ABSTRACT
Aim To explore the mechanism of Huoxue Rongluo reeipe in promoting angiogenesis after ischemic stroke based on the correlation between mir-370-3p and JAK2/STAT3 pathway.Methods Hats were randomly divided into six groups.MCAO/R method was used to establish the model.After seven days of intragastrie administration,the expressions of CD31 ,vWF and vascular endothelial growth factor ( VEGF) in brain tissue were observed by immunofluorescence stai- ning; the expression of JAK2 ,p-jak2,STAT3 and p- STAT3 in brain tissue was detected by Western blot; J the expressions of JAK2,STAT3 mRNA and mir-370- 3p in brain tissue were detected by real-time PCR f RT-PCR) ; the correlation between mir-370-3p and JAK2/STAT3 pathway was analyzed by Pearson correlation ; the expressions of lncma-hl9 and mir-370-3p were detected by real-time quantitative PCR ( RT qPCR) ; the targeting relationship between lncrna-hl9 and mir-370-3p was detected by luciferase reporter assay.Results Huoxue Rongluo decoction could increase the microvessel density and average fluorescence intensity of VEGF,up-regulate JAK2 and STAT3 mR- NA,down-regulate the expression of mir-37()-3p,and promote the expressions of JAK2,p-jak2 ,STAT3 and p- STAT3.Mir-370-3p was highly negatively correlated with JAK2 and STAT3 mRNA respectively,which could be reversed by stattic,an inhibitor of STAT3 SH2 domain.Conclusions Huoxue Rongluo recipe may stimulate angiogenesis after ischemic stroke by down- regulating the expression of mir-370-3p,activating JAK2 / STAT3 pathway and promoting the expression of downstream VEGF,so as to improve the symptoms of neurolo.
ABSTRACT
【Objective】 To investigate the effects and mechanism of STAT3 inhibitor Stattic combined with arsenic trioxide (ATO) on the survival and apoptosis of acute myeloid leukemia (AML) THP1 cells. 【Methods】 CCK8 assay was used to detect the effects of Stattic combined with ATO on cell viability, flow cytometry was used to detect cellular apoptosis and ROS levels, and Caspase 3/7 Glo assay was used to determine Caspase 3/7 activity. qPCR was used to detect mRNA expression levels of GCLM, GCLC and HO-1 genes, and Western blotting was used to detect protein expression levels of P-STAT3, STAT3 and Nrf2. 【Results】 Stattic significantly inhibited the level of phosphorylated STAT3, aggravated the inhibitory effect of ATO on THP1 cell viability, and enhanced the apoptosis and reactive oxygen species (ROS) induced by ATO. Stattic significantly inhibited the expression of ATO-upregulated Nrf2 and the expression of Nrf2 downstream genes including HO-1, GCLM and GCLC. 【Conclusion】 Stattic can enhance the effects of ATO-mediated viability inhibition and apoptosis. The mechanism may be related to the increased ROS via inhibition of Nrf2 and Nrf2 downstream gene expression.
ABSTRACT
Objective@#To elucidate the role of signal transducer and activator of transcription 3 on orthodontic tooth movement, aiming at providing evidence for improving orthodontic bone modeling and remodeling.@*Methods@#Orthodontic tooth movement (OTM) models were established in 8-week-old Wistar rats, which were divided into 2 groups: the control group (tooth movement) and the test group (tooth movement with local injection of STAT3 inhibitor stattic). Rats were sacrificed on day 7 and 14. Micro-CT scanning was conducted to measure bone volume/tissue volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and bone mineral density (BMD), and the amount of tooth movement of the specimens. The mouse preosteoblastic cell line MC3T3-e1 and mononuclear macrophagic leukemia cell line RAW264.7 were cocultured in Transwell® culture plates and divided into the control group (blank) and the test group (STAT3 inhibitor stattic was added). Alkaline phosphatase (ALP) staining and tartrate-resistant acid phosphatase (TRAP) staining were carried out to reveal osteoblastic and osteoclastic differentiation, respectively. qRT-PCR was performed to evaluate mRNA expression levels of the receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) in the MC3T3-e1 cells.@*Results @#Compared with the control group, in the test group, the alveolar bone at the OTM site showed a significant decrease in the BV/TV, Tb.N, Tb.Th, and BMD indexes and a significant increase in Tb.Sp on day 14, while there was no significant difference in the above indexes between the two groups on day 7. The amount of tooth movement was significantly smaller in the test group on day 7 but showed no difference on day 14. ALP staining and TRAP staining revealed weakened osteoblastic and osteoclastic differentiation in the test group. qRT-PCR demonstrated the inhibitor inhibited the mRNA expression of RANKL and OPG and increased the mRNA ratio of RANKL/OPG in osteogenic precursor cells.@*Conclusion@#Suppression of STAT3 activation leads to inhibition of both osteoblastic and osteoclastic differentiation, resulting in lowered tooth movement and catabolic effects on alveolar bone. STAT3 may play an important role in orthodontic bone modeling and bone remodeling.
