In vitroPreprint
This study investigated whether Tβ4-17, a small bioactive peptide derived from thymosin β4 and identified via iTRAQ technology, could enhance the sensitivity of cisplatin (DDP)-resistant ovarian cancer cells to chemotherapy. Using in vitro models of DDP-resistant ovarian cancer cell lines, the researchers examined the effects of Tβ4-17 alone and in combination with DDP on cell proliferation, migration, and apoptosis. Multiple assays were employed, including CCK8 viability assays, EDU fluorescence proliferation assays, cell scratch (wound healing) assays, qRT-PCR, and Western blot. The study found that Tβ4-17 combined with DDP significantly inhibited proliferation and migration of resistant cells and promoted apoptosis compared to either agent alone. Mechanistically, the researchers reported that NF-κB p65 was highly expressed in DDP-resistant cells, and that Tβ4-17 down-regulated NF-κB p65 protein expression. Use of NF-κB inhibitors and activators further supported this proposed pathway. Key limitations include the exclusive use of cell-line models with no animal or human data, the preprint status of the work, and the absence of in vivo validation. Findings are preliminary and require further study.
Unknown journal · Jul 2025DOI ↗ Animal only
This controlled animal trial investigated the comparative effects of three butyrate-based dietary supplements — tributyrin with copper and essential oils (TB-500), di- and tri-butyrin (TB-300), and coated sodium butyrate (SB-500) — versus a control diet in 1,000 Arbor Acres broiler chicks across four treatment groups (250 birds each, six replicates). Over 35 days, researchers measured growth performance, carcass traits, serum biochemistry, immune markers, gene expression (mTOR, TLR4, NBN), intestinal histomorphometry, caecal microbiota, and litter hygiene. The study found that TB-300 was associated with improved body weight (+4.6%), feed conversion ratio (–5.2%), and European Production Efficiency Factor (+14.9%). SB-500 was linked to reduced litter Clostridia and aerobic bacterial counts. All butyrate treatments were associated with improved intestinal villus morphology, elevated serum total proteins and digestive enzymes (lipase and protease), and upregulated TLR4 gene expression. TB-300 and SB-500 were associated with reduced serum lipids, urea, and AST, alongside enhanced mTOR and NBN gene expression. Limitations include that the study was conducted exclusively in broiler chickens, findings are not directly translatable to humans, and it did not include a blinded assessment of outcomes.
Scientific reports · Jul 2025DOI ↗ Limited · human
This observational study investigated the relationship between serum Thymosin β4 (Tβ4) levels and Kawasaki disease (KD) in children, with a specific focus on coronary artery lesions (CALs). Researchers measured serum Tβ4 concentrations via ELISA in children diagnosed with KD and age-matched healthy controls, further subdividing the KD group into those with and without CALs. The study found that serum Tβ4 levels were significantly lower in children with KD compared to healthy controls, and were reduced even further in KD patients who developed CALs. Following intravenous immunoglobulin (IVIG) treatment, Tβ4 levels increased significantly. Correlation analyses revealed negative associations between Tβ4 and several cytokines, including pro-inflammatory markers (TNF-α, IL-1β) and anti-inflammatory markers (IL-4, IL-10). The authors suggest that Tβ4 may play a role in KD's inflammatory pathogenesis and the progression of coronary artery involvement, proposing it as a potential diagnostic or therapeutic target. Key limitations include the observational design, which precludes causal inference, the pediatric-specific and single-condition focus, and the reliance on serum biomarker associations without mechanistic validation in this population.
