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【Objective】 To investigate the regulatory effect of JAK2/STAT3 signaling pathway inhibitor AG490 on the functional phenotype of M2-like macrophages and its effects on proliferation, apoptosis, migration, and invasion of gastric cancer cells. 【Methods】 Human mononuclear cell line THP-1 was induced to differentiate into M0 macrophages by PMA in vitro. The M1-like phenotype was induced by LPS and IFN-γ, and M2-like phenotype was induced by IL-4 and IL-13, respectively, and identified by immunofluorescence labeling CD68, CD86 and CD206. The mRNA expressions of CD163, Arg1, CCL22, PPARγ, IL-10, IL-20 and TNF-α were determined by RT-qPCR. The expressions of key proteins in JAK2/STAT3 signaling pathway were detected by Western blotting. M2-like macrophages were treated with JAK2/STAT3 inhibitor (AG490) to observe the expression level of marker genes for M2 like phenotype. Macrophages were co-cultured with gastric cancer cells, and the effects of the macrophages on proliferation, migration, invasion, and apoptosis of gastric cancer cells were detected by CCK-8 method, healing assay, transwell intracellular Matrigel invasion assay, and flow cytometry. The xenograft tumor model of MKN45 gastric cancer in nude mice was prepared, and the tumor size and quality were observed for 20 days after the model was established. 【Results】 THP-1 cells were induced into M1-like macrophages and M2-like macrophages. M1-like marker (CD86) and M2-like marker (CD206) were identified by flow cytometry. The P38MAPK, JAK2, p-STAT3/STAT3 protein levels of M2-like macrophages treated with AG490 were significantly reduced. The mRNA expression levels of Arg1, CCL22, PPARγ and IL-10 were significantly reduced in the group of M2-like macrophages treated with AG490. Co-culture of M2-like macrophages with gastric cancer cells could promote gastric cancer cell viability, increase migration and invasion ability, and inhibit apoptosis. When the group of M2-like macrophages treated with AG490 was co-cultured with gastric cancer cells, the proliferation activity of MKN-45 cells and MGC823 was significantly lower than that in M2 group (1.047±0.062 vs. 1.426±0.076, 1.149±0.006 vs. 1.301±0.015). Compared to M2 group, the migration (100.0%±5.73% vs. 72%±3.85%) and invasion ability (100.0%±7.40% vs. 60%±6.54%) of MGC823 gastric cancer cells in AG490 treatment group were significantly reduced. The apoptosis rate of MGC823 cells in the AG490 treated group was significantly higher than that in M2 group (27.51%±0.70% vs. 20.82%±0.92%). In the nude mouse xenograft tumor model, the volume and weight of the transplanted tumor collected at day 20 were significantly lower in AG490 treated group than in M2 group (736.04±182.34 vs. 1 080.5±250.57)mm3, (0.64±0.11 vs. 0.87±0.17)g. 【Conclusion】 AG490 downregulates the activation level of JAK2/STAT3 signaling pathway in M2-like macrophages, inhibits M2-type polarization, partially reverses the cancer-promoting function of M2-like macrophages, inhibits proliferation, migration and invasion of gastric cancer cells, and induces apoptosis. The JAK2/STAT3 signaling pathway can be further studied as a potential therapeutic target for gastric cancer.
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Objective To investigate the influence of limonin on the malignant biological behavior of non-small cell lung cancer (NSCLC) cells by regulating the protein tyrosine kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway. Methods CCK-8 method was applied to detect the survival rate of A549 cells treated with different concentrations of limonin (0, 5, 10, 25, 50, 75, 100 μmol/L). A549 cells were separated into normal culture (NC) group, low-dose limonin group (treatment with 10 μmol/L limonin for 24 h), medium-dose limonin group (treatment with 25 μmol/L limonin for 24 h), high-dose limonin group (treatment with 50 μmol/L limonin for 24 h), coumermycin A1 group (treatment with 10 μmol/L JAK2 activator coumermycin A1+50 μmol/L limonin for 24 h), and AG490 group (treatment with 10 μmol/L JAK2 inhibitor AG490+50 μmol/L limonin for 24 h). Clone formation assay was applied to detect the clones of each group of cells. Transwell assay was applied to detect cell migration and invasion, and flow cytometry was applied to detect apoptosis. Western blot analysis was applied to detect the protein expression levels of JAK2, p-JAK2, STAT3, p-STAT3, E-cadherin, N-cadherin, and vimentin in each group. Results The viability of A549 cells decreased significantly in a limonin concentration-dependent manner (P < 0.05), with IC50 of 45.16±1.66 μmol/L. Concentrations of 10, 25, and 50 μmol/L were selected for subsequent experiments. The numbers of clones, migration, and invasion of A549 cells and the protein expression levels of IL-6, p-JAK2, p-STAT3, N-cadherin, and vimentin in the low-, medium-, and high-dose limonin groups significantly decreased, compared with those in the NC group, and the apoptosis rate and E-cadherin protein expression significantly increased (P < 0.05). The JAK2 activator coumermycin A1 attenuated the ability of limonin to inhibit the proliferation, migration, invasion, and other malignant biological behavior of A549 cells and attenuated the apoptosis ability. The JAK2 inhibitor AG490 enhanced the ability of limonin to inhibit the proliferation, migration, invasion, and other malignant biological behavior of A549 cells and enhanced the apoptosis ability. Conclusion Limonin can inhibit the malignant biological behavior of NSCLC cells, such as proliferation, migration, and invasion, by inhibiting the JAK2/STAT3 pathway.
