Protein Expression Of p-JAK2 Research Articles

Jiajiejian gel ameliorates thyroid nodules through regulation of thyroid hormones and suppression of the (IL-6, TNF-α, IL-1β)/JAK2/STAT3/VEGF pathway

BackgroundThe high incidence of thyroid nodules and their rapid growth in recent years have become an important issue affecting public health. Traditional Chinese medicine (TCM) external treatments have unique advantages in treating this disease, but the currently available external preparations are relatively few and the therapeutic mechanism is unclear. Jiajiejian gel (JJJG) is a TCM external preparation developed by our team for the thyroid nodule treatment, which has been preliminarily proven to be safe and effective in clinical practice.ObjectiveThe current study was aimed to elucidate the therapeutic effects and the underlying mechanisms of JJJG on thyroid nodules in rats.MethodsThe contents of paeonol and forsythoside A in JJJG were determined by HPLC. The thyroid nodules rat model was established through oral gavage of 0.1% propylthiouracil (PTU) for 6 weeks and meanwhile the rats were treated with external JJJG (0.26, 0.52, 1.04 g/kg). Subsequently, the therapeutic effect of JJJG was observed by means of ultrasonic examination, morphology observation, organ coefficients determination and histopathological analysis. Mechanismlly, the levels of FT3, FT4 and TSH in serum were measured and transcriptomics methods were used to analyse and screen the key targets and pathways of alleviating thyroid nodules by JJJG. Further, gene and protein expression levels of key factors in the pathways were measured and validated using quantitative real-time PCR, ELISA, western blotting and immunofluorescence, so as to clarify the therapeutic mechanism.ResultsThe contents of the paeonol and forsythoside A were 1.160 and 0.608 mg/g, respectively. JJJG reduced thyroid swelling, improved nodular lesions, decreased thyroid coefficients, and inhibited abnormal nodular hyperplasia of follicular epithelial cells. In terms of mechanism, JJJG significantly increased the levels of FT3 and FT4 and decreased TSH level in serum (P < 0.05). Transcriptomics suggested that the (IL-6, TNF-α, IL-1β)/JAK2/STAT3/VEGF pathway may be one of the key mechanisms in the treatment of thyroid nodules by JJJG. Further validation experiments demonstrated that JJJG significantly reduced the mRNA expression and protein content of IL-1β, IL-6 and TNF-α in thyroid tissue, as well as the mRNA expression of JAK2, STAT3 and VEGF and the protein expression of p-JAK2/JAK2, p-STAT3/STAT3 and VEGF (P < 0.05).ConclusionThis study indicates that JJJG efficiently ameliorates thyroid nodules by regulating the levels of FT3, FT4 and TSH in serum and suppressing (IL-6, TNF-α, IL-1β)/JAK2/STAT3/VEGF pathway in thyroid tissue, providing a potential therapeutic approach for thyroid nodules.

Effects of Ruxolitinib on Immune Checkpoint Molecule Expression in JAK2 V617F-Positive Cells.

This study aimed to explore the clinical significance of ruxolitinib and its effects on the proliferation and apoptosis of human erythroleukemia (HEL) cells and the expression of immune checkpoint molecules programmed death-1 (PD-1), programmed death-ligand 1 (PD-L1), and regulatory T cells (Tregs) in HEL cells and JAK2 V617F-positive patients with myeloproliferative neoplasms (MPNs). JAK2 V617F-positive patients with MPNs admitted to the Baoding No. 1 Hospital from January 2016 to September 2023 were recruited, including 30 patients for the newly diagnosed group and 10 for the treatment group. Additionally, 15 healthy volunteers were selected as the control group. JAK2 V617F mutation was detected by using fluorescence quantitative PCR, and the expression levels of phosphorylated JAK2 (p-JAK2), PD-1, and PD-L1 in fresh bone marrow were examined by immunohistochemistry. HEL cells were treated with ruxolitinib at different concentrations (0, 50, 100, 250, 500, and 1,000 nmol/L). Cell viability was detected by CCK-8 assay. The mRNA expression levels of JAK2, PD-1, and PD-L1 were determined by using fluorescence quantitative PCR. The protein expression of p-JAK2 was detected by Western blot and those of PD-1 and PD-L1 were evaluated by flow cytometry. The expression of PD-1, PD-L1, and Tregs after the 48-hour co-culture of primary bone marrow cells and HEL cells were also analyzed by flow cytometry. In the newly diagnosed group, the bone marrow myeloid cells highly expressed p-JAK2, PD-1, and PD-L1. The Tregs expression in their peripheral blood increased and was significantly higher than those in the treatment and control groups (all p < 0.05). Ruxolitinib at different concentrations could inhibit the proliferation of HEL cells and was positively correlated with treatment time and dose. Additionally, ruxolitinib could reduce p-JAK2, PD-1, and PD-L1 expression in HEL cells and Tregs expression. Ruxolitinib reduces the expression of p-JAK2, PD-1, and PD-L1 in JAK2 V617F-positive cells by specifically inhibiting the JAK2 signaling pathway, thereby suppressing the progression of MPNs.

