TB-500 THYMOSIN BETA-4 PEPTIDE

Main Research Findings

1) Tβ4 has potential wound healing properties as a result of several biological activities such as down-regulation of inflammatory chemokines and cytokines, as well as the promotion of cell migration, blood vessel formation, cell survival, and stem cell maturation.

2) Treatment with Tβ4 elicits neuroprotective effects that reduces pathology related to Alzheimer’s disease and altered neurogenesis.

 

Selected Data

1) The article completed by researchers Philp et al synthesizes findings from numerous animal studies investigating the therapeutic potential of Tβ4 across various models of disease, injury, and repair.
For topical administration, Tβ4 was utilized in both skin and eye models. In ocular studies, alkali injury and heptanol debridement models were established in mice and rats. Tβ4 was applied as liquid drops to the eye at a concentration of 5 µg in 5 µL, administered twice daily. The duration of treatment in some eye studies extended for up to 30 days. For dermal wounds, full-thickness punch biopsies were created in rats and mice. These included models of normal wound healing, as well as impaired healing models such as diabetic mice, aged mice, and steroid-treated rats. Tβ4 was applied topically either in PBS or incorporated into a hydrogel, with doses ranging from 5 to 50 µg in 50 µL per wound. The frequency of application varied; in some cases, it was applied at the time of wounding on day 0, and then after 48 hours, while in others, daily application was employed, particularly when bandages were used. Studies also investigated hair growth, where Tβ4 was applied topically every other day to shaved or depilated rats and mice at concentrations of 2.5-5.0 µg in 50 µL of hydrogel. Both synthetic and recombinant forms of Tβ4 were used in these dermal wound models, and one study even employed a Tβ4-derived peptide specifically containing its actin-binding site [1].
Systemic administration of Tβ4 encompassed a wider array of animal models, including cardiovascular disease, neural damage, dermal injury, and septic shock, utilizing animals such as pigs, mice, rats, and even monkeys for toxicity studies. Doses varied significantly depending on the animal and application, ranging from as low as 400 ng/animal for intracardiac injection in mice, to 15 mg/animal for retroinfusion in 25 kg German pigs, and up to 60 mg/kg for larger animals in toxicity assessments.
For cardiovascular models, Tβ4 was delivered via retroinfusion into the anterior interventricular vein in a hypoxia-reoxygenation pig model, or through intracardiac or intraperitoneal injection in murine permanent ligation models. Doses for these systemic cardiac applications included 15 mg for pigs and 400 ng/animal intracardiac or 150 µg/animal intraperitoneal for mice. The frequency of administration varied from a single dose to every three days in permanent ligation models. In neurological models, kainic acid was administered to rats via a cannula in the lateral cerebral ventricle to induce neurotoxicity. Tβ4 was then administered intraventricularly at a concentration of 10 µM twice a day for five days [1].
For models of experimental autoimmune encephalomyelitis (EAE) in mice, which is a model for multiple sclerosis, Tβ4 was administered intraperitoneally at 6 mg/kg every three days for five treatments, starting 24 hours after infection. In a model of septic shock, rats were given a lethal dose of endotoxin (LPS LD50), and Tβ4 was administered via intraperitoneal injection at 100 µg/animal immediately following endotoxin and again at 2 and 4 hours after the dose. Systemic application for dermal wound healing included intraperitoneal injection of 60 µg/rat at day 0 and day 2. Across all these systemic studies, Tβ4 was generally dissolved in appropriate vehicles for injection. Furthermore, extensive nonclinical pharmacology and toxicology studies were conducted in dogs, rats, and monkeys to assess safety and tolerability. These involved intravenous administration at high doses, up to 60 mg/kg for dogs and rats, and 50-250 mg/kg for rats and monkeys, to confirm the absence of toxic effects [1].

2) The study performed by the research team of Zeng et al aimed to identify intervention targets for Alzheimer’s disease (AD) using human brain organoids and further validate them in an animal model. The foundational methodology involved generating and characterizing cerebral organoids from induced pluripotent stem cells (iPSCs), employing various genomic, proteomic, and imaging techniques, followed by in vivo validation in a mouse model [2].

The researchers utilized three distinct iPSC lines. One served as a healthy control (CTRL), while the other two carried mutations associated with familial AD (fAD). Specifically, one fAD iPSC line (APP2) had an APP gene duplication, and the other (HVRD) harbored an APPV717I mutation. These mutations are known to be linked to increased Aβ production and AD pathology. Cerebral organoids were generated from these iPSC lines following a previously established protocol, which likely involved sequential differentiation through various stages to mimic brain development in vitro. Organoids were maintained and analyzed at specific developmental time points, notably Day 30, Day 60, and Day 90, to observe dynamic cellular changes and maturation [2].

