Thymosins are a class of water-soluble, biologically active molecules with hormone-like effects. Thymosins consist of a series of peptides with varying amino acid sequences and functions, categorized into three superfamilies based on their isoelectric points: α, β, and γ. Thymosins with isoelectric points <5 are classified as α-thymosins, those with isoelectric points between 5 and 7 are β-thymosins, and those with isoelectric points >7 are γ-thymosins. β-thymosins, composed of 40-44 amino acid residues, can specifically bind to G-actin to promote cell migration, attracting significant research interest. To date, three β-thymosins have been identified in humans: thymosin β4, thymosin β10, and thymosin β15, all sharing notable amino acid sequence homology. Thymosin β4, a short peptide consisting of 44 amino acids, was isolated in 1981 using combined column chromatography and gel filtration methods from bovine thymus extracts. It is the most abundant member of the β-thymosins, constituting 70-80% of the total β-thymosin content. The molecular structure of thymosin β4 is highly conserved and encoded by the TMSB4X gene on the X chromosome. This molecule is widely present in various cells except for mature red blood cells, and it is highly expressed in platelets, lymphocytes, neutrophils, and macrophages. It is also found in various body fluids and exudates, such as tears, saliva, and wound exudates, exhibiting multiple biological functions.
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Biological Active Sites and Functions of Thymosin Beta 4
Thymosin β4 is a multifunctional protein with at least four biologically active sites, playing various roles in injury repair, antifibrosis, and tumor metastasis.
Amino Acids 1-4
The first four amino acids of thymosin β4 can be degraded by prolyl oligopeptidase or N-asparaginyl endopeptidase into the tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP). Initially, researchers observed that Ac-SDKP could promote the proliferation of vascular endothelial cells in BALB/c mice. Subsequent studies confirmed that Ac-SDKP could promote angiogenesis in tumor spheroid models, rat corneas, and myocardium injected with Ac-SDKP. Ac-SDKP also possesses anti-inflammatory activity, reducing inflammation by inhibiting the differentiation of bone marrow stem cells into macrophages, macrophage activation and migration, TNF-α release, and lymphocyte proliferation. Additionally, Ac-SDKP has antifibrotic activity in various organs, blocking the Ras-related C3 botulinum toxin substrate 1 (Rac1) signaling pathway, thus inhibiting the TGF-β/Smad pathway and reducing fibroblast proliferation and collagen deposition. However, research indicates that Ac-SDKP does not exert its physiological effects in the intact thymosin β4 molecule but only after thymosin β4 is cleaved, possibly due to the active site being hidden within the structure of thymosin β4.
Fig. 1 Amino acid sequence of thymosin β4 (Tβ4) (Kassem K.M, et al. 2019).
Amino Acids 1-15
The peptide segment formed by amino acids 1-15 contains anti-apoptotic and cell-protective active sites. Chlorhexidine, a strong broad-spectrum bactericide, has certain cytotoxicity. At a concentration of 0.002%, it can induce apoptosis in mouse fibroblast L929 cells, and at 0.016%, it can cause necrosis. Studies have shown that amino acids 1-15 of thymosin β4 can protect human fibroblasts by blocking the cytotoxic effects of chlorhexidine solutions at concentrations of 0.002% and 0.005%, but not at 0.01%. This peptide segment also provides cells with some resistance to hydrogen peroxide, benzalkonium chloride, and benzethonium chloride.
Amino Acids 17-23
Actin, a major component of the cytoskeleton, is present in almost all eukaryotic cells in two forms: monomeric G-actin and polymeric F-actin (microfilaments). The continuous polymerization and depolymerization of G-actin and microfilaments are involved in important cellular activities such as apoptosis, proliferation, migration, phagocytosis, immune regulation, and tumor immune evasion. Thymosin β4 is a primary G-actin binding molecule, with its actin-binding sequence formed by amino acids 17-23, which binds to G-actin, inhibiting microfilament depolymerization, thus playing a role in dermal injury repair, mast cell exocytosis, and angiogenesis.
Carboxy-terminal Peptide
The carboxy-terminal peptide of thymosin β4 is conserved across different thymosins. This peptide can enhance myocardial cell activity, promote angiogenesis, and reduce inflammation via the protein kinase B (Akt) signaling pathway, though its role in skin wound healing requires further investigation.
Mechanism of Thymosin Beta 4 in Wound Healing
Thymosin β4 is a widely acting endogenous repair factor, with studies showing its activity in all stages of skin wound healing. The following describes the mechanisms of thymosin β4 in wound healing through the four stages.
Coagulation Stage
After skin injury, exposed vascular endothelial cells and tissue factors stimulate a large aggregation of platelets at the wound site. Thymosin β4, which is abundant in platelets, is released into the wound with platelet destruction. Thymosin β4 selectively cross-links with actin, collagen, and fibrin under the catalysis of transglutaminase, promoting clot formation.