Effects of Ruxolitinib on Myeloproliferative Neoplasms via the Negative Regulators.

We evaluated the JAK2V617F mutation and p-JAK2, SOCS-1, SHP-1 expression in JAK2V617F positive myeloproliferative neoplasms (MPNs) patients and the role of JAK/STAT pathway in human erythroleukemia (HEL) cells, which had JAK2V617F mutation. Protein expression of p-JAK2, SOCS-1, SHP-1 in bone marrow biopsies (BMBs) were detected by immunohistochemical staining methods. Cell apoptosis and cell cycle were detected by flow cytometry and Caspase 3/7 assay kits. 1. The p-JAK2, SOCS-1, and SHP-1 expressions were significantly different between JAK2V617F positive MPN and control patients (p < 0.01); 2. After being treated for 3 months, the p-JAK2, SOCS-1, and SHP-1 expressions were significantly different compared with newly diagnosed patients (p < 0.01). 3. HEL cell viabilities were significantly different after being treated with different concentrations of ruxolitinib. Ruxolitinib had a significant effect on the cell apoptosis, viability, and the protein activity of caspase-3 and -7 of HEL cells. 3. The mRNA and protein expressions of JAK2 and the protein expression of p-JAK2 were gradually decreased (p ＜ 0.01, p ＜ 0.05), while the mRNA and protein expressions of SOCS1 and SHP1 were gradually increased (all p ＜ 0.01).

Gastrodin ameliorates Concanavalin A-induced acute hepatitis via the IL6/JAK2/STAT3 pathway

Aims Gastrodin, the main active ingredient of Gastrodia elata Blume, has been shown to protect against many inflammatory diseases. Our study aimed to investigate the anti-inflammatory role of gastrodin in concanavalin A (ConA)-induced acute hepatitis in mice and to explore its precise mechanism. Methods C57BL/6 mice were administered with gastrodin (50 or 100mg/kg) for 3 days prior to intravenous injection of ConA to induce acute autoimmune hepatitis (AIH). Serum aminotransferases levels and cytokine levels were measured. Liver tissue histology was conducted to assess the degree of liver injury. Splenocytes pretreated with gastrodin were stimulated with ConA to observe splenocyte proliferation. Results Gastrodin greatly reduced the level of serum aminotransferases, inflammatory cytokine such as IL-6 and TNF-α and histopathological damage in ConA-induced hepatitis. Besides, gastrodin had an inhibitory effect on liver apoptosis, and autophagy. Furthermore, gastrodin inhibited the proliferation of splenocytes in vitro. The protein expression of p-JAK2 and p-STAT3 was markedly affected by gastrodin pretreatment. Conclusions The present study indicated that gastrodin pretreatment exerted protective effects against ConA-induced acute hepatitis, partly through the inhibition of the IL6/JAK2/STAT3 pathway. Further studies are recommended to determine the potential therapeutic role of gastrodin in acute AIH.

LINC00870 regulates Th1/Th2 via the JAK/STAT pathway in peripheral blood mononuclear cells infected with Mycobacterium tuberculosis

Long, noncoding RNAs reportedly play vital roles in tuberculosis (TB). For example, upregulation of LINC00870 has been observed in tuberculosis, though its role and underlying mechanism remains unclear. In this study, we investigated the expression and effect of LINC00870 in Mycobacterium tuberculosis (MTB) infection by comparing MTB-infected peripheral blood mononuclear cells (PBMCs) with controls. The results showed LINC00870 was significantly increased in MTB infected PBMCs. Additionally, LINC00870 overexpression inhibited Th1-secreted cytokines while promoted Th2-secreted cytokine in MTB-infected PBMCs. Furthermore, LINC00870 promoted p-STAT5 and p-JAK2 protein expression, thus activating JAK/STAT signaling in MTB-infected PBMCs. Sputum and plasma samples were obtained from TB, latent tuberculosis infection (LTBI) patients and healthy controls. The qRT-PCR results showed higher levels of LINC00870 in the sputum and plasma from TB and LTBI patients compared to healthy controls. In addition, LINC00870 were decreased in both TB and LTBI patients after three month of therapy, respectively. The results showed a correlation between LINC00870 inhibition and Th1/Th2, as well as JAK/STAT signaling pathway in PBMCs from active TB patients. In conclusion, higher levels of LINC00870 in tuberculosis might be associated with Th1/Th2-related immune responses by activating JAK/STAT signaling. LINC00870 thus may be a novel biomarker for diagnosing and treating tuberculosis.