To investigate the role of Tβ4 and the effects of Aβ, specific treatments were applied to the organoids. For Tβ4 treatment, control and APP2 cerebral organoids received 0.5 µg/mL of Tβ4 in their Maturation Medium. This treatment commenced at Day 20 of differentiation and was reapplied every five days. Organoids were then examined at Day 60 and Day 90 to assess the therapeutic effects. To simulate AD pathology in control organoids, Aβ peptide was added to the Maturation Medium at a concentration of 5 µM, starting at Day 20 and replaced every five days, with sampling for analysis at Day 60.

A variety of techniques were employed for detailed characterization. First, single-cell RNA sequencing (scRNA-seq) was a cornerstone technique, performed on organoid samples collected at Day 30 and Day 60. This allowed for the identification of various cell types, assessment of cell composition and heterogeneity, differential gene expression (DEG) analysis, and construction of cellular pseudotime trajectories to understand differentiation pathways. Next, organoids underwent immunostaining to visualize key markers of AD pathology and neuronal development. Specifically, β-amyloid was stained at Day 90 to quantify plaque-like pathology. Markers for mature neurons (NeuN) and sixth-layer neurons (TBR1) were used at Day 30 and Day 60 to assess neuronal maturation and density. Cleaved Caspase-3 (c-CASP3) and TUNEL staining were utilized to detect apoptosis and cell death. These immunofluorescence images were quantitatively analyzed for intensity and cell density [2].

Additionally, bulk RNA-seq was performed on Tβ4-treated APP2 organoids at Day 30 to investigate gene expression changes induced by Tβ4. The scRNA-seq and bulk RNA-seq data underwent extensive bioinformatic processing, including dimensionality reduction (UMAP), cell-type clustering, identification of DEGs, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and pseudotime trajectory analysis to map cellular differentiation and pathological progression. The results from organoids were also compared with publicly available single-nucleus RNA sequencing (snRNA-seq) datasets from AD patients (GSE157827 and GSE174367) for cross-validation and identification of commonly affected genes.

A 5xFAD mouse model, which overexpresses five familial AD mutations and develops significant AD pathology, was then used for in vivo validation. An adeno-associated virus (AAV) vector, specifically AAV-PhP.eB capsid, was engineered to deliver human TMSB4X (encoding Tβ4) under the control of a human synapsin (hSyn) promoter, ensuring neuron-specific overexpression. The construct also included a HA tag and EGFP for detection. A control AAV-GFP vector was also prepared. The AAV vectors were delivered via intrathecal injection into the L4-L5 or L3-L4 vertebral space of 5xFAD mice, following established protocols. Mice were monitored for 1.5 months post-injection [2].

After the treatment period, mouse brains were analyzed. Amyloid plaques were visualized and quantified using Thioflavin S staining. Microglial activation was assessed using IBA1 immunostaining, and astrocytic gliosis by GFAP immunostaining. Quantitative PCR was used to measure inflammatory cytokine expression (TNF-α, IL-6) in the hippocampus. Soluble Aβ levels (Aβ1-40 and Aβ1-42) were measured. To assess neuronal hyperexcitability, action potentials (APs) were recorded from cortical excitatory neurons of 6- to 7-month-old WT and 5xFAD mice (both AAV-GFP and AAV-TMSB4X injected). Parameters such as AP half-width, threshold potential, and amplitude were quantified. Finally, RNA sequencing was performed on brain samples from AAV-GFP and AAV-TMSB4X injected 5xFAD mice to identify gene expression changes underlying the observed phenotypic rescue [2].

 

Discussion

1) The results of the review completed by Philp et al reported that animal studies performed with Tβ4 consistently demonstrated its remarkable multifunctional capabilities in tissue repair and regeneration across a wide range of injury and disease models, supporting its therapeutic potential. The observed results highlight Tβ4’s roles in promoting cell migration, reducing inflammation, fostering angiogenesis, enhancing cell survival, differentiating stem cells, and providing neuroprotection [1].

