OBJECTIVE: To investigate the role and mechanism of thymosin beta 4 (Tβ4) in oxidative stress injury of spinal cord-derived neural stem/progenitor cells (NSPCs) induced by hydrogen peroxide (H₂O₂). METHODS: NSPCs were isolated from Sprague-Dawley (SD) adult male rats, and divided into control group (untreated NSPCs cells), H₂O₂ group (NSPCs cells damaged by 500 μM H₂O₂), Tβ4 -3 groups (NSPCs were treated with 1, 2.5, 5 μg/ml Tβ4 on the basis of H₂O₂ treatment) and TAK-242 group [NSPCs were treated with 5 μg/ml Tβ4 and Toll-like receptor 4(TLR4) inhibitor TAK-242 on the basis of H₂O₂ treatment]. NSPCs were transfected with lentivirus vector of myeloid differentiation factor 88(MyD88) to construct MyD88-overexpressing cell lines, which were treated with H₂O₂ and Tβ4. The expression of Tβ4, TLR4, MyD88 were detected by qRT-PCR and Western blot. Cell viability was detected by MTT assay and lactate dehydrogenase(LDH) assay kit. Ca2+ concentration was detected by Fluo-3/AM probe method. The apoptosis of NSPCs was detected by flow cytometry and Caspase-3 and Caspase-9 kits;reactive oxygen species (ROS), superoxi dedismu-tase dismutase(SOD) activity and glutathione (GSH) content were detected by corresponding kits. Interleukin(IL)-6 and IL-1β were detected by enzyme-linked immunosorbent assay. RESULTS: The expression of Tβ4 was decreased in H₂O₂ injured NSPCs(P<0.05). Compared with H₂O₂ group, the cell viability and Ca2+ concentration was significantly increased, release of LDH and apoptosis were significantly decreased, production of ROS and pro-inflammatory cytokines were significantly decreased, and the expression levels of TLR4 and MyD88 protein were significantly decreased in Tβ4-3 groups and TAK-242 group (P<0.05). After overexpression of MyD88, cell viability, SOD activity and GSH content of NSPCs decreased, LDH release and apoptosis increased significantly (P<0.05), while after treatment with Tβ4, cell viability, SOD activity and GSH content increased, LDH release and apoptosis decreased (P<0.05). CONCLUSION Tβ4 attenuates H₂O₂-induced NSPCs oxidative stress, apoptosis and inflammation in NSPCs via inhibiting TLR4 and MyD88 pathways.
Animals ; Apoptosis ; Calcium/pharmacology* ; Cell Survival ; Hydrogen Peroxide/pharmacology* ; Male ; Myeloid Differentiation Factor 88/pharmacology* ; Oxidative Stress ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species/pharmacology* ; Spinal Cord Injuries/drug therapy* ; Stem Cells ; Superoxide Dismutase/pharmacology* ; Thymosin/metabolism* ; Toll-Like Receptor 4/metabolism*

With the aging of population and the development of social economy, the incidence of chronic wounds is increasing day by day, while the incidence of burns and trauma remains at a high level, making wound repair an increasingly concerned area in clinical practice. Thymosin β4 is a naturally occurring small molecule protein in vivo, which is widely distributed in a variety of body fluids and cells, especially in platelets. Thymosin β4 has biological activities of promoting angiogenesis, anti-inflammation, anti-apoptosis, and anti-fibrosis, and has many important functions in wound repair. Thymosin β4 has been observed to promote the healing of various wounds, such as burns, diabetic ulcers, pressure ulcers. This paper will review the molecular structure, mechanism of wound healing promotion, pharmacokinetics, and clinical application of thymosin β4, aiming to introduce its potential in wound treatment and the shortcomings of current researches.
Burns/drug therapy* ; Humans ; Pressure Ulcer ; Thymosin/therapeutic use* ; Wound Healing/physiology*