Effect of matrine on JAK2/STAT3 signaling pathway and brain protection in rats with cerebral ischemia-reperfusion.

Ischemic encephalopathy is a common clinical disease. The main treatment goal is to achieve vascular recanalization. However, after vascular recanalization, the reperfusion of fresh blood can change local cell metabolism, thus adversely affecting cell structure and function, which can result in reperfusion injury. To explore the effect of matrine intervention of different concentrations on JAK2/STAT3 signaling pathway and brain protection in rats with cerebral ischemia-reperfusion. Healthy male Sprague Dawley rats were divided into a blank control group (20 rats), a model group (80 rats) and a sham group (20 rats). In the model group, the middle cerebral artery was occluded with suture method to establish cerebral ischemia-reperfusion model rats, which were subdivided into cerebral ischemia-reperfusion group, and 5, 10 and 20 mg/kg matrine groups, with 20 rats in each group. Indicators including neurological function score, brain infarct size, brain water content, lactic dehydrogenase activity, protein expressions of p-JAK2 and p-STAT3, as well as superoxide dismutase activity and malondialdehyde content were evaluated. Compared with cerebral ischemia-reperfusion group, all the indicators were significantly improved in the 3 matrine treatment groups in a dose-dependent manner, and protein expressions of p-JAK2 and p-STAT3 in the brain tissue and brain cell apoptosis rate were decreased with the increase of matrine concentration (all p < 0.05). Matrine can significantly ameliorate the neurological function and brain edema of rats with cerebral ischemia-reperfusion, and improve superoxide dismutase, malondialdehyde and lactic dehydrogenase levels in the brain tissue and brain cell apoptosis rate. The mechanism of matrine may be related to the inhibition of abnormal JAK2/STAT3 signaling pathway activation.

JLX001 improves myocardial ischemia-reperfusion injury by activating Jak2-Stat3 pathway

AimsTo investigate the preclinical pharmacodynamics and mechanism of JLX001 against myocardial ischemia reperfusion (MI/R) for clinical application. Materials and methodsIn vivo, SD rats were given intragastric administration for 5 days, and the MI/R model was established by ligating/releasing the left anterior descending coronary artery. In vitro, the oxygen-glucose deprivation/reperfusion (OGD/R) model was established after the drug was pre-incubated for 24 h in H9C2 cells. The infract size was determined by TTC staining. Left ventricular function of MI/R rats was detected by echocardiography. The level of histopathological score was determined by hematoxylin-eosin (HE) staining. The level of superoxide dismutase (SOD), malondialdehyde (MDA), creatine kinase (CK), lactic dehydrogenase (LDH), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) were determined by relevant kits. The level of apoptosis was measured by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and Hoechst staining. The expression of p-Jak2, p-Stat3, Bax, Bcl-2, TNF-α, IL-1β protein were determined by western blot. Key findingsJLX001 can significantly improve left ventricular function, reduce myocardial infract size, histopathological score, the level of MDA, CK, LDH, TNF-α, IL-1β and the expression of Bax protein, significantly increase the activity of SOD, Bcl-2 protein expression, p-Jak2 protein expression, p-Stat3 protein expression in rat heart tissues and H9C2 cells. These effects can be reversed by AG490 which is a specific inhibitor of Jak2-Stat3 pathway. SignificanceJLX001 can alleviate MI/R injury by inhibiting myocardial apoptosis, inflammation, and oxidative stress via Jak2-Stat3 pathway in vivo and in vitro.

