An Investigation on the Therapeutic Effect of Thymosin <svg xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg" style="vertical-align:-4.268501pt" id="M1" height="16.5463pt" version="1.1" viewBox="-0.0657574 -12.2778 10.5522 16.5463" width="10.5522pt"><g transform="matrix(.017,0,0,-0.017,0,0)"><path id="g5-224" d="M584 573C584 646 528 703 441 703C387 703 327 679 274 632C202 568 166 493 132 302L57 -122C43 -201 35 -214 21 -227L25 -246C54 -244 122 -236 156 -203C168 -191 175 -162 195 -40L261 369C291 556 318 652 391 652C431 652 465 620 465 543C465 497 449 456 408 422C395 411 364 405 340 403L309 336C386 319 447 279 447 188C447 105 419 50 329 50C290 50 253 63 226 77L216 55C218 23 252 -16 286 -16C324 -16 369 3 419 31C488 69 575 149 575 238C575 325 516 369 437 400C516 443 584 486 584 573Z"/></g></svg>4 and Its Expression Levels in Streptozotocin-Induced Diabetic Mice

Cho, Kyung Sook; Kim, Dong-Jin; Shim, Bomee; Kim, Jung Yeon; Kang, Jun Mo; Park, Seon Hwa; Lee, Sang-Ho; Yang, Hyung-In; Kim, Kyoung Soo

doi:https://doi.org/10.1155/2018/3421568

BioMed Research International

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Abstract Introduction Materials and Methods Results Discussion Conclusions Data Availability Ethical Approval Consent Conflicts of Interest Authors’ Contributions Acknowledgments References Copyright Related Articles

Research Article | Open Access

Volume 2018 | Article ID 3421568 | https://doi.org/10.1155/2018/3421568

An Investigation on the Therapeutic Effect of Thymosin 4 and Its Expression Levels in Streptozotocin-Induced Diabetic Mice

Kyung Sook Cho,¹Dong-Jin Kim,²Bomee Shim,¹Jung Yeon Kim,³Jun Mo Kang,⁴Seon Hwa Park,²Sang-Ho Lee,²Hyung-In Yang,^5,6and Kyoung Soo Kim^1,6

Academic Editor: Shoichiro Ono

Received08 Jun 2018

Revised01 Aug 2018

Accepted12 Aug 2018

Published27 Aug 2018

Abstract

Thymosin β4 (Tβ4) treatment was known to show the potential therapeutic effects on diabetic complications. This study was performed to determine if Tβ4 expression is changed in both serum and tissues under diabetic conditions and can be a serum biomarker. Type 1 diabetic mice were induced in C57/BL6J mice by intraperitoneal injection of streptozotocin (STZ) at a dose of 50 mg/kg body weight. The mice were sacrificed at 16 weeks after STZ injection. Tissues and plasmas were obtained to determine the expression levels of Tβ4 using ELISA, real time RT-PCR, and immunohistochemistry. The average serum glucose level was increased to approximately 400 mg/dL beginning 2 weeks after the five injections of STZ and lasting for at least 13 weeks until sacrifice. The plasma and tissue levels of Tβ4 in the age-matched control mice were not significantly different from those of the diabetic mice. In conclusion, the Tβ4 expression level in the plasmas and tissues of diabetic mice was not affected by diabetic conditions. It indirectly suggests that the therapeutic effect of Tβ4 on diabetic complications is due to its regenerative effects on damaged tissue but not to the changed expression level of Tβ4 in plasma and tissues of diabetes.

1. Introduction

Thymosin β4 (Tβ4), a 4.9 kDa peptide with 43 amino acids, has a number of active sites. The Tβ4 containing amino acids 1-4 has anti-inflammatory activity, residues 1-15 have anti-apoptotic and cytoprotective activity, and residues 17-23 affect cell migration, actin biding, dermal wound healing, angiogenesis, and hair growth [1]. Specifically, it binds to G-actin to regulate the formation of F-actin, which in turn regulates the dynamics of cytoskeletal rearrangement [2], leading to effects on cell mobility, cell division, and differentiation. Tβ4 is secreted into bodily fluids at a relatively high concentration even though there are no known signals for its secretion, and it has numerous physiological and pathological effects on cells and organs although it has no known receptors. For instance, it aids in wound healing [3], tumor metastasis, angiogenesis [4], cell migration [5], and stem cell differentiation [6]. Because increased Tβ4 expression may worsen clinical outcomes from tumors [7], Tβ4 is a potential molecular target for tumor treatment [8]. In contrast, decreased levels of Tβ4 in the kidneys accelerated glomerular disease in global Tβ4 knockout mice [9]. In addition, Tβ4 treatment has been used to alleviate the symptoms of various diseases of animal models. Thus, Tβ4 serum level has been measured for developing potential biomarker in various diseases such as cardiovascular, infectious, and autoimmune disease [10].