ABSTRACT
Objective To evaluate the function of regulatory T (Treg) cells in peripheral blood from patients with rheumatoid arthritis (RA),and to explore the possible role of the signal transducer and activator of transcription 3 (STAT3) signaling pathway in Treg cell dysfunction.Methods Totally,60 patients with rheumatoid arthritis,were enrolled into this study.Sixty healthy blood donors served as the control group.Peripheral heparin anticoagulant venous blood was collected from 60 patients with RA and 60 healthy controls respectively.Flow cytometry was used to detect the proportion of Treg cells in peripheral blood.Mixed culture of lymphocytes in vitro was used to detect the proliferation of Treg cells and the inhibition of effective T cells (Tresp) in peripheral blood of RA patients and healthy controls.Phosphorylated STAT3 protein and secretion of pro-inflammatory factors (IFN)-γ,Tumor necrosis factors (TNF)-α and interleukin-17A (IL-17A) in Treg cells were tested by flow cytometry and Quantitative Real-time polymerase chain reaction (qRT-PCR).Treg cells of RA patients were handled with STAT3 pathway inhibitor StatticV to observe the recovery of proliferation and inhibition function and the change of secretion of pro-inflammatory factors.The differences between groups were analyzed by analysis of Variance,and Dunnett's test was used to compare the mean of multiple samples.Results Significant differences were observed in the proportion of Treg cells in peripheral blood between the patient group and the control group [(6.51±0.24)％ vs (2.23±0.18)％,t=4.22,P＜0.05].Compared with controlderived Treg cells,the patient-derived Treg cells showed significantly decreased proli-ferative activity and inhibitory effects on Tresp cells,but increased propòrtion of cells secreting p-STAT3,IFN-γ TNF-α and IL-17A (all P＜0.05).After treated with 50 μg/L Stattic V,a significant increase was observed in the inhibitory effect of patient-derived Treg cells on Tresp cells [inhibition rate:(61.24±4.62)％ vs (28.15±10.37)％,P＜0.05],but a significant decrease in the mRNA expressions of IFN-γ(2-△△Ct:(1.65±0.88) vs (23.32±6.71),P＜0.05],TNF-α (0.85±0.71) vs (4.85±1.53),P＜0.05) and IL-17A (0.57±0.14) vs (3.10±0.62),all P＜0.05] in patientderived Treg cells compared with untreated patient-derived Treg cells.Conclusion The negative regulatory effect of Treg cells on Tresp cells is decreased in patients with rheumatoid arthritis,which may be associated with abnormal activation of the STAT3 signaling pathway,and inhibition of the pathway may restore the function of Treg cells to a certain extent.
ABSTRACT
Objective To investigate the radiosensitization effect of stattic on the xenograft of esophageal squamous cell carcinoma ( ESCC ) ECA109 cells in nude mouse and explore the underlying mechanisms. Methods Male nude mice inoculated subcutaneously with ECA109 cells were used to examine the radiosensitization effect of stattic in vivo. When average volume of tumors was about 150 mm3 , mice were randomly divided into 4 groups:control group, 25 mg/kg stattic alone group, ionizing radiation (IR) (6 Gy) alone group, and 25 mg/kg stattic plus IR group. During the term of treatment, the volume of tumors was measured every 2 days. On 25 days after treatment, the mice were killed and the expressions of pSTAT3, STAT3, HIF-1α and VEGF in ECA109 xenografts were assessed by Western blot and immunohistochemistry analysis. Results The volume of tumor in nude mice was (705. 1 ± 75. 5) mm3 in stattic plus IR group, which was significantly smaller than that in IR group (113. 5 ± 101. 4) mm3 and stattic alone group(1696.5 ±100.6)mm3(t=4.35, 14.14,P<0.0). The inhibition rate was (66.1 ± 3. 2)％ in stattic plus IR group. The expression levels of pSTAT3, HIF-1α and VEGF were obviously decreased in the stattic plus IR group compared with other groups (t=17. 07, 5. 05, 3. 54, P<0. 05). Conclusions Stattic play a radiosensitization role in the xenograft of esophageal carcinoma in nude mice probably by inhibiting the signaling pathways of pSTAT3, HIF-1α and VEGF.