Ephedrine causes retinal damage in SD rats associating with JAK2/STAT3 pathway

Purpose Ephedrine has various side effects in the cardiovascular and nervous systems. However, the cellular mechanism of toxicity remains unknown, specifically on the retina. This study was to investigate effects of ephedrine on the retina and explore the underlying mechanisms. Methods Sprague Dawley rats were treated with ephedrine (n = 10) or saline (n = 10) by oral gavage for seven days. The retinal morphology was evaluated by Toluidine blue staining. Apoptosis-related markers were detected in the retinal lysate. Enzyme-linked immunosorbent assays were used to measure neurotransmitters and oxidative stress markers. Real-time PCR and western blot were used to measure gene and protein expression, respectively. Results Our results demonstrated that ephedrine induced apoptosis in the retina, increased dopamine level as well as oxidative stress, and down-regulated the Jak2/Stat3 gene expression as well as protein expression of p-JAK2/p-STAT3. Conclusions Our study indicated that ephedrine treatment caused retinal damage in SD rats, which may be associated with the JAK2/STAT3 pathway.

The Effects of Sidt2 on the Inflammatory Pathway in Mouse Mesangial Cells.

In patients with chronic kidney disease, the abnormal activation of inflammatory pathways is usually an important factor leading to renal fibrosis and further deterioration of renal function. Finding effective intervention targets of the inflammatory signaling pathway is an important way to treat chronic kidney disease. As a newly discovered lysosomal membrane protein, the correlation between SID1 transmembrane family member 2 (Sidt2) and the inflammatory signaling pathway has not been reported. The aim of this study was to investigate the effect of Sidt2 on inflammation by inhibiting the expression of the Sidt2 gene in a mouse mesangial cell line mediated by a lentiviral CRISPR/Cas9 vector. Hematoxylin and eosin staining and microscopy found that the mesangial cells lost their normal morphology after inhibiting the expression of Sidt2, showing that the cell body became smaller, the edge between the cells was unclear, and part of the nucleus was pyknotic and fragmented, appearing blue-black. The expressions of IKK β, p-IKK α/β, NF-κB p65, p-NF-κB p65, p-IκBα, IκBα, and TNF-α in the NF-κB pathway of the Sidt2−/− group were higher than those of the Sidt2+/+ group. p-Jak2 and IL6 increased in the Jak/Stat pathway, and p-ERK and p-P38 increased in the MAPK pathway. The expressions of IKK β, p-IKK α/β, NF-κB p65, p-NF-κB p65, p-IκBα, IκBα, and TNF-α in the NF-κB pathway of the Sidt2+/++LPS group were significantly higher than those in the Sidt2+/+ group. The expressions of IKK β, p-IKK α/β, NF-κB p65, p-NF-κB p65, p-IκBα, IκBα, and TNF-α in the Sidt2−/−+LPS group were higher than those in the Sidt2−/− group. The expressions of p-IKK α/β, NF-κB p65, p-NF-κB p65, p-IκBα, IκBα, and TNF-α in the Sidt2−/−+LPS group were higher than those in the Sidt2+/++LPS group. In the Jak/Stat pathway, the protein expressions of p-Jak2 and IL6 in the Sidt2+/++LPS group were higher than those in the Sidt2+/+ group. The expressions of p-Jak2 and IL6 in the Sidt2−/−+LPS group were higher than those in the Sidt2−/− group. The expressions of p-Jak2 and IL6 in the Sidt2−/−+LPS group were higher than those in the Sidt2+/++LPS group. The expressions of p-JNK, p-ERK, p-P38, and ERK in the MAPK pathway in the Sidt2+/++LPS group were higher than those in the Sidt2+/+ group. The expressions of p-JNK, p-ERK, p-P38, and ERK in the Sidt2−/−+LPS group were higher than those in the Sidt2−/− group. The expressions of p-JNK, p-ERK, p-P38, and ERK in the Sidt2−/−+LPS group were higher than those in the Sidt2+/++LPS group. These data suggested that deletion of the Sidt2 gene changed the three inflammatory signal pathways, eventually leading to the damage of glomerular mesangial cells in mice.

