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Objective @# To investigate the effect of pathogenic bacterium-Porphyromonas gingivalis (P.g) on the proliferation and inflammatory factor expression of human colorectal cancer Caco-2 cells, to determine whether the Janus kinase 2-signal transducers and activators of transcription 3 (JAK2-STAT3) pathway is involved in the regulation of Caco-2 cell proliferation by P.g and to provide an experimental basis for further exploring the relationship between P.g and colorectal cancer. @*Methods @# Caco-2 cells were cultured in vitro, and P.g at different multiplicities of infection (MOIs) (0, 1, 10, 25) was selected to stimulate for 12, 24 and 48 h. The effect of P.g on the proliferation of Caco-2 cells was detected by CCK8. The stimulation time was set as 12, 24 and 48 h. MOI=0 was the control group, and MOI=1, 10 and 25 comprised the experimental group. qRT-PCR and Western blot were used to detect the changes in interleukin-6 (IL-6), interleukin-10(IL-10), JAK2 and STAT3 gene and protein (phosphorylated protein) levels in each group. @*Results @# After P.g infection of Caco-2 cells, P.g had a sustained stimulatory effect on the cells for 12, 24 and 48 h at MOI=1 and MOI=10 compared with the control group. Compared with that in the control group, the expression of pro-inflammatory factor IL-6 and related proliferative pathway protein JAK2 and STAT3 in Caco-2 cells with P.g infection increased in a concentration- and time-dependent manner (P<0.05). Additionally, the expression of IL-10, an anti-inflammatory factor, in Caco-2 cells infected with P.g decreased (P<0.05). After the addition of the JAK2 inhibitor AZ960, the proliferation of Caco-2 cells infected with P.g decreased, and the mRNA expression of STAT3 and JAK2 and the protein expression of p-STAT3 and p-JAK2 decreased (P<0.05). @*Conclusion @#P.g can promote the proliferation of the colorectal cancer cell line Caco-2, and the effect of P.g on Caco-2 cells may promote cell proliferation through the JAK2-STAT3 pathway while promoting the expression of the proinflammatory factor IL-6 and inhibiting the expression of the anti-inflammatory factor IL-10, creating an inflammatory environment conducive to cell proliferation, which may be the mechanism by which P.g affects the proliferation of Caco-2 cells.
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OBJECTIVES@#Glioma is the most common primary intracranial tumor and there is still no ideal treatment at present. Gene therapy, as one of the new methods for treating glioma, has attracted attention in recent years. But its application in treating glioma is very limited due to lack of effective delivery vectors. This study aims to investigate the feasibility of biomimetic nanomaterials made from glioma cells-derived extracellular vesicles (EV) for targeted delivery of signal transducers and activators of transcription 3 (STAT3)-small interfering RNA (siRNA) in treating glioma.@*METHODS@#First, U251 glioma cells-derived extracellular vessel (EVU251) was extracted by ultra-centrifugal method. Nanoparticle tracking analysis was used to characterize the particle size distribution, the transmission electron microscope was used to analyze the morphology, and Western blotting was used to verify the expression of srface characteristic protein. The homing ability was verified by cell uptake assay after labeling EVU251 with membrane dye kit PKH67; the EVU251 contents were removed by a low permeability method and then EVMU251 was prepared through a microporous membrane. Finally, the biomimetic nanomaterials EVMU251@STAT3-siRNA were prepared by loading STAT3-SiRNA with electro-dyeing method. The real-time quantitative PCR was used to quantify the successful encapsulation of siRNA, and the encapsulation and drug loading rate was calculated; then Cy5-labeled siRNA was used to evaluate the ability of biomimetic nanomaterials (EVMU251@CY5-siRNA) to target U251 cells. Lysosomal escape ability of the biomimetic nanomaterial was evaluated by lysosomal dye lyso-tracker green. At last, the ability of EVMU251@STAT3-siRNA to knock down STAT3 gene and selective killing of U251 cells was detected by cell experiments in vitro.@*RESULTS@#The size of EVU251 ranged from 50 nm to 200 nm with a natural disc shape. The expression of extracellular vesicle marker proteins could be detected on the membrane of EVU251. The cell uptake assay demonstrated that it had homing ability to target U251 cells. After EVU251 was prepared as EVMU251@STAT3-siRNA, the particle size was (177.9±5.0) nm, the siRNA loading rate was (33.5±2.2)% and the drug loading rate was (3.24±0.21)%. The biomimetic nanomaterial EVMU251@STAT3-siRNA still had the ability to target U251 cells and successfully deliver siRNA to the cytoplasm without lysosomal degradation. The EVMU251@STAT3-siRNA can effectively knock down the expression of STAT3 gene and produce selective killing ability in U251 cells.@*CONCLUSIONS@#The biomimetic nanomaterials EVMU251@STAT3-siRNA made from glioma U251 cells-derived extracellular vesicles can knock down STAT3 gene of U251 cells and produce selective killing effect, which can provide a new idea for the treatment of glioma.
