Application Value of Serum STAT3 and SMAD4 Expression Levels in Early Diagnosis and Staging of Primary Glaucoma Patients / 现代检验医学杂志

Objective To explore the application of serum signal transducers and activators of transduction 3(STAT3)and SMAD4 expression levels in the early diagnosis and clinical staging of primary glaucoma patients.Methods 86 patients with primary glaucoma admitted to Cangzhou Eye Hospital from August 2021 to May 2023 were selected as the study group,according to the clinical symptoms and visual examination results of the research group,they were divided into mild injury stage(n=30),moderate injury stage(n=34)and severe injury stage(n=22).Another 86 healthy individuals who underwent physical examinations in Cangzhou Eye Hospital during the same period were collected as the control group.Enzyme linked immunosorbent assay(ELISA)was applied to detect the expression levels of serum STAT3 and SMAD4.Multivariate Logistic regression was applied to analyze the relevant factors affecting clinical staging of primary glaucoma,receiver operating characteristic(ROC)curve was applied to analyze the diagnostic value of serum STAT3 and SMAD4 in patients with moderate/severe primary glaucoma injury.Results The expression levels of serum STAT3(13.96±3.45 ng/ml)and SMAD4(11.23±2.85 ng/ml)in the study group were obviously higher than those in the control group(9.83±1.72 ng/ml,7.78±1.95 ng/ml),the differences were statistically significant(F=13.085,17.513,all P＜0.05).The expression levels of serum STAT3(11.88±2.52 ng/ml,13.85±3.51 ng/ml,16.96±4.63 ng/ml)and SMAD4(9.15±1.95 ng/ml,11.23±2.83 ng/ml,14.08±4.12 ng/ml)in patients with primary glaucoma in mild,moderate and severe injury groups were gradually increased,the differences were statistically significant(F=13.085,17.513,all P＜0.05).There was a statistically obvious difference in intraocular pressure among patients with mild,moderate(24.21±5.03 mmHg,28.16±6.31 mmHg,32.26±7.57mmHg),and severe injuries(F=10.577.P＜0.05).serum STAT3[OR(95%CI)=2.728(1.409～5.281)],SMAD4[OR(95%CI)=2.849(1.507～5.387)],and intraocular pressure[OR(95%CI)=2.435(1.094～5.417)]were risk factors affecting clinical staging of primary glaucoma(all P＜0.05).The area under the curve(AUC)of the combined diagnosis of serum STAT3 and SMAD4 for moderate/severe injury in patients with primary glaucoma was 0.963(95%CI:0.899～0.992),which was superior to their respective individual diagnoses(Z =2.558,1.961;P=0.010,0.049),their sensitivity and specificity were 96.43%and 83.33%,respectively.Conclusion The higher the expression levels of STAT3 and SMAD4 in serum,the more severe the clinical symptoms in patients.The combined detection of the two has good diagnostic value for patients with moderate/severe injury.

Porphyromonas gingivalis enhances the proliferation of colorectal cancer Caco-2 cells via the JAK2-STAT3 pathway / 口腔疾病防治

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.

Erythrocyte membrane-associated protein inhibiting Th17 cell differentiation via IL-6 / STAT3 / ROR-γt signaling pathway in experimental autoimmune encephalomyelitis mice / 解剖学报

Objective To explore the effect of exogenous and endogenous erythrocyte membrane-associated protein (ERMAP) on helper T cell 17 (Th17) cell differentiation through interleukin 6 / signal transducers and activators of transcription 3 / retionoid-related orphan nuclear receptor-γt(IL-6 / STAT3 / ROR-γt) signal pathway in the mouse model of experimental autoimmune encephalomyelitis (EAE) . Methods Using flow cytometry to verify the function of ERMAP-Ig fusion protein at different concentrations; Agarose gel electrophoresis was performed to identify ERMAP knockout mice. Flow cytometry was performed to detect the effect of ERMAP-Ig fusion protein on Th17 cell differentiation in vitro. Forty 6-week-old normal C57BL / 6 mice were randomly divided into 2 groups to establish EAE models, control-Ig and ERMAP-Ig groups, with 20 mice in each group; Clinical scores were recorded; Flow cytometry was performed to detect Th17 cell differentiation in EAE mice in vivo. Forty 6-week-old identified wild-type and ERMAP knockout mice were divided into 2 groups to establish EAE models. Identified wild-type and ERMAP knockout mice were divided into 2 groups to establish EAE models, ERMAP

Role of biomimetic nanomaterials made from glioma cell- derived extracellular vesicles in targeted delivery of STAT3-siRNA / 中南大学学报(医学版)

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.