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OBJECTIVE@#To investigate the effect of scutellarin (SCU) on proliferation, cell cycle and apoptosis of acute myeloid leukemia (AML) cells and its related molecular mechanism.@*METHODS@#Human AML HL-60 cells were cultured in vitro. The cells were treated with SCU at the concentration of 0, 2, 4, 8, 16, 32, 64 μmol/L, and the inhibition rate of cell proliferation was detected by CCK-8 method. Then HL-60 cells were treated with SCU at the concentration of 4, 8, 16 μmol/L, and the negative control group (NC group) was set. The cell cycle distribution and apoptosis were detected by flow cytometry, and the expression of cell cycle, apoptosis and JAK2/STAT3 pathway related proteins were detected by Western blot.@*RESULTS@#SCU significantly inhibited the proliferation of HL-60 cells in a concentration- and time-dependent manner(r =0.958,r =0.971). Compared with NC group, the proportion of cells in G0/G1 phase and apoptosis rate of HL-60 cells in 4, 8, 16 μmol/L SCU group were significantly increased, and the proportion of cells in S phase was significantly decreased (P <0.05). The relative protein expression levels of p21, p53, caspase-3 and Bax were significantly increased, while the relative protein expression levels of CDK2, cyclin E and Bcl-2 were significantly decreased (P <0.05). The ratio of p-JAK2/JAK2 and p-STAT3/STAT3 were significantly decreased (P <0.05). The changes of above-mentioned indexes were concentration dependent.@*CONCLUSION@#SCU can inhibit the proliferation of AML cells, induce cell cycle arrest and apoptosis, and its mechanism may be related to the regulation of JAK2/STAT3 signaling pathway.
Subject(s)
Humans , Apoptosis , Signal Transduction , Leukemia, Myeloid, Acute , HL-60 Cells , Cell Proliferation , Cell Line, TumorABSTRACT
Based on the Janus kinase 2/signal transducer and activator of transcription 3(JAK2/STAT3) signaling pathway, this study investigated the effect of medicated serum of Sparganii Rhizoma(SR) and Curcumae Rhizoma(CR) on the proliferation, apoptosis, migration, and secretion of inflammatory factors of ectopic endometrial stromal cells(ESCs). Specifically, human ESCs were primary-cultured. The effect of different concentration(5%, 10%, 20%) of SR-, CR-, and SR-CR combination-medicated serum, and AG490 solution(50 μmol·L~(-1)) on the proliferation of ESCs was detected by methyl thiazolyl tetrazolium(MTT) assay, and the optimal dose was selected accordingly for further experiment. The cells were classified into normal serum(NS) group, SR group(10%), CR group(10%), combination(CM) group(10%), and AG490 group. The apoptosis level of ESCs was detected by flow cytometry, and the migration ability was examined by wound healing assay. The secretion of interleukin(IL)-1β, IL-6, and tumor necrosis factor(TNF)-α was determined by enzyme-linked immunosorbent assay(ELISA). The protein levels of cysteinyl aspartate specific protei-nase-3(caspase-3), B-cell lymphoma(Bcl-2), and Bcl-2-associated X protein(Bax) and the levels of phosphorylated(p)-JAK2 and p-STAT3 were detected by Western blot. The results showed that the viability of ESCs cells was lowered in the administration groups compared with the blank serum group(P<0.01), especially the 10% drug-medicated serum, which was selected for further experiment. The 10% SR-medicated serum, 10% CR-medicated serum, and 10% CM-medicated serum could increase the apoptosis rate(P<0.01), up-regulate the protein expression of caspase-3 and Bax in cells(P<0.05 or P<0.01), down-regulate the expression of Bcl-2(P<0.01), decrease the cell migration rate(P<0.05 or P<0.01), and reduce the secretion levels of IL-1β, IL-6, and TNF-α(P<0.05 or P<0.01), and levels of p-JAK2 and p-STAT3(P<0.05 or P<0.01). Compared with the SR and CR groups, CM group showed low cell viability(P<0.01), high protein expression of caspase-3 and Bax(P<0.05 or P<0.01), and low protein expression of Bcl-2 and p-JAK2(P<0.05). After incubation with CM, the apoptosis rate was higher(P<0.05) and the migration rate was lower(P<0.01) than that of the CR group. The p-STAT3 protein level of CM group was lower than that of the RS group(P<0.05). The mechanism of SR, CR, and the combination underlying the improvement of endometriosis may be that they blocked JAK2/STAT3 signaling pathway, inhibited ESC proliferation, promoted apoptosis, weakened cell migration, and reduced the secretion of inflammatory factors. The effect of the combination was better than that of RS alone and CR alone.