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Humans , RNA, Small Interfering/genetics , Biomimetics , Cell Line, Tumor , Glioma/therapy , Nanostructures , Cell Proliferation , STAT3 Transcription Factor/metabolismABSTRACT
ObjectiveThis study aims to investigate the efficacy and underlying mechanism of Da Chaihutang （DCHT） in treating hepatocellular carcinoma （HCC） in vitro and in vivo. MethodWe employed methyl thiazolyl tetrazolium （MTT） assay and crystal violet staining to observe the proliferation of Hepa1-6 liver cancer cells treated with DCHT at different doses （0， 125， 250， 500， 1 000 mg·L-1） for different time periods （1， 2， 4， 8 days）. The orthotopic liver cancer model was established by injection of 1×106 Hepa1-6 cells into mouse， and then the model mice were randomly assigned into six groups： blank， model， DCHT （0.21， 0.625， 1.875 g·kg-1， ig， qd）， and positive control （5-fluorouracil， 25 mg·kg-1， ip， qod）. After 14 days of administration， the mice were sacrificed， and the liver samples were collected and fixed in 4% paraformaldehyde for hematoxylin-eosin （HE） staining. The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP）， Cytoscape 3.7.2， STRING， and DAVID were used for the searching of the key targets of DCHT in treating HCC， the construction of protein-protein interaction （PPI） network， and the Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment. Quantitative real-time PCR was performed to determine the mRNA level of interleukin-6 （IL-6） in Hepa1-6 cells and liver tissue. Western blotting was employed to measure the protein levels of the proteins involved in the mitogen-activated protein kinase （MAPK） and signal transducers and activators of transcription 3 （STAT3） signaling pathways. ResultDCHT （500， 1 000 mg·L-1） treatment for 4 and 8 days inhibited the proliferation of Hepa1-6 cells in a dose- and time-dependent manner （P<0.05）. The in vivo assay showed that DCHT （high dose， 1.875 g·kg-1） treatment for 14 days led to high differentiation and unobvious heterogeneity of HCC cells and small necrotic area compared with the model group. Network pharmacology analysis predicted that the potential targets of DCHT in the treatment of HCC were mainly the inflammation cytokines such as IL-6， interleukin-1β （IL-1β）， and tumor necrosis factor-alpha （TNF-α） in HCC microenvironment. The potential signaling pathways involved in the treatment were mainly associated with HCC growth and differentiation， including MAPK and STAT3 signaling pathways. Compared with the blank group， DCHT （1 000 mg·L-1） treatment for 1， 2， 4， and 8 days down-regulated the mRNA level of IL-6 in Hepa1-6 cells （P<0.05）. Similar results were observed in the livers of mice treated with DCHT （0.625， 1.875 g·kg-1）. The in vitro assay demonstrated that DCHT （1 000 mg·L-1） treatment for 4 and 8 days and DCHT （500， 1 000 mg·L-1） treatment inhibited the phosphorylation of extracellular signal-regulated kinases 1/2 （ERK1/2）， c-Jun NH2-terminal kinase/stress-activated protein kinase （JNK）， p38 MAPK， and STAT3 in a dose- and time-dependent manner （P<0.05）. The in vivo assay showed that DCHT （0.625 and 1.875 g·kg-1） treatment only inhibited the phosphorylation of p38 MAPK and STAT3 （P<0.05）. ConclusionThe present study indicates that DCHT can inhibit liver cancer cell proliferation by regulating p38 MAPK/IL-6/STAT3 signaling pathway.
