity":"74e66d58-2ad4-40c1-8307-90de3501fefa","added_by":"auto","created_at":"2026-03-13 15:21:14","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":4275409,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e4-IPP suppress the expression of TXNIP/NLRP3 pathway in skeletal muscle. \u003c/strong\u003e(A-D) Representative immunohistochemical staining images (left panels) show the expression of TXNIP, NLRP3, caspase-1, and IL-1β in gastrocnemius muscle sections, with immunopositive areas (%) quantified using ImageJ software (right panels); scale bar 100 μm. Data are expressed as mean ± standard deviation. *P \u0026lt; 0. 05, **P \u0026lt; 0. 01, ***P \u0026lt; 0. 001 compared to the DEX group\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8979207/v1/3006900b5d88960fecec88f6.png"},{"id":104782244,"identity":"a49bc83c-3bbb-44ef-9d21-eb54d5e22b4d","added_by":"auto","created_at":"2026-03-17 07:57:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":22276601,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8979207/v1/31b1dc4b-d7f2-46ff-a137-e7b820993045.pdf"},{"id":104583471,"identity":"43289b6b-6444-4434-abb6-3a5b38703c7d","added_by":"auto","created_at":"2026-03-13 15:21:14","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6437855,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-8979207/v1/fbc51773a10c49c3c3c576eb.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"4-IPP Alleviates Dexamethasone-Induced Muscle Atrophy by Targeting the MIF/TXNIP/NLRP3 Axis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSarcopenia is a common, progressive musculoskeletal disorder marked by age-related declines in skeletal muscle mass, strength, and function. It is associated with an increased risk of adverse outcomes, including falls, functional impairment, frailty, and mortality, thereby substantially diminishing quality of life and imposing a significant socioeconomic burden [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Epidemiological research indicates that sarcopenia affects about 5\u0026ndash;13% of people aged 60\u0026ndash;70, with prevalence rising to 11\u0026ndash;50% in individuals aged 80 and above. With the ongoing trend of global population aging, the economic burden associated with sarcopenia is expected to escalate further [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, despite its clinical significance, effective therapeutic strategies for sarcopenia remain limited.\u003c/p\u003e \u003cp\u003eThe core pathological feature of sarcopenia is an imbalance between protein synthesis and proteolysis. Under physiological conditions, skeletal muscle maintains mass and function through balanced protein synthesis and degradation [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The ubiquitin-proteasome system (UPS) is essential in proteolytic processes, with E3 ubiquitin ligases Atrogin-1 and MuRF-1 serving as key regulators of muscle atrophy [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Bodine et al. Overexpression of Atrogin-1 causes atrophy in cultured myotubes, whereas genetic deficiency in either ligase provides resistance to atrophy in mice [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Research indicates that Atrogin-1 and MuRF1 expression is significantly increased by various catabolic stimuli, such as oxidative stress, inflammatory cytokines, and glucocorticoids, which are associated with enhanced muscle degradation [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Atrogin-1 and MuRF1 are involved in muscle wasting associated with various common human diseases, including cancer, diabetes, chronic kidney disease (CKD), chronic obstructive pulmonary disease (COPD), and age-related sarcopenia [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe pathogenesis of sarcopenia involves complex physiological processes, with systemic chronic inflammation considered a critical contributing factor [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Research indicates that inflammatory factors such as TNF-α, IL-6, IL-1β, and IFN-γ are notably increased in sarcopenia patients and closely linked to reduced muscle strength. Chronic inflammation leads to the substantial release of pro-inflammatory cytokines into the bloodstream, which directly affect the immune system and facilitate muscle protein degradation through metabolic pathway modulation. TNF-α induces atrophy by activating the IKKβ/NF-κB signaling pathway, which upregulates the expression of the ubiquitin carrier protein UbcH2 and thereby activates the UPS. Alternatively, it can directly induce the expression of the E3 ubiquitin ligase MuRF1, facilitating the ubiquitin-mediated degradation of myosin heavy chain (MyHC) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. IL-6 activates NF-κB via the gp130/JAK/STAT3 pathway, leading to increased expression of E3 ubiquitin ligases and subsequent muscle wasting. Concomitantly, skeletal muscle-specific IL-6 knockout effectively attenuates this pathological process [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Furthermore, IL-1β activates p38 MAPK and NF-κB signaling through mechanisms independent of oxidative stress and the AKT/Foxo pathway. It specifically upregulates the Atrogin-1 and MuRF1, thereby accelerating myofibrillar protein degradation and contributing to muscle atrophy [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Inflammatory mechanisms are pivotal in sarcopenia pathogenesis, making chronic inflammation a potential therapeutic target.\u003c/p\u003e \u003cp\u003eMacrophage migration inhibitory factor (MIF) is a widely distributed pleiotropic proinflammatory cytokine produced by various cell types, playing a role in inflammatory responses through multiple signaling pathways. MIF is associated with the development and advancement of inflammation-related diseases, such as glomerulonephritis, inflammatory bowel disease, and pancreatitis, as well as immune-mediated disorders like rheumatoid arthritis, systemic lupus erythematosus, and spondyloarthritis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. MIF, upon binding to its receptor CD74 and coreceptors, activates signaling pathways such as NF-κB, MAPK, and PI3K/Akt. These pathways trigger the release of pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6, which enhance localized and systemic inflammation [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Previous research indicates that MIF stimulates reactive oxygen species (ROS) production and autophagy in hepatocellular carcinoma cell lines [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Moreover, MIF-mediated activation of ROS signaling has been implicated, at least in part, in promoting breast cancer metastasis [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Current research on MIF in the muscular system has primarily focused on its cardioprotective effects in myocardial ischemia and its involvement in anti-senescence mechanisms [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. However, its specific role and underlying mechanisms in sarcopenia remain largely unexplored. Investigating MIF's role in inflammatory signaling and chronic diseases may reveal new therapeutic targets for age-related muscle atrophy by clarifying its specific function in muscle wasting.