Treatment with Tβ4 or its active fragment, N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP), helps to recover function following a brain injury by inhibiting the TGF-β1/NF-κB signaling pathway [11]. It also aids in dermal healing because of its ability to stimulate tissue repair and regeneration[12]. Thus, Tβ4 therapeutic treatments have been tested in phase 2 clinical trials of patients with pressure ulcers, stasis ulcers, and epidermolysis bullosa wounds [12]. In addition, the therapeutic effects of Tβ4 have been widely studied in the field of diabetes mellitus. In particular, Tβ4 treatment showed therapeutic effects on diabetic peripheral nephropathy [13] and neuropathy [14, 15] in diabetic mice. Coadministration of thymosin fraction 5 (TF5), which contains Tβ4 [16], with a subdiabetogenic dose of streptozotocin (STZ, 35 mg/kg) blocked the initiation of insulitis and hyperglycemia in mice [17]. A high dose of TF5 (0.1 mg/day) maintained normal blood glucose levels in diabetic mice, but treatment with low-dose TF5 (0.01 mg/kg) had no effect on blood glucose levels. The protective effects of Tβ4 could be partly induced by modulating the immune-regulatory or immunostimulating effects on suppressor T cells [18].

Despite extensive studies on Tβ4 treatment and the potential therapeutic effects of Tβ4 on diabetic complications, the blood and tissue expression levels of Tβ4 during the progression of diabetes mellitus have not been thoroughly investigated. In this study, we determined if Tβ4 expression is changed in both plasma and tissues under diabetic conditions using an STZ-induced type 1 diabetic mouse model.

2. Materials and Methods

2.1. Cell Culture of Isolated Splenocytes

Splenocytes were isolated from the spleens of C57/BL6J mice as described previously [19]. The splenocytes were seeded at 3 × 10⁶ cells/well in 6-well plates. The cells were stimulated with a high concentration of glucose (20-60 mM) in 2 ml of DMEM for 24 hrs. The culture supernatants were harvested for the analysis of Tβ4 protein levels using ELISAs. Total RNA was extracted from the cells for the analysis of Tβ4 mRNA levels using real time PCR.

2.2. Preparation of Diabetic Mice

Diabetes was induced in 8-week-old male C57/BL6J mice as described previously [20]. Briefly, C57BL6J mice (n=9) were intraperitoneally injected with streptozotocin (STZ) (50 mg/kg/day), which was dissolved in 50 mM Na citrate buffer (pH 4.5), for 5 consecutive days. Blood glucose levels were checked every 7 days using a blood glucose meter (G-Doctor, Allmedicus, Anyang, Korea) and blood from the tail, and body weights were also measured weekly. Mice with blood glucose levels less than 250 mg/dL at 2 weeks after the final injection of STZ were excluded from the experiment. The mice were sacrificed 16 weeks after the final administration of STZ, and an age-matched control group (n=6) was also sacrificed at the same time. Mice were anesthetized with the mixture of Zoletil (30mg/kg) and xylazine (10mg/kg). The blood was collected from the inferior vena cava into heparinized syringes. Before collecting the blood, heparin (5000IU/ml, JW-Pharma, Seoul, Korea) was sucked into a syringe and the syringe was moved repeatedly up and down to coat the heparin. Then heparin was removed from the syringe. After blood collection, remained blood was removed through cardiac perfusion with cold saline. The blood was centrifuged (2000xg, 20 minutes, 4°C) and plasma was collected and stored in deep-freezer before analysis. Also, the organs from both groups were obtained. All the samples were kept at −80°C or in 10% buffered formalin for mRNA and immunochemistry analysis. Two separate replicate experiments were done, and all experiments were performed with approval (KHNMC AP 2016-007) according to the guidelines of the Gangdong Animal Research Ethics Committee of Kyung Hee University Hospital.