In topical applications, Tβ4 showed significant efficacy in wound healing. For corneal injuries, such as heptanol debridement or alkali burns in mice and rats, Tβ4 treatment led to rapid re-epithelialization and accelerated corneal repair. In the alkali injury model, Tβ4 also markedly decreased inflammation, evidenced by reduced levels of inflammatory chemokines and cytokines, and inhibition of matrix metalloproteinase (MMP) activity, NF-kappaB activation, and TNF-alpha stimulated cytokine release. The peptide further increased laminin-5 production, which aids in cell migration and cell-cell contacts, and reduced apoptosis in eye wounds. Interestingly, despite its pro-angiogenic properties elsewhere, Tβ4 did not induce angiogenesis in the avascular cornea, which is vital for maintaining vision. In dermal full-thickness punch wounds in rats and mice, Tβ4 accelerated wound closure, promoted keratinocyte migration, enhanced collagen deposition, and stimulated angiogenesis. These benefits were observed in both normal and impaired healing models, including diabetic and aged mice, and steroid-treated rats, with both synthetic and recombinant Tβ4 proving effective. An unexpected but consistent finding was the acceleration of hair growth in the wound areas and even in shaved/depilated animals, resulting in thicker and darker hair. This effect was attributed to increased migration and differentiation of hair follicle stem cells from the bulge region, activating existing follicles rather than generating new ones [1].

For systemic applications, Tβ4 exhibited profound therapeutic effects across various organ systems. In cardiovascular models, Tβ4 demonstrated potent cardioprotective effects. In a murine permanent ligation model, it enhanced myocyte survival, improved cardiac function, and reduced fibrosis. In a hypoxia-reoxygenation pig model, retroinfusion of Tβ4 significantly decreased infarct size and inflammatory cell infiltration, mimicking the effects of embryonic endothelial progenitor cell administration. The mechanism involved Tβ4 activating integrin-linked kinase (ILK) and Akt activity, promoting cardiac cell migration and survival, and stimulating stem cell activation and differentiation. This multifaceted action resulted in improved myocardial function and enhanced survival, distinguishing Tβ4 as a significant agent for cardiac repair [1].

In neurological models, Tβ4 proved to be neuroprotective. Systemic administration in mice with EAE, a model for multiple sclerosis, led to reduced inflammation and an increase in mature oligodendrocytes, resulting in functional neurological recovery. In rats treated with kainic acid, Tβ4 administered intracerebroventricularly prevented the loss of hippocampal neurons, demonstrating its ability to counteract excitotoxicity. In vitro studies further supported Tβ4’s neuroprotective role by shielding cortical neurons and hippocampal slices from glutamate-induced toxicity, and cerebral cortex astrocytes from ethanol toxicity. Its natural induction in the brain following ischemia suggests its endogenous role as a repair factor.

Beyond specific organ systems, Tβ4 consistently demonstrated its anti-inflammatory and anti-apoptotic properties. In a septic shock model induced by endotoxin (LPS) in rats, Tβ4 administration significantly reduced lethality and downregulated inflammatory mediators, suggesting it could be a crucial component of the host’s natural response to sepsis and a potential therapeutic. The article emphasized that Tβ4 does not function as a classical growth factor, as it does not promote general cell growth, does not bind to heparin, and is much smaller. Its broad actions on many cell types, including being anti-apoptotic, antimicrobial, and antifibrotic, further distinguish it. Finally, comprehensive nonclinical pharmacology and toxicology studies in dogs, rats, and monkeys revealed that systemic administration of Tβ4, even at very high doses, was safe and well-tolerated across all species, paving the way for clinical investigations [1].

2) This study completed by Zeng et al successfully developed and characterized familial Alzheimer’s disease (fAD) brain organoids, identified Tβ4 as a potential therapeutic target, and demonstrated its efficacy in both organoid and in vivo mouse models of AD. The results highlight Tβ4’s multifaceted role in mitigating neurodevelopmental defects and AD pathology [2].

The fAD organoids, derived from iPSCs with APP gene duplication (APP2) and APPV717I mutation (HVRD), exhibited key AD-like pathological features. Immunofluorescence analysis at Day 90 showed a significant increase in β-amyloid intensity in fAD organoids compared to controls. Soluble Aβ analysis at Day 60 also revealed a significantly decreased Aβ1-42/Aβ1-40 ratio in fAD organoids, consistent with AD progression. Single-cell RNA sequencing (scRNA-seq) at Day 30 and Day 60 revealed substantial alterations in cell composition and maturation. There was a notable reduction in the proportion of mature neurons (MN) and an increase in young neurons (YN), BMP-related cells (BRC), and astrocytes (ASC) at Day 60 in fAD organoids. Specifically, markers for sixth-layer neurons (TBR1) and general mature neurons (NeuN) were significantly decreased in fAD organoids. Transcriptomic analysis indicated that YN in fAD organoids were enriched in AD pathways, suggesting early onset of AD molecular features. Furthermore, fAD organoids exhibited signs of neuronal apoptosis, with increased levels of cleaved Caspase-3 (c-CASP3) and TUNEL signals, and mitochondrial dysfunction markers (NDUFA4, NDUFA6, NDUFB11) in Day 60 fAD organoids [2].