Subject(s)
Female , Humans , Janus Kinase 2 , Caspase 3 , bcl-2-Associated X Protein , Interleukin-6/genetics , Apoptosis , Signal Transduction , Cell Proliferation , STAT3 Transcription Factor/geneticsABSTRACT
Dexmedetomidine (DEX) is known to provide neuroprotection against cerebral ischemia and reperfusion injury (CIRI), but the exact mechanisms remain unclear. This study was conducted to investigate whether DEX pretreatment conferred neuroprotection against CIRI by inhibiting neuroinflammation through the JAK2/STAT3 signaling pathway. Middle cerebral artery occlusion (MCAO) was performed to establish a cerebral ischemia/reperfusion (I/R) model. Specific-pathogen-free male Sprague-Dawley rats were randomly divided into Sham, I/R, DEX, DEX+IL-6, and AG490 (a selective inhibitor of JAK2) groups. The Longa score, TTC staining, and HE staining were used to evaluate brain damage. ELISA was used to exam levels of TNF-α. Western blotting was used to assess the levels of JAK2, phosphorylated-JAK2 (p-JAK2), STAT3, and phosphorylated-STAT3 (p-STAT3). Our results suggested that both pretreatment with DEX and AG490 decreased the Longa score and cerebral infarct areas following cerebral I/R. After treatment with IL-6, the effects of DEX on abrogating these pathological changes were reduced. HE staining revealed that I/R-induced neuronal pathological changes were attenuated by DEX application, consistent with the AG490 group. However, these effects of DEX were abolished by IL-6. Furthermore, TNF-α levels were significantly increased in the I/R group, accompanied by an increase in the levels of the p-JAK2 and p-STAT3. DEX and AG490 pretreatment down-regulated the expressions of TNF-α, p-JAK2, and p-STAT3. In contrast, the down-regulation of TNF-α, p-JAK2, and p-STAT3 induced by DEX was reversed by IL-6. Collectively, our results indicated that DEX pretreatment conferred neuroprotection against CIRI by inhibiting neuroinflammation via negatively regulating the JAK2/STAT3 signaling pathway.
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@#BACKGROUND: Exosomes and exosomal microRNAs have been implicated in tumor occurrence and metastasis. Our previous study showed that microRNA-761 (miR-761) is overexpressed in hepatocellular carcinoma (HCC) tissues and that its inhibition affects mitochondrial function and inhibits HCC metastasis. The mechanism by which exosomal miR-761 modulates the tumor microenvironment has not been elucidated. METHODS: Exosomal miR-761 was detected in six cell lines. Cell counting kit-8 (CCK-8) and transwell migration assays were performed to determine the function of exosomal miR-761 in HCC cells. The luciferase reporter assay was used to analyze miR-761 target genes in normal fibroblasts (NFs). The inhibitors AZD1480 and C188-9 were employed to determine the role of the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling pathway in the transformation of cancer-associated fibroblasts (CAFs). RESULTS: In this study, we characterized the mechanism by which miR-761 reprogrammed the tumor microenvironment. We found that HCC-derived exosomal miR-761 was taken up by NFs. Moreover, HCC exosomes affected the tumor microenvironment by activating NFs via suppressor of cytokine signaling 2 (SOCS2) and the JAK2/STAT3 signaling pathway. CONCLUSIONS: These results demonstrated that exosomal miR-761 modulated the tumor microenvironment via SOCS2/JAK2/STAT3 pathway-dependent activation of CAFs. Our findings may inspire new strategies for HCC prevention and therapy.