2.3. Real Time Polymerase Chain Reaction (PCR)

Total RNA was extracted from splenocytes and the tissue of each organ using Trizol (Thermo Fisher Scientific Korea, Seoul). The cDNA of the RNA was synthesized using a commercial cDNA synthesis kit (Thermo Fisher Scientific Korea, Seoul) according to the manufacturer’s instructions. Real time PCR was performed using SYBR Green PCR mater mix (Applied Biosystems, Carlsbad, CA, USA) and the following primer sequences: Tβ4-forward 5′-CTC GGC TCC TTC CAG CAA C-3′, Tβ4-reverse 5′-TCG CCA GCT TGC TTC TCT TG-3′, 18S-forward 5′-GTA ACC CGT TGA ACC CCA TT-3′, and 18S-reverse 5′-CCA TCC AAT CGG TAG TAG CG -3′.

2.4. Tβ4 ELISA

The Tβ4 protein levels in the plasma and the cell culture supernatants were measured using a commercial ELISA kit from Peninsula Laboratories (San Carlos, CA, USA). The ELISA was performed according to the manufacturer’s instructions. The intra- and interassay coefficients of variation (CV) are less than 10% and 15%, respectively.

2.5. Histopathological Analysis and Immunohistochemistry

We harvested the internal organs (i.e., the stomach, intestines, pancreas, liver, spleen, kidney, thymus, lungs, and heart) from diabetic mice (n=5) and control mice (n=3). The organs were dissected, fixed with 10% buffered formalin, embedded in paraffin, and prepared into 4 μm thick sections. The sections were either stained with hematoxylin and eosin (H&E) or used for immunohistochemical staining in an automated system (Leica Biosystems, Newcastle, UK) using anti-Tβ4 antibodies (Peninsula Laboratories International Inc. San Carlos, CA). The antigens were retrieved with epitope retrieval solution 1 (Leica Biosystems). The slides were incubated with each antibody at room temperature for 20 minutes then with a biotinylated secondary antibody for 8 minutes. The resulting complexes were detected using avidin-peroxidase conjugate polymer (Leica Biosystems), and the color was developed using 3,3′-diaminobenzidine (Leica Biosystems). Mayer’s hematoxylin (Leica Biosystems) was used as a counterstain. Positive- and negative-control staining was also used for each assay run.

2.6. Statistical Analysis

GraphPad Prism software, version 5 (San Diego, CA), was used for the statistical analysis and graphing. The two-tailed Mann–Whitney test was used to compare the Tβ4 serum levels between the diabetic and control mice. The Tβ4 mRNA expression levels from triplicate tissue samples from control and diabetic mice were assessed using the Mann–Whitney test. P values <0.05 were considered to be statistically significant. All data are expressed as the mean ± standard error of the mean (SEM).

3. Results

3.1. Comparison of Thymosin β4 (Tβ4) Levels in the Plasma of Diabetic and Control Mice

To determine how hyperglycemia affects the expression level of Tβ4 in plasma and tissues, test mice received five consecutive injections of STZ (50 mg/kg) and were maintained for 16 weeks without any therapeutic treatments. The body weights were significantly lower in the STZ-injected mice compared to the control mice as shown in Figure 1(a). The blood glucose levels (> 400 mg/dL) in the STZ-injected mice remained significantly higher than those in the control mice beginning 2 weeks after the final STZ injection (Figure 1(b)) until sacrifice at week 16. However, the Tβ4 plasma expression levels were not significantly different between the diabetic mice (129.3 ± 22.2 ng/mL) and the control mice (123.3 ± 18.2 ng/mL) (Figure 2). This suggests that the expression level of Tβ4 is not changed in plasma of STZ-induced type 1 diabetic mice.

(a)

(b)