\u003c/p\u003e \u003cp\u003eThis study investigates the role of MIF in muscle wasting and assesses the therapeutic potential of 4-IPP, a specific MIF inhibitor, through the use of \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e muscle atrophy models. The research improves our understanding of inflammation-induced sarcopenia by clarifying MIF-mediated pathological pathways, providing new insights for clinical intervention.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eReagents\u003c/h2\u003e \u003cp\u003eFetal bovine serum (FBS), horse serum, DMEM, and streptomycin-penicillin (SP) solution were sourced from Thermo Fisher Scientific (Waltham, MA, USA). Recombinant Mouse MIF was obtained from R\u0026amp;D Systems (Minneapolis, MN, USA). Dexamethasone (DEX) was acquired from Sigma-Aldrich (St. Louis, MO, USA), and 4-iodo-6-phenylpyrimidine (4-IPP) was procured from MedChemExpress (Monmouth Junction, NJ, USA). Primary antibodies against MuRF1, Atrogin-1, NLRP3, Caspase-1, and IL-1β were procured from Proteintech (Rosemont, IL, USA). Antibodies against TXNIP and GAPDH were obtained from Cell Signaling Technology (Danvers, MA, USA) and Abcam (Cambridge, UK), respectively. Species-matched secondary antibodies (anti-rabbit and anti-mouse) were acquired from Cell Signaling Technology.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHuman subjects\u003c/h3\u003e\n\u003cp\u003e The Ethics Committee of Lishui Central Hospital, Zhejiang Province, approved this retrospective study ( approval number 2020-17). Sarcopenia was classified according to the Asian Working Group on Sarcopenia 2019 (AWGS 2019) criteria, which include low grip strength (male\u0026thinsp;\u0026lt;\u0026thinsp;28 kg, female\u0026thinsp;\u0026lt;\u0026thinsp;18 kg), low calf circumference (male\u0026thinsp;\u0026lt;\u0026thinsp;34 cm, female\u0026thinsp;\u0026lt;\u0026thinsp;33 cm), and low skeletal muscle index (SMI; male\u0026thinsp;\u0026lt;\u0026thinsp;7. 0 kg/m\u0026sup2;, female\u0026thinsp;\u0026lt;\u0026thinsp;5. 4 kg/m\u0026sup2;) as measured by dual-energy X-ray absorptiometry (DXA). Plasma samples were collected from the antecubital vein between 6:00 and 8:00 a. m., then centrifuged and stored at -80\u0026deg;C. Written informed consent was obtained from all participants. Patients were excluded from the study if they had chronic diseases (such as COPD, CHF, diabetes mellitus, or cancer), autoimmune disorders (including rheumatoid arthritis, psoriasis, multiple sclerosis, systemic sclerosis, or inflammatory bowel disease), a recent infection (within the past week), or had used corticosteroids in the past month to eliminate the impact of these factors on MIF levels.\u003c/p\u003e\n\u003ch3\u003eELISA (Enzyme-linked immunosorbent assay)\u003c/h3\u003e\n\u003cp\u003eVenous blood samples from patients were collected into anticoagulant tubes between 6:00\u0026ndash;8:00 AM and processed immediately. Plasma was obtained by centrifugation at 3, 000\u0026times;g for 10 min at 4\u0026deg;C. Cell culture supernatants were centrifuged at 1, 000\u0026times;g for 5 minutes to eliminate cellular debris, then aliquoted and stored consistently. MIF, TNF-α, IL-6, and IL-1β concentrations were quantified using ELISA kits from Proteintech (Group, Inc., Rose, USA.).\u003c/p\u003e\n\u003ch3\u003eCytotoxicity assay\u003c/h3\u003e\n\u003cp\u003eThe cytotoxic effect of 4-IPP on C2C12 cells was assessed using the CCK-8 cell proliferation and cytotoxicity assay kit (Dalian, Liaoning, China). Cells were initially seeded at a density of 5\u0026times;10^3 per well in a 96-well plate. After 24 hours of culture to allow cell adhesion, the cells were further cultured until reaching 80% confluence. The growth medium was subsequently substituted with a differentiation medium containing 2% horse serum to promote myotube formation over a period of 6 days. Post-differentiation, cells were exposed to 4-IPP at concentrations of 0 to 640 \u0026micro;M, alongside DEX, for 48 hours. Each concentration was tested in five replicate wells. In the last hour of incubation, 10 \u0026micro;L of CCK-8 reagent was added to each well with 100 \u0026micro;L of DMEM and incubated at 37\u0026deg;C. Absorbance at 450 nm was recorded with a TECAN Infinite M200 microplate reader (TECAN, M\u0026auml;nnedorf, Switzerland).\u003c/p\u003e\n\u003ch3\u003eExtraction of RNA and quantitative real-time PCR (qPCR)\u003c/h3\u003e\n\u003cp\u003eCells were plated in a 12-well dish to facilitate adhesion. Cells were subjected to total RNA extraction utilizing the RNA Rapid Extraction Kit from Yishan Biotech, Shanghai, China. cDNA was synthesized via reverse transcription using the Strand cDNA Synthesis SuperMix for qPCR kit (Yishan Biotech, Shanghai, China). qPCR was used to evaluate relative mRNA expression levels. Detection was performed using qPCR SYBR Green Master Mix (Yishan Biotech, Shanghai, China) and an ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The cycling protocol involved initial denaturation at 95℃, followed by 40 amplification cycles at 60℃. The 2\u0026thinsp;\u0026minus;\u0026thinsp;ΔΔCT method was employed to calculate the results. The figure below presents the primer sequences.\u003c/p\u003e \u003cp\u003eMouse MuRF1 primers: Forward 5\u0026prime;-TGGAGGTCGTTTCCGTTGC-3\u0026prime;, Reverse 5\u0026prime;-CTGAGGTTCTGTCTGCGGTAG-3\u0026prime;.\u003c/p\u003e \u003cp\u003eMouse Atrogin-1 primers: Forward 5\u0026prime;-CAGCTTCGTGAGCGACCTC-3\u0026prime;, Reverse 5\u0026prime;-GGCAGTCGAGAAGTCCAGTC-3\u0026prime;.\u003c/p\u003e \u003cp\u003eMouse GAPDH primers: Forward 5\u0026prime;-TAGGGCCTCTCTTGCTCAGT-3\u0026prime;, Reverse 5\u0026prime;-TGTCAAGCTCATTTCCTGGT-3\u0026prime;.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eTotal protein was extracted by lysing cells using RIPA lysis buffer (Solarbio Science \u0026amp; Technology Co., Ltd., Beijing, China) with added protease inhibitors. Protein levels were measured with a BCA Protein Assay Kit from Beyotime Biotechnology (Shanghai, China).