BACKGROUND: Thymosin β₄ is a multi-functional hormone-like polypeptide, being involved in cell migration, angiogenesis, and tumor metastasis. This study was undertaken to clarify the clinicopathologic implications of thymosin β₄ expression in human colorectal cancers (CRCs). METHODS: We investigated tissue sections from 143 patients with CRC by immunohistochemistry. In addition, we evaluated the expression patterns and the clinico-pathological significance of thymosin β₄ expression in association with hypoxia inducible factor-1α (HIF-1α) expression in the CRC series. RESULTS: High expression of thymosin β₄ was significantly correlated with lymphovascular invasion, invasion depth, regional lymph node metastasis, distant metastasis, and TNM stage. Patients with high expression of thymosin β₄ showed poor recurrence-free survival (p = .001) and poor overall survival (p = .005) on multivariate analysis. We also found that thymosin β4 and HIF-1α were overexpressed and that thymosin β₄ expression increased in parallel with HIF-1α expression in CRC. CONCLUSIONS: A high expression level of thymosin β₄ indicates poor clinical outcomes and may be a useful prognostic factor in CRC. Thymosin β₄ is functionally related with HIF-1α and may be a potentially valuable biomarker and possible therapeutic target for CRC.
Anoxia* ; Cell Movement ; Colorectal Neoplasms* ; Humans* ; Immunohistochemistry ; Lymph Nodes ; Multivariate Analysis ; Neoplasm Metastasis ; Thymosin*

Dysfunction or loss of blood vessel causes several ischemic diseases. Although endothelial progenitor cells (EPCs) are a promising source for cell-based therapy, ischemia-induced pathophysiological condition limits the recovery rate by causing drastic cell death. To overcome this issue, we attempted to develop a cell-targeted peptide delivery and priming system to enhance EPCbased neovascularization using an engineered M13 bacteriophage harboring nanofibrous tubes displaying ∼ 2700 multiple functional motifs. The M13 nanofiber was modified by displaying RGD, which is an integrin-docking peptide, on the minor coat protein, and bymutilayering SDKPmotifs,which are the key active sites for thymosin b4, on themajor coat protein. The engineered M13 nanofiber dramatically enhanced ischemic neovascularization by activating intracellular and extracellular processes such as proliferation, migration, and tube formation in the EPCs. Furthermore, transplantation of the primed EPCs with the M13 nanofiber harboring RGD and SDKP facilitated functional recovery and neovascularization in a murine hindlimb ischemia model. Overall, this study demonstrates the effectiveness of theM13 nanofiber-based novel peptide deliveryandprimingstrategy inpromotingEPC bioactivity and neovessel regeneration. To our knowledge, this is first report onM13 nanofibers harboring dual functional motifs, the use of which might be a novel strategy for stem and progenitor cell therapy against cardiovascular ischemic diseases.
Animals ; Bacteriophages ; Blood Vessels ; Catalytic Domain ; Cell Death ; Endothelial Progenitor Cells* ; Hindlimb ; Ischemia ; Nanofibers* ; Regeneration ; Stem Cells ; Thymosin

Thymosin β4 (Tβ4) is a key factor in cardiac development, growth, disease, epicardial integrity, blood vessel formation and has cardio-protective properties. However, its role in murine embryonic stem cells (mESCs) proliferation and cardiovascular differentiation remains unclear. Thus we aimed to elucidate the influence of Tβ4 on mESCs. Target genes during mESCs proliferation and differentiation were detected by real-time PCR or Western blotting, and patch clamp was applied to characterize the mESCs-derived cardiomyocytes. It was found that Tβ4 decreased mESCs proliferation in a partial dose-dependent manner and the expression of cell cycle regulatory genes c-myc, c-fos and c-jun. However, mESCs self-renewal markers Oct4 and Nanog were elevated, indicating the maintenance of self-renewal ability in these mESCs. Phosphorylation of STAT3 and Akt was inhibited by Tβ4 while the expression of RAS and phosphorylation of ERK were enhanced. No significant difference was found in BMP2/BMP4 or their downstream protein smad. Wnt3 and Wnt11 were remarkably decreased by Tβ4 with upregulation of Tcf3 and constant β-catenin. Under mESCs differentiation, Tβ4 treatment did not change the expression of cardiovascular cell markers α-MHC, PECAM, and α-SMA. Neither the electrophysiological properties of mESCs-derived cardiomyocytes nor the hormonal regulation by Iso/Cch was affected by Tβ4. In conclusion, Tβ4 suppressed mESCs proliferation by affecting the activity of STAT3, Akt, ERK and Wnt pathways. However, Tβ4 did not influence the in vitro cardiovascular differentiation.
Animals ; Cell Cycle ; drug effects ; genetics ; Cell Differentiation ; drug effects ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Extracellular Signal-Regulated MAP Kinases ; genetics ; metabolism ; Gene Expression Regulation ; drug effects ; JNK Mitogen-Activated Protein Kinases ; genetics ; metabolism ; Mice ; Mouse Embryonic Stem Cells ; cytology ; drug effects ; metabolism ; Myocytes, Cardiac ; cytology ; drug effects ; metabolism ; Nanog Homeobox Protein ; genetics ; metabolism ; Octamer Transcription Factor-3 ; genetics ; metabolism ; Patch-Clamp Techniques ; Primary Cell Culture ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; Proto-Oncogene Proteins c-fos ; genetics ; metabolism ; Proto-Oncogene Proteins c-myc ; genetics ; metabolism ; STAT3 Transcription Factor ; genetics ; metabolism ; Signal Transduction ; Thymosin ; pharmacology