Inflammatory Stage
Thymosin β4 protects damaged tissue through three mechanisms: anti-inflammatory, anti-apoptotic, and reduction of oxidative radicals. It alleviates inflammation by inhibiting the activation of the nuclear factor κB pathway, reducing the release of inflammatory cytokines like IL-1 and TNF-α. Thymosin β4 also prevents apoptosis by downregulating the expression of caspase-3 and reducing the Bax/Bcl-2 ratio, which aids in the survival of cells and tissues at the wound site. Hypoxia in the wound increases thymosin β4 expression, which then reduces oxidative stress by inhibiting inducible NOS and cyclooxygenase-2, leading to decreased reactive oxygen species, nitric oxide secretion, and prostaglandin E2 levels, thus accelerating wound healing. The anti-inflammatory and anti-apoptotic effects of thymosin β4 protect damaged tissues from further injury caused by necrotic cell debris.
Proliferation Stage
Angiogenesis is crucial for wound healing as the blood supply provides necessary nutrients and removes necrotic cell debris, improving the wound microenvironment. Thymosin β4 promotes angiogenesis through several mechanisms:
1. Binding to G-actin, thymosin β4 promotes endothelial cell proliferation and migration, forming new capillaries.
2. Upr2.egulating VEGF expression, indirectly promoting angiogenesis.
3. Activating pro-angiogenic pathways such as the PI3K/Akt pathway and the Notch/NF-κB pathway. Thymosin β4 induces the expression of phosphorylated Akt protein in myocardial infarction rat models, activating downstream factors and promoting angiogenesis. It also upregulates the Notch/NF-κB signaling pathway, promoting capillary formation in limb ischemia mouse models.
4. Promoting the migration of endothelial progenitor cells (EPCs) and inhibiting EPC apoptosis, thus fostering angiogenesis. EPCs, a subtype of hematopoietic stem cells, can differentiate into endothelial cells. Thymosin β4 enhances EPC migration isolated from the peripheral blood of healthy volunteers and dose-dependently upregulates the phosphorylation of Akt and eNOS signaling pathways, inhibiting EPC apoptosis by upregulating the Bcl-2/Bax ratio, stabilizing mitochondrial membrane potential, and reducing cytochrome C release. High glucose environments reduce EPC numbers and impair their function. Thymosin β4 can restore the nitric oxide synthesis capacity of EPCs inhibited by high glucose via the Akt/eNOS pathway, potentially aiding in the treatment of diabetic ulcers.
Remodeling Stage
In the early stages of wound healing, thymosin β4 promotes the synthesis of proteins related to wound repair, such as laminin 5 (LN-5), LN-322, hepatocyte growth factor, and matrix metalloproteinases (MMPs), accelerating wound closure. After wound healing, thymosin β4 and its degradation product Ac-SDKP can not only reduce fibrosis/scar formation but even reverse this process. Studies comparing thymosin β4 expression levels in human keloids, hypertrophic scars, foreskin, and normal skin tissues (excluding foreskin) showed that thymosin β4 content was lowest in keloids, followed by hypertrophic scars, normal skin tissues (excluding foreskin), and foreskin tissues, suggesting a dose-effect relationship between thymosin β4 concentration and scar formation. High local concentrations of thymosin β4 inhibit scar formation, while reduced concentrations may lead to scar proliferation. Adding thymosin β4 to human hypertrophic scar fibroblasts reduces the expression of collagen types I and III and connective tissue growth factor while increasing MMP-2 and MMP-9 expression. MMP-2 and MMP-9 are gelatinases that can degrade various collagens, elastin, and fibronectin, further confirming the scar-reducing effect of thymosin β4. Additionally, thymosin β4 reduces the formation of myofibroblasts, which may explain its ability to reduce fibrosis/scar formation.
Conclusion
Thymosin β4 plays a multifaceted role in wound healing through its involvement in coagulation, inflammation, proliferation, and remodeling stages. Its ability to promote angiogenesis, reduce inflammation and apoptosis, and inhibit fibrosis makes it a promising therapeutic agent for enhancing wound healing and treating chronic wounds and fibrotic diseases. Further research into its mechanisms and potential clinical applications could lead to significant regenerative medicine and wound care advancements.
References
- Kassem K.M., et al. Tβ4-Ac-SDKP pathway: Any relevance for the cardiovascular system?. Canadian Journal of Physiology and Pharmacology. 2019, 97 (7): 589-99.
- Kleinman H.K., Sosne G. Thymosin β4 promotes dermal healing. Vitamins and Hormones. 2016, 102: 251-75.
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