Pseudotime trajectory analysis confirmed that differentiated neurons in fAD organoids trended towards a cell death state. Glial cells in fAD organoids also displayed AD-like features, with upregulation of disease-associated astrocyte (DAA) gene signatures and enrichment in AD and PI3K-Akt signaling pathways. Differentially expressed mitochondrial genes were also observed in astrocytes. Crucially, genes like RTN3 and RTN4, known to be associated with increased BACE1 production and APP processing, were found to be downregulated in fAD organoids’ glial cells.

A comparative analysis of gene expression profiles between fAD cerebral organoids, control organoids, and human AD patient snRNA-seq data highlighted TMSB4X (encoding Tβ4) as a consistently downregulated gene in excitatory neurons across both fAD organoids and AD patients. This suggested a potential neuroprotective role for Tβ4. Further investigation revealed that treating control organoids with 5 µM Aβ led to a significant downregulation of TMSB4X expression, thereby establishing a functional link between Aβ pathology and reduced Tβ4 levels. GO and KEGG pathway analysis associated Tβ4 with apoptosis, AD, cytoskeleton regulation, and neurotrophin signaling [2].

Treatment of fAD organoids (APP2 line) with 0.5 µg/mL Tβ4 from Day 20 significantly rescued several pathological phenotypes. Tβ4 treatment markedly increased the density of mature neurons (NeuN+) in both control and APP2 Day 60 organoids. It also notably inhibited TUNEL intensity in APP2 Day 60 cerebral organoids, indicating reduced cell death. Moreover, Tβ4 significantly decreased the β-amyloid levels at Day 90 and improved the Aβ1-42/Aβ1-40 ratio at Day 60 in APP2 organoids. Bulk RNA-seq of Tβ4-treated APP2 organoids at Day 30 showed upregulation of genes involved in neurodevelopmental pathways such as axonogenesis and calcium ion detection, as well as downregulation of genes associated with immune response and protein phosphorylation. Specifically, Tβ4 downregulated APH1B, a subunit of γ-secretase, which could inhibit APP processing, and reduced the expression of other amyloid-beta formation-related genes [2].

In vivo validation using 5xFAD mice demonstrated that neuron-specific overexpression of TMSB4X via intrathecal AAV injection effectively mitigated AD pathology. Treated 5xFAD mice showed a significant reduction in amyloid plaques in both the cortex and hippocampus. Soluble Aβ analysis also confirmed a decrease in Aβ1-40 levels and an increased Aβ1-42/Aβ1-40 ratio, indicating a shift towards a less pathogenic Aβ profile. AAV-TMSB4X injection also inhibited microglial proliferation (IBA1+) and astrocytic gliosis (GFAP+) and suppressed the expression of inflammatory cytokines TNF-α and IL-6 in the hippocampus. Crucially, electrophysiological recordings revealed that Tβ4 overexpression alleviated neuronal hyperexcitability in cortical excitatory neurons of 5xFAD mice, evidenced by reduced action potential half-width, increased threshold potential, and decreased amplitude. Bulk RNA-seq from the brains of AAV-TMSB4X-treated 5xFAD mice showed upregulation of genes in neural signaling pathways, correlating with the improved neuronal excitability. Downregulation of small GTPase and Ras protein signaling pathways was also observed, which aligns with previous findings where Ras deletion ameliorates AD pathology.

In conclusion, the study identified Tβ4 as a potent neuroprotective factor that can effectively rescue neurodevelopmental deficits, reduce Aβ pathology, and normalize neuronal excitability in both human fAD brain organoids and 5xFAD mice, positioning Tβ4 as a promising therapeutic target for Alzheimer’s disease intervention [2].

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Philp D, Kleinman HK. Animal studies with thymosin beta, a multifunctional tissue repair and regeneration peptide. Ann N Y Acad Sci. 2010;1194:81-86. doi:10.1111/j.1749-6632.2010.05479.x

[2] Zeng PM, Sun XY, Li Y, et al. Thymosin beta 4 as an Alzheimer disease intervention target identified using human brain organoids. Stem Cell Reports. 2025;20(9):102601. doi:10.1016/j.stemcr.2025.102601

Thymosin β-4 Effects on Wound Healing

Thymosin β-4 is a peptide that is found to have many regenerative properties. Various animal studies have shown that when using Tβ4 in mammalian cells it led to wound healing in dermal, corneal, and cardiac cells. In addition to its wound healing properties, Tβ4 has been shown to improve cell survival, formation of blood vessels, and maturation of stem cells as well as decrease levels of inflammatory cytokines and chemokines

These claims are supported in a study conducted by Phlip and Kleinman in which they applied a topical treatment of Tβ4 to both alkali and heptanol related injuries in the eyes and the skin of rats and mice.