Edaravone attenuates myocyte apoptosis through the JAK2/STAT3 pathway in acute myocardial infarction

Basic and clinical studies have demonstrated that the free radical scavenger edaravone has cytoprotective effects on acute myocardial infarction (AMI) but the underlying mechanism is not fully understood. The aim of this research is to explore the effect of edaravone on the apoptotic process involving the JAK2/STAT3 signaling pathway. AMI in rats was established by left anterior descending coronary artery ligation. Two hours after AMI model established rats were treated with edaravone, edaravone plus AG490, physiological saline, respectively. We detected antioxidant effects by reduced glutathione (GSH), glutathione S-transferase (GST), and Glutathione Peroxidase (GSHPx) Activity. The expressions of t-JAK2, p-JAK2, t-STAT3, p-STAT3 and cleaved caspase-3 were examined by western blot. The mRNA levels for Bcl-2, Bax, Fas, and FasL were measured by RT-PCR and apoptosis was assessed by TUNEL. Edaravone significantly improved hemodynamics after AMI (p < 0.05) and reduced the total infarct volumes (p < 0.05). Compared with Sham rats, the mRNA of Bax, Fas, and FasL increased in different degrees in the AMI group, however, the mRNA of Bcl-2 and the ratio of Bcl-2/Bax decreased, especially the myocardial apoptosis index significantly increased in AMI hearts (all p < 0.05). After treatment with edaravone, the mRNA levels of Bcl-2 and the ratio of Bcl-2/Bax significantly upregulated whereas Bax, Fas, FasL apparently decreased, and the protein expressions of p-JAK2 and p-STAT3 dramatically increased (p < 0.05). In addition, cotreatment with JAK2 inhibitor AG490 abolished the effects of edaravone. We conclude that edaravone attenuated myocardial apoptosis induced by AMI via JAK2/STAT3 signaling pathway.

Losartan promotes myocardial apoptosis after acute myocardial infarction in rats through inhibiting Ang II-induced JAK/STAT pathway.

To study the pro-apoptotic effect of Losartan on myocardial cells after acute myocardial infarction (AMI) in rats. Before intervention, a total of 48 male Wistar rats were randomly divided into the Sham group (n=12), AMI group (n=12), 5 mg/kg Losartan group (n=12) and 1 mg/kg AG-490 group (n=12). The rats in the Sham group and AMI group received gavage with normal saline, those in the Losartan group received gavage with Losartan for 7 d and those in the AG-490 group were intravenously injected with AG-490 at 30 min before the operation. At 4 d after drug administration, the anterior descending coronary artery was ligated to establish the AMI model in the AMI group and Losartan group, while the same operation was performed, and the anterior descending coronary artery was only threaded in the Sham group. The rats were sacrificed at 24 h after operation. Then, the myocardial apoptosis was detected via terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, and the protein expressions of Janus kinase 2 (JAK2), the signal transducer and activator of transcription 3 (STAT3), the B-cell lymphoma-2 (Bcl-2) and the Bcl-2 associated X protein (Bax) were detected via immunohistochemistry and Western blotting. Moreover, the myocardial cells of rats were incubated with angiotensin II (Ang II) at the same concentration at different time points and blocked with Losartan. Finally, the changes in protein expressions of p-JAK2, p-STAT3, Bax, and Bcl-2 were detected via Western blotting. Losartan treatment could increase the number of apoptotic myocardial cells after AMI in rats. In Losartan group, the protein expressions of JAK2, STAT3, and Bcl-2 declined, while the protein expression of Bax was increased, and the Bax/Bcl-2 ratio was also increased, which are consistent with the conditions under the treatment with the JAK-STAT pathway inhibitor AG-490. With the prolonged time of stimulation (5 min, 30 min, 2 h, and 24 h) in myocardial cells using 1.0×10-6 mol/L Ang II, the protein expressions of p-JAK2 and p-STAT3 were increased and reached the peak at 24 h. After the application of Losartan, the increased protein expressions of p-JAK2 and p-STAT3 returned to normal levels, and the protein expression of Bax was increased, while that of Bcl-2 was decreased. Losartan promotes myocardial apoptosis after AMI in the rats through inhibiting the Ang II-induced JAK/STAT pathway.

MiR-203 regulates proliferation and apoptosis of ovarian cancer cells by targeting SOCS3.

