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Objective To investigate the protective effect and mechanisms of thymosin beta 4 (Tß4) on oxygen-glucose deprivation/reoxygenation (O G D / R) injury in rat cortical neurons. Methods Primary cultured cortical neurons were isolated and identified; Ischemia-reperfusion injury model of rat cortical neurons was established (oxygen glucose deprivation 6 h, reoxygenation 12 h) by OGD / R. The rats were divided into the control group,the model group and the treatment group (addition of thymosinß4 2 h before modeling); The cell counting Kit-8 (CCK8) was used to determine the optimal concentration of Tß4. Flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) were used to detect apoptosis. Western blotting was used to detect the expression of 78 000 glucose-regulated protein 78 (GRP 78), CCAAT/enhancer binding protein-homologous protein (CHOP), B-cell lymphoma-2 (Bel-2) and Bax. Expression levels were compared among groups. One-way analysis of variance was used to compare the normally distributed measurement data among three groups with the SPSS19. 0 software. Results Cortical neurons were separated correctly. The optimal concentration of Tß4 was 10 (ig/L. Comparing the model group with the control group, the survival rate of cortical neurons decreased significantly (P = 0. 002), the apoptotic rate increased significantly (P < 0. 01), the expression of GRP78,CHOP and Bax was up-regulated significantly (P values were 0. 034,0 and 0. 045 .respectively),and the expression of Bcl-2 was reduced significantly (P = 0. 006). Comparing the treatment group(10 p.g/L exogenous Tß4) with the model group, cell viability increased significantly (P = 0. 008), the apoptotic rate decreased significantly (P = 0. 002),the expression of GRP78,CHOP and Bax decreased significantly (P values were 0. 032,0. 027 and 0. 019, respectively),and the expression of Bcl-2 increased significantly (P = 0.028) .The differences were statistically significant. Conclusions Tß4 inhibits OGD/R-induced endoplasmic reticulum stress-dependent apoptosis. These results provide a theoretical basis for the application of Tß4 in the treatment of cerebral ischemia-reperfusion injury.
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Objective: To explore the changes of levels of endogenous N acetyl seryl aspartyl lysylproline (AcSDKP) and its regulatory factors in liver tissue of rats with liver fibrosis induced by bile duct ligation, and to elucidate the effect of endogenous AcSDKP in the pathologic process of liver fibrosis. Methods: Forty-five healthy male rats were randomly divided into model group (the liver fibrosis rat models were set up by bile duct ligation), blocking group (the liver fibrosis rat models were treated with angiotensin-converting enzyme inhibitor before) and control group (the rats received laparotomy but not bile duct ligation); each group had 15 rats. The serum and liver tissue of the rats in various groups were gained at the 1st week, 2nd week and 4th week. The differences in the indexes of liver function, the liver fibrosis degrees, and the AcSDKP levels in liver tissue of the rats in various groups were analyzed. The levels of thymosin beta 4 (Tβ4), prolyl oligopeptidase (POP) and angiotensin converting enzyme (ACE) in liver tissue of the rats in various groups were detected by Western blotting. Results: The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (TB) of the rats in model group were higher than those in other two groups from the 1st week (P0. 05). The ALB and the AcSDKP level in liver tissue of the rats in model group were lower than those in other two groups from the 2nd week (P0. 05). The levels procollagen type IE (PCIE), collagen type IV (CKO and hyaluronic acid (HA) in three groups had no differences at the 1st week (P>0. 05). The levels of PCIII, CIV, and HA of the rats in model group were higher than those in other two groups from the 2nd week (P 0. 05). The levels of Tβ4 and ACE in liver tissue of the rats in model group were higher than those in other two groups from the 2nd week (P 0. 05). The liver tissue of the rats in model group was basically normal at the 1st week, showed the focal necrosis at the 2nd week, and showed the flake necrosis at the 4th week. The liver tissue of the rats in blocking group and control group were basically normal at all time points. Conclusion: The endogenous AcSDKP level in the liver fibrosis rats induced by bile duct ligation is down-regulated through the axis of Tβ4-POP-AcsDKP.