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Objective: To investigate the effects of β-arrestin 1 (ARRB1) on apoptosis and proliferation of non-small cell lung cancer (NSCLC) cells, and to explore the underlying molecular mechanism. Methods: In NSCLC cell lines A549 and H1650, the expression of ARRB1 was knocked down by transfection of siRNA or over-expressed by transfection of Flag-ARRB1 recombinant plasmids, which was verified by real-time fluorescent quantitative PCR and Western blotting. The effects of down-regulating and up-regulating ARRB1 expression on the transcription and secretion of interleukin-6 (IL-6) were detected by real-time fluorescent quantitative PCR and ELISA method, respectively. The interaction between ARRB1 and p300 was detected by protein immunocoprecipitation. The recruitment of p300 and the acetylation of histone in IL-6 promoter region after ARRB1 knock-down or overexpression were detected by chromatin immunocoprecipitation. The proliferation and apoptosis of ARRB1 silencing A549 cells were detected by CCK-8 assay and FCM, respectively. The expression and activation of IL-6/signal transducer and activator of transcription 3 (STAT3) signaling pathway-related molecules in ARRB1 silencing or overexpression NSCLC cells were investigated by Western blotting. Results: In ARRB1-silenced or overexpressed NSCLC cell lines A549 and H1650, ARRB1 enhanced the transcription and production of IL-6 (all P < 0.05). The interaction of ARRB1 and p300 was confirmed by forward and reverse immunocoprecipitation. After ARRB1 knockdown or overexpression, it was found that ARRB1 enhanced the recruitment of p300 in IL-6 promoter region (both P < 0.01) and increased the acetylating of IL-6 promoter (both P < 0.05). Moreover, ARRB1 could facilitate the growth (P < 0.01) and apoptosis inhibition of NSCLC cells. ARRB1 could promote the phosphorylation of STAT3 and the expressions of c-Myc and Bcl-2 proteins. Conclusion: In NSCLC cells, ARRB1 interacts with p300, facilitates the recruitment of p300 to IL-6 promoter, and up-regulates the acetylation of histone H3 and H4 in IL-6 promoter, leading to transcriptional activation of IL-6. So that ARRB1 positively regulates the activation of IL-6/STAT3 signaling through promoting the phosphorylation of STAT3 and the expressions of c-Myc and Bcl-2 proteins, contributing to the growth and anti-apoptosis of NSCLC cells.
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Objective: To investigate the effect of Sijunzi Decoction extract (SDE) on the growth of human triple negative breast cancer (TNBC) cell line MDA-MB-468. Methods: MDA-MB-468 cells were treated with different concentrations of SDE. The effect of SDE on the proliferation and migration of the cells were detected by CCK-8 assay and the cell wound healing assay. The colony formation assay was performed to analyze the effect on the ability of colony formation of the cells with SDE. Hoechst 33342 staining technique and flow cytometry (FCM) were used to detect apoptosis and cell cycle of the cells. Western blotting was used to detect the expression levels of STAT3, which was related with the proliferation and apoptosis of cells. Results: Compared with the control group, SDE had a certain inhibitory effect on MDA-MB-468 cells (P < 0.05), and it was dependent on the concentration and time. Cloning formation experiments showed that SDE inhibited the clonality of the cells. The cell migration experiment showed that the wound healing ability of the cells could be weakened by the extract with the medium and high dosage (P < 0.001). The results of FCM showed that the apoptosis rate of all SDE dosage increased gradually in a dose-dependent manner. And SDE with the medium and high dosage induced apoptosis of the cells significantly (P < 0.01 and 0.001). Cell cycle was affected by SDE with the obvious reduction of the cells in G2 phase (P < 0.01). The results of Western blotting showed that the expression level of STAT3 was decreased significantly. Conclusion: SDE inhibited the proliferation and clonal formation of MDA-MB-468 cells, inhibited migration, promoted apoptosis and decreased the cells of G2 phase. which may be related to the regulation of STAT3 pathway.
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Objective::To observe the effect of Shenling Baizhusan(SBS)on the mammalian target of rapamycin complex 1 (mTORC1)/signal transducers and activators of transcription 3 (STAT3) pathway in liver hepatocyte of nonalcoholic fatty liver disease(NAFLD)rats induced by high fat diet, in order to reveal the mechanism of SBS against rat NAFLD from the perspective of inflammation. Method::Totally 80 SD rats were randomly divided into 4 groups, normal control group, model group, high-dose SBP group(30 g·kg-1), and low-dose SBS group(10 g·kg-1), with 20 rats in each group. The rats of NAFLD model were established by being fed with high-fat diets for 8 weeks, and the treatment groups were fed with high or low dose of SBS respectively. After treatment for 8 weeks, blood and liver samples of rats were collected. Alanine aminotransferase (ALT), aspartate aminotransferase(AST), total cholesterol (TC), triglyceride(TG), high-density lipoprotein cholesterol(HDL-C)and low-density lipoprotein cholesterol(LDL-C)levels in blood serum were detected with automatic biochemical analyzer. The liver tissues were observed by oil red O and hematoxylin-eosin (HE) staining. Hepatocytes were isolated by type Ⅳ collagenase perfusion in vitro. Tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-5 and IL-6 in hepatocytes were detected by enzyme-linked immunosorbent assay (ELISA), and the relevant gene and proteins expressions of mTORC1 and STAT3 in hepatocytes were detected by Real-time fluorescent quantitative polymerase chain reaction (Real-time PCR) and Western blot detection respectively. Result::Compared with the normal control group, the serum levels of TG, TC, AST, ALT and LDL-C were increased significantly, the levels of TNF-α, IL-1β, IL-5 and IL-6 in hepatocytes were increased significantly, and the expression levels of mTORC1, STAT3 mRNA and proteins in hepatocytes were increased significantly(P<0.01). Compared with the model group, the hepatic lipid accumulation of the medicine intervention group was relieved significantly, the serum levels of AST, ALT, TG and LDL-C were decreased significantly, the expression levels of TNF-α, IL-1β, IL-5 and IL-6 of hepatocytes were decreased significantly, and the expressions of mTORC1, STAT3 mRNA and proteins in hepatocytes were decreased significantly(P<0.05, P<0.01). In the high-dose SBS group, the effects in improving the lipid accumulation and inhibiting the inflammatory reaction were better than those of the low-dose SBS group, and the expressions of mTORC1 and STAT3 genes and proteins in hepatocytes were significantly decreased (P<0.05, P<0.01). Conclusion::SBS can improve the fat metabolism disorder and reduce liver lipid accumulation and inflammatory reaction in NAFLD rats induced by high-fat diet. The mechanism may be correlated with the inhibition of mTORC1/STAT3 pathway relating to genes and protein expression in hepatocytes.
