STAT3 activation of tumor-associated macrophages is associated with cytokines of tumor microenvironment and prognostic factors in breast cancer

Objective:The aim of this study was to investigate the correlation of STAT3 activation of tumor-associated macrophages(TAMs) and local cytokines(IL-1β,TNF-α,TGF-β and IL-12) and prognostic factors in breast cancer.Methods:TAMs in 50 primary breast cancers and macrophages in 15 normal breasts were examined by immunohistochemistry.And STAT3 DNA-binding activity of TAMs in 33/50 primary breast cancers was measured by transcription factor DNA-binding ELISA.In addition,the concentrations of IL-1β,TNF-α,TGF-β and IL-12 were measured in the 33 primary breast cancers extracts by ELISA.The correlation between STAT3 activity of TAMs and concentrations of IL-1β,TNF-α,TGF-β and IL-12 were analyzed.The correlation between STAT3 activity of TAMs and conventional clinicopathologic parameters were also evaluated.Results:The macrophages density showed a significant increase in primary breast cancers compared to normal breasts.STAT3 DNA-binding activity of TAMs in breast cancer was significantly higher than that of monocytes/macrophages from peripheral blood of the patients.Furthermore,STAT3 activity of TAMs was correlated significantly with the levels of IL-1β,TNF-α and TGF-β in breast cancer tissues.But an inverse association was observed between STAT3 activity of TAMs and IL-12.In addition,STAT3 activity of TAMs was higher in high histological type than in low histological type,and STAT3 activity of TAMs was higher in CerBb-2 positive than CerBb-2 negative.Conclusion:STAT3 activation of TAMs may be associate with increasing of IL-1β,TNF-α and TGF-β and decreasing of IL-12 in breast cancer.STAT3 activation of TAMs may also be correlated with histological grade and CerBb-2 status of breast cancer.

Tyrosine 23 Phosphorylation of Annexin A2 Promotes Proliferation,Invasion,and Stat3 Phosphorylation in the Nucleus of Human Breast Cancer SK-BR-3 Cells

Objective To investigate the role of tyrosine 23 （Tyr23） phosphorylation of Annexin A2 （Anxa2） in regulating the proliferation and invasion of human breast cancer SK-BR-3 cells. Methods A panel of lentivirus plasmids expressing Anxa2-wide type （Ana2-WT）, Anxa2-Y23A, and Anxa2-Y23D was generated and infected with SK-BR-3 cells. The monoclonal strains were screened. The expression of Anxa2-WT, Anxa2-Y23A, and Anxa2-Y23D was determined by Western blot analysis. The ability of the cells to proliferate was detected through an MTT [3-（4,5-Dimethylthiazol- 2-yl）-2,5-diphenyltetrazolium bromide] test. Boyden chamber assays were employed to examine migration and invasion abilities. The interaction between Anxa2 and Stat3 was analyzed by immunoprecipitation analyses. Nucleoprotein and cytosolic protein were extracted from SK-BR-3, Anxa2-WT, Anxa2-Y23A, and Anxa2-Y23D cells to analyze the expression and localization of Stat3 phosphorylation. Results The monoclonal strains constitutively expressing Anxa2-WT, Anxa2-Y23A, and Anxa2-Y23D were screened. Both Anxa2-WT and Anxa2-Y23D enhanced the proliferation, migration and invasion abilities of SK-BR-3 cells （P〈0.05）. Immunoprecipitation analysis revealed that Anxa2 and Stat3 interacted with each other, and the expression of Stat3 phosphorylation in the nucleus was enhanced by Anxa2-Y23D. Conclusions Tyr23 phosphorylation of Anxa2 promotes the proliferation and invasion of human breast cancer SK-BR-3 cells and the phosphorylation of Stat3 in the nucleus.

Danhongqing formula alleviates cholestatic liver fibrosis by downregulating long non-coding RNA H19 derived from cholangiocytes and inhibiting hepatic stellate cell activation