3.2. Comparison of Tβ4 Levels in the Tissues of Diabetic and Control Mice

Next, the relative Tβ4 mRNA expression levels were measured in various tissues such as the liver, stomach, thymus, spleen, and kidneys. The mRNA expression level in the spleen was approximately 20-fold higher than in the other tissues (Figure 3(a)). Also, the Tβ4 mRNA expression level was not affected in each of the tissues of the diabetic mice (Figure 3(b)). To further study Tβ4 expression at the protein level, tissues samples from the diabetic and control mice were compared using histology and immunohistochemistry. The histology assay of the diabetic mice showed a higher degree of damage or loss of islet cells in the pancreas and mild lobular disarray in the liver compared to the control mice (Figure 4(a)). The other tissues were not significantly different between the two groups in terms of damage or loss secondary to hyperglycemia. For immunohistochemical staining, cells of moderate or strong nuclear and/or cytoplasmic staining were determined as a percentage and scored as follows: 0 (staining in less than 10% of cells); 1 (staining in 10-50% of cells); and 2 (staining in more than 50% of cells). Cases with score 1 or 2 were classified as positive. Tβ4 staining was positive for the lungs, liver, thymus, spleen, stomach, and small intestine (score 1), but not in the heart and pancreas (score 0) (Figure 4(b)), and revealed no difference in Tβ4 expression between the diabetic and control mice. Specifically, the lung samples had Tβ4-positive staining cells in the interstitial tissue of both the diabetic and the control mice. The Kupffer cells and sinusoidal cells in the liver were stained for Tβ4 in both the diabetic and control mice, but the number of positive-stained cells was higher in the diabetic mice than in the control mice. The pancreas showed a few positive-stained cells only in the diabetic mice (score 0). The thymus revealed scattered Tβ4-positive staining in both the cortex and the medulla, and Tβ4-positive cells were noted in both the white pulp and the red pulp of the spleen. Taken together, these results indirectly suggest that diabetic conditions such as hyperglycemia do not significantly affect the serum and tissue levels of Tβ4 in type 1 diabetic mice.

(a)

(b)

Figure 3

Comparison of Tβ4 mRNA levels in the tissues of diabetic and control mice. STZ-induced type 1 diabetic mice and age-matched control mice were sacrificed at 16 weeks after the final injection of STZ. Each organ was harvested and the total RNA was extracted using Trizol. The Tβ4 transcriptional level was measured using real time PCR as described in the Materials and Methods. (a) In the control mice, the spleen had the highest expression level of Tβ4 among the various organs in relative to liver. A similar pattern was observed in the diabetic mice. The data represent triplicate samples of each organ of the control mice. (b) The Tβ4 expression level of each organ of the control mice was compared with the diabetic mice. There was no significant difference in Tβ4 expression between the control and diabetic mice. The representative results are shown. NS: not significant.

(a) Control mice

(b) Diabetic mice

Figure 4

Comparison of Tβ4 protein levels in the tissues of diabetic and control mice. The difference in Tβ4 expression level and the degree of tissue damage between the two groups was compared using (a) H&E staining (×100) and (b) immunohistochemistry (×200) (scale bar, 100 μm). The spleen had a higher expression level than the other tissues comparable with the difference in transcription levels. There were no significant differences between the two groups in Tβ4 protein expression level or degree of tissue damage.

3.3. Effects of High-Glucose Concentrations on the Expression of Tβ4 in Splenocytes In Vitro

To determine how high-glucose concentration affects the in vitro expression of Tβ4 in immune cells that highly express Tβ4, splenocytes were treated with high concentrations of glucose for 24 hrs. The mRNA expression level of Tβ4 did not significantly increase in the glucose-treated group compared to the control group. The protein level of Tβ4 also was not increased by the high-glucose treatment (Figure 5). This suggests that high-glucose concentrations did not affect the in vitro expression of Tβ4 in immune cells that had a high basal level of Tβ4 expression.

(a)

(b)

Figure 5

Effects of glucose concentration on the in vitro expression of Tβ4 in splenocytes. Immune cells from the spleen were isolated and treated with high-glucose concentrations for 24 hrs. Subsequently, the total RNA was extracted from the cells for real time PCR analysis (a) and the cell culture supernatant was obtained to determine the Tβ4 levels using ELISA (b). Three independent experiments were performed using triplicates of each sample for the real time PCR and ELISA. Similar results were obtained from each experiment, and representative results are shown. NS: not significant.

4. Discussion

In this study, the expression levels of Tβ4 in plasma and tissues from STZ-induced diabetic mice were investigated to know if its expression could be changed in diabetic conditions. This can indirectly explain if the therapeutic effects of Tβ4 on diabetic complications could result from ameliorating the decreased expression of Tβ4. The STZ-induced type 1 diabetic mice were under hyperglycemic conditions for at least 13 weeks because they were not treated with any blood glucose lowering agents and were sacrificed 16 weeks after the final injection of STZ. Using real time PCR, ELISA, and immunohistochemistry, we determined that the expression of Tβ4 in the plasma and tissues of the diabetic mice was not significantly different than in the age-matched control mice. Marked tissue damage was also not observed in the diabetic mice, except for that in the pancreas. Taken together, these data suggest that hyperglycemia was not a factor that had some impact on Tβ4 plasma level in this model, but the Tβ4 released from damaged tissues was not able to affect the overall Tβ4 serum level.