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Interleukin-6 (IL-6)/janus kinase (JAK)/signal transducers and activators of transcription 3 (STAT3) is a pivotal signaling pathway in the regulation of cell proliferation, survival, differentiation and T cell activation. Aberration of this pathway is involved in multiple autoimmunity diseases and cancers, therefore the pathway is considered as a hot target for drug development. In our study, we validated a cell-based model of IL-6/JAK/STAT3 and used it in screening of its inhibitors. HEK-Blue IL-6 cells of Invivogen Inc. were used to stably express IL-6 receptor and STAT3-induced secreted embryonic alkaline phosphatase (SEAP) report gene. After stimulation by IL-6, SEAP was secreted from cells and reacted with QUANTI-Blue. The product can be detected at 655 nm. The inhibitory effect of compounds on STAT3 signaling showed as IC50 was calculated by OD value. The results shown that IL-6 specifically activated the cells, which could be applied to screen the inhibitors for IL-6/JAK/STAT3 signaling pathway. The optimized screening conditions were described as below:50 000 cells/well, 1 ng·mL-1 IL-6 incubation for 20 h and reaction with QUANTI-Blue for 1 h. Based on this condition, we screened 14 natural products based on this cell model and arctigenin, cryptotanshinone and curcumin showed potential inhibitory activities on STAT3 signaling pathway with IC50 of 1.28, 2.96 and 6.61 μmol·L-1. Our study suggests that HEK-Blue IL-6 cells were suitable for screening inhibitors for the IL-6/JAK/STAT3 signaling pathway.
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Objective To investigate the mechanism of interleukin-22(IL-22) induced the secretion of vas-cular endothelial growth factor A(VEGF-A) in gastric cancer cell line AGS .Methods Gastric cancer cell line AGS were cultured in vitro ,and recombination cytokine IL-22 were added ,or signal pathway inhibitor were pre-incubated with AGS for 1 hour and then IL-22 were added ,the level of VEGF-A were detected by enzyme-linked immunosorbent assay .Results Compared with the unstimulated group ,the secretion of VEGF-A in IL-22-stimulated group was significantly increased ,the difference was statistically significant (P＜0 .05) ,which was in a dose and time dependent manner .In addition ,IL-22-stimulated the secretion of VEGF-A by AGS was significantly decreased while pre-incubated by the signal transducers and activators of transcription 3(STAT3) inhibitor ,the difference was statistically significant (P＜0 .05) ,but such effect was not observed while AGS were pre-incubated with the nuclear factor kappa B inhibitor ,c-Jun N-terminal kinase inhibitor ,mitogen-acti-vated protein kinase kinase 1/2 inhibitor and p38 mitogen-activated protein kinase inhibitor ,there was no sta-tistical significance(P＞0 .05) .Conclusion IL-22 could induce the secretion of VEGF-A in gastric cancer cell line AGS via STAT3 signal pathway ,which may contribute to tumor progression .