BACKGROUNDThymosin beta-4 (TB-4) is considered key roles in tissue development, maintenance and pathological processes. The study aimed to prove TB-4 positive biological function on nucleus pulposus (NP) cell apoptosis and slowing the process of cell aging while increasing the cell proliferation.

METHODSTB-4 recombinant adeno-associated virus (AAV) was constructed and induced to human NP cells. Cell of same group were cultured without gene modification as controlled group. Proliferation capacity and cell apoptosis were observed during 6 passages of the cells. Morphology and expression of the TB-4 gene were documented as parameter of cell activity during cell passage.

RESULTSNP cells with TB-4 transfection has normal TB-4 expression and exocytosis. NP cells with TB-4 transfection performed significantly higher cell activity than that at the control group in each generation. TB-4 recombinant AAV-transfected human NP cells also show slower cell aging, lower cell apoptosis and higher cell proliferation than control group.

CONCLUSIONSTB-4 can prevent NP cell apoptosis, slow NP cell aging and promote NP cell proliferation. AAV transfection technique was able to highly and stably express TB-4 in human NP cells, which may provide a new pathway for innovation in the treatment of intervertebral disc degenerative diseases.

Apoptosis ; genetics ; physiology ; Cell Line ; Cell Proliferation ; genetics ; physiology ; Cells, Cultured ; Cellular Senescence ; genetics ; physiology ; Dependovirus ; genetics ; Humans ; Immunohistochemistry ; Intervertebral Disc ; metabolism ; pathology ; Male ; Thymosin ; genetics ; metabolism

Vascular endothelial growth factor (VEGF) is an important regulator of neovascularization. Hypoxia inducible nitric oxide (NO) enhanced the expression of VEGF and thymosin beta-4 (Tbeta4), actin sequestering protein. Here, we investigated whether NO-mediated VEGF expression could be regulated by Tbeta4 expression in HeLa cervical cancer cells. Hypoxia inducible NO production and VEGF expression were reduced by small interference (si) RNA of Tbeta4. Hypoxia response element (HRE)-luciferase activity and VEGF expression were increased by the treatment with N-(beta-D-Glucopyranosyl)-N2-acetyl-S-nitroso-D, L-penicillaminamide (SNAP-1), to generate NO, which was inhibited by the inhibition of Tbeta4 expression with Tbeta4-siRNA. In hypoxic condition, HRE-luciferase activity and VEGF expression were inhibited by the treatment with N(G)-monomethyl-L-arginine (L-NMMA), an inhibitor to nitric oxide synthase (NOS), which is accompanied with a decrease in Tbeta4 expression. VEGF expression inhibited by L-NMMA treatment was restored by the transfection with pCMV-Tbeta4 plasmids for Tbeta4 overexpression. Taken together, these results suggest that Tbeta4 could be a regulator for the expression of VEGF via the maintenance of NOS activity.
Actins ; Anoxia ; Nitric Oxide Synthase ; Nitric Oxide* ; omega-N-Methylarginine ; Plasmids ; Response Elements ; RNA ; Thymosin* ; Transfection ; Uterine Cervical Neoplasms* ; Vascular Endothelial Growth Factor A*

OBJECTIVETo study the effect of acupuncture on the immune function of sepsis patients.

METHODSNinety sepsis patients were assigned to the control group, the thymosin a1 group, and the acupuncture treatment group according to random digit table, 30 patients in each group. Patients in the control group were treated according to the guideline of Surviving Sepsis Campaign (SSC). Patients in the control group received routine treatment. Those in the thymosin alpha1 group additionally received subdermal injection of thymosin alpha1 (1.6 mg), once per day for 6 successive days. Needling at related points such as Zusanli (ST36), Yanglingquan (GB34), Neiguan (PC6), Guanyuan (RN4), and so on, was performed in patients of the acupuncture treatment group, once per day for 6 successive days. T cell subgroups (CD3+, CD4+, CD8+, CD4+ /CD8+) and immunoglobulin levels (IgG, IgA, IgM) were detected. The length of ICU hospital stay, hospital readmission rate, and 28-day mortality were compared among the three groups.