Cytokine signal transduction inhibitor 3 (SOCS3) negatively regulates Janus kinases (JAK) - signal transducer and activator of transcription (STAT) pathway. Bioinformatics analysis revealed a targeted relationship between miR-203 and SOCS3 mRNA. This study investigated the role of miR-203 in ovarian cancer cell proliferation and apoptosis. Ovarian cancer tissues and adjacent tissues were collected to detect the expression of miR-203 and SOCS3. Ovarian cancer HO8910 cells were divided into miR-NC group, miR-203 inhibitor group, and miR-203 mimic group followed by the analysis of the expression of miR-203 and SOCS3 mRNA by quantitative Reverse Transcription-PCR (qRT-PCR), protein expression of p-JAK2 and p-STAT3 by Western blot, cell apoptosis by flow cytometry, and proliferation by 5-Ethynyl-2'-deoxyuridine (EdU) staining chronologically. Compared with adjacent tissues, miR-203 expression was significantly increased in tumor tissues and SCOS3 mRNA expression was decreased. Compared with those with lower miR-203 expression, the prognosis of patients with higher expression of miR-203 was significantly worse. There was a targeted regulatory relationship between miR-203 and SOCS3 mRNA. Compared with IOSE80 cells, miR-203 expression in HO8910 and SKOV3 cells was increased, and its expressions of SOCS3 mRNA and protein were decreased. Compared with miR-NC group, the transfection of miR-203 inhibitor significantly increased SOCS3 expression, and decreased the expression of p-JAK2 and p-STAT3 protein. We draw the conclusion that miR-203 increased cell apoptosis and decreased cell proliferation. However, opposite results were observed after the transfection of miR-203 mimic. Abnormal miR-203 and SOCS3 expression are related to the pathogenesis of ovarian cancer. MiR-203 affects the proliferation of JAK-STAT pathway and regulates the proliferation and apoptosis of ovarian cancer cells by targeting the inhibition of SOCS3 expression.

Effect of propofol on myocardial ischemia/reperfusion injury in rats through JAK/STAT signaling pathway.

The aim of this study was to investigate the influences of propofol on myocardial ischemia/reperfusion injury in rats through the Janus kinase/signal transducers and the activators of transcription (JAK/STAT) signaling pathway. A total of 48 Sprague-Dawley (SD) rats were randomly divided into four groups, including: the sham-operation group (n=12), the model group (n=12), the propofol group (n=12) and the inhibitor group (n=12). The rats in the sham-operation group only received thoracotomy, without the modeling of the ischemia/reperfusion injury. The model of myocardial ischemia/reperfusion injury was established in the rats of the model group, and the rats were given normal saline for intervention. The rats in the propofol group were utilized to prepare the model of myocardial ischemia/reperfusion injury and were intervened with propofol. Meanwhile, the rats in the inhibitor group received intervention with AG490 after the establishment of myocardial ischemia/reperfusion injury model. Immunohistochemistry was applied to detect the expressions of B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax). Western blotting was utilized to measure the relative protein expressions of phosphorylated JAK2 (p-JAK2) and p-STAT3. The messenger ribonucleic acid (mRNA) expressions of Bax and Bcl-2 were determined via quantitative Polymerase Chain Reaction (qPCR). Furthermore, cell apoptosis was examined using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Immunohistochemistry results showed that compared with the sham-operation group, the positive expression of Bax remarkably increased (p<0.05), while Bcl-2 notably decreased (p<0.05) in the model group, propofol group, and inhibitor group. The propofol group and inhibitor group showed a significant lower positive expression of Bax (p<0.05) and evident higher positive expression of Bcl-2 (p<0.05) when compared with the model group. However, there were no significant differences in the positive expressions of Bax and Bcl-2 between the propofol group and inhibitor group (p>0.05). According to the results of Western blotting, the relative protein expression levels of p-JAK2 and p-STAT3 proteins were remarkably elevated in the model group, propofol group and inhibitor group in comparison with those in the sham-operation group (p<0.05). Propofol group and inhibitor group exhibited remarkably lower protein expression levels of p-JAK2 and p-STAT3 compared with the model group (p<0.05). However, no significant differences were observed in the protein expressions of p-JAK2 and p-STAT3 between propofol group and inhibitor group (p>0.05). The results of qPCR manifested that the mRNA expression of Bax was notably higher (p<0.05), whereas Bcl-2 was significantly lower (p<0.05) in the model group, propofol group and inhibitor group than those of the sham-operation group. Compared with the model group, the mRNA expression of Bax was evidently declined (p<0.05), while Bcl-2 was significantly elevated (p<0.05) in the propofol group and inhibitor group. Meanwhile, there were no evident differences in the mRNA expressions of Bax and Bcl-2 between the propofol group and inhibitor group (p>0.05). Subsequent TUNEL assay indicated that the model group, propofol group, and inhibitor group showed remarkably higher apoptosis rate than the sham-operation group (p<0.05). Moreover, the apoptosis rate was remarkably reduced in the propofol group and inhibitor group in comparison with the model group (p<0.05). However, no significant difference was observed in the apoptosis rate between propofol group and inhibitor group (p>0.05). Propofol inhibits myocardial cell apoptosis after myocardial ischemia/reperfusion injury by repressing the JAK/STAT signaling pathway.