Objective:This study explores the mechanism of action of Danhongqing formula(DHQ),a compoundbased Chinese medicine formula,in the treatment of cholestatic liver fibrosis.Methods:In vivo experiments were conducted using 8-week-old multidrug resistance protein 2 knockout(Mdr2-/-)mice as an animal model of cholestatic liver fibrosis.DHQ was administered orally for 8 weeks,and its impact on cholestatic liver fibrosis was evaluated by assessing liver function,liver histopathology,and the expression of liver fibrosis-related proteins.Real-time polymerase chain reaction,Western blot,immunohistochemistry and other methods were used to observe the effects of DHQ on long non-coding RNA H19(H19)and signal transducer and activator of transcription 3(STAT3)phosphorylation in the liver tissue of Mdr2-/-mice.In addition,cholangiocytes and hepatic stellate cells(HSCs)were cultured in vitro to measure the effects of bile acids on cholangiocyte injury and H19 expression.Cholangiocytes overexpressing H19 were constructed,and a conditioned medium containing H19 was collected to measure its effects on STAT3 protein expression and cell activation.The intervention effect of DHQ on these processes was also investigated.HSCs overexpressing H19 were constructed to measure the impact of H19 on cell activation and assess the intervention effect of DHQ.Results:DHQ alleviated liver injury,ductular reaction,and fibrosis in Mdr2-/-mice,and inhibited H19expression,STAT3 expression and STAT3 phosphorylation.This formula also reduced hydrophobic bile acid-induced cholangiocyte injury and the upregulation of H19,inhibited the activation of HSCs induced by cholangiocyte-derived conditioned medium,and decreased the expression of activation markers in HSCs.The overexpression of H19 in a human HSC line confirmed that H19 promoted STAT3 phosphorylation and HSC activation,and DHQ was able to successfully inhibit these effects.Conclusion:DHQ effectively alleviated spontaneous cholestatic liver fibrosis in Mdr2-/-mice by inhibiting H19 upregulation in cholangiocytes and preventing the inhibition of STAT3 phosphorylation in HSC,thereby suppressing cell activation.

Effect of RNA Interference Targeting STAT3 Gene Combined with Ultrasonic Irradiation and SonoVue Microbubbles on Proliferation and Apoptosis in Keratinocytes of Psoriatic Lesions

Background： Signal transducer and activator of transcription 3 （STAT3） was strongly expressed and activated in psoriatic keratinocytes （KCs） and correlated with the severity of psoriasis. The study aimed to investigate the effects of STAT3 small interfering RNA （siRNA） combined with ultrasonic irradiation and SonoVue microbubbles on the proliferation and apoptosis in KCs of psoriatic lesions and the relative mechanisms. Methods： Psoriatic KCs were transfected under four experimental conditions： （1） STA T3 siRNA carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles （LUS group）; （2） STA T3 siRNA only carried by Lipofectamine 3000 （L group）; （3） the negative control ofsiRNA carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles （siRNA-NC）; （4） not treated as Blank. Cell Counting Kit-8 assay was used to evaluate the cell proliferation. Cell cycle analysis was detected with cycle test Plus DNA reagent kit associated with flow cytometer. FITC Annexin V apoptosis detection kit associated with flow cytometer was applied for apoptosis analysis. Fluo calcium indicator associated with flow cytometer was used to analyze intracellular free calcium concentration （[Ca^2＋）. The expressions of cyclin D1 and Bcl-xL were detected both at the mRNA level by real-time reverse transcription-polymerase chain reaction （RT-PCR） and at the protein level by Western blotting. The obtained data were statistically evaluated by two-way analysis of variance. Results： STAT3 siRNA inhibited the growth of KCs in a time-dependent manner showing the highest proliferation inhibition in LUS group with proliferation ratio of 45.38% ± 5.85% at 72h （P 〈 0.05 vs. L group, siRNA-NC, or Blank）. STAT3 siRNA induced an altered cell cycle distribution of KCs showing the highest increases in G2/M-phase population up to 18.06% ± 0.36% in LUS group （P 〈 0.05 vs. L group, siRNA-NC, or Blank）. ST,4 T3 siRNA induced late apoptosis of KCs with the highest late apoptosis percentage of 22.87% ± 1.28% in LUS group （P 〈 0.05 vs. L group, siRNA-NC, or Blank）. STAT3 siRNA induced the elevation of [Ca^2＋] of KCs with the highest calcium fluorescence intensity mean of 1213.67 ±60.51 in LUS group （P 〈 0.05 vs. L group, siRNA-NC, or Blank）. STA T3 siRNA induced the downregulation ofcyclin D 1 and Bcl-xL expressions of KCs at mRNA and protein levels with the lowest expressions in LUS group with cyclin D1 expression of 51.81% ± 9.58% and 70.17% ± 4.22% at mRNA level and at protein level, respectively, and with Bcl-xL expression of 37.58%±4.92% and 64.06% ±7.78% at mRNA level and at protein level, respectively （P 〈 0.05 vs. L group, siRNA-NC, or Blank）. Conclusions： STA T3 siRNA inhibited the growth and induced the apoptosis in psoriatic KCs likely partly through altering cell cycle distribution, elevating [Ca^2＋], and downregulating cyclin DI and Bcl-xL expressions. Silencing the target gene STAT3 in psoriatic KCs with siRNA combined with ultrasonic irradiation and microbubbles would contribute to a significant innovation as a new clinical therapy for psoriasis.