The Tβ4 serum level has been documented to change significantly according to disease state. In our previous studies, Tβ4 level was significantly increased in the serum and synovial fluid of patients with rheumatoid arthritis [21, 22]. The Tβ4 level was significantly increased in the serum of patients with inflammatory bowel disease [23], and the level was high in human wounds and blister fluid after dermal injury [24]. This increase in Tβ4 may be due to tissue damage or cell lysis, or it may be increased by a host defense system due to Tβ4’s anti-inflammatory effects [25, 26]. However, a conflicting study showed that the serum level of Tβ4 was significantly decreased in patients with liver failure caused by chronic hepatitis B virus (HBV) [27]. In diabetic mice in the current study, serum Tβ4 level was not increased or decreased compared to that of control mice. This suggests that blood glucose level did not affect Tβ4 serum level and that Tβ4 was not released into serum from damaged cells or cell lysis. Chronic hyperglycemia also induces an inflammatory state [28, 29]. Nonenzymatically glycosylated proteins interact with their receptors to induce oxidative stress and proinflammatory responses. Hyperglycemia also enhances inflammation by promoting cytokine secretion in monocytes, adipocytes, and other cells. The hyperglycemia-induced inflammation produced in this study may not have been sufficient to induce the expression of Tβ4 or the release of Tβ4 from cells through cell damage or lysis. Furthermore, we may consider the possibility that Tβ4 level might decrease (or increase) during the initial stage and gradually return to baseline as hyperglycemia continues to develop. In addition, a great amount of Tβ4 is released from platelet at sites of injury for wound healing. The serum level can be influenced by various factors [30]. However, we do not provide a detailed temporal profiling on the expression of this peptide.

A radioimmunoassay for Tβ4 demonstrated that it was ubiquitously present in most rat and mouse tissues although the spleen had higher concentrations than other tissues such as the brain, kidneys, liver, and testes [31]. In this study, the effects of hyperglycemia on Tβ4 expression in tissues were investigated using quantitative real time PCR and immunohistochemistry. As with the serum expression, Tβ4 tissue expression was not affected by hyperglycemia. Among the organ tissues of the control mice, the spleen had the highest transcriptional expression, which was comparable with the previous report [9]. The heart and pancreas had very low Tβ4 expression on both the transcriptional and translational levels, and the other tissues had moderate expression of Tβ4. In contrast, human pancreatic islet cells highly express Tβ4 in previous study [32]. In mouse islet cells, however, Tβ4 was not expressed as much as human islets in this study. This may be partly due to the different reactivity of antibodies against Tβ4 between mouse and human tissue. Peritoneal macrophages have high concentrations of Tβ4, and the spleen has various immune cells containing macrophages or macrophage-like cells [16], possibly explaining why it has the highest expression of Tβ4 among the various tissues. Additionally, it has been suggested that Tβ4’s presence in other tissues may be related to the presence of macrophages or macrophage-like cells in those tissues [16]. Tβ4 expression is also significantly increased in the retinas of patients with proliferative diabetic retinopathy (PDR)[33]. The vitreous and plasma Tβ4 concentrations in the excised membranes of patients with PDR who underwent a pars plana vitrectomy (PPV) were significantly higher than those in the control group. The mRNA levels of both Tβ4 and VEGF were also significantly increased in the diabetic membranes compared to the nondiabetic membranes, and their increase appears to play a role in angiogenesis in the retina. In contrast, high-glucose concentrations in our study did not affect the in vitro transcriptional and translational expression of Tβ4 in splenocytes. This suggests that the Tβ4 expression level changed minimally in response to the change in glucose concentration.

The tissue repair activity of Tβ4 towards diabetic complications, such as neuropathy and nephropathy, does not appear to be related to the decreased Tβ4 expression in the serum and tissues of diabetic animals. Rather, Tβ4 seems to help tissue regeneration by inducing growth factors that are responsible for tissue repair. For instance, it induces insulin-like growth factor-1 (IGF-1), which modulates cell survival, metabolism, and glucose homeostasis in high glucose- (HG-) human umbilical vein endothelial cells (HUVECs) [34]. Also, Tβ4 is a chemotactic factor to migrate satellite cell-derived myoblasts and myocytes into muscle injury site [35]. Muscle damage promotes the local production of Tβ4, which chemoattracts myoblasts to regenerate muscle [36]. In addition, Tβ4 reverses the decreased expression of angiopoiein-1 (Ang1) in endothelial and Schwan cells in diabetic sciatic nerves under high-glucose concentrations through the PI3K/Akt signaling pathway [14]. Additionally, Tβ4 improves glucose intolerance and ameliorates insulin resistance in mice by acting as an insulin sensitizer through the increased phosphorylation of Akt signaling level [37].