The inhibition of miR-126 in cell migration and invasion of cervical cancer through regulating ZEB1

BackgroundCervical cancer is a malignancy that’s common in female with high incidence and mortality worldwide. MicroRNAs (miRNAs) act a pivotal part in human cancer development. Our aim was to investigate the effect of miR-126 on cervical cancer and its underlying molecular mechanism.ResultsFirstly, RT-qPCR assay revealed that the expression of miR-126 was significantly downregulated in cervical cancer tissues and cell lines, compared with that in normal adjacent tissues and normal cervical epithelial cell line (Ect1/E6E7), respectively. Then, ZEB1 was verified as a target of miR-126 by using luciferase reporter assay. Inversely, the expression of ZEB1 was markedly upregulated in tumor tissues, and its mRNA level was negatively regulated by miR-126 expression in SiHa and Hela cells. Moreover, the capability of cell proliferation, migration and invasion was analyzed by CCK-8, wound healing assay and transwell assay, respectively. The results demonstrated that overexpression of miR-126 inhibited SiHa and Hela cell proliferation, migration and invasion, while ZEB1 abolished the inhibition induced by miR-126. Additionally, miR-126 suppressed MMP2 and MMP9 in mRNA and protein levels, as well as inhibited the protein expression of p-JAK2 and p-STAT3 in both SiHa and Hela cells, while ZEB1 rescued miR-126-induced suppression.ConclusionmiR-126 functions as a tumor suppressor in cervical cancer cells in vitro, which inhibits the proliferation, migration and invasion by suppressing MMP2, MMP9 expression and inactivating JAK2/STAT3 signaling pathway through targeting ZEB1, suggesting that miR-126 might be a novel potential target for the diagnosis and treatment of patients with cervical cancer.

PO-183 Prolactin activation of JAK2/STAT3 signalling pathway through GHR in NSCLC

IntroductionMilk secretion is stimulated by prolactin, a hormone secreted from anterior pituitary gland. It is known that prolactin could lead breast and prostate cancer progression. Recent reports point out the concentration of serum prolactin in non small cell lung cancer (NSCLC) patients were significantly higher than health people. The patients with higher concentration of serum prolactin have lower survival rate than normal level on serum prolactin patients. PRL receptor (PRLR) expression level, however, was not relatived to paitients’ survival rate. It is unclear whether prolactin promoted lung cancer progression and the action mechanism of prolactin in lung cancer.Material and methodsCells were seeded in 96 well plate with complete medium. After 24 hour of plating, cells were serum starved for 16 hour and then treatment with PRL. MTT assay was applied for cell proliferation. The protein level of JAK2/STAT3 and VEGF were determined by western blot. The mRNA level was measured by quantitative real time polymerase chain reaction (q-PCR).Results and discussionsOur data show that prolactin promoted cell proliferation in NSCLC cells. Expressed growth hormone receptor (GHR), not prolactin receptor (PRLR), was observed in all NSCLC cell lines. Increased expression of p-JAK2 and p-STAT3 were found in cells treated with prolactin. Treatment with prolactin also increased STAT3-regulated downstream gene VEGF mRNA and protein expressions. In contrast, the protein expression of p-JAK2 was decreased after inhibition of GHR.ConclusionProlactin binds to and activates GHR downstream signalling pathway JAK2/STAT3. Activated STAT3 translocates to nucleus and increases downstream gene VEGF transcription activity. Also, prolactin promotes cell proliferation and VEGF expression in NSCLC cells.

JAK2/STAT3 Pathway Mediates Protection of Metallothionein Against Doxorubicin-Induced Cytotoxicity in Mouse Cardiomyocytes.