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Background Studies showed that inflammation is associated with the pathogenesis and development of dry eyes,and tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) are key inflammatory factors.Thymosin β4 (Tβ4) plays a promoting effect on the migration of epithelial cells and anti-inflammatory action.However,the influences of Tβ4 on the repair of ocular surface in dry eyes are unelucidated.Objective This study was to investigate the regulation of Tβ4 to the expressions of TNF-o and IFN-γand its effect on the recovery of ocular surface in rat dry eye models.Methods The dry eye models were induced by topically administered of benzalkonium chloride (BAC) for consecutive 7 days in the left eyes of 50 SPF male SD rats,and 36 successful models were used in the experiment.Tβ4 solution (9 μg/ml),recombinant human epithelial growth factor (rhEGF)and sterile PBS at 5 μl was topically administered three times for consecutive 7 days in the Tβ4 group,rhEGF group and PBS group,and no drug was used in the model control group.The normal right eyes of rats served as the normal control group.The break-up time of tear film (BUT),corneal fluorescein staining score and Schirmer Ⅰ test (S I t)were examined and evaluated in the rats on the seventh day after administration of drugs.Then the rats were sacrificed by excessive anesthesia and the sections of the ocular surface were prepared.The morphology of the specimens was examined by hematoxylin and eosin staining,and the number of conjunctival gobelt cells was counted by periodic acidschiff staining.The ultrastructure of the corneal and conjunctival cells was examined under the transmission electron microscope.The expressions of TNF-α mRNA and IFN-γ mRNA and their proteins in conjunctiva tissue were quantified by quantitative real-time PCR and Western blot,respectively.The use and care of the animals followed by Regulation for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.Results The BUT was (10.42±0.66),(7.46±0.49),(8.71±0.50),(9.59±0.35) and (8.63± 0.68) seconds in the normal control group,model control group,rhEGF group,Tβ4 group and BUT group,showing a significant difference among the groups (F =5.65,P =0.00),and the BUT was evidently shortened in the model control group compared with the normal control group,while the BUT was significantly extended in the rhEGF group and Tβ4 group in comparison with the model control group (all at P＜0.05).No significant differences were found in the corneal fluorescence score and S I t among the groups (F =0.42,P =0.79;F =136.77,P =0.00).The corneal and conjunctival epithelium defect and corneal stromal edema were seen in the model control group,and the proliferation of the epithelial cells were found in the rhEGF group and Tβ4 group,with the irregulated arrangement of the cells.A considerable difference was seen in the number of conjunetival goblet cells among the groups (F=3.16,P =0.04),and the number of eonjunctival goblet cells in the rhEGF group and model control group was significantly less than that in the normal control group (all at P＜0.05),and no statistically significant difference was seen between Tβ4 group and normal control group (P ＞ 0.05).The swelling,mergence,crispation,rupture and decrease of the microvilli and micro fold were found in the model control group,and the repair of the cell microvilli was seen in the Tβ4 group.The expressions of the TNF-α mRNA,IFN-γ mRNA and their proteins in the conjunctiva were significantly different among the groups (F =43.08,371.69,34.27,43.52,all at P =0.00),the expressions of the inflammatory factors were significantly higher in the model control group compared with the normal control group,and these expressions were evidently lower in the Tβ4 group in comparison with the model control group and rhEGF group (all at P＜0.05).Conclusions The topical administration of Tβ4 solution can promote the repair of ocular surface by down-regulating the expression of TNF-α and IFN-γ in conjunctiva and stablize the tear film in rat dry eyes.