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ObjectiveTo investigate the effect of pulsatilla saponin A （PSA） on proliferation and apoptosis of human Burkitt lymphoma （BL） cell line Raji cells and expression of related pathway proteins. MethodWith Raji cells as the research object， the cell proliferation was detected by cell counting kit-8 （CCK-8） method， and the half-maximal inhibitory concentration （IC50） values of 24 h， 48 h and 72 h were calculated to be 19.77， 18.31， 16.70 μmol·L-1， respectively. In subsequent related experiments， 0， 8， 16， 32 μmol·L-1 PSA were selected according to the IC50 value of Raji cells treated with PAS for 72 h. After 0， 8， 16， 32 μmol·L-1 PSA acted on Raji cells for 24， 48， 72 h， the optical density values of cell growth curve were detected by CCK-8 method. The zymogen activities of cysteine aspartate-specific protease （Caspase）-3， Caspase-8 and Caspase-9 in Raji cells treated with 0， 8， 16 and 32 μmol·L-1 PSA for 24 h were measured by Caspase-3， Caspase-8 and Caspase-9 colorimetric assay kit. The apoptosis rate and cell cycle of Raji cells treated with different concentrations of PSA after 24 h were detected by flow cytometry. The expression of B-cell lymphoma 2 （Bcl-2）， Bcl-2-associated X protein （Bax）， cleaved poly（ADP-ribose） polymerase （cleaved PARP）， cleaved cysteinyl aspartate-specific protease-3 （cleaved Caspase-3） apoptosis related protein and Janus kinase 2 （JAK2）， signal transducer and activator of transcription 3 （STAT3）， phosphorylated-JAK2 （p-JAK2）， and phosphorylated- STAT3 （p-STAT3） pathway proteins in Raji cells after 24 h of treatment with 0， 8， 16 and 32 μmol·L-1 PSA were tested by Western blot. ResultCompared with control group， decreased cell survival rate， inhibited cell proliferation， activated zymogens of Caspase-3， Caspase-8 and Caspase-9 （P<0.01）， increased apoptosis （P<0.05， P<0.01）， and enhanced cell cycle arrest in Gap phase 2 （G2） were observed in 8， 16 and 32 μmol·L-1 PSA groups（P<0.05， P<0.01）. Compared with control group， cells treated with 8， 16 and 32 μmol·L-1 PSA had lower expression of Bcl-2， p-JAK2， p-STAT3 proteins （P<0.05， P<0.01）， and higher expression of Bax， cleaved PARP and cleaved Caspase-3 protein （P<0.01）， while no significant change was found in the expression of JAK2 and STAT3 proteins. ConclusionPSA could inhibit proliferation and induce apoptosis of Raji cells， and its potential mechanism might be related to the regulation of JAK2/STAT3 signaling pathway.
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OBJECTIVE：To st udy the effects of α7 nicotinic acetylcholine receptor agonists （PNU282987）on improving cardiac remodeling of mice and Janus kinase 2/signal transducer and activator of transcription 3（JAK2/STAT3）signaling pathway. METHODS：Male Kunming mice were randomly divided into normal control group ，model group ，propranolol group （positive control，i.g. 40 mg/kg）and PNU 282987 low-dose，medium-close and high-dose groups （intraperitoneal injection of 0.5，1.0，3.0 mg/kg），with 10 mice in each group. Except for the normal control group ，mice in the other groups were given isoproterenol （ISO，30 mg/kg） subcutaneously for 7 days to induce the cardiac remodeling model. After 30 minutes of ISO injection ， administration groups were given relevant liquid ，once a day ，for 7 consecutive days. Twelve hours after last administration ，the left ventricular ejection fraction （EF）and left ventricular short axis shortening rate （FS）of mice in each group were measured ，and the whole heart mass index （HMI）was calculated ；the pathological changes of myocardium were observed. The serum contents of lactate dehydrogenase （LDH），creatine kinase （CK），tumor necrosis factor α（TNF-α），interleukin 6（IL-6），the protein expression of intercellular adhesion molecule 1（ICAM-1）and adhesion molecule 1（VCAM-1）were also determined. The ratios of p-JAK2/JAK2，p-STAT3/STAT3 in myocardial tissue were detected. RESULTS ：Compared with normal control group ，EF and FS of model group were significantly reduced ，HMI，the contents of LDH，CK，TNF-α and IL-6，the protein expression of ICAM- 1 and VCAM- 1，the ratio of p-JAK 2/JAK2 and p-STAT 3/STAT3 were increased significantly （P＜0.05 or P＜0.01）； blue collagen deposition in the interstitium of myocardium was obvious，and the degree of fibrosis was severe. Compared with model group ， the EF and FS of the mice in the medium-dose and high-dose groups were increased significantly ， HMI （except for PNU 282987 medium-dose group ），the contents of LDH （except for PNU 282987 medium-dose group ），CK，TNF-α and IL-6，the protein expression of ICAM- 1 and VCAM- 1，the ratio of p-JAK 2/JAK2 and p-STAT 3/STAT3 were decreased significantly （P＜0.05 or P＜ 0.01）；blue collagen deposition in the myocardial interstitium was significantly reduced ，and the degree of myocardial fibrosis was significantly reduced. There was no significant difference in the comparison of the above indicators in PNU 282987 low-dose group （P＞0.05）. CONCLUSIONS ：PNU282987 can improve cardiac remodeling of mice ，the mechanism of which may be associated with inhibiting JAK 2/STAT3 signaling pathway.