\u003c/p\u003e \u003cp\u003eFor the Western blot experiment, 0 \u0026micro;g of protein samples were separated using SDS‒PAGE and then transferred onto a PVDF membrane (Bio-Rad Laboratories, Hercules, CA, USA). Membranes were incubated in 5% non-fat milk in TBST at room temperature for 1\u0026ndash;2 hours to block them. After blocking, membranes were briefly rinsed with TBST and cut into strips according to the molecular weight markers to isolate the regions containing the proteins of interest. The membrane strips were incubated with primary antibodies at 4\u0026deg;C for 12\u0026ndash;16 hours overnight. The following day, membranes underwent three 5-minute washes with TBST and were then incubated with species-specific secondary antibodies (anti-mouse or anti-rabbit IgG, 1:5, 000 dilution) for 1\u0026ndash;2 hours at room temperature. After three additional 5-minute TBST washes, protein bands were detected using an Enhanced ECL Ultra-Sensitive Chemiluminescence Kit (Yeasen Biotech, Shanghai, China) and captured with an Invitrogen iBright 1500 Imaging System (Thermo Fisher Scientific, Waltham, MA, USA). Band intensity was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The protein bands in the figure represent typical outcomes from three independent experiments, with quantitative data presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGiemsa Staining\u003c/h3\u003e\n\u003cp\u003eCells were seeded into 6-well plates and differentiated into multinucleated myotubes. After treatment for 48h, removing the culture medium, cells were gently washed twice with PBS. Fixation was performed by incubating cells with 70% ethanol at room temperature for 10 minutes. Fixed cells were then stained with the freshly prepared modified Giemsa working solution (1\u0026times;) (Beyotime Biotechnology, Shanghai, China) After staining for 45 minutes. Unbound dye was removed by thorough rinsing with distilled water until the effluent became clear. Plates were air-dried, and myotube morphology images were captured using an inverted light microscope with a digital camera. Myotube diameters were quantified from ten random fields per well using ImageJ software.\u003c/p\u003e\n\u003ch3\u003eAnimal model and treatment\u003c/h3\u003e\n\u003cp\u003eEight-week-old C57BL/6J mice were obtained from HangSi Biotech in Hangzhou, China. Animal experiments received approval from the Ethics Committee of Lishui Central Hospital (Research Ethics Committee Number 2024YD0161) and were conducted following the Guidelines for Animal Treatment and the NIH Guide for the Care and Use of Experimental Animals.\u003c/p\u003e \u003cp\u003eAn 8-week-old mouse model for DEX-induced muscle atrophy was developed using 24 male C57BL/6J mice, divided into four groups for daily intraperitoneal injections over 15 days. The Control group received 30% PEG400 in 0. 9% saline (10 mL/kg body weight). The DEX group was administered DEX (25 mg/kg) in 30% PEG400 in 0. 9% saline. The DEX\u0026thinsp;+\u0026thinsp;4-IPP (Low-dose) group received DEX with 4-IPP (1 mg/kg) in 10% DMSO in corn oil. The DEX\u0026thinsp;+\u0026thinsp;4-IPP (High-dose) group received DEX with 4-IPP (5 mg/kg) in 10% DMSO in corn oil. Throughout the treatment period, grip strength and body weight were monitored. Twenty-four hours post-final injection, mice were euthanized, and their gastrocnemius muscles, tibiae, and organs (heart, liver, spleen, lungs, and kidneys) were collected and fixed in 4% paraformaldehyde (PFA) for histological analysis.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eFour-Limb Grip Strength Test\u003c/h2\u003e \u003cp\u003eA digital grip strength meter (model ZX-ZL; Zhixiang, Shanghai Yuyan Scientific Instruments Co., Ltd., China) was used to evaluate four-limb grip strength of mice. During testing, the mouse was placed on the grip strength meter, ensuring all four limbs firmly grasped the horizontal bar. The tail was gently pulled horizontally until the mouse released its grip, and the device's maximum force value was recorded. Each mouse underwent five consecutive trials with 10-second rest intervals between measurements. After excluding the highest and lowest values, the average maximal grip strength was determined by calculating the mean of the remaining three measurements.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eHaematoxylin\u0026ndash;eosin (H\u0026amp;E) staining\u003c/h2\u003e \u003cp\u003eTissue sections embedded in paraffin were deparaffinized using xylene and then rehydrated with a series of graded ethanol. Following this, sections were stained with haematoxylin and eosin and subsequently dehydrated through an ascending ethanol gradient. Sections were ultimately cleared in xylene and mounted using neutral balsam. Images were captured with a Leica DMi8 confocal microscope (Leica, Wetzlar, Germany). ImageJ software was utilized to quantify muscle fiber cross-sectional area (CSA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemical staining\u003c/h2\u003e \u003cp\u003eGastrocnemius muscles were fixed in 4% PFA, embedded in paraffin, and sectioned to a thickness of 4 \u0026micro;m. Following deparaffinization in xylene and a graded ethanol series, antigen retrieval was conducted using heat in 10 mM citrate buffer (pH 6. 0) at 95\u0026deg;C for 20 minutes. Endogenous peroxidase activity was quenched with 3% H₂O₂ in methanol for 30 min at room temperature. Sections were blocked with 5% normal goat serum for 1 hour and incubated overnight at 4\u0026deg;C with primary antibodies: anti-TXNIP (1:200), anti-NLRP3 (1:300), anti-caspase-1 (1:200), and anti-IL-1β (1:100). Following PBS washes, a 1:500 dilution of HRP-conjugated secondary antibody was applied for 1 hour at room temperature. Signal detection used 3, 3'-diaminobenzidine (DAB) chromogen with hematoxylin counterstaining. Images were captured with a Leica DMi8 confocal microscope. An investigator, blinded to group allocation, quantified the positive staining area (%) from five random fields per section at 200\u0026times; magnification using ImageJ.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eExperiments were conducted in triplicate, yielding consistent results across independent replicates. Data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) or shown as representative images from a minimum of three independent experiments. GraphPad Prism 10 (GraphPad Software, La Jolla, CA, USA) was used for statistical analyses. Group differences were evaluated using one-way ANOVA with post-hoc tests or Student's t-test for pairwise comparisons. A P-value less than 0. 