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Aim To explore the effects of miR-7 on astrocyte activation and the underlying mechanisms. Methods Following isolation and culturing, astro-cytes extracted from rat cortex were treated with culture solution (control group), ciliary neurotrophic factor (CNTF, an agonist of astrocyte activation), miR-7 mimic+CNTF, miR-7 mimic control+CNTF, miR-7 inhibitor+CNTF and miR-7 inhibitor control+CNTF, respectively. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the mRNA ex-pression of glial fibrillary acidic protein (GFAP) and epidermal growth factor receptor(EGFR). The protein expression of GFAP, EGFR, signal transducers and activators of transcription 3(STAT3) and phosphoryla-ted STAT3 (p-STAT3) was measured using Western blot. Wild type pGL3-EGFR and mutant pGL3-EGFR-m recombinant plasmids were constructed and then co-transfected with miR-7 mimic into HEK293T cells,re-spectively. The luciferase activity of reporter gene was measured. In addition,astrocytes were treated with ei-ther EGFR siRNA or S31-201 (an inhibitor of STAT3),followed by the incubation with miR-7 inhib-itor and CNTF. Both qRT-PCR and Western blot were subsequently used to detect the mRNA and protein lev-els of GFAP. Results The expression levels of GFAP and EGFR as well as p-STAT3/STAT3 ratio in CNTF group were higher than those in control group (P <0.01). When compared with CNTF group,GFAP and EGFR levels and p-STAT3/STAT3 ratio significantly decreased in miR-7 mimic+CNTF group but increased in miR-7 inhibitor+CNTF group(P<0.01). In com-parison with control group, transfection with miR-7 mimic markedly reduced the luciferase activity of wild type EGFR (P <0.01). Moreover, miR-7 inhibitor-induced up-regulation of GFAP expression was almost completely reversed by either EGFR siRNA or S31-201 pretreatment (P<0.01). Conclusion miR-7 antag-onizes the activation of astrocytes from rats by inhibi-ting the EGFR/STAT3 signaling pathway.
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Objective To investigate the molecular mechanism of calpain in regulating macrophage polarization.Methods Macrophages (RAW264.7) were transfected with siRNA by Lipofectamine 2000 to konckdown calpain1 and calpain2,respectively.The mRNA levels of markers in M1 (IL-23,TNF-α and iNOS) and M2 (IL-10,TGF-β and Arg-1) were measured by qRT-PCR.The levels of proteins in signaling pathways (NF-κB/STAT3) were measured by Western blot.The ability of macrophage migration was detected by Transwell assay.Results The expression level of calpain1 was lower in M1 than that in M2 (P＜0.05),but the expression level of calpain2 was significantly higher in M 1 than that in M 2 (P＜0.05).In calpain1-siRNA group,the mRNA levels of M1-type macrophages markers and M2-type macrophages markers were decreased by lipopolysaccharide (LPS) stimulation;The mRNA levels of M1-type macrophages markers in calpain2-siRNA group were significantly reduced (P ＜ 0.05),while the mRNA levels of M2-type macrophages markers were significantly increased (P ＜ 0.05).In calpain2-siRNA intervention group,the total phosphorylation inhibitor of nuclear factor kappa-B kinase (p-IKK) protein level of LPS-induced macrophages was decreased;in IL-4-induced macrophages,the protein level of plasmic signal transducers and activators of transcription 3 (STAT3) was also decreased,but there was no significant difference in total level of phosphorylation-p65 (p-p65).It was also found that the ability of migration was reduced by the interventions of calpain1-siRNA and calpain2-siRNA as compared with control-siRNA intervention (P＜0.05).Conclusions Calpain2 may potentially promote M1 polarizations by NF-κB and STAT3 signaling pathways,and inhibit the ability of its migration by the interventions of calpain1-siRNA and calpain2-siRNA.
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@#Objective To evaluate the effect of left atrial enlargement on atrial myocardial fibrosis degree and levels of the angiotensinⅡ (AngⅡ)/Rac GTPase activating protein 1 (Rac1)/signal transducersand activators of transcription 3 (STAT3) signaling pathways expressing in patients with persistent atrial fibrillation and rheumatic heart disease (RHD). Methods From March to December 2011, 30 patients with RHD who underwent prosthetic valve replacement in our hospital were enrolled, including 16 males and 14 females, aged 42-70 (56.9±6.8) years. Twenty RHD patients with persistent atrial fibrillation as a research group and ten RHD patients with sinus rhythm as a control group (group A) underwent transthoracic echocardiography and right atrial appendage (RAA) tissue samples were obtained from these patients during mitral/aortic valve replacement operation. The research group according to left atrial diameter (LAD) was divided into two groups, ten patients in each group: a group B with LAD of 50–65 mm and a group C with LAD of LAD>65 mm. For each sample, histological examination was performed by hematoxylin-eosin and Masson’s trichrome staining. Light-microscopic pictures of atrial tissues samples were stained and tissue fibrosis degree in each group was analyzed. AngⅡ concentration was measured by enzyme linked immunosorbent assay. Rac1 and STAT3 were measured by western blotting. Results LAD was significantly greater in AF patients with RHD than in the control group. Hematoxylin-eosin staining demonstrated highly organized arrangement of atrial muscles in the control group and significant derangement in both group B and group C with reduced cell density and increased cell size. Moreover, Masson’s trichrome staining showed that atrial myocytes were surrounded by large trunks of collagen fibers in both group B and group C, but not in the group A. There was a positive correlation between atrial tissue fibrosis and LAD. AngⅡ content was positively correlated with LAD. Similarly, Rac1 and STAT3 protein levels were found considerably higher in the group C and group B than in the group A with excellent correlation to LAD. Conclusion In patients with RHD complicated with persistent atrial fibrillation, the degree of atrial fibrosis and the expression level of AngⅡ/Rac1/STAT3 signaling pathways significantly increase with the left atrialenlargement.