ABSTRACT
Objective To explore the role of signal transducer and activator of transcription 3 (STAT3) phosphorylation in the pathogenesis of Behcet's disease (BD),and to investigate the association between STAT3 phosphorylation and disease activity in BD patients.Methods Peripheral blood mononuclear cell (PBMC) was isolated from 15 mL peripheral boood of 10 active BD patients (BD-A),10 BD patients in remission (BD-R) and 10 healthy controls (HC) respectively.The blockade of STAT3 phosphorylation was performed by Stattic.The PBMC was divided into Stattic subgroup (treated with 2.5 μmol/L stattic and 1 640 medium,5 mL) and blank control subgroup (treated with 5 mL 1 640 medium),respectively.The protein levels of phosphorylated STAT3 (pSTAT3) and STAT3 were examined by flow cytometry and Western blot.The protein and mRNA levels of TNF-α,IFN-γ and IL-17 were tested by RT-PCR and ELISA.Two-way ANOVA and Bonferroni post hoc test were used to analyze the results.Results Compared with HC,the BD patients showed higher protein levels of pSTAT3 and STAT3,and higher protein and mRNA levels of TNF-α,IFN-γ and IL-17;compared with blank control subgroup,the protein levels of pSTAT3 and STAT3,and protein and mRNA levels of TNF-α,IFN-γ and IL-17 decreased in Stattic subgroup.In the BD-A group,the protein level of pSTAT3,and protein and mRNA levels of TNF-α,IFN-γ and IL-17 were significantly higher than those in the BD-R group.Conclusions An increased activation of the STAT3 pathway may contribute to the pathogenesis of BD and relate to disease activity in BD patients by inducing TH1 and Th17 cells activation.
ABSTRACT
Objective To investigate the radiosensitivity of ESCC by signal transducer and activator of transcription 3 (STAT3) inhibitor Stattic,since the radioresistance of esophageal squamous cell carcinoma(ESCC) remains an obstacle for the effective radiotherapy of ESCC.Methods ECA109 cell line was exposed to hypoxia and treated with Stattic or radiation,alone or in combination.Cell proliferation,colony formation,apoptosis,and double-stranded DNA breaks (DSBs) were examined.The levels of STAT3,pSTAT3,hypoxiainduciblefactor1α (HIF-1α),and vascular endothelial growthfactor (VEGF) in ESCC cells were detected by Western blot.Results Stattic efficiently inhibited the proliferation of ECA109 cells in time-dependent and dose-dependent fashions with an IC50 of 5.499 μmol/ L.Clonogenic survival assay showed that stattic (1.0 μmol/L) sensitized ECA109 cells to ionizing radiation and its SERDo was 1.20 (in normoxia) or 1.28 (in hypoxia).Under hypoxic condition,stattic combined with IR disrupted the repair of DSBs and increased the apoptosis(t =7.33,P ＜ 0.05) in ESCC cells compared to that of radiation treatment alone.Moreover,Western blot assay showed that stattic inhibited STAT3 activation and downregulated the expression level of pSTAT3 and HIF-1α and VEGF.Conclusions Stattic confers radiosensitivity in ESCC cells in vitro and is a potential adjuvant for the radiotherapy of ESCC in the clinical setting.