05 was deemed statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e1. Elevated MIF and cytokines correlate with muscle weakness in sarcopenic patients\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess inflammatory levels and differences in MIF between groups, participants were stratified into two categories according to AWGS2019: Sarcopenia and Healthy controls (Figure 1A). Demographic parameters showed no significant differences\u0026nbsp;between the two groups (Figure 1B). However, the sarcopenia group exhibited significantly lower calf circumference (Male: 35.89±1.21 cm vs 30.79±1.07 cm, p\u0026lt;0.001；Female: 34.41±1.04 cm vs 27.83±2.33 cm, p\u0026lt;0.001; Figure 1C), grip strength(Male: 32.39±3.42 kg vs 15.86±1.98 kg , p\u0026lt;0.001；Female: 22.52±2.45 kg vs 13.01±3.72 kg, p\u0026lt;0.001; Figure 1D), and skeletal muscle parameters (Male: 7.43±0.35 kg/m²\u0026nbsp;vs 6.72±0.18 kg/m², p\u0026lt;0.01；Female: 6.08±0.60 kg/m²\u0026nbsp;vs 4.91±0.37 kg/m², p\u0026lt;0.001; Figure 1E) compared to the healthy control group.\u0026nbsp;In contrast\u0026nbsp;, plasma levels of MIF (1.47±0.41 pg/ml vs 2.11±0.42 pg/ml, p\u0026lt;0.001), TNF-α (133.2±33.47 pg/ml vs 233.4±61.67 pg/ml, p\u0026lt;0.001), IL-6 (159.0±34.27 pg/ml vs 291.6±65.72 pg/ml, p\u0026lt;0.001), and IL-8 (259.1±70.35 pg/ml vs 546.1±127.60 pg/ml, p\u0026lt;0.001) were notably higher in the sarcopenia group than in the healthy controls (Figure 1F).\u003c/p\u003e\n\u003cp\u003eCorrelation analyses revealed significant negative correlations between plasma MIF concentrations and both skeletal muscle index (p=0.0038, Figure 1G) and muscle strength (p=0.0016, Figure 1H). These results suggest that elevated MIF levels are associated with reduced muscle function in sarcopenia patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. Exogenous MIF induces myotube atrophy and upregulates MuRF1 and Atrogin-1 expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe role of MIF in C2C12 myotubes was further investigated. C2C12 myoblasts were differentiated into myotubes by treating them with 2% horse serum for 6 days. Differentiated myotubes were then exposed to increasing concentrations of murine recombinant MIF protein (0, 6.25, 12.5, 25, 50, 100 ng/ml) for 48 hours to evaluate its effects. qPCR analysis showed that MIF treatment significantly upregulated the expression of atrophy-related markers, \u003cem\u003eMuRF1\u003c/em\u003e and \u003cem\u003eAtrogin-1\u003c/em\u003e, in a dose-dependent manner, with the effect becoming more pronounced at higher concentrations (Figure 2A). Consistent with these results, western blot analysis also confirmed the dose-dependent increase in marker expression (Figure 2B-C). Morphologically, Giemsa staining demonstrated dose-dependent myotube thinning with increasing MIF concentrations (Figures 2D-E).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTaken together, these data demonstrate that exogenous MIF induces myotube atrophy through the activation of proteolytic pathways.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. 4-IPP reverses DEX-induced myotube atrophy, reduces MuRF1 and Atrogin-1 expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo confirm the role of MIF in muscle atrophy and assess the therapeutic potential of inhibiting MIF in sarcopenia, we used the specific inhibitor 4-IPP for further experiments. C2C12 myotubes were exposed to 20 μM DEX to establish an \u003cem\u003ein vitro\u003c/em\u003e model of muscle atrophy. To determine the optimal 4-IPP concentration, C2C12 myotubes were co-treated with DEX and increasing 4-IPP doses (0–640 μM) for 48 hours. CCK-8 assay results indicated that cell viability remained unaffected at concentrations ≤80 μM but significantly decreased in a dose-dependent manner at higher doses (\u0026gt;80 μM) (Figure 3A).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eqPCR analysis revealed that DEX significantly increased the expression of MuRF1 and Atrogin-1, serving as evidence of the atrophy model. This upregulation progressively decreased as the concentration of 4-IPP increased (Figure 3B). Western blot analysis confirmed these results, further validating the inhibitory effect of 4-IPP on DEX-induced upregulation (Figure 3C). \u0026nbsp;Consistently, Giemsa staining confirmed the morphological rescue effects of 4-IPP in a concentration-dependent manner (Figure 3D-E). These results collectively confirm the pathogenic role of MIF in sarcopenia and provide preliminary evidence for the therapeutic potential of 4-IPP in alleviating muscle wasting.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. MIF drives ROS production and activates TXNIP/NLRP3 inflammasome\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInflammation and oxidative stress are closely interconnected, and their vicious cycle serves as a critical driver of sarcopenia pathogenesis. To explore the role of MIF in this context, We treated C2C12 myotubes with increasing concentrations of murine recombinant MIF protein (0, 6.25, 12.5, 25, 50, 100 ng/ml) and evaluated intracellular ROS generation in myotubes. DCFH-DA fluorescence shows MIF exposure markedly elevated ROS production in a concentration-dependent manner (Figures 4A-B). Subsequently, we performed Western blot analysis to examine the key components of the TXNIP/NLRP3 inflammasome pathway. MIF treatment increased the expression of TXNIP, NLRP3, Caspase-1, and mature IL-1β (Figures 4C-D). ELISA analysis of culture supernatants revealed that TNF-α, IL-1β secretion increased significantly and in a concentration-dependent manner following MIF treatment (Figure 4E).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCollectively, these findings demonstrate that MIF mediates myotube degeneration through ROS-dependent activation of the TXNIP/NLRP3 inflammasome pathway, thereby triggering inflammatory responses characterized by intracellular inflammasome assembly and extracellular cytokine release.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5. 4-IPP blocks ROS/NLRP3 axis and reduces IL-1β secretion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further investigate the mechanistic role of MIF in muscle atrophy, we established the\u0026nbsp;\u003cem\u003ein vitro\u003c/em\u003e atrophy model as previously described by treating C2C12 myotubes with 20 μM DEX. Concomitantly, myotubes were co-treated with low-dose (10 μM) or high-dose (40 μM) 4-IPP. DCFH-DA fluorescence showed that DEX treatment induced excessive ROS production in myotubes, while co-treatment with 4-IPP effectively attenuated this ROS accumulation (Figures 5A-B). Western blot analysis confirmed that DEX increased the expression of TXNIP, NLRP3, Caspase-1, leading to elevated intracellular expression of mature IL-1β. Conversely, high-dose 4-IPP (40 μM) significantly suppressed the expression of TXNIP, NLRP3, Caspase-1 and reduced IL-1β production (Figures 5C-D). ELISA analysis indicated that DEX significantly increased the level of MIF, TNF-α, and IL-1β in culture supernatants, while high-dose 4-IPP substantially decreased these inflammatory mediators (Figure 5E). These findings substantiate the pathogenic role of MIF in the development of sarcopeniaand further suggest a mechanistic cascade wherein DEX induces MIF secretion, leading to intracellular ROS elevation and upregulating TXNIP/NLRP3 inflammasome expression, ultimately driving muscle atrophy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6. 