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Signal transducers and activators of transcription 3 generally located in cytoplasm is a key intra-cellular transcription. So far, STAT3 has been known that could be activated by several different upstream kinase within cell, the most common one of which is Janus kinase. The aberrant activation of STAT3 induces tumorigenesis and promotes tumor development. Consequently, researchers have always been committed to developing novel anti-tumor drugs targeting JAK/STAT3 pathway signaling. In this review, researches as well as clinical trials of JAK/STAT3 inhibitors published over the past several years are collected and the targets, mechanism and pharmacological properties of certain inhibitors are summarized.
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Aim To investigate the effects of metformin on phospho-signal transducers and activators of transcription 3(p-STAT3) expression in the spinal dorsal horn in rats with bone cancer pain(BCP).Methods Healthy female SD rats weighing 200~220 g were randomly divided into four groups(n=12 each): Sham+NS, Sham+Metformin, BCP+NS, BCP+Metformin groups.Normal saline or metformin(200 mg·kg-1) was given intraperitoneally once a day at 7~14 day after surgery in four groups.Mechanical withdrawal threshold(MWT) was measured on-1, 3, 5, 7, 10, 12, 14 day after BCP.The L4-6 segments of spinal cords were used to detect the expression of p-STAT3 by Western blot and immunohistochemical staining technique after the behavioral test was completed on day 14.Results Intraperitoneal injection of metformin reversed hyperalgesia and suppressed the expression of p-STAT3 in BCP rats.Conclusion Metformin can attenuate BCP by inhibiting the expression of p-STAT3 in the spinal dorsal horn.
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Objective To study the expression and significance of HMGB1,TLR4 and STAT3 in the mouse model of anti glomerular basement membrane glomerulonephritis.Methods Totally 30 mice were randomly divided into normal group(control group),anti GBM glomerulonephritis model group(experimental group),PAS staining was used to observe the changes of glomerular basement membrane,HMGB1,p-STAT3 mRNA in renal tissue were detected by RT-PCR,HMGB1,TLR4,STAT3,p-STAT3 in renal tissue was detected by Western blot method.Results Compared with the control group,the experimental group of mice glomerular basement membrane thickening;the relative expression of HMGB1,TLR4,STAT3,p-STAT3 and HMGB1 mRNA,p-STAT3 mRNA in the kidney tissue of experimental group were significantly higher than the control group,the difference was statistically significant(P<0.05).The expression level of HMGB1 and TLR4,TLR4 and p-STAT3,HMGB1 and p-STAT3,HMGB1 mRNA and p-STAT3 mRNA in renal tissues of mice in experimental group,there were a positive correlation(r=0.401,P=0.005;r=0.399,P=0.005;r=0.412,P=0.004;r=0.398,P=0.005).Conclusion The inflammatory effect of HMGB1 in mice with anti glomerular basement membrane glomerulonephritis can activate STAT3 by binding to its receptor TLR4,so as to achieve anti glomerular basement membrane glomerulonephritis.