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JAK2-STAT3 signaling pathway, as the main chain of intracellular signal transmission, plays an important role in cell proliferation, apoptosis, invasion, migration and immune response. Triggered by cytokines and interferon, this pathway can quickly transduce extracellular signals into the nucleus, and it has abnormal expression in various tumors, such as squamous cell carcinoma of the head and neck, lung cancer, esophageal cancer, gastric cancer, liver cancer, breast cancer and myeloproliferative neoplasms. Further understanding of the carcinogenic mechanism of JAK2-STAT3 signaling pathway can provide new ideas for clinical treatment of tumors and prognosis.
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OBJECTIVE：To compare the an ti-hepatocarcinoma effects of curcumin （CUR）and its derivative hydrazincurcumin （HZC），and to explore the mechanism. METHODS ：MTT assay was used to detect the effects of CUR or HZC （2.5，5，10，20， 40，80 μmol/L）on the proliferation of HepG 2 cells. Flow cytometry was used to detect the effects of CUR or HZC （10，20，40 μmol/L）on cell cycle distribution and apoptosis of HepG 2 cells. Western blotting assay was used to detect the effects of CUR or HZC（10，20，40 μmol/L）on the expression of apoptosis-related protein in HepG 2 cells. The male SD rats were randomly divided into normal control group （n＝10），CUR control group （n＝10），HZC control group （n＝10），model group （n＝30），CUR protection group （n＝30）and HZC protection group （n＝30）. CUR control group and HZC control group were given CUR 85917439。E-mail：zhaoji-an-88@163.com or HZC （80 mg/kg） intraperitoneally. Model group ，CUR protection group and HZC protection group were given diethylnitrosamine （50 mg/kg）intraperitoneally to establish hepatocarcinoma model ；at the same time ，2 protection groups were given CUR or HZC （80 mg/kg）intraperitoneally，twice a day，for consecutive 12 weeks. During medication ，the change of body weight and death of rats were recorded. Twenty four weeks later，liver index of rats was calculated and appearance was observed ；the number of cancer nodules was counted ；HE staining was used to observe the pathological changes of liver tissue and calculate the nuclear division index of hepatocarcinoma ；the proliferating cell nuclear antigen （PCNA）index was detected by immunohistochemistry. RESULTS ：CUR and HZC could increase the inhibitory rate of HepG 2 cells（P＜0.05），and increased the percentage at G 0/G1 phase and apoptotic rate of HepG 2 cells（P＜ 0.05）. CUR and HZC could significantly decrease the protein expression of p-JAK 2，p-STAT3，Bcl-2 and Bcl-xl ，while increased the protein expression of Bax ，Cyt-c，Caspase-9，Caspase-3 and PAPR （P＜0.05）. Above effects of HZC were significantly better than those of CUR （P＜0.05）. The results of animal experiment showed that there was no death ，no liver canceration and no pathological changes in liver appearance and tissue section of the three control groups ；there was no statistical significance in body weight and its increased weight ，liver index ，nuclear division index of carcinoma or PCNA index （P＞0.05）. Compared with model group， survival rate of rats were increased significantly in CUR protection group and HZC protection group ， while hepatocarcinoma incidence and the number of cancer nodules were decreased significantly （P＜0.05）；body weight and its increased weight were increased significantly ，while liver index ，nuclear division index of carcinoma and PCNA index were decreased significantly （P＜0.05）. There were some pathological changes in liver appearance and tissue section ；cancerous lesions with focal necrosis or cancerous lesions with patchy necrosis were observed. There was no statistical significance in the improvement of above indexes in 2 protection groups （P＞0.05）. CONCLUSIONS ：HZC could inhibit the proliferation and induce apoptosis of HepG 2 cells by inhibiting JAK 2/STAT3 signaling pathway and regulating the activation of mitochondrial endogenous pathway，which shows stronger anti-hepatocarcinoma effect in vitro than CUR. On the other hand ，there was no significant difference in the improvement of liver caner indexes in hepatic cancer model rats between HZC and CUR.