4-IPP Mitigates DEX-Induced muscle atrophy and functional decline \u003cem\u003ein vivo\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe further validated 4-IPP efficacy\u0026nbsp;\u003cem\u003ein vivo\u003c/em\u003e. Eight-week-old male C57BL/6J mice were used to establish the DEX-induced muscle atrophy model and were divided into four groups for daily intraperitoneal injections over 15 days: (1) Control group receiving PEG400 vehicle (10 mL/kg), (2) DEX group receiving 25 mg/kg DEX in PEG400, (3) DEX+4-IPP Low-dose group receiving DEX with 1 mg/kg 4-IPP in 10% DMSO/90% corn oil, and (4) DEX+4-IPP High-dose group receiving DEX with 5 mg/kg 4-IPP. Body weight and grip strength were monitored daily throughout the treatment period. Following euthanasia on day 15, gastrocnemius muscles and tibiae were dissected for morphometric analysis (Figure 6A).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLongitudinal monitoring revealed distinct treatment effects on body weight and muscle function. DEX administration induced progressive body weight loss, which was attenuated by 4-IPP co-treatment (Figure 6B). Concurrently, DEX-treated mice exhibited significantly impaired grip strength, which was substantially rescued by 4-IPP intervention (Figure 6C). Terminal measurements on day 15 demonstrated significantly reduced body weight in the DEX group compared to controls. While statistical significance was not reached, both low-dose and high-dose 4-IPP groups showed higher body weights than DEX-treated animals (Figure 6D). Conversely, grip strength analysis confirmed marked impairment in DEX-treated mice versus controls，with both 4-IPP doses significantly restoring muscle function \u0026nbsp;(Figure 6D). Additionally, morphological assessment of gastrocnemius muscles showed visible atrophy in DEX-treated mice, while the 4-IPP cohorts maintained relatively preserved muscle volume (Figure 6E). Quantitative analysis demonstrated significantly lower gastrocnemius weights in the DEX group compared to controls, with 4-IPP treatment restoring muscle mass to near-normal levels (Figure 6F). Furthermore, H\u0026amp;E staining confirmed a significant reduction in muscle cross-sectional area in DEX-treated mice, an atrophic phenotype that was notably attenuated by 4-IPP co-treatment (Figures 6G-H). These\u0026nbsp;\u003cem\u003ein vivo\u003c/em\u003e results demonstrate that systemic administration of 4-IPP effectively mitigates dexamethasone-induced muscle atrophy in mice.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e7. 4-IPP suppresses inflammasome activation in skeletal muscle\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we explored the mechanism of 4-IPP \u003cem\u003ein vitro\u003c/em\u003e. Our study shows that DEX-induced muscle atrophy in mice significantly activates the TXNIP/NLRP3 inflammasome pathway, as evidenced by increased expression of TXNIP, NLRP3, caspase-1, and IL-1β in gastrocnemius muscle tissues (Figure 7A-D). Immunohistochemical analysis revealed a significant increase in the positive staining area (%) of the TXNIP/NLRP3 inflammasome pathway in the DEX group compared to controls. Notably, 4-IPP treatment inhibited pathway activation in a dose-dependent manner, with the high-dose group (5 mg/kg) showing nearly complete suppression of TXNIP, NLRP3, and caspase-1 expression, alongside reduced IL-1β levels. These results suggest that pharmacological inhibition of MIF may offer a novel therapeutic strategy to alleviate sarcopenia-associated inflammation and muscle wasting. Further research is required to evaluate the clinical potential of targeting MIF.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eSarcopenia is a clinical syndrome characterized by a reduction in muscle mass and strength, typically associated with aging but also observed in other pathological conditions. With the global aging population, sarcopenia has become a significant public health issue. This study specifically focused on the inflammatory mediator MIF to elucidate its mechanistic role in sarcopenia. Ultimately, we used a MIF inhibitor in in vivo experiments to treat sarcopenia.\u003c/p\u003e \u003cp\u003eInflammation plays a crucial role in the development of sarcopenia. It disrupts muscle metabolic homeostasis by inhibiting protein synthesis, activating the ubiquitin-proteasome system and autophagic pathways for protein degradation, impairing muscle stem cell function, and inducing insulin resistance [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Numerous studies have demonstrated elevated levels of various inflammatory cytokines in sarcopenia patients, indicative of a chronic inflammatory state. Previous research investigating MIF levels in patients with sarcopenia comorbid with COPD or CHF revealed significantly increased MIF concentrations in sarcopenic individuals, which correlated significantly with reduced muscle strength [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. However, these studies did not account for the potential confounding effects of COPD and CHF on MIF levels. Consequently, during clinical plasma sample collection for the present study, sarcopenia patients with comorbid COPD or CHF were excluded. Our results align with prior findings: MIF levels were significantly elevated in sarcopenia patients, and associated inflammatory cytokines were also markedly increased. The results indicate MIF's involvement in sarcopenia development.