ABSTRACT
Objective To evaluate the function of regulatory T (Treg)cells in peripheral blood from patients with psoriasis, and to explore the possible role of the STAT3 signaling pathway in Treg cell dysfunction. Methods Totally, 81 patients with psoriasis vulgaris, who all presented with chronic plaques and had a psoriasis area and severity index (PASI)score of 10 - 30, were enrolled into this study. Forty-six healthy blood donors served as the control group. Venous blood samples were collected from these subjects followed by isolation of Treg cells and responder T (Tresp)cells. Flow cytometry was performed to determine the proportion of Treg cells in peripheral blood as well as that of cells secreting phosphorylated-STAT3(p-STAT3), interferon γ(IFN-γ), tumor necrosis factor α(TNF-α)and interleukin 17(IL-17)in Treg cells, and quantitative real-time PCR (qRT-PCR)to measure the expression levels of IFN-γ, TNF-α and IL-17 mRNAs in Treg cells. Some Treg cells and Tresp cells were cultured in vitro alone or in combination, and flow cytometry was conducted to estimate cellular proliferative activity on day 7 after stimulation with IL-2. Some patient-derived Treg cells were classified into several groups to be cultured alone or in combination with Tresp cells with or without the presence of the STAT3 pathway inhibitor, Stattic V (10 or 50 μg/L), for 7 days. Subsequently, flow cytometry was performed to evaluate the proliferative activity of Tresp cells, and qRT-PCR to measure the expression levels of IFN-γ, TNF-α and IL-17 mRNAs in Treg cells. Results No significant differences were observed in the proportion of Treg cells in peripheral blood between the patient group and control group (6.437% ± 0.186% vs. 6.812% ± 0.241%, t = 1.224, P >0.05). Compared with control-derived Treg cells, the patient-derived Treg cells showed significantly decreased proliferative activity and inhibitory effects on Tresp cells, but increased proportion of cells secreting p-STAT3, IFN-γ, TNF-α and IL-17 (all P < 0.05). After the treatment with 50 μg/L Stattic V, a significant increase was observed in the inhibitory effect of patient-derived Treg cells on Tresp cells (inhibition rate: 61.670% ± 4.640% vs. 28.820% ± 11.490%, P < 0.05), but a significant decrease in the mRNA expressions of IFN-γ (2-△△C t: 1.654 ± 0.879 vs. 23.350 ± 6.721, P <0.05), TNF-α(0.850 ± 0.705 vs. 4.847 ± 1.525, P < 0.05)and IL-17(0.572 ± 0.135 vs. 3.095 ± 0.650, all P < 0.05)in patient-derived Treg cells compared with untreated patient-derived Treg cells. Conclusions The negative regulatory effect of Treg cells on Tresp cells is decreased in patients with psoriasis, which may be associated with abnormal activation of the STAT3 signaling pathway, and inhibition of the pathway may restore the function of Treg cells to a certain extent.
ABSTRACT
Objective To study the effects and preliminary mechanism of Stattic (Y705),an inhibitor of the signal transducer and activator of transcription-3 (STAT3),on the growth,migration,invasion and radiosensitization of hepatocellular carcinoma cell line Bel-7402.Methods Bel-7402 cells were divided to four groups:blank control group,Stattic treatment group,radiation group,and Stattic combined with radiation group.The cell growth and proliferation were detected by using CCK8 kit.The influence of Stattic on radiation sensitivity of Bel-7402 cells was determined by clone formation assay.The cell migration and invasion ability were tested by scratch migration assay and transwell assay,respectively.The protein expressions of STAT3,p-STAT3,Bax,Bcl-2,Caspase-3,Cleaved Caspase-3,MMP-2 and MMP-9 were quantified by Western blotting assay.Results Stattic significantly inhibited the growth of human hepatocellular carcinoma Bel-7402 cells with a dose-depended manner.The IC50 of Stattic after 48 h treatment was 2.5 μ mol/L.When 1.0 μmol/L Stattic was combined with 8 Gy X-rays,there was a synergistic effect in inhibition of cell proliferation with a inhibition rate of (15.00 ± 1.87) ％ (F =63.30,P ＜ 0.05).Scratch migration assay and transwell invasion assay showed that the migration and invasion abilities of the combination group were significantly reduced.In addition,compared with the radiation group,the SF2,D0and Dq values obtained from survival curve were decreased (t =4.20,6.92,9.32,P ＜0.05),the protein expressions of p-STAT3,MMP-2,MMP-9 were reduced (t =5.32,6.02,13.26,P ＜0.05),the protein expressions of Bax and Cleaved Caspase-3 were increased in the combination group(t =-7.82,-14.09,P ＜ 0.05),meanwhile the protein expressions of Bcl-2 was decreased (t =18.43,P ＜ 0.05).When the concentration of Stattic was 0.5 μmol/L,the radiation sensitization ratio at 2 Gy (SERSF2) was 1.22.Conclusions By inhibiting the activation of the p-STAT3 in Bel-7402 cells,stattic could induce cell apoptosis and increase the radiosensitivity,down regulate MMP-2 and MMP-9 and thereby reduce the invasion and migration of tumor cells.