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Objective To investigate the roles of cytokeratin 18 (CK18) M30 and M65,thymosin beta 4 (Tβ4) and tumor necrosis factor (TNF)-α in hepatic steatosis and development of inflammatory and fibrosis in chronic hepatitis B (CHB) patients with nonalcoholic fatty liver disease (NAFLD).Methods A total of 46 CHB patients with NAFLD and 42 CHB patients were collected.Serum CK-18 M30,M65,Tβ4 and TNF-α levels were measured by enzyme linked immunosorbent assay (ELISA) in two groups.The associations between inflammatory factors levels and biochemical or pathological indicators were analyzed.The statistical analysis was conducted by t test and chi square test of two independent samples.The correlation analysis was performed by Pearson and Logistic regression analysis.Results The mean serum CK 18 M30 level in CHB with NAFLD group was (614.48±471.43) U/L,which was significantly higher than that in CHB group (374.50±231.04) U/L (t=2.988,P＜0.01).The mean levels of CK18 M65,Tβ4 and TNF-α in CHB with NAFLD group were (369.41±262.21) U/L,(0.80±0.32) mg/L and (54.87±20.36) ng/L,respectively,and those in CHB group were (296.50±231.44) U/L,(0.68±0.30) mg/L and (51.88± 20.60) ng/L,respectively.There were no difference between CHB with NAFLD group and CHB group (t=1.378,1.810 and 0.685,respectively,all P＞0.05).In CHB with NAFLD patients,the CK-18 M30 level was positively correlated with alanine aminotransferase,triglyceride,fasting blood glucose,histology inflammation score,fibrosis score and steatosis (r=0.507,0.456,0.384,0.551,0.458 and 0.457,respectively,all P＜0.01).Tβ4 level was negatively correlated with inflammation and fibrosis score (r=0.371 and-0.308,respectively,P＜0.05).TNF-α level was positively correlated with inflammation score and steatosis (r=0.570 and 0.441,respectively,P＜0.01).CK-18 M30,Tβ4 and TNF-α were independent predictors of CHB combined with NAFLD,progressive inflammatory fibrosis and severe steatosis.Conclusions Serum CK-18 M30,Tβ4 and TNF-α levels are associated with hepatic steatosis,development of inflammation and fibrosis in CHB with NAFLD patients.
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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.
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Actinas , Hipoxia , Óxido Nítrico Sintasa , Óxido Nítrico , omega-N-Metilarginina , Plásmidos , Elementos de Respuesta , ARN , Timosina , Transfección , Neoplasias del Cuello Uterino , Factor A de Crecimiento Endotelial VascularRESUMEN
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.
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Animales , Ratones , Cavidad Abdominal , Aorta , Biopsia , Western Blotting , Fibrosis , Cabello , Miembro Posterior , Isquemia , Óxido Nítrico Sintasa de Tipo III , Enfermedades Vasculares Periféricas , Músculo Cuádriceps , TimosinaRESUMEN
Objective To investigate the expression of thymosin beta4 ( Tβ4 ) in the liver tissues of children with hepatoblastoma ( HB) , and further study the function of Tβ4 in HB metastasis in vitro. Methods Immunohisto-chemistry (IHC) was used to determine expression of Tβ4, E-cadherin andβ-catenin in liver tissues from 19 chil-dren with HB,and further to analyze its function in metastasis of HB cells. Results The positive rate of Tβ4 ( P<0.01 ) in HB liver tissues was significantly higher than it in the adjacent tissues. The positive rates of Tβ4 and nu-clearβ-catenin in HB with lymphnode metastasis were significantly higher than in HB without lymphnode metastasis ( P <0.01 ) . While expression of E-cadherin in HB with lymphnode metastasis was lower than in HB without lymphnode metastasis ( P<0.01 ) . Scratch-wound assay showed that HepG2 cells with Tβ4 knockdown had signifi-cantly lower metastatic ability (P<0.05). Conclusion Tβ4 plays an important role in HB metastasis, suggesting it is a potential target for HB metastasis.