\u003c/p\u003e \u003cp\u003eMIF is a highly conserved, pleiotropic pro-inflammatory molecule implicated in numerous infectious, inflammatory, and autoimmune diseases. MIF influences the release of various cytokines, such as TNF-α, IFN-γ, IL-2, IL-6, and IL-8, through both direct and indirect pathways, playing a role in the development of inflammatory disorders [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. This study found that adding exogenous MIF to C2C12 myoblast cell lines increased TNF-α and IL-1β levels in the cell culture supernatant, suggesting that MIF triggers extracellular inflammatory responses. In a DEX-induced muscle atrophy model, MIF and its related inflammatory factors, TNF-α and IL-1β, showed significant increases. The specific MIF inhibitor 4-IPP significantly decreased inflammatory cytokine levels in the supernatant, reinforcing MIF's role in promoting their release. Exposure of myoblast cell lines to MIF significantly enhanced the gene and protein expression of MuRF1 and Atrogin-1, essential E3 ubiquitin ligases in the UPS. These enzymes are established core mediators of sarcopenia and are upregulated in multiple muscle atrophy models [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The upregulation of MuRF1 and Atrogin-1 expression induced by MIF was inhibited by 4-IPP, indicating that MIF promotes muscle atrophy through inflammation and activation of the UPS pathway.\u003c/p\u003e \u003cp\u003eOxidative stress refers to an imbalance between pro-oxidant production and antioxidant defenses, resulting in the accumulation of oxidatively damaged molecules and the generation of ROS and their derivatives [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In humans, the mitochondrial respiratory chain is the primary source of ROS; as a high-energy-demand organ, skeletal muscle is rich in mitochondria [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Oxidative stress occurs when ROS production within the muscle exceeds its clearance capacity. TXNIP naturally inhibits thioredoxin (Trx), a protein that scavenges reactive oxygen species (ROS). Excessive ROS generation causes TXNIP to detach from the TXNIP-Trx complex, subsequently binding to NLRP3 and recruiting ASC and procaspase-1 to assemble the NLRP3 inflammasome. This process initiates inflammatory responses and pyroptosis [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Previous studies have reported that in tumor cells, MIF can bind to TXNIP, thereby alleviating TXNIP-mediated suppression of the NF-κB pathway and consequently promoting inflammation [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In this study, we demonstrated in muscle cells that MIF significantly elevates intracellular ROS levels. The increase in elevation leads to TXNIP protein expression, facilitating NLRP3 inflammasome assembly and boosting IL-1β expression and release. Furthermore, in a subsequent DEX-induced muscle atrophy model, we validated that inhibiting MIF effectively reduces ROS levels in myotubes and suppresses the activation of the TXNIP/NLRP3 pathway. The direct binding of MIF to TXNIP in muscle cells and its role in mediating the inflammatory response is yet to be clarified.\u003c/p\u003e \u003cp\u003eIn this study, we employed the classic glucocorticoid dexamethasone to establish both cellular and murine models of muscle atrophy. MIF and glucocorticoids exhibit a bell-shaped dose-response relationship, where high glucocorticoid levels inhibit MIF secretion, while low physiological levels promote its release from monocytes, macrophages, and T cells [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In vitro, MIF counteracts the suppressive impact of glucocorticoids on the secretion of pro-inflammatory cytokines, such as TNF, IL-1, IL-6, and IL-8, by activated macrophages. In vivo, within a lethal endotoxemia inflammation model, MIF completely counteracts the protective effects of glucocorticoids. Although glucocorticoids are generally known to suppress MIF expression at high doses, our experiments revealed that treatment of C2C12 myoblasts with 20 \u0026micro;M DEX markedly increased MIF concentration in the cell culture supernatant. Studies further confirm that DEX administration significantly upregulates MIF protein expression in tissues sensitive to glucocorticoid-induced growth inhibition, including immune/endocrine organs, skin, and skeletal muscle [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. This paradoxical upregulation suggests a potential tissue-specific regulatory mechanism in skeletal muscle and indicates that MIF may serve as a key mediator in DEX-induced muscle atrophy.\u003c/p\u003e \u003cp\u003e4-IPP is a suicide substrate that irreversibly inhibits MIF's biological activity via covalent binding. Current studies demonstrate that 4-IPP has application value in tumor treatment and inflammation regulation by inhibiting MIF activity [\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Zheng et al. Research indicates that 4-IPP inhibits MIF expression in HOS/143B cells [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In this study, treatment with a relatively high concentration of 4-IPP (40 \u0026micro;M) effectively counteracted DEX-induced upregulation of MIF protein levels, which is consistent with previous research findings. Through concentration-gradient experiments conducted in DEX-induced C2C12 myotube atrophy models, it was further verified that 4-IPP can effectively alleviate intracellular oxidative stress in myotubes and downregulate the activation level of the oxidative stress-associated TXNIP/NLRP3 pathway. In DEX-induced murine muscle atrophy models, 4-IPP significantly improved muscle mass and contractile strength in mice, with these results aligning with the cellular experimental conclusions. Although no direct reports exist on interactions between 4-IPP and DEX, whether synergistic or antagonistic effects exist between them requires further investigation.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study elucidates the mechanistic role of MIF in sarcopenia pathogenesis through integrated in vitro and in vivo investigations. We demonstrate that MIF directly induces excessive ROS generation, thereby activating the TXNIP/NLRP3 inflammasome pathway and amplifying inflammatory cascades, ultimately driving muscle atrophy in vitro. Pharmacological inhibition of MIF using the specific inhibitor 4-IPP significantly improves muscle mass and strength in a murine sarcopenia model. These findings establish MIF as a pivotal mediator of inflammatory muscle wasting and validate its therapeutic targeting for sarcopenia intervention.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the Lishui Key Research and Development Program (Grant No. 2022ZDYF10) and the Zhejiang Province Medical and Health Science and Technology Projects (Grant No. 2024KY565).\u0026nbsp;\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original contributions presented in the study are included in the article and supplementary material, further inquiries can be directed to the corresponding authors.\u0026nbsp;\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research was designed by Weiqing Li, Zhenzhong Chen, and Dengwei He. Weiqing Li, Yahong Lu and Yu Bai performed the experiments and acquired the data. Weiqing Li, Ye lin, Haiwei Ma, Zhiguo Zhou and Chendi Wang analyzed the data. Dengwei He and Hehuan Lai supervised the project. All authors participated in the creation of the article and consented to its submission.