Qingnaoyizhi decoction suppresses the formation of glial fibrillary acidic protein-positive cells in cultured neural stem cells by inhibiting the Janus kinase 2/signal transducer and activator of transcription 3 signaling pathway

OBJECTIVE: Inactivation of the Janus kinase 2(JAK2)/signal transducer and activator of transcription 3(STAT3) signaling axis plays a crucial role in determining the fate of neural stem cells(NSCs).Qingnaoyizhi decoction(QNYZD) has been used for the treatment of vascular dementia and has shown to improve synaptic remodeling. The aim of this study was to evaluate the effect of cerebrospinal fluid(CSF) containing QNYZD(CSF-QNYZD) on the differentiation of cultured NSCs and the involvement of the JAK2/STAT3 pathway.METHODS: The protein expression levels of glial fibrillary acidic protein(GFAP), tubulin, drosophila mothers against decapentaplegic protein(SMAD-1), STAT3, and phosphorylated-STAT3 were detected by western immunoblot analysis in the groups: control, CSF, JAK/STAT inhibitor(AG490),CSF-QNYZD, and CSF-XDZ(CSF-Xidezhen). The differentiation of NSCs was determined by immunofluorescence staining. The proliferation of NSCs was measured using the Cell Counting Kit-8 proliferation assay.RESULTS: Compared with the control group,CSF-QNYZD and AG490 significantly increased the number and expression of tubulin-positive cells, reduced the number and expression of GFAP-positive cells, and down-regulated the expression of p-STAT3. However, CSF-QNYZD also decreased the expression of SMAD-1 and STAT3.CONCLUSION: Enhanced neuronal differentiation may be associated with the down-regulation of glial differentiation instead of promoting proliferationin treated NSCs. Furthermore, QNYZD may play a direct role in suppressing the formation of GFAP-positive cells and enhancing neuronal differentiation by inhibiting JAK2/STAT3 activation. Overall, these results provide insights into the possible mechanism underlying QNYZD-mediated neurogenesis.

Expression of interleukin-12 and its signaling molecules in peripheral blood mononuclear cells in systemic lupus erythematosus patients

Objective To determine the in vitro expression of interleukin-12 (IL-12) and its effect on signal transducers and activators of transcription (STAT) signaling molecules in peripheral blood mononuclear cells (PBMCs) in patients with systemic lupus erythematosus (SLE).Methods Peripheral blood mononuclear cells in 39 patients with definite systemic lupus erythematosus and 11 healthy volunteers were collected. Expression of IL-12 P40mRNA in PBMCs was determined with reverse transcription-polymerase chain reaction (RT-PCR). Quantity of IL-12 protein supernatant was measured by enzyme-linked immunosorbent assay (ELISA). The levels of phosphorylated STAT3 and STAT4 signaling molecules in PBMCs were detected by immunoblot. Results Levels of IL-12 protein and mRNA expression in patients with active or inactive SLE were significantly higher than those in controls. Phytohemagglutinin (PHA) may promote the expression of IL-12. IL-12 alone induced the phosphorylation of STAT3 and STAT4 in PBMCs from patients with SLE, especially in active SLE. However it had no obvious effect on normal PBMCs. Phosphorylated STAT3 and STAT4 might be observed in normal PBMCs treated with IL-12 plus PHA.Conclusion IL-12 is produced aberrantly in patients with SLE. IL-12 might exert its biological role in SLE via the aberrantly phosphorylated STAT3 and STAT4 signaling molecules.

Effects of peroxisome proliferator-activated receptor-β/δ on sepsis induced acute lung injury