Da Chaihutang Inhibits Hepatocellular Carcinoma by Regulating p38 MAPK/IL-6/STAT3 Signaling Pathway / 中国实验方剂学杂志

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.

Regulatory Mechanism and Role of STAT3 Signaling Pathway in Multiple Myeloma / 中国生物化学与分子生物学报

Multiple myeloma (MM) is a kind of plasma cell tumor, characterized by clonal expansion of malignant plasma cells in the micro-environment of bone marrow, production of monoclonal immunoglobulins and dysfunction of related organs. In recent years, with the introduction of autologous stem cell transplantation and the application of Lenalidomide and Bortezomib, the traditional treatment of myeloma had been changed and the overall survival of the patients was prolonged. Although significant progresses have been made in treatment, multiple myeloma is still incurable, mainly due to primary drug resistance and disease recurrence. Signal transducers and activators of transcription 3 (STAT3) is a kind of signal transcription factor, which is involved in diverse cellular processes including the differentiation, proliferation and angiogenesis in normal cells. Recently, it had been found that the high expression of STAT3 in tumors was closely related to the occurrence, development, invasion and metastasis of malignant tumors. STAT3 also played a key role in the occurrence and progression of multiple myeloma. In this paper, we reviewed the molecular structure, signal pathway, activation, regulation and basic biological functions of STAT3, and found that non coding RNA, heat shock protein 90 (Hsp90), heme oxygenase-1 (HO-1) and other factors play important roles in the occurrence, survival and immune escape of multiple myeloma through STAT3 pathway whose activation is related to the resistance of multiple myeloma cells to Bortezomib, Lenalidomide and other conventional drugs. Therefore, STAT3 can be used as a potential target for multiple myeloma. This review provides a basis for accurate diagnosis and treatment of MM and a reference for STAT3 as a potential prognostic marker.

Effect of Shenling Baizhusan on mTORC1/STAT3 Pathway in Hepatocytes of Nonalcoholic Fatty Liver Disease Rats / 中国实验方剂学杂志

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.

Effect of Huangqi Jianzhongtang on JAK2/STAT3 Signal Pathway in Rats with Spleen-Stomach Deficiency Cold Type Gastric Ulcer / 中国实验方剂学杂志

Objective:To investigate the effect of Huangqi Jianzhongtang on Janusprotein tyrosine kinase 2/signal transducers and transcriptional activator protein 3 (JAK2/STAT3) signal pathway in rats with spleen-stomach deficiency cold type gastric ulcer (GU). Method:A total of 60 SPF level Wistar rats were randomly divided into two groups: blank group and model group. Model rats were used to reconstruct the spleen-stomach deficiency cold type GU model by comprehensive modeling method. Model rats were divided into model group, Anweiyang group and high, medium and low-dose Huangqi Jianzhongtang groups according to the random number table, with 10 rats in each group. Rats in blank group and model group were given 10 mL·kg-1·d-1 distilled water, and 16, 8, 4 g·kg-1·d-1 Huangqi Jianzhongtang, respectively. Rats in the positive control group were given 0.14 g·kg-1·d-1 Anweiyang for 21 days. The gene expressions of JAK2 and STAT3 in the ulcer tissue were detected by Real-time fluorescent quantitative polymerase chain reaction (Real-time PCR), the protein expressions and phosphorylation levels of JAK2 and STAT3 in the ulcer tissue were detected by Western blot, and the contents of interleukin-10（IL-10）and interleukin-17（IL-17）in the gastric tissue of each group were detected by enzyme-linked immunosorbent assay (ELISA). Result:Compared with the blank group, the general survival condition of the model group was worse, the content of IL-10 in gastric homogenate was significantly reduced, while the content of IL-17 was significantly increased (P<0.05), the protein expressions of JAK2 and STAT3 in gastric tissue was not significantly increased, whereas the gene expressions and phosphorylation levels of JAK2 and STAT3 were significantly increased (P<0.05). Compared with the model group, the content of IL-10 increased, but the content of IL-17 decreased, the gene expressions of JAK2 and STAT3 and the level of protein phosphorylation decreased in the treatment group, especially in the high-dose Huangqi Jianzhongtang group, with statistically significant differences (P<0.05). Conclusion:Huangqi Jianzhongtang can improve the survival condition of rats with spleen stomach deficiency cold type gastric ulcer, and its mechanism may be related to the intervention of gastric mucosal immune barrier dysfunction mediated by JAK2/STAT3 pathway activation.