\u0026nbsp;\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eDisclosures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have stated that there are no conflicts of interest related to this article. \u003cstrong\u003e\u003c/strong\u003e\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCruz-Jentoft, A. J. et al. 2019. \u003cem\u003eSarcopenia: revised European consensus on definition and diagnosis Age Ageing\u003c/em\u003e 48(1):16\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFielding, R. A. et al. 2011. Sarcopenia: an undiagnosed condition in older adults. Current consensus definition: prevalence, etiology, and consequences. International working group on sarcopenia. \u003cem\u003eJournal Of The American Medical Directors Association\u003c/em\u003e 12(4):249\u0026ndash;256.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, D. et al. 2024. Frontiers in sarcopenia: Advancements in diagnostics, molecular mechanisms, and therapeutic strategies. \u003cem\u003eMolecular Aspects Of Medicine\u003c/em\u003e 97:101270.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSandri, M. 2013. Protein breakdown in muscle wasting: role of autophagy-lysosome and ubiquitin-proteasome. \u003cem\u003eInternational Journal Of Biochemistry \u0026amp; Cell Biology\u003c/em\u003e 45(10):2121\u0026ndash;2129.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAttaix, D. et al. 2005., \u003cem\u003eThe ubiquitin-proteasome system and skeletal muscle wasting.\u003c/em\u003e Essays Biochem, 41: pp. 173\u0026thinsp;\u0026ndash;\u0026thinsp;86.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBodine, S. C. et al. 2001. \u003cem\u003eIdentification of ubiquitin ligases required for skeletal muscle atrophy Science\u003c/em\u003e 294(5547):1704\u0026ndash;1708.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGlass, D. J. 2005. \u003cem\u003eSkeletal muscle hypertrophy and atrophy signaling pathways.\u003c/em\u003e Int. \u003cem\u003eJ Biochem Cell Biol\u003c/em\u003e 37(10):1974\u0026ndash;1984.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRom, O., and A. Z. Reznick. 2016. The role of E3 ubiquitin-ligases MuRF-1 and MAFbx in loss of skeletal muscle mass. \u003cem\u003eFree Radical Biology And Medicine\u003c/em\u003e 98:218\u0026ndash;230.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChang, K. V. et al. 2023., \u003cem\u003eEnhanced serum levels of tumor necrosis factor-alpha, interleukin-1beta, and \u0026ndash;\u0026thinsp;6 in sarcopenia: alleviation through exercise and nutrition intervention.\u003c/em\u003e Aging (Albany NY), 15(22): pp. 13471\u0026ndash;13485.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFranceschi, C., and J. Campisi. 2014. Chronic inflammation (inflammaging) and its potential contribution to age-associated diseases. \u003cem\u003eJournals Of Gerontology. Series A, Biological Sciences And Medical Sciences\u003c/em\u003e 69(Suppl 1):S4\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCai, D. et al. 2004., \u003cem\u003eIKKbeta/NF-kappaB activation causes severe muscle wasting in mice.\u003c/em\u003e Cell, 119(2): pp. 285\u0026ndash;298.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBonetto, A. et al. 2012., \u003cem\u003eJAK/STAT3 pathway inhibition blocks skeletal muscle wasting downstream of IL-6 and in experimental cancer cachexia.\u003c/em\u003e Am J Physiol Endocrinol Metab, 303(3): pp. E410-21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBonetto, A. et al. 2011. STAT3 activation in skeletal muscle links muscle wasting and the acute phase response in cancer cachexia. \u003cem\u003ePLoS One\u003c/em\u003e 6(7):e22538.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZanders, L. et al. 2022., \u003cem\u003eSepsis induces interleukin 6, gp130/JAK2/STAT3, and muscle wasting.\u003c/em\u003e J Cachexia Sarcopenia Muscle, 13(1): pp. 713\u0026ndash;727.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, W. et al. 2009. Interleukin-1 stimulates catabolism in C2C12 myotubes. \u003cem\u003eAmerican Journal Of Physiology. Cell Physiology\u003c/em\u003e 297(3):C706\u0026ndash;C714.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarris, J. et al. 2019. Rediscovering MIF: New Tricks for an Old Cytokine. \u003cem\u003eTrends In Immunology\u003c/em\u003e 40(5):447\u0026ndash;462.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCalandra, T., and T. Roger. 2003. Macrophage migration inhibitory factor: a regulator of innate immunity. \u003cem\u003eNature Reviews Immunology\u003c/em\u003e 3(10):791\u0026ndash;800.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLue, H. et al. 2002. Macrophage migration inhibitory factor (MIF): mechanisms of action and role in disease. \u003cem\u003eMicrobes And Infection\u003c/em\u003e 4(4):449\u0026ndash;460.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOsipyan, A., D. Chen, and F. J. Dekker. 2021. \u003cem\u003eEpigenetic regulation in macrophage migration inhibitory factor (MIF)-mediated signaling in cancer and inflammation.\u003c/em\u003e Drug Discov Today, 26(7): pp. 1728\u0026ndash;1734.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChuang, Y. C. et al. 2012. Macrophage migration inhibitory factor induces autophagy via reactive oxygen species generation. \u003cem\u003ePLoS One\u003c/em\u003e 7(5):e37613.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLv, W. et al. 2016., \u003cem\u003eMacrophage migration inhibitory factor promotes breast cancer metastasis via activation of HMGB1/TLR4/NF kappa B axis.\u003c/em\u003e Cancer Lett, 375(2): pp. 245\u0026ndash;255.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhuang, L. et al. 2020., \u003cem\u003eExosomal LncRNA-NEAT1 derived from MIF-treated mesenchymal stem cells protected against doxorubicin-induced cardiac senescence through sponging miR-221-3p.\u003c/em\u003e J Nanobiotechnology, 18(1): p. 157.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, H. et al. 2024., \u003cem\u003eA small molecule macrophage migration inhibitory factor agonist ameliorates age-related myocardial intolerance to ischemia-reperfusion insults via metabolic regulation.\u003c/em\u003e Metabolism, 153: p. 155792.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu, W. et al. 2021., \u003cem\u003eMacrophage migration inhibitory factor facilitates the therapeutic efficacy of mesenchymal stem cells derived exosomes in acute myocardial infarction through upregulating miR-133a-3p.