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OBJECTIVE: The aim of this study was to determine thymosin beta4 expression in epithelial ovarian cancer compared to normal ovarian tissue. METHODS: Normal and pathologic ovarian tissues were obtained from healthy women (n=18), and from patients with ovarian cancer (n=27). The expression of thymosin beta4 mRNA was examined by quantitative competitive polymerase chain reaction (QC PCR). Thymosin beta4 mRNA expression was examined with angiopoietic factors such as vascular endothelial growth factor, angiopoietin-1 and 2. RESULTS: The expression of thymosin beta4 mRNA in epithelial ovarian cancer was higher than that in the normal ovary (p<0.05). Thymosin beta4 mRNA expression was not correlated with ovarian cancer stages, pathologic types, preoperative CA125 levels, or metastasis to lymph nodes but was correlated with the expression vascular endothelial growth factor and angiopoietin-2 (p<0.05). CONCLUSIONS: Our results suggest that overexpression of thymosin beta4 mRNA may be a biologic marker to differentiate epithelial ovarian cancer from normal ovary and it may play a role in angiogenesis of epithelial ovarian cancer.
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Femenino , Humanos , Inductores de la Angiogénesis , Angiopoyetina 1 , Angiopoyetina 2 , Biomarcadores , Ganglios Linfáticos , Metástasis de la Neoplasia , Neoplasias Glandulares y Epiteliales , Neoplasias Ováricas , Ovario , Reacción en Cadena de la Polimerasa , ARN Mensajero , Timosina , Factor A de Crecimiento Endotelial VascularRESUMEN
OBJECTIVE: The aim of this study was to compare the expression of annexin I and thymosin beta4 in invasive cervical cancer including normal cervix and CIN. METHODS: In Ewha Womans University Mokdong Hospital, normal cervical tissues were obtained from healthy women (n=10), from patients with cervical intraepithelial neoplasia (CIN, n=10) and from patients with cervical cancer (n=33). The expressions of annexin I and thymosin beta4 mRNA and protein were examined by quantitative competitive-PCR and by western blot analysis. The expressions of annexin I and thymosin beta4 protein were measured by western blot analysis with thymosin beta4 peptides non treated and treated SiHa cells. RESULTS: The expression of thymosin beta4 mRNA and protein in cervical cancer were higher than that in normal cervix (p0.05). But thymosin beta4 and annexin I protein expressions were increased according to the cancer stage. The expression of annexin I was slightly higher in thymosin beta4 treated SiHa cells than that in nontreated SiHa cells. CONCLUSIONS: Our results suggest that overexpression of thymosin beta4 and annexin I may play roles in progression of invasive cervical cancer. Thymosin beta4 upregulates expression of annexin I in invasive cervical cancer. Therefore, thymosin beta4 and annexin I may be biological markers in detecting the progression of invasive cervical cancer, and their interaction is important in invasive cervical cancer.
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Femenino , Humanos , Anexina A1 , Biomarcadores , Western Blotting , Displasia del Cuello del Útero , Cuello del Útero , Péptidos , ARN Mensajero , Timosina , Neoplasias del Cuello UterinoRESUMEN
Background : Thymosin- 4 is an actin-sequestering protein that regulates actin polymerization. It is known to be associated with cell migration, angiogenesis and wound healing, as well as with tumor metastasis. Methods : We immunohistochemically evaluated the thymosin- 4 expression in gastric adenocarcinoma specimens, the relationship between this protein and the pathologic features and other tumor-related proteins, and its influence on the patient outcome. Results : We demonstrated that 40 specimens (26.3%) of 152 gastric adenocarcinomas showed positivity for thymosin- 4. The thymosin- 4 expression was statistically associated with advanced tumor stage (p=0.010), the nodal stage (p=0.029), the TNM stage (p=0.008), and the presence of lymphovascular invasion (p=0.009). The thymosin- 4 protein expression was closely related to the positivity for VEGF (p=0.000), c-Myc (p=0.007), and cyclin D1 (p=0.005), but it was not associated with the E-cadherin (p=0.861) or -catenin (p=0.640) expressions. The median survival and disease relapse time of patients showing thymosin-4 immunoreactivity were statistically shorter than those of patients without expression. Multivariate analysis showed that the tumor stage (p=0.003), nodal stage (p=0.005), thymosin- 4 expression (p=0.019) and Lauren's classification (p=0.037) were statistically important prognostic factors for gastric adenocarcinomas. Conclusions : The thymosin- 4 expression might be associated with disease progression of gastric adenocarcinomas and it should be regarded as an important prognostic factor for estimating patient survival.