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Hypoxic-ischemic encephalopathy (HIE) is the leading cause of neonatal death and neurodevelopmental disorders in infants. Part of patients have different degrees of neurological sequelae, such as cerebral palsy, cognitive and motor function development disorders. Hypoxia-ischemia may activate JAK2/STAT3 signaling pathway, which leads to the microglia activation and neuroinflammation. Down-Regulating JAK2/STAT3 signaling pathway can inhibit microglia activation and regulate the inflammatory injury of nervous system. At present, the treatment of hypoxic ischemic encephalopathy is limited, so the study of regulatory mechanism about microglia activation has important value for the treatment of hypoxic-ischemic encephalopathy. This paper summarizes the role of JAK2/STAT3 signaling pathway in microglia activation and analyzes the relationship between them, in order to provide new ideas and strategies for treatment on hypoxic-ischemic encephalopathy.
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Aim To investigate the anti-neuropathic pain effect of DXL-A-22 and further to explore the potential mechanisms. Methods The anti-neuropathic pain effect was evaluated by chronic constriction injury (CCI) model. The potential anti-neuropathic pain mechanisms of DXL-A-22 was studied by Western blot and qPCR. The acute toxicity was evaluated by ultimate test. Results DXL-A-22 (2,1,0. 5 mg . kg-1 ,i. g.) dose-dependently elevated the mechanical withdrawal threshold (MWT) and the paw withdrawal latency (PWL) in CCI rats (P < 0. 05, P < 0. 01), the percentage of pain threshold elevation (PTE%) and the percentage of Maximal Possible Effect (MPE%) was 108%,86%,71% and 77%,56%,43% respectively on day 7 post-operation. DXL-A-22 (2 mg . kg-1 ,i. g.) significantly reduced the expression of p-CaMK II α, p-CREB, p-JAK2, p-STAT3 proteins and TNF-α mRNA, c-Fos mRNA in DRG (P < 0. 05, P < 0.01), and the percent inhibition was 37%, 48%, 35%,58%, 39% and 32% respectively. The expression of TNF-α mRNA and c-Fos mRNA in spinal pord was reduced by 47% and 72% respectively in CCI rats (P <0. 01). Acute toxicity test showed that DXL-A-22 had no obvious toxicity reaction. Conclusions Spirocyclopiperazinium salt compound DXL-A-22 exerts significant antinociceptive effect on CCI model. The anti-neuropathic pain effect of DXL-A-22 may be related to the inhibition of CaMK II α/CREB and JAK2/STAT3 signaling pathways, and the inhibition of the mRNA expression of TNF-α and c-Fos.
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Objective: To detect the effects of dicentrine on the migration and invasion of human hepatocellular carcinoma MHCC97-H cells and potential mechanisms. Methods: MHCC97-H cells were cultured in vitro and cultured at logarithmic growth stage. The cells were treated with 5, 10, 25, 50, 100 μmol/L dicentrine for 24, 48, 72 h, and cell proliferation was determined by MTT assay. The cells were treated with 5, 10, 25 μmol/L dicentrine for 24 h, the cell adhesion, cell migration, cell invasion, gene expression of VEGF, MMP-2 and MMP-9, and protein expression of p-JAK2 and p-STAT3 were determined by MTT, scratch, Transwell, real-time qPCR, and western blot assay, respectively. Results: The cell viability of MHCC-97H cells treated with 25 μmol/L dicentrine at 48 and 72 h, and 50 and 100 μmol/L dicentrine at 24, 48, and 72 h was decreased significantly, which was significantly different from that of the control group (P < 0.05 or P < 0.01). Compared with control group, cell adhesion ability, wound healing ability, cell penetration modulus, gene expression of VEGF, MMP-2 and MMP-9, and protein expression of p-JAK2 and p-STAT3 of MHCC-97H cells treated with 5, 10 and 25 μmol/L dicentrine at 24 h were decreased significantly (P < 0.05 or P < 0.01). Conclusion: Dicentrine can inhibit the migration and invasion of human hepatocellular carcinoma MHCC97-H cells, which is related to the down-regulation of VEGF, MMP-2, and MMP-9 gene expression and inhibition of JAK2/STAT3 signaling pathway activation.