5. Conclusions

Tβ4 expression levels in the plasma and tissues of diabetic mice were not changed compared to the control mice, while the Tβ4 level was known to change significantly according to disease state such as tumor and inflammatory disease. Also, hyperglycemia seemed not to modulate the expression of Tβ4 in STZ-induced diabetic mice. Thus, all these results indirectly suggest that the molecular mechanism by which Tβ4 treatment alleviates hyperglycemia-induced tissue damage in diabetic mice may not be due to replenishing Tβ4 levels that may have been decreased under hyperglycemic conditions. Rather, the diverse biological activities of Tβ4, such as regenerative and angiogenetic activities, may contribute to tissue repair by inducing growth factors, promoting cell migration and wound healing, exerting anti-inflammatory effects, and inhibiting apoptosis.

Data Availability

The data used to support the findings of this study are available from the corresponding author upon request.

Ethical Approval

All experiments were performed with approval (KHNMC AP 2016-007) according to the guidelines of the Gangdong Animal Research Ethics Committee of Kyung Hee University Hospital.

Only mice were included in the study.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Authors’ Contributions

Kyung Sook Cho and Dong-Jin Kim equally contributed to this work.

Acknowledgments

This research was supported by a grant from the Korean Health Technology R&D Project through the Korean Health Industry Development Institute (KHIDI) funded by the Ministry of Health & Welfare, Republic of Korea (Grant no. HI17C0658). This research was also supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (Grant nos. 2017R1A1B03031409 and 2018R1D1A1B07048706) and partly supported by a grant from Kyung Hee University in 2016 (KHU-20161392).