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Objective Inflammation is a defensive reaction of body , but excessive inflammatory response can lead to physi-cal injury.The aim of this study was to explore the effects of luteolin on the secretion of inflammatory cytokines from lipopolysaccharide (LPS) and interferon-g(IFN-γ) activated RAW264.7 cells. Methods RAW264.7 cells were divided into 5 groups: control group (without any medicine), M1 group (polarized M1 cells activated by final concentration of 10 ng/mL LPS+20 ng/mL IFN-γ), M1+5L group (simultaneous activation of LPS and IFN-γplus final concentration of 5μmol/L luteolin), M1+10L group(simultaneous activa-tion of LPS and IFN-γplus 10μmol/L luteolin), M1+20L group(simultaneous activation of LPS and IFN-γplus 20μmol/L luteolin). The cell morphological transformation was observed by laser confocal microscope ;the mRNA levels of iNOS , IL-1βand IL-6 were test-ed by real-time quantitative PCR respectively;the secretion levels of TNF-αand IL-6 in culture supernatant were detected by ELISA;the changes of p-STAT3 (ser727) protein pathways were examined by western blot. Results Cellular morphology of activated RAW 264.7 cells changed obviously .Compared with the control group , the mRNA levels of iNOS, IL-1βand IL-6 decreased significantly in the other 4 groups(P<0.05).The iNOS level in M1+20L group significantly de-creased compared with M1 group[(29.52±3.07) vs (98.91±10.65), P<0.01].As to IL-1βlevel, it decreased significantly in M1+10L group(78.38±8.65) and M1+20L group(41.59±6.80) compared with M1 group(110.69±4.12)(P<0.05).While the IL-6 levels decreased significantly in M1+5L group(177.51±19.28), M1+10L group (106.14±5.63), M1+20L group(27.15±1.26), compared with M1 group(394.10±33.47)(P<0.05).LPS+IFN-γcould induce in-creased p-STAT3 (ser727) expression in M1 phenotype of RAW264.7 cells which was proved by its significant increase in M 1 group, M1+5L group and M1+10L group compared with control group (P<0.05).In comparison to M1 group, p-STAT3-ser expression in M1 phenotype downregulated in M1+5L group, M1+10L group, M1+20L group(P<0.05), along with dose-dependent characteristic.Com-pared with control group, the levels of IL-6 and TNF-αincreased significantly in M1 group, M1+5L group and M1+10L group.Com-pared with M1 group, the levels of IL-6 and TNF-αdecreased significantly in M1+5L group, M1+10L group and M1+20L group(P<0.05) , in which IL-6 showed concentration independence and TNF-αshowed no concentration independence . Conclusion Luteolin inhibits the secretion of pro-inflammatory cytokines through the down-regulation of p-STAT3 so as to exert anti-inflammatory effects .
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Cerulein-induced pancreatitis is similar to human edematous pancreatitis, characterized by the dysregulation of digestive enzyme production, edema formation, and an infiltration of inflammatory cells into the pancreas. We previously showed that the Janus kinase 2 (JAK2)/STAT3 pathway mediates inflammatory signaling in cerulein-stimulated pancreatic acinar cells. PPAR-γ has been implicated in the regulation of inflammatory responses in several cells. In the present study, we investigated the role of PPAR-γ in cerulein-induced activation of JAK2/STAT3 in pancreatic acinar cells. Treatment with cerulein induced the activation of JAK2/STAT3 and PPAR-γ expression in AR42J cells. Cerulein-induced PPAR-γ expression was inhibited by AG490, a JAK2/STAT3 inhibitor, in AR42J cells. An immunoprecipitation analysis showed that PPAR-γ binds to STAT3 in cerulein-stimulated AR42J cells. Down-regulation of PPAR-γ by siRNA increased STAT3 phosphorylation in AR42J cells stimulated with cerulein. These results show that PPAR-γ inactivates STAT3 by directly interacting with STAT3 in cerulein-stimulated pancreatic acinar cells. Overexpression of PPAR-γ may be beneficial for preventing pancreatitis by suppressing the activation of STAT3 in pancreatic acinar cells.
Subject(s)
Humans , Acinar Cells , Ceruletide , Down-Regulation , Edema , Immunoprecipitation , Janus Kinase 2 , Pancreas , Pancreatitis , Peroxisomes , Phosphorylation , RNA, Small InterferingABSTRACT
Aim To observe the expression of epider-mal growth factor receptor ( EGFR) in cerebral tissues around hematomas after intracerebral hemorrhage, and explore the effects of EGFR on activation of astrocytes derived from rats and the involved mechanisms. Meth-ods The specimens of cerebral tissues around hemo-tomas after intracerebral hemorrhage undergoing hemo-tomas removal operation were collected and then divid-ed into 4 groups according to the time of intracerebral hemorrhage: 10 d groups. Each group included 20 cases. At the same time, 20 dropped brain tissues distant to hemorrhage in the operative process were collected as control group. Immunohistostaining and Western blot were used to measure the expression of EGFR. After isolation and culturing, the astrocytes of rat cortex were treated with culture solution ( control group) , CNTF that was used to activate astrocytes, scramble siRNA + CNTF and EGFR siRNA +CNTF for 24h, respectively. The ex-pression of glial fibrillary acidic protein ( GFAP) mR-NA was detected through fluorescence real-time quanti-tative PCR. In addition, the protein levels of GFAP, signal transducers and activators of transcription 3 ( STAT3 ) and phosphorylated STAT3 ( p-STAT3 ) were examined using Western blot. Results With the ex-tension of intracerebral hemorrhage time, positive sig-nal index and protein expression levels of EGFR gradu-ally elevated, reached the peak on 6 ~10d, and then decreased after 10 d. There was statistical difference ( P0. 05 ) . Conclusions EGFR expression is upregulated in the cerebral tissues around hemotomas after intracerebral hemorrhage. Gene silence of EGFR contributes to suppressing the activation of astrocytes derived from rats, which may be involved in the block-ade of STAT3 phosphorylation.