Journal of inflammation research · Jul 2025DOI ↗ Animal only
This study developed a multifunctional injectable hydrogel platform — called Exo@Tβ4/HAMA — designed to accelerate bone repair by simultaneously promoting stem cell recruitment, angiogenesis, neurogenesis, and osteogenesis. The hydrogel was fabricated by grafting Thymosin β4 (Tβ4), a short tissue-repair peptide, onto methylmalonic anhydride-modified hyaluronic acid (HAMA) via photo-cross-linking, then loading it with bone marrow mesenchymal stem cell (BMSC)-derived exosomes. In vitro experiments showed the hydrogel had favorable mechanical properties, good biocompatibility, and could recruit BMSCs, enhance tube formation in human umbilical vein endothelial cells (HUVECs), and promote osteogenic differentiation. In vivo rat cranial defect models demonstrated that the hydrogel promoted new bone formation, vascularization, and nerve ingrowth. The study identified the ERK1/2-dependent RUNX2 signaling pathway as a likely mechanistic contributor to osteogenesis. Key limitations include exclusive use of rat models with no human data, a relatively short observation window, and lack of comparison to current clinical gold-standard grafts. The findings suggest promise as a cell-free, injectable bone regeneration scaffold, but clinical translation requires further validation.
Limited · humanPreprint
This observational study examined the relationship between salivary oxidized forms of thymosin β4 (Tβ4) and thymosin β10 (Tβ10) and oxidative stress-related diseases in preterm infants born before 30 weeks of gestation. Researchers collected 149 saliva samples from 18 infants and analyzed the intact salivary proteome using nano-HPLC-ESI-MS, with relative quantification based on extracted ion current (XIC) peak area. The study found that post-menstrual age correlated significantly with total Tβ4, oxidized Tβ4 percentage, and total Tβ10. Higher fraction of inspired oxygen (FiO₂) values were associated with lower levels and percentages of oxidized Tβ10. Thymosin levels did not differ between infants with or without retinopathy of prematurity; however, higher oxidized Tβ10 levels were observed in infants who did not develop bronchopulmonary dysplasia (BPD), leading the authors to suggest a possible protective role for oxidized Tβ10 in inflammation and tissue repair. Key limitations include the very small sample size (18 infants), observational design, and the inability to establish causality. The authors propose saliva as a non-invasive matrix for future monitoring of oxidative stress in neonates.
Unknown journal · May 2025DOI ↗ Limited · human
This study investigated the role of CCN5, a matricellular protein, in preventing vascular restenosis after stent implantation (in-stent restenosis, ISR). Using RNA sequencing of stent-implanted porcine coronary arteries and single-cell RNA sequencing of mouse femoral artery injury models, the researchers found that CCN5 expression was reduced in vascular smooth muscle cells (VSMCs) following injury but elevated in regenerating endothelial cells (ECs). In ISR patients, plasma CCN5 levels were significantly lower and correlated inversely with restenosis severity. Using cell-type-specific loss- and gain-of-function mouse models, the study found that EC- and VSMC-specific deletion of CCN5 worsened neointimal hyperplasia, while CCN5 overexpression was protective. Mechanistically, CCN5 was found to interact with thymosin β4 (Tβ4) in ECs, promoting endothelial repair via the cleavage product Ac-SDKP, and also interacted with CD9 to support EC recovery. A CCN5 recombinant protein (CCN5rp)-coated stent deployed in a porcine model significantly increased endothelial strut coverage and reduced neointimal formation. Limitations include the translational gap between animal models and humans, and the observational nature of the patient plasma data.
European heart journal · May 2025DOI ↗ Animal only
This study investigated whether Thymosin β4 (Tβ4), a bioactive polypeptide, could regulate liver inflammation in nonalcoholic fatty liver disease (NAFLD) by influencing macrophage polarization. Researchers used a mouse model of NAFLD induced by a methionine and choline-deficient (MCD) diet in C57 mice, with liver Tβ4 knocked down via tail-vein-injected siRNA. Macrophage involvement was assessed using clodronate liposome depletion. Additionally, in vitro experiments co-cultured THP-1 macrophage cells with oleic acid-treated LO2 hepatocytes at varying Tβ4 concentrations. The study found that Tβ4 treatment was associated with reduced liver inflammation and steatosis in mice, while Tβ4 knockdown worsened steatosis. Tβ4 appeared to shift macrophages toward an M2 (anti-inflammatory) phenotype, reduce M1 marker expression, decrease hepatocyte apoptosis, downregulate STAT1 phosphorylation, and increase SOCS1/3 expression. A publicly available dataset was also used to assess Tβ4 expression in hepatocellular carcinoma-adjacent fatty tissue. Limitations include reliance primarily on animal and in vitro models, a relatively small experimental scope, and no direct human clinical data, leaving the translational relevance of these findings uncertain.