Doxorubicin (Dox) is one of the most important anticancer agents; however, its clinical application is limited by its severe cardiotoxicity. In our previous study, we found that the gene expression levels of the Janus-activated kinase/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway were different between MT(-/-) cardiomyocytes and MT(+/+) cardiomyocytes when they were treated with Dox. Thus, this study was intended to investigate the role of JAK2/STAT3 pathway in metallothionein (MT) protection of Dox-induced cardiotoxicity. Tyrphostin AG490 (α-cyano-(3,4-dihydroxy)-N-benzylcinnamide) is a synthetic protein tyrosine kinase inhibitor which at first has been considered as a specific JAK2 inhibitor and can inhibit the JAK2/STAT3 signaling pathway. In the present study, AG490 was used to assess the role of JAK2/STAT3 in MT protection against Dox-induced cardiotoxicity. The AG490 can attenuate the MT protection by increasing lactate dehydrogenase and the number of apoptotic cells. Interestingly, pretreated with AG490, MT(-/-) cardiomyocytes were more sensitive than MT(+/+) to Dox-induced cytotoxicity as measured by reactive oxygen species generation, lipid peroxidation, and protein carbonylation. Metallothionein 1 and MT-2 messenger RNA were upregulated by Dox, and AG490 decreased the protein expression of MT-1 and MT-2. After Dox treatment, the protein expression of p-Jak2 and p-Stat3 levels was significantly increased in MT(+/+) cardiomyocytes, suggesting that the JAK2/STAT3 pathway was partially involved in MT protection against Dox-induced cardiotoxicity.

Da-Huang-Fu-Zi-Tang attenuates liver injury in rats with severe acute pancreatitis

Ethnopharmacological relevanceDa-Huang-Fu-Zi-Tang (DHFZT) is a famous traditional Chinese prescription with strong anti-inflammatory effects. Our previous work found that DHFZT could act against pancreatic injury in rats with severe acute pancreatitis (SAP) via inhibiting the Janus kinase 2/signal transducers and activators of transcription 3 (JAK2/STAT3) signaling pathway in pancreatic tissues. Aim of the studyTo investigate the therapeutic effects of DHFZT on liver injury in SAP rats, and the effects on JAK2/STAT3 signaling in liver tissue and Kupffer cells (KCs). Materials and methodsFifty SD male rats were randomly divided into five groups: sham operation group (SO), SAP model group, DHFZT treatment groups (12, 24, and 48mg/kg body weight). The model of SAP was constructed by injecting sodium taurocholate (3.5%) into pancreatic and biliary ducts. One hour before constructing the model, DHFZT was perfused into the stomach. All rats were sacrificed after 24h following the operation; livers were examined with hematoxylin and eosin staining. The protein expression of pJAK2 and pSTAT3 in liver tissue was detected by immunohistochemical staining. The activity of ALT, IL-6 and TNF-α in serum was detected. KCs of each group were isolated. After culture for 4h, the protein expression of JAK2, pJAK2, STAT3 and pSTAT3, the mRNA expression of IL-6 and TNF-α in KCs were examined. ResultsSodium taurocholate induced liver injury concomitant with increased expression of pJAK2 and pSTAT3 in liver tissue and KCs. Pretreatment with DHFZT significantly attenuated liver injury induced by SAP, and concurrently, effectively lowered the serum ALT level. Furthermore, our studies showed that DHFZT obviously decreased the expression of pJAK2 and pSTAT3 in liver tissue and KCs. ConclusionsDHFZT could ameliorate liver injury in rats with SAP.

Therapeutic effect of 7, 3′-dimethoxy hesperetin on adjuvant arthritis in rats through inhibiting JAK2-STAT3 signal pathway

In our previous study, we have demonstrated that 7, 3′-dimethoxy hesperetin (DMHP), an active derivative of hesperidin, showed pro-apoptotic effect on synoviocytes in vitro. The present study was to investigate the potential therapeutic effect of DMHP on adjuvant arthritis (AA) in rat and its possible mechanisms. Freund's complete adjuvant was used to induce AA in rats. DMHP were administered intragastrically once a day from days 12 to 21 after AA induction. Secondary paw swelling, arthritis index, and pathological assessments were observed. IL-6 production in serum and IL-6 mRNA expression in synovium was detected by ELISA and real-time RT-PCR respectively. The expression of mRNA (JAK2, STAT3) and protein (JAK2, p-JAK2, STAT3, p-STAT3) in synovium were determined. We found that DMHP significantly inhibited hind paw swelling and arthritis index, and ameliorated pathological changes of ankle joint in AA rats. DMHP suppressed the level of IL-6 in serum and the expression of IL-6 mRNA in synovium of AA rats in a dose-dependent manner. DMHP apparently decreased mRNA expression of JAK2 and STAT3 as well as protein expression of p-JAK2 and p-STAT3 in the synovium of the AA rats. Correlation analysis indicated that p-JAK2 or p-STAT3 protein expression was highly correlated with joint damage severity. In conclusion, DMHP has a powerful therapeutic effect on AA in rats and its mechanisms might be partly related to inhibiting excessive activation of JAK2-STAT3 pathway.