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OBJECTIVE: To further define the modulation effect of signal transducers and activators of transcription 3 (STAT3) in adriamycin-resistant breast cancer and to promote the clinical application of the inhibitors of STAT3 in reversing multidrug resistance in cancer. METHODS: Firstly, the levels of STAT3 and phosphorylated STAT3 (pSTAT3) expression in clinical breast cancer tissue samples were determined by Western blotting. The expression of STAT3 and pSTAT3 in adriamycin-sensitive and adriamycin-resistant breast cancer cell lines was evaluated by RT-PCR and Western blotting. Secondly, the expression of STAT3 was detected by Western blotting after blocking the STAT3 signal pathway with small interfering RNA targeting STAT3 (STAT3-siRNA). Finally, after the expression of STAT3 was blocked by STAT3-siRNA, immunofluorescence was performed to study the proliferation activity of breast cancer cells and MTT was used to determine the IC50 of the cells in order to observe whether STAT3-siRNA had any inhibitory effects on the growth of breast cancer cells and whether it could promote the efficacy of adriamycin in breast cancer cells. RESULTS: STAT3 and pSTAT3 were highly expressed both in clinical breast cancer tissue samples and in adriamycin-sensitive-and-resistant breast cancer cells. The expression of pSTAT3 in adriamycin-resistant breast cancer cells was significantly higher than in adriamycin-sensitive breast cancer cells. STAT3-siRNA conspicuously decreased the expression of STAT3 protein and inhibited the growth of adriamycin-sensitive breast cancer cells. Compared with the single application of adriamycin, IC50 of adriamycin-resistant breast cancer cells decreased by 4 fold when adriamycin was used in combinaiton with STAT3-siRNA. Meanwhile, an inhibition of the expression of the anti-apoptotic protein mediated by STAT3 was observed in adriamycin-resistant breast cancer cells. CONCLUSION: This study reveals that the development of breast cancer is related to the activation of STAT3. The activation of STAT3 in adriamycin-resistant breast cancer cells was more notable than in adriamycin-sensitive cells. The inhibition of the STAT3 pathway could improve the adriamycin sensitivity of adriamycin-resistant breast cancer cells and lead to their apoptosis. The RESULTS of this study explores the feasibility of the reversal of drug resistance in cancer by blocking STAT3 pathway and establishes the experimental basis for promoting the clinical use of the inhibitors of STAT3 pathway and the chemotherapeutics to overcome the multidrug resistance in cancer.
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Objective To construct recombinant lentivirus with the gene STAT3 of the Mus musculus,measure the expres-sion of STAT3,and conduct lentivirus packing and identification.Methods The mRNA of mouse myoblast was extracted and transformed into STAT3 cDNA by the special primer.then,STAT3 cDNA was amplified and reclaimed and inseted into pLVX-IRES-ZsGreen1 vector.Cleavage map and sequencing analysis were used for identification of the recombinant lentivirus vector (pLVX-IRES-ZsGreen1-STAT3).293 T cells were transfected with main vector pLVX-IRES-ZsGreen1-STAT3.and 48 h later, Western blott detected the expression of STAT3 protein.Lentiviral vectors were packaged and the titer was determined.Results The lentiviral vector plasmid pLVX-IRES-ZsGreen1-STAT3 was identified correctly by cleavage map and Co-transfection of 293 T cells with 48 h,the expression of STAT3 was significantly enhanced by western blot.And DNA sequencing analysis confirmed that STAT3 gene sequencing was exactly the same with that reported by genbank.Conclusion Lentiviral vector carrying STAT3 was successfnlly constructed and could express STAT3 with high efficiency,and can be used in further study.
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JAK/STAT includes many types of cell factors. Growth factor signal transduction is an important pathway and widely participates in cell multiplication, differentiation, and immunity adjustment. Aberrant-activated JAK/STAT signaling pathway is in-volved in carcinogenesis. Recent studies demonstrated that abnormal expression and activation of STAT3 were found in lymphoma. Constitutive activation of STAT3 promotes development, invasion, and metastasis of cancer. AG490, a JAK2 inhibitor, can block the JAK/STAT3 signal pathway and reduce the pathway's downstream STAT3 expression. Several studies showed that AG490 can inhibit cell proliferation and promote apoptosis in lymphoma. AG490 combined with chemotherapy drugs could improve sensitivity. In our study, we reviewed the potential role of JAK/STAT signaling pathway and the blocker AG490 in lymphoma.