Journal of inflammation research · Apr 2025DOI ↗ Animal only
This study investigated whether inhaled recombinant human thymosin beta 4 (rhTβ4), delivered via nebulization, could reduce pulmonary fibrosis in a mouse model. Researchers induced pulmonary fibrosis in mice using bleomycin and then administered rhTβ4 aerosols at three different time points: early, mid-term, and late stages of fibrosis development. Efficacy was assessed through hydroxyproline content (a marker of collagen deposition), lung function measurements, and histopathological examination of lung tissue. The study found that nebulized rhTβ4 was associated with reduced fibrosis markers across all three dosing strategies. In parallel cell-based experiments, rhTβ4 appeared to suppress lung fibroblast proliferation, migration, and activation, and to inhibit epithelial-mesenchymal transition (EMT) in pulmonary epithelial cells, with both effects linked to modulation of the TGF-β1 signalling pathway. Limitations include the exclusive use of a mouse bleomycin model (which incompletely recapitulates human IPF), the absence of human clinical data, and the lack of direct mechanistic confirmation in vivo. The authors conclude that nebulized rhTβ4 warrants further investigation as a potential therapy for idiopathic pulmonary fibrosis.
The Journal of pharmacy and pharmacology · Apr 2025DOI ↗ Preclinical
This study investigated the role of plasmacytoid dendritic cells (pDCs) during the active effector phase of allergic asthma, moving beyond their previously known role in immune tolerance. Using BDCA2-DTR transgenic mice in which pDCs can be selectively depleted with diphtheria toxin, researchers sensitized and challenged animals with house dust mite allergen. They found that pDC depletion worsened asthmatic inflammation by increasing CCR2-dependent recruitment of inflammatory monocyte-derived cells. RNA sequencing of lung pDCs revealed that the small anti-inflammatory peptide thymosin β4 (Tβ4) was among the most upregulated genes in asthmatic conditions. IL-33 released by airway epithelial cells was found to drive Tβ4 expression in pDCs, which represented the dominant pulmonary source of Tβ4. Mechanistically, Tβ4 suppressed IL-4/IL-13-induced JAK1/STAT6/EGR2 signaling in alveolar macrophages, reducing CCL2 production and monocyte recruitment. Tβ4 supplementation reversed disease exacerbation in pDC-depleted mice. Notably, reduced serum Tβ4 levels were observed in both asthmatic mice and a human cohort with active allergic asthma. Limitations include the predominantly murine mechanistic data and a small, cross-sectional human serum dataset that limits causal interpretation in humans.
The Journal of allergy and clinical immunology · Feb 2025DOI ↗ Preclinical
This study developed and validated a simultaneous analytical method using ultra-high-performance liquid chromatography coupled with high-resolution Orbitrap mass spectrometry (UHPLC-Q-Exactive MS/MS) to quantify TB-500 (Ac-LKKTETQ), a synthetic peptide derived from the active site of thymosin β4 (Tβ4), alongside its metabolites. Metabolism was studied in human serum, multiple in-vitro enzyme systems, and rat urine samples following TB-500 administration. Researchers synthesized authentic standards of the metabolites to enable structural identification. Key findings included that Ac-LK was the predominant short-term metabolite (0–6 hours), while Ac-LKK was detectable as a longer-term metabolite up to 72 hours. In fibroblast cell culture experiments, neither TB-500 nor its metabolites demonstrated cytotoxicity. Notably, among all compounds tested, only the metabolite Ac-LKKTE showed statistically significant wound-healing activity compared to controls, leading the authors to suggest that previously reported wound-healing effects attributed to TB-500 in the literature may actually be mediated by this metabolite rather than the parent peptide. Limitations include that biological activity testing was confined to cell-based assays, and no human pharmacokinetic or efficacy data were generated.