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Objective: To observe the effect of STC-1 gene expression was inhibited on the apoptosis, IL-1β and TNF-α expression and JAK2/STAT3 signal in esophageal cancer cells. Methods: Compared with normal human esophageal squamous epithelial cells Het-1 A, STC-1 expression was detected in human esophageal squamous cell carcinoma KYSE170, Eca109, TE1 and TE10 cells by RTPCR and Western blot; the siRNA sequence that the synthesized STC-1 and the si NRA sequence without interference were transfected into Eca109 cells, which were labeled as STC-1-siRNA group and NC group, and the blank control group was set, cells were transfected for 48 h, the expression of STC-1 were detected by RT-PCR and Western blot. Eca109 cell viability and apoptosis rate were detected by CCK8 and flow cytometry. IL-1β and TNF-α expression were detected by RT-PCR; the expression of Ki67, p53, p-JAK2 and p-STAT3 protein were detected by Western blot. Results: Compared with Het-1 A cells, expression of STC-1 mRNA and protein in KYSE170, Eca109, TE1 and TE10 cells were increased significantly ( P＜0. 05); compared with the control group, STC-1 expression was decreased significantly in STC-1-siRNA group, cell viability was decreased significantly in STC-1-siRNA group, the apoptosis rate was increased significantly in STC-1-siRNA group; IL-1β, TNF-α, Ki67, p-JAK2 and p-STAT3 expression were decreased significantly in STC-1-siRNA group, p53 expression was increased significantly in STC-1-siRNA group ( P＜0. 05). Conclusion: STC-1 was highly expressed in esophageal cancer cells; inhibiting of STC-1 expression could significantly reduce the activity of cancer cells, and increase apoptosis rate, this effect may be related to the inhibition of JAK2/STAT3 signaling pathways and inflammatory factors as IL-1β and TNF-α.
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Objective@#To investigate the effects of sulforaphane on lipopolysaccharide (LPS)- induced cardiac myocytes hypertrophy of rats and study its possible mechanism.@*Methods@#The hypertrophic primary cardiac cells of neuonatal rats were divided into control group, LPS-induced group, JAK2 inhibitor group, low-, middle-, and high-dose sulforaphane groups. The cells in all groups, except for control group, were intervened by LPS (1 mg/L). The low-, middle-, and high-dose sulforaphane groups were treated with 10, 20, 40 μg/ml sulforaphane respectively. The JAK2 inhibitor group was treated with 10 nmol/L of JAK2 inhibitor for 24 h. Computer photograph analysis system was used to determine the cardiomyocyte volume, BCA kit was used to analyze the total protein concentration, ELISA kit was used to observe the expression levels of inflammatory cytokine (including IL-6 and TNF-α), and the Western Blot technology was used to analyze the expression levels of the related proteins in JAK2/STAT3 signaling pathway.@*Results@#Compared with control group, the cell volume (1 635.71 ± 154.84 μm3, 1 450.06 ± 140.13 μm3, 1 194.51 ± 134.76 μm3 vs. 1 854.28 ± 181.37 μm3) and the levels of protein (20.14 ± 2.11 μg, 17.59 ± 1.64 μg, 14.27 ± 1.47 μg vs. 23.17 ± 2.56 μg) in low-, medium-, high- dose of sulforaphane groups were significantly decreased (P<0.05). The levels of IL-6 (1 410.08 ± 255.17 pg/ml, 1 065.61 ± 210.37 pg/ml, 790.09 ± 140.28 pg/ml vs. 1 804.07 ± 275.34 pg/ml) and TNF-α (164.16 ± 27.51 pg/ml, 130.13± 22.08 pg/ml, 92.27 ±12.16 pg/ml vs. 182.42 ± 30.37 pg/ml) in low-, medium-, high- dose of sulforaphane groups were significantly decreased (P<0.05). The expression levels of p-JAK2/JAK2 (0.39 ± 0.05, 0.27 ± 0.04, 0.21 ± 0.03 vs. 0.54 ± 0.07) and p-STAT3 (0.47 ± 0.05, 0.25 ± 0.03, 0.18 ± 0.03 vs. 0.53 ± 0.06) in low-, medium-, high- dose of sulforaphane groups were also significantly decreased (P<0.05).@*Conclusions@#Sulforaphane has a protective effect on LPS-induced cardiac hypertrophy, which is partially via inhibiting inflammatory response via suppressing the activation of JAK2/STAT3 signaling pathway.