Effect of Qingzao Jiufei Tang on JAK2/STAT3 Signaling Pathway and Expression of Downstream Apoptosis-related Proteins in Lung Cancer / 中国实验方剂学杂志

Objective::To observe the effect of Qingzao Jiufei Tang on apoptosis of lung cancer, Janus protein tyrosine kinase 2/signal transducers and transcriptional activator protein 3 (JAK2/STAT3) signaling pathway, as well as the expressions of downstream apoptosis-related proteins Bcl-2-associated X (Bax) and Cyclin D1. Method::Totally 50 male C57BL/6J mice were randomly divided into five groups: chemotherapy group (CTX), model group, high-dose Qingzao Jiufei Tang group, middle-dose Qingzao Jiufei Tang group and low-dose Qingzao Jiufei Tang group, with 10 mice in each group. The model of lung cancer was established by injecting Lewis lung cancer cells into the right axillary of mice. High-dose, middle-dose and low-dose Qingzao Jiufei Tang groups were orally given drugs (11, 5.5, 2.75 g·kg-1·d-1) two weeks before the modeling. Chemotherapy group was administered intraperitoneally at a dose of 50 mg·kg-1·(2 d)-1, while model group was administered intragastrically with the equal volume of normal saline. After inoculation, the mice in each group were continued to be administered. Two weeks later, the mice in each group were killed, and the tumors were collected. Then the JAK2 protein phosphorylation level was detected by immunohistochemistry (IHC). STAT3, Bax and Cyclin D1 protein expression levels were detected by Western blot, and apoptosis of lung cancer cells was observed by transmission electron microscopy. Result::Compared with model group, the phosphorylation levels of JAK2 and STAT3 protein in lung cancer cells were significantly decreased, the expression of Bax protein was significantly increased, and the expression of Cyclin D1 protein was significantly decreased in high-dose Qingzao Jiufei Tang group, middle-dose Qingzao Jiufei Tang group and chemotherapy group, with statistically significant differences (P<0.05, P<0.01). The results of transmission electron microscopy showed significant apoptotic phenomena in high-dose Qingzao Jiufei Tang group, middle-dose Qingzao Jiufei Tang group, low-dose Qingzao Jiufei Tang group and chemotherapy group compared with the model group. Conclusion::Qingzao Jiufei Tang had an obvious effect in promoting the apoptosis of lung cancer cells. Its mechanism may be related to the inhibition of the phosphorylation of JAK2 and STAT3 protein, the promotion of its downstream Bax protein expression and the inhibition of its downstream Cyclin D1 protein expression.

Inhibition of Sijunzi Decoction extract on human breast cancer cells MDA-MB-468 / 中草药

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.

β-arrestin 1 facilitates acetylation of IL-6 gene promoter to affect proliferation and apoptosis of non-small cell lung cancer cells / 肿瘤

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.