Twice per day 5 micrograms/5 microliters were administered to the mice as liquid eye drops. As the drops were administered the epithelium of the cornea rapidly moved over the surface of the cornea in order to repair the damaged tissue from the alkali injury. Since the alkali injury is considered a burn there is a considerable amount of inflammation in the surrounding areas. The topical Tβ4 drops work to alleviate the inflammation by decreasing the levels of matrix metalloproteinases, activating nF kappa b, and blocking TNF cytokine release. Additionally, Tβ4 increases the synthesis of laminin-5 which helps to promote cell contact and migration in the corneal epithelium as well as decrease levels of apoptosis and increase the rate of angiogenesis in assays.

The researchers also tested the effects that Tβ4 had on the healing process of dermal wounds. Rats and mice with full thickness punch dermal wounds were treated with either a PBS form or a hydrogel of Tβ4 in doses varying from 5 to 50 micrograms/50 microliters/ wound. In regards to dermal wound healing, the application of Tβ4 led to an increase in the rate of collagen deposition, keratinocyte migration, and angiogenesis. Branches of this same study conducted on rats and mice with various forms of impaired healing such as diabetes or advanced age reported that the use of Tβ4 accelerated the healing process in the test subjects (https://pubmed.ncbi.nlm.nih.gov/20536453/).

Effects of Thymosin β4 on the Cardiovascular System

Tβ4 has been shown to have positive effects in various animals when treating cardiovascular disease, nerve damage, septic shock, and dermal injury. For intracardiac injection of Tβ4, the typical dose was 400 ng, while retroinfusion doses ranged around 15 mg.

Tβ4 is considered ‘cardioprotective’, in the coronary ligation models in mice Tβ4 was shown to promote repair throughout the cardiac muscle. Additionally in the refusion injury model of sheep it was shown to prevent damage to the vascular system. Tβ4 works in the heart by activating integrin-linked kinase and promoting cell migration, survival, and activation thus leading to improved function of the heart, lower levels of fibrosis, and improved cell survival rates.

Effects of Thymosin β4 on Sepsis

As it was stated above, Tβ4 helps to lower inflammation in the body and helps to reduce levels of proinflammatory cytokines. Since proinflammatory cytokines play a role in septic shock, Phlip and Kleinman injected an endotoxin into rats in order to examine the anti-inflammatory properties of Tβ4. After the endotoxin was administered, Tβ4 was administered immediately as well as at 2 hours and at 4 hours after the initial administration.

After dosing with Tβ4 it was found that the lethality of endotoxin-induced septic shock was greatly reduced as well as the inflammatory mediators that are found in association with septic shock. The researchers noted an interesting phenomenon that when inducing sepsis in rats, levels of Tβ4 were naturally reduced. Reduced levels of Tβ4 were also found in rats with non-induced septic shock as well as when injected with the endotoxin, thus indicating that Tβ4 can play an important role in treating sepsis (http://www.paulinamedicalclinic.com/wp-content/uploads/2019/11/Animal-studies-with-thymosin-beta4-a-multifunctional-tissue-repair-and-regeneration-peptide-1.pdf).

Effects of Thymosin β4 on the Nervous System

Similar to the cardiovascular system, Tβ4 is considered protective in the nervous system as well both in vivo and in vitro. Rats were injected with kainic acid in order to induce hippocampal neuron loss. After the kainic acid treatment the rats were administered Tβ4 twice a day for 5 days which combated the loss of neurons.

Furthermore, mice experiencing autoimmune encephalomyelitis were treated with 6mg/kg of Tβ4 24 hour after the infection was induced and every 3 days after that. Treatment with Tβ4 was shown to help improve functionality as well as increase the amount of mature oligodendrocytes and reduced overall inflammation. Additionally, Tβ4 was shown to protect against glutamate-induced toxicity and ethanol toxicity in vitro (http://www.paulinamedicalclinic.com/wp-content/uploads/2019/11/Animal-studies-with-thymosin-beta4-a-multifunctional-tissue-repair-and-regeneration-peptide-1.pdf).

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