RESULTSAfter six days of treatment, CD3+, CD4+, CD8+, IgG, IgA, IgM, and CD4+ /CD8+ ratio of three groups were all significantly increased (P < 0.01). Of them, CD3+, CD4+, CD8+, IgG, IgA, and IgM increased more significantly in the thymosin alpha1 group and the acupuncture treatment group (P < 0.01). Compared with the control group, the ICU hospitalization length was significantly shortened, the hospital readmission rate and the 28-day mortality were lower in the thymosin alpha1 group and the acupuncture treatment group (P < 0.05, P < 0.01). There was no statistical difference in each index between the thymosin alpha1 group and the acupuncture treatment group (P > 0.05).

CONCLUSIONAcupuncture could adjust the immune function of sepsis patients, improve their immunological indicators and prognoses.

Acupuncture Therapy ; CD4-CD8 Ratio ; Humans ; Length of Stay ; Prognosis ; Sepsis ; diagnosis ; immunology ; therapy ; T-Lymphocyte Subsets ; Thymosin ; analogs & derivatives

Recombinant thymosin beta4 (rTbeta4) has been reported to migrate and promote vascularization, wound-healing, and hair growth in a mouse hindlimb ischemia model of peripheral vascular disease. C57BL/6 mice (11-weeks-old) were anesthetized and an ischemic model was made by cutting the right aorta femoralis. The ischemic group was intraperitoneally administered with saline (300 microL/mouse) and the muscular administration group received rTbeta4 (150 microg in 300 microL of saline) or rTbeta4 (150 microg in 300 microL saline) to the abdominal cavity at 3-day intervals for 21 days. Myoatrophy of the ischemic group was observed compared to the normal control group. Generation of adjacent vessels was carried out in the rTbeta4 administration group compared to the ischemic group. The biopsy results showed significant fibrosis around the muscular undersurface and perimysium in the musculus quadriceps femoris of the ischemic group, whereas partial fibrosis was observed in the perimysium and endomysium in the rTbeta4 administration group. Immunostaining indicated that expression levels of hypoxiainducible factor-1alpha (HIF-1alpha), vascular endothelial growth factor-1 (VEGF-1), and endothelial nitric oxide synthase (eNOS) in the rTbeta4 group were higher than those of the ischemic group. Western blotting showed that expression levels of HIF-1alpha, VEGF-1, and eNOS in the rTbeta4 group were higher than those of the ischemic group. In conclusion, rTbeta4 increases expression levels of HIF-1alpha, VEGF-1, and eNOS, resulting in angiogenesis.
Abdominal Cavity ; Animals ; Aorta ; Biopsy ; Blotting, Western ; Fibrosis ; Hair ; Hindlimb* ; Ischemia* ; Mice* ; Nitric Oxide Synthase Type III ; Peripheral Vascular Diseases ; Quadriceps Muscle ; Thymosin*

OBJECTIVETo evaluate the therapeutic effect of ulinastatin combined with thymosin α1 in the treatment of severe sepsis in rats.

METHODSNormal Wistar rats were subject to cecal ligation and puncture (CLP) to establish models of severe sepsis. The rats were then randomized into 4 groups for treatment with saline (control), ulinastatin, thymosin α1, or the combination of the latter two injected through the caudal vein or subcutaneously at 6, 24, 48 and 72 h after modeling. The mortality rate was recorded daily and the rats were executed at 24, 48, 72 and 96 h after CLP to harvest the heart, liver, spleen, lung, kidney and small intestines for pathological examination. The spleen of the rats were taken for detection of apoptosis of the spleen cells.

RESULTSThe mortality rate of the septic rats in the combined treatment group was decreased significantly (P=0.0325). The control group showed the most severe organ damage, which was moderate in single drug treatment group and the mildest in combined treatment group. Obvious spleen cell apoptosis was found in the control group, and was significantly ameliorated in the combined treatment group[(47.4∓10.9)% vs (39.3∓11.4)%, P=0.0000].

CONCLUSIONCombined treatment with ulinastatin and thymosin α1 can significantly improve the prognosis and ameliorate organ damage and spleen cell apoptosis in rats with sever sepsis.

Animals ; Apoptosis ; Drug Therapy, Combination ; Glycoproteins ; therapeutic use ; Male ; Rats ; Rats, Wistar ; Sepsis ; drug therapy ; pathology ; Spleen ; cytology ; pathology ; Thymosin ; analogs & derivatives ; therapeutic use