\u003c/em\u003e J Nanobiotechnology, 19(1): p. 61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, Y. P. et al. 2003. TNF-alpha increases ubiquitin-conjugating activity in skeletal muscle by up-regulating UbcH2/E220k. \u003cem\u003eThe Faseb Journal\u003c/em\u003e 17(9):1048\u0026ndash;1057.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKwak, J. Y. et al. 2018. Prediction of sarcopenia using a combination of multiple serum biomarkers. \u003cem\u003eScientific Reports\u003c/em\u003e 8(1):8574.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQaisar, R. et al. 2021. \u003cem\u003ePrediction of sarcopenia using a battery of circulating biomarkers Sci Rep\u003c/em\u003e 11(1):8632.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu, H. et al. 2021. Muscle mitochondrial catalase expression prevents neuromuscular junction disruption, atrophy, and weakness in a mouse model of accelerated sarcopenia. \u003cem\u003eJ Cachexia Sarcopenia Muscle\u003c/em\u003e 12(6):1582\u0026ndash;1596.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTschopp, J., and K. Schroder. 2010. NLRP3 inflammasome activation: The convergence of multiple signalling pathways on ROS production? \u003cem\u003eNature Reviews Immunology\u003c/em\u003e 10(3):210\u0026ndash;215.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim, M. J. et al. 2017., \u003cem\u003eMacrophage migration inhibitory factor interacts with thioredoxin-interacting protein and induces NF-kappaB activity.\u003c/em\u003e Cell Signal, 34: pp. 110\u0026ndash;120.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeng, L. et al. 2009., \u003cem\u003eGlucocorticoid-induced MIF expression by human CEM T cells.\u003c/em\u003e Cytokine, 48(3): pp. 177\u0026ndash;185.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCalandra, T. et al. 1995. MIF as a glucocorticoid-induced modulator of cytokine production. \u003cem\u003eNature\u003c/em\u003e 377(6544):68\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFingerle-Rowson, G. et al. 2003. Regulation of macrophage migration inhibitory factor expression by glucocorticoids in vivo. \u003cem\u003eAmerican Journal Of Pathology\u003c/em\u003e 162(1):47\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng, Y. et al. 2016., \u003cem\u003eRole of Myeloma-Derived MIF in Myeloma Cell Adhesion to Bone Marrow and Chemotherapy Response\u003c/em\u003e. \u003cem\u003eJournal Of The National Cancer Institute\u003c/em\u003e, 108(11).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWinner, M. et al. 2008. A novel, macrophage migration inhibitory factor suicide substrate inhibits motility and growth of lung cancer cells. \u003cem\u003eCancer Research\u003c/em\u003e 68(18):7253\u0026ndash;7257.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBozzi, F. et al. 2017., \u003cem\u003eMIF/CD74 axis is a target for novel therapies in colon carcinomatosis.\u003c/em\u003e J Exp Clin Cancer Res, 36(1): p. 16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng, L. et al. 2022. Destabilization of macrophage migration inhibitory factor by 4-IPP reduces NF-kappaB/P-TEFb complex-mediated c-Myb transcription to suppress osteosarcoma tumourigenesis. \u003cem\u003eClin Transl Med\u003c/em\u003e 12(1):e652.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"inflammation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ifla","sideBox":"Learn more about [Inflammation](https://www.springer.com/journal/10753)","snPcode":"10753","submissionUrl":"https://submission.nature.com/new-submission/10753/3","title":"Inflammation","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Sarcopenia, MIF, ROS, TXNIP/NLRP3, 4-IPP","lastPublishedDoi":"10.21203/rs.3.rs-8979207/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8979207/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSarcopenia, an age-related degenerative skeletal muscle disorder, is strongly associated with adverse clinical outcomes including recurrent falls, functional disability, frailty, and increased mortality. Emerging evidence suggests that systemic chronic inflammation plays a central role in muscle wasting. Although macrophage migration inhibitory factor (MIF) is a known pro-inflammatory cytokine in various inflammatory diseases, its role in sarcopenia development remains unclear. We found significantly elevated plasma levels of MIF, TNF-α, IL-6, and IL-8 in sarcopenia patients, which were inversely correlated with muscle strength. \u003cem\u003eIn vitro\u003c/em\u003e, we demonstrated that MIF exposure directly induced atrophy in C2C12 myotubes. Furthermore, our study showed that MIF induces excessive ROS production, activating the TXNIP/NLRP3 inflammasome pathway and subsequent pro-inflammatory responses. Consistent with these findings, \u003cem\u003ein vivo\u003c/em\u003e administration of 4-IPP significantly alleviated muscle mass loss and functional decline in a Dexamethasone (DEX)-induced murine sarcopenia model. Our findings demonstrate that MIF is a key regulator of inflammatory muscle atrophy, supporting its potential as a therapeutic target for sarcopenia.\u003c/p\u003e","manuscriptTitle":"4-IPP Alleviates Dexamethasone-Induced Muscle Atrophy by Targeting the MIF/TXNIP/NLRP3 Axis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-13 15:21:05","doi":"10.21203/rs.3.rs-8979207/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-08T17:24:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-07T19:13:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"189139168443634564428116155419368883866","date":"2026-04-02T14:23:27+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-22T08:55:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"317257093736000680024709868777688782890","date":"2026-03-12T05:29:21+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-09T16:49:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-02T10:20:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-02T10:20:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"Inflammation","date":"2026-02-26T14:48:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"inflammation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ifla","sideBox":"Learn more about [Inflammation](https://www.springer.com/journal/10753)","snPcode":"10753","submissionUrl":"https://submission.nature.com/new-submission/10753/3","title":"Inflammation","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9f1e5b6f-8ad1-4202-83de-fcc2650adab3","owner":[],"postedDate":"March 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-04-08T17:39:43+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-13 15:21:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8979207","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8979207","identity":"rs-8979207","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}