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences · Feb 2024DOI ↗ Limited · human
This study developed and validated an analytical method for detecting a broad panel of 18 performance-enhancing peptides (molecular weight <2 kDa) in human urine, as defined by the World Anti-Doping Agency (WADA) prohibited list. The method uses direct urine injection—bypassing complex sample preparation—coupled with liquid chromatography and ion mobility time-of-flight mass spectrometry (IM-TOFMS). The researchers reported limits of detection (LOD) ranging from 50 to 500 pg/mL, well below WADA's minimum required performance level of 2 ng/mL. The method demonstrated acceptable precision (imprecision <20%) and linearity across a 0–10 ng/mL working range. Stability testing identified –20°C as the appropriate storage temperature for urine samples. As a proof-of-concept, the method was applied to real elimination study urine samples from individuals who had administered GHRP-2, GHRP-6, or LHRH, successfully detecting these compounds. Key limitations include the small number of human subjects used in the elimination studies, which were primarily intended to demonstrate analytical feasibility rather than investigate pharmacokinetics or clinical effects. The study is a methodological/analytical validation paper focused on anti-doping screening, not a clinical or therapeutic investigation.
Journal of separation science · Dec 2015DOI ↗ Review
This review paper surveys the current landscape of analytical methods used in sports anti-doping laboratories to detect peptide-based drugs, drug candidates, and their analogs in biological specimens such as blood, serum, and urine. The authors describe the broad range of peptidic compounds subject to anti-doping scrutiny, spanning low-molecular-mass peptides (e.g., growth hormone-releasing peptides, ARA-290, TB-500, AOD-9604, CJC-1295, desmopressin) to intermediate-mass proteins (e.g., insulins, IGF-1, growth hormone, erythropoietin) and higher-molecular-mass biologics (e.g., stamulumab). The review outlines detection approaches including chromatographic-mass spectrometric, electrophoretic, immunological, and combined methodologies, emphasizing the challenge of proving exogenous origin at very low concentrations in limited sample volumes. A central finding is that a meaningful gap remains between what is technically achievable in detection and what is routinely practiced in day-to-day analytical workflows. Limitations of this paper include its nature as a narrative review rather than an original experimental study, meaning it synthesizes existing literature without generating new empirical data. It does not evaluate clinical outcomes or therapeutic efficacy of any compound discussed.
Expert review of proteomics · Nov 2014DOI ↗ Review
This review summarizes five years of published literature on the analytical detection of emerging and non-approved performance-enhancing substances in the context of sports anti-doping controls. The authors examine a broad range of compounds classified as doping agents by the World Anti-Doping Agency (WADA), spanning both peptidic substances—such as modified insulin-like growth factor-1 (IGF-1), TB-500 (thymosin beta-4 fragment), peginesatide/hematide, growth hormone releasing peptides, and AOD-9604—and non-peptidic substances, including selective androgen receptor modulators (SARMs), hypoxia-inducible factor (HIF) stabilizers, siRNA, S-107, and aladorian (ARM036). Inorganic substances such as cobalt are also discussed. The review focuses specifically on the analytical challenges posed by these compounds, including their physicochemical properties, low concentrations in biological matrices (blood and urine), metabolic transformation, and suitability for chromatographic–mass spectrometric or alternative detection methods. The study does not present original experimental data or human clinical outcomes, but rather synthesizes existing literature to guide development of detection strategies. A key limitation is that, as a review, it does not independently validate any analytical method and reflects the state of the field only up to its publication date.
Journal of pharmaceutical and biomedical analysis · May 2014DOI ↗