References

G. Sosne, P. Qiu, A. L. Goldstein, and M. Wheater, “Biological activities of thymosin β4 defined by active sites in short peptide sequences,” The FASEB Journal, vol. 24, no. 7, pp. 2144–2151, 2010.
View at: Publisher Site | Google Scholar
M. C. Sanders, A. L. Goldstein, and Y. L. Wang, “Thymosin beta 4 (Fx peptide) is a potent regulator of actin polymerization in living cells.,” Proceedings of the National Acadamy of Sciences of the United States of America, vol. 89, no. 10, pp. 4678–4682, 1992.
View at: Publisher Site | Google Scholar
K. M. Malinda, G. S. Sidhu, H. Mani et al., “Thymosin β4 accelerates wound healing,” Journal of Investigative Dermatology, vol. 113, no. 3, pp. 364–368, 1999.
View at: Publisher Site | Google Scholar
H. J. Cha, M. J. Jeong, and H. K. Kleinman, “Role of thymosin beta4 in tumor metastasis and angiogenesis,” JNCI: Journal of the National Cancer Institute, vol. 95, no. 22, pp. 1674–80, 2003.
View at: Google Scholar
Z. Piao, C. Hong, M. Jung, C. Choi, and Y. Park, “Thymosin β4 induces invasion and migration of human colorectal cancer cells through the ILK/AKT/β-catenin signaling pathway,” Biochemical and Biophysical Research Communications, vol. 452, no. 3, pp. 858–864, 2014.
View at: Publisher Site | Google Scholar
D. Philp, S. St-Surin, H.-J. Cha, H.-S. Moon, H. K. Kleinman, and M. Elkin, “Thymosin beta 4 induces hair growth via stem cell migration and differentiation,” Annals of the New York Academy of Sciences, vol. 1112, pp. 95–103, 2007.
View at: Publisher Site | Google Scholar
S. Y. Lee, M. J. Park, H. K. Lee et al., “Increased Expression of Thymosin β4 Is Independently Correlated with Hypoxia Inducible Factor-1α (HIF-1α) and Worse Clinical Outcome in Human Colorectal Cancer,” Journal of Pathology and Translational Medicine, vol. 51, no. 1, pp. 9–16, 2017.
View at: Publisher Site | Google Scholar
Y. Xiao, Y. Chen, J. Wen, W. Yan, K. Zhou, and W. Cai, “Thymosin β4: A Potential Molecular Target for Tumor Therapy,” Critical Reviews in Eukaryotic Gene Expression, vol. 22, no. 2, pp. 109–116, 2012.
View at: Publisher Site | Google Scholar
E. Vasilopoulou, M. Kolatsi-Joannou, M. T. Lindenmeyer et al., “Loss of endogenous thymosin β4 accelerates glomerular disease,” Kidney International, vol. 90, no. 5, pp. 1056–1070, 2016.
View at: Publisher Site | Google Scholar
W. K. Tan, K. Purnamawati, L. S. Pakkiri et al., “Sources of variability in quantifying circulating thymosin beta-4: literature review and recommendations,” Expert Opinion on Biological Therapy, vol. 18, no. sup1, pp. 141–147, 2018.
View at: Publisher Site | Google Scholar
Y. Zhang, Z. G. Zhang, M. Chopp et al., “Treatment of traumatic brain injury in rats with N-acetyl-seryl-aspartyl-lysyl-proline,” Journal of Neurosurgery, vol. 126, no. 3, pp. 782–795, 2017.
View at: Publisher Site | Google Scholar
H. Kleinman and G. Sosne, “Thymosin β4 Promotes Dermal Healing,” in Thymosins, vol. 102 of Vitamins and Hormones, pp. 251–275, Elsevier, 2016.
View at: Publisher Site | Google Scholar
J. Zhu, L. Su, Y. Zhou, L. Ye, K. Lee, and J. Ma, “Thymosin β4 Attenuates Early Diabetic Nephropathy in a Mouse Model of Type 2 Diabetes Mellitus,” American Journal of Therapeutics, vol. 22, no. 2, pp. 141–146, 2015.
View at: Publisher Site | Google Scholar
L. Wang, M. Chopp, A. Szalad et al., “Thymosin β4 promotes the recovery of peripheral neuropathy in type II diabetic mice,” Neurobiology of Disease, vol. 48, no. 3, pp. 546–555, 2012.
View at: Publisher Site | Google Scholar
L. Wang, M. Chopp, L. Jia et al., “Therapeutic benefit of extended thymosin β4 treatment is independent of blood glucose level in mice with diabetic peripheral neuropathy,” Journal of Diabetes Research, vol. 2015, Article ID 173656, 13 pages, 2015.
View at: Publisher Site | Google Scholar
E. Hannappel, G. J. Xu, J. Morgan, J. Hempstead, and B. L. Horecker, “Thymosin beta 4: a ubiquitous peptide in rat and mouse tissues.,” Proceedings of the National Acadamy of Sciences of the United States of America, vol. 79, no. 7, pp. 2172–2175, 1982.
View at: Publisher Site | Google Scholar
T. Tabata, Y. Kinoshita, S. Fujii et al., “Protection by thymosin fraction 5 from streptozotocin-induced diabetes in mice,” Cell Mol Biol, vol. 35, no. 2, p. 121, 1989.
View at: Google Scholar
S. H. Wang, G. Chen, and K. Zhang, “Effects of thymosin and insulin on suppressor T cell in type 1 diabetes,” Diabetes Res 19(1, p. 21, 1992.
View at: Google Scholar
E. K. Park, M. H. Ryu, Y. H. Kim et al., “Anti-inflammatory effects of an ethanolic extract from Clematis mandshurica Rupr,” Journal of Ethnopharmacology, vol. 108, no. 1, pp. 142–147, 2006.