Background Acute lung injury （ALI） and acute respiratory distress syndrome （ARDS） are the first steps in the development of multiple organ failure induced by sepsis.A systemic excessive inflammatory reaction is currently the accepted mechanism of the pathogenesis of sepsis.Several studies have suggested a protective role of the peroxisome proliferator activated receptor-β/δ （PPAR-β/δ） in related inflammatory diseases.But the role of PPARβ/δ in ALI remains uncertain.The aim of this study was to investigate the role and possible mechanism of PPARβ/δ in ALI induced by sepsis.Methods Cecal ligation and puncture （CLP） was used as a sepsis model.Rats were randomly divided into four groups,the control group （CON,n=6）,sham-operation group （SHAM,n=12）,cecal ligation and puncture group （CLP,n=30）,GW501516 group （CLP＋GW,n=25）,which underwent CLP and were subcutaneously injected with the PPAR-β/δ agonist GW501516 （0.05 mg/100 g body weight）.Survival was monitored to 24 hours after operation.Blood pressure,serum creatinine,blood urea nitrogen,aspartate aminotrasferase and alanine aminotrasferase were measured after CLP.Concentrations of tumor necrosis factor α （TNF-α） and interleukin （IL）-1β in serum were detected by enzyme linked immunosorbent assay （ELISA） kits.Lung tissue samples were stained with H＆E and scored according to the degree of inflammation.Bacterial colonies were counted in the peritoneal fluid.Alveolar macrophages were cultured and incubated with GW501516 （0.15 μmol/L） and PPARβ/δ adenovirus and then treated with Lipopolysaccharide （2 μg/ml） for 2 hours.The TNF-α,IL-1β and IL-6 RNA in lung and alveolar macrophages were determined by real-time PCR.Phosphorylation of signal transducer and activator of transcription 3 （STAT3） in lung and alveolar macrophages was detected by Western blotting.Results GW501516 significantly increased the survival of septic rats,decreased histological damage of the lungs,reduced inflammatory cytokines in serum and lung tissues of septic rats and did not increase counts of peritoneal bacteria.In vitro,GW501516 and over-expression of PPARβ/δ attenuated gene expression of TNF-α,IL-1β and IL-6 in alveolar macrophages.Both in vivo and in vitro,PPARβ/δ inhibited the phosphorylation of STAT3.Conclusion PPARβ/δ plays a protective role in sepsis induced ALI via suppressing excessive inflammation.

Zebrafish facilitates drug screening: potential of 3-deoxy-andrographoside from Chuanxinlian(Herba Andrographitis Paniculatae) as an anti-inflammatory agent

OBJECTIVE: To systematically evaluate the in vivo antiinflammatory potential of diterpene lactones from Chuanxinlian(Herba Andrographitis Paniculatae)(AP). METHODS: We firstly adopted zebrafish, a novel and ideal animal model for high-throughput drug screening, to investigate the in vivo anti-inflammatory activities of 17 diterpene lactones isolated from AP.RESULTS: The results showed that most of diterpene lactones displayed significant anti-inflammatory effects in lipopolysaccharide microinjection-, copper sulfate exposure-or tail transection-induced zebrafish inflammation models. Moreover, diterpene lactone 3-deoxy-andrographoside(AP-5) was firstly found to attenuate inflammatory responses, which was closely associated with the myeloid differentiation primary response 88/nuclear factor-kappa B and signal transducer and activator of transcription 3 pathways. CONCLUSION: Our research sheds light on the inestimable roles of zebrafish in high-throughput drug screening, elucidates the potent inhibitory effects of diterpene lactones against inflammation and indicates that AP-5 may serve as a potential alternative agent for the treatment of inflammatory diseases.

Tiaobu Feishen therapy inhibits inflammation induced by cigarette smoke extracts in a human monocyte/macrophage cell line

OBJECTIVE: To study the mechanistic effects of Tiaobu Feishen therapy(TBFS) on inflammation induced by cigarette smoke extract(CSE) in a human monocyte/macrophage cell line.METHODS: The human monocyte/macrophage cell line THP-1 was stimulated with 10% CSE in the presence or absence of Bufei Yishen formula(BYF),Bufei Jianpi formula(BJF) and Yiqi Zishen formula(YZF). All formulations contained serum. Pro-inflammatory cytokines were measured in the supernatants using enzyme-linked immunosorbent assay.The activity of STAT3 DNA binding was detected using electrophoretic mobility shift assay and janus kinase/signal transducer and activator of transcription(JAK/STAT) pathway activation was assessed using Western blotting.RESULTS: The results showed that BYF, BJF and YZF treatment strongly decreased the CSE-induced secretion of interleukin(IL)-6, IL-8, tumor necrosis factor-α and matrix metalloproteinase-9 by THP-1 cells. Furthermore, BYF, BJF and YZF treatment attenuated STAT3 DNA binding capacity and JAK2 and STAT3 were shown to be phosphorylated.CONCLUSION: The data revealed that BYF, BJF and YZF effectively inhibited a CSE-induced inflammatory response in THP-1 cells by limiting activation of the JAK2/STAT3 pathway.