Effect of recombinant human IL-17A on growth of colon cancer cells and its mechanism / 吉林大学学报(医学版)

Objective: To explore the effect of recombinant human interleukin-17AI (IL-17A) on the growth of colon cancer cells, and to investigate the related mechanism. Methods: The colon cancer SW480 cells were divided into control group and experimental group. The SW480 cells in control group were untreated and the SW480 cells in experimental group were added with 50 fig • L_1IL-17A. The proliferation abilities of SW480 cells in two groups were detected by CKK-8 method. The levels of IL-17A in the SW480 cells in two groups were detected by ELISA, and the expression levels of signal transducers and activators of transcription 3 (STAT3) and p-signal transducers and activators of transcriptions 3 (p-STAT3) were examined by Western blotting methed. Results: Compared with control group, the proliferation ability of the SW480 cells in experimental group was increased (P < 0 . 05). The level of IL-17A in the SW480 cells in experimental group was significantly higher than that in control group (P< 0. 01). Compared with control group, the expression levels of STAT3 and p-STAT3 proteins in the SW480 cells in experimental group were significantly higher than those in control group (P < 0 . 01). Conclusion: Recombinant protein IL-17A can stimulate the growth of colon cancer SW480 cells, which may be related to the activation of STAT3 signaling pathway.

Research progress of janus kinase - Signal transducers and activators of transcription signal transduction pathway and neurogenesis / 解剖学报

Janus kinase-signal transducers and activators of transcription (JAK-STAT) signal transduction pathway is related to cellular biological activities and occurrence and development of many diseases. In recent years, JAK-STAT signal transduction pathway has been found in the central nervous system to regulate neurodegenerative diseases and nerve regeneration after nerve injury: however, promoting endogenous neurogenesis as a new direction of nerve regeneration research is also closely related to the positive or negative regulation of JAK-STAT signal transduction pathway. This article reviews the research progress of JAK-STAT signal transduction pathway, neurogenesis, and JAK-STAT signal transduction pathway regulating neurogenesis.

JAK/STAT3 Signaling Pathway and Its Inhibitors in Tumor Therapy / 中国药学杂志

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.

miR-7 inhibits activation of astrocytes derived from rats via silencing of EGFR / 中国药理学通报

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.

The mechanism of interleukin-22 induced the secretion of vascular endothelial growth factor A in gastric cancer cell line AGS / 国际检验医学杂志

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 .

Molecular mechanism of calpain in regulating macrophage polarization mediated by NF-κB/STAT3 signaling pathways / 复旦学报（医学版）

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.

Effect of left atrial enlargement on expression of the angiotensinⅡ, signal transducers and activators of transcription 3 and Rac GTPase activating protein 1 signaling pathways in patients with persistent atrial fibrillation and rheumatic heart disease / 中国胸心血管外科临床杂志

@#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.

A novel TNS4 mutant in prostate cancer cells and its mechanism / 肿瘤

Objective: To investigate the expression and mutation status of tensin family member 4 (TNS4) gene in various prostate cancer cell lines, and to explore its potential molecular mechanism. Methods: The expression of TNS4 gene in several prostate cancer cell lines was detected by RT-PCR. The full-length cDNA of TNS4 gene was amplified for the restriction endonuclease hydrolysis and DNA sequencing analysis. Meantime, the recombinant plasmids carrying wild- or mutant-type TNS4 gene were constructed and transfected into prostate cancer PC-3 cells, then the effects of TNS4 over-expression on the mRNA and protein expressions as well as the localization of signal transducer and activator of transcription 1 (STAT1) were detected by RT-PCR, Western blotting and immunofluorescence microscopy, respectively. Results: As compared with benign prostatic hyperplasia BPH1 cells, TNS4 gene was lowly expressed in prostate cancer cell lines DU145, LNCaP and 22Rv1 (all P 0.05). Additionally, the over-expression of mutant TNS4 significantly promoted nuclear translocation of STAT1 protein (P < 0.01). Conclusion: TNS4 gene is lowly expressed in prostate cancer cell lines, and there is a novel TNS4 mutant (S143P). TNS4 over-expression can up-regulate the expression of STAT1 protein, furthermore the mutant TNS4 can alter the location of STAT1 protein.

Validation and application a cell-based screening model for IL-6/JAK/STAT3 inhibitor / 药学学报

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.