View at: Publisher Site | Google Scholar
S. Y. Lee, J. M. Kang, D. J. Kim et al., “PGC1alpha Activators Mitigate Diabetic Tubulopathy by Improving Mitochondrial Dynamics and Quality Control,” Journal of Diabetes Research, vol. 2017, Article ID 6483572, 15 pages, 2017.
View at: Publisher Site | Google Scholar
R. Song, H. M. Choi, H. Yang, M. C. Yoo, Y. Park, and K. S. Kim, “Association between serum thymosin β4 levels of rheumatoid arthritis patients and disease activity and response to therapy,” Clinical Rheumatology, vol. 31, no. 8, pp. 1253–1258, 2012.
View at: Publisher Site | Google Scholar
H. M. Choi, Y. Lee, H. Yang, M. C. Yoo, and K. S. Kim, “Increased levels of thymosin β4 in synovial fluid of patients with rheumatoid arthritis: association of thymosin β4 with other factors that are involved in inflammation and bone erosion in joints,” International Journal of Rheumatic Diseases, vol. 14, no. 4, pp. 320–324, 2011.
View at: Publisher Site | Google Scholar
M. G. Mutchnick, H. H. Lee, D. I. Hollander, G. D. Haynes, and D. C. Chua, “Defective in vitro gamma interferon production and elevated serum immunoreactive thymosin β4 levels in patients with inflammatory bowel disease,” Clinical Immunology and Immunopathology, vol. 47, no. 1, pp. 84–92, 1988.
View at: Publisher Site | Google Scholar
M. Frohm, H. Gunne, A.-C. Bergman et al., “Biochemical and antibacterial analysis of human wound and blister fluid,” European Journal of Biochemistry, vol. 237, no. 1, pp. 86–92, 1996.
View at: Publisher Site | Google Scholar
S. M. Lunin and E. G. Novoselova, “Thymus hormones as prospective anti-inflammatory agents,” Expert Opinion on Therapeutic Targets, vol. 14, no. 8, pp. 775–786, 2010.
View at: Publisher Site | Google Scholar
S. Bollini, P. R. Riley, and N. Smart, “Thymosin beta4: multiple functions in protection, repair and regeneration of the mammalian heart,” Expert Opin Biol Ther 15, supplement 1, pp. S163–74, 2015.
View at: Google Scholar
T. Han, “Serum thymosin β4 levels in patients with hepatitis B virus-related liver failure,” World Journal of Gastroenterology, vol. 16, no. 5, p. 625, 2010.
View at: Publisher Site | Google Scholar
L. M. Roman-Pintos, G. Villegas-Rivera, A. D. Rodriguez-Carrizalez, A. G. Miranda-Diaz, and E. G. Cardona-Munoz, “Diabetic Polyneuropathy in Type 2 Diabetes Mellitus: Inflammation, Oxidative Stress, and Mitochondrial Function,” Journal of Diabetes Research, vol. 2016, Article ID 3425617, 16 pages, 2016.
View at: Publisher Site | Google Scholar
D. Aronson, “Hyperglycemia and the pathobiology of diabetic complications,” Advances in Cardiology, vol. 4, pp. 1–16, 2008.
View at: Google Scholar
H. Kaur and B. Mutus, “Platelet function and thymosin beta4,” The Journal of Biological Chemistry, vol. 393, no. 7, pp. 595–598, 2012.
View at: Google Scholar
G. J. Goodall, J. L. Hempstead, and J. I. Morgan, “Production and characterization of antibodies to thymosin beta 4,” The Journal of Immunology, vol. 131, no. 2, pp. 821–825, 1983.
View at: Google Scholar
S. Nemolato, T. Cabras, F. Cau et al., “Different thymosin beta 4 immunoreactivity in foetal and adult gastrointestinal tract,” PLoS ONE, vol. 5, no. 2, 2010.
View at: Google Scholar
J. Wang, Q. Lu, Y. Tao, Y. Jiang, and J. B. Jonas, “Intraocular expression of thymosin β4 in proliferative diabetic retinopathy,” Acta Ophthalmologica, vol. 89, no. 5, pp. e396–e403, 2011.
View at: Publisher Site | Google Scholar
S. Kim and J. Kwon, “Effect of thymosin beta 4 in the presence of up-regulation of the insulin-like growth factor-1 signaling pathway on high-glucose-exposed vascular endothelial cells,” Molecular and Cellular Endocrinology, vol. 401, pp. 238–247, 2015.
View at: Publisher Site | Google Scholar
T. Hara, “Thymosins and Muscle Regeneration,” Vitamins & Hormones, vol. 87, pp. 277–290, 2011.
View at: Publisher Site | Google Scholar
Y. Tokura, Y. Nakayama, S.-I. Fukada et al., “Muscle injury-induced thymosin β4 acts as a chemoattractant for myoblasts,” The Journal of Biochemistry, vol. 149, no. 1, pp. 43–48, 2011.
View at: Publisher Site | Google Scholar
J. Zhu, L.-P. Su, L. Ye, K.-O. Lee, and J.-H. Ma, “Thymosin beta 4 ameliorates hyperglycemia and improves insulin resistance of KK Cg-Ay/J mouse,” Diabetes Research and Clinical Practice, vol. 96, no. 1, pp. 53–59, 2012.
View at: Publisher Site | Google Scholar

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Copyright © 2018 Kyung Sook Cho et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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