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AIM:To investigate the effect of homeodomain-interacting protein kinase 2 (HIPK2) on the viabi-lity, apoptosis and JAK2/STAT3 signaling pathway in NRK-52E renal tubular epithelial cells induced by hypoxia and reox-ygenation (H/R). METHODS:HIPK2 small interfering RNA (siRNA) was transfected into NRK-52E cells by Lipo-fectamineTM 2000, and normal control group (control group) and negative control group (HIPK2-NC group) were set up. After H/R, the cell viability was measured by CCK-8 assay, the apoptotic rate and Ca2+ fluorescence intensity were ana-lyzed by flow cytometry, and the protein levels of Ki67, cleaved caspase-3, caspase-12, Bcl-2, Bax, p-JAK2 and p-STAT3 were determined by Western blot. RESULTS:Compared with control group, the protein expression of HIPK2 in the NRK-52E cells was significantly decreased after transfection with HIPK2 siRNA (P<0.05). Compared with control group, the cell viability and the protein expression of Ki67 and Bcl-2 in H/R group were also significantly decreased, and the apoptotic rate, the Ca2+ fluorescence intensity and the protein levels of cleaved caspase-3, caspase-12, Bax, p-JAK2 and p-STAT3 were significantly increased (P<0.05). Compared with H/R group, the cell viability and the protein expression of Ki67 and Bcl-2 in HIPK2-siRNA+H/R group were significantly increased, while the apoptotic rate, the Ca2+ fluorescence inten-sity and the protein levels of cleaved caspase-3, caspase-12, Bax, p-JAK2 and p-STAT3 were significantly decreased (P<0.05). CONCLUSION:Inhibition of HIPK2 gene expression promotes H/R-induced growth of NRK-52E renal tubular epi-thelial cells, and reduces the apoptosis. The mechanism is related to down-regulating the JAK2/STAT3 signaling pathway.
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Objective To investigate whether the JAK2/STAT3 signaling pathway is involved in the epithelial-mesenchymal transition (EMT) of peritoneal mesothelial cells in uremic peritoneal dialysis (PD) rats.Methods A total of 48 male Sprague-Dawley (SD) rats were randomly separated into six groups:normal control group (NC group,n=8),sham group (n=8),uremic group (n=8),PD group (n=8),S3I-201 control group (n=8) and S3I-201 group (n=8).Uremic model generated by 5/6 nephrectomy surgery in rats was established.The rats of PD group,S3I-201 control group and S3I-201 group received daily infusion of 4.25％ glucose-based peritoneal dialysate fluid (3 ml/100 g) from PD catheters for 28 days.Rats of S3I-201 group were injected with STAT3 inhibitor S3I-201 (2.5 mg/kg) solution from the catheters every other day;the same dose of the solvent of S3I-201 was simultaneously given to S3I-201 control group rats.After PD for 28 days,peritoneal function,pathologic changes,and microvessel density (MVD) were evaluated.Creatinine,urea nitrogen and interleukin-6 (IL-6) concentration in blood and dialysate,and protein and mRNA levels of phospho-JAK2 (p-JAK2),phospho-STAT3 (p-STAT3),E-cadherin,alpha-smooth muscle actin (α-SMA) and vascular endothelial growth factor (VEGF) in peritoneum were determined.Results Uremia and peritoneal dialysate could aggravate the peritoneal function and elevate peritoneal thickness and MVD.They could also increased the concentration of IL-6 in blood and dialysate and the expression levels of α-SMA,VEGF,p-JAK2 and p-STAT3 in peritoneum,while lowering E-cadherin expression in peritoneum.These manifestations were even more remarkable in PD group compared to those in uremic group.There was no statistical difference between the S3I-201 control group and the PD group as regards all the index (all P ＞ 0.05).Compared with the S3I-201 control group,the rats treated with S3I-201 showed better peritoneal function.S3I-201 could reduce peritoneal thickness (P＜0.05),MVD (P＜0.05),the concentration of IL-6 in blood and dialysate,the mRNA and protein expression of α-SMA,VEGF,p-JAK2 and p-STAT3 (all P ＜ 0.05),while enhance the mRNA and protein expression of E-cadherin (all P ＜ 0.05).Conclusions After STAT3 is inhibited,the peritoneal thickness,MVD and IL-6 concentration in PD rats are decreased,and EMT is also inhibited,while peritoneal function is improved.The JAK2/STAT3 signaling pathway may thus be involved in the process of EMT of peritoneum in uremic peritoneal dialysis rats by regulating the expression of IL-6.
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Objective: To observe the effect and explore the mechanism of Tribulus terrestris (TT) on kidney of rats with obesity-related hypertension through leptin mediated JAK2/STAT3 pathway. Methods: To establish the model of rats with obesity-related hypertension by high-fat diet. The model rats were randomly divided into three groups: TT group (eight rats, 17.2 g/kg), Telmisartan group (eight rats, 3.4 mg/kg), and model group (eight rats, normal saline 2 mL/d). Rats were ig given drugs or saline for 12 weeks. The body weights and blood pressure were measured regularly. At the end of the study, the rats were sacrificed and the levels of serum lipid and angiotensinII (AngII) and β2-microglobulin (β2-MG) were determined by ELISA. Morphological changes of adipose tissue and kidney were observed by HE staining. The density of LepR in kidney was observed by immunohistochemical staining. Levels of mRNA and protein expression of JAK2 and STAT3 in kidney were determined by quantitive real-time PCR (qRT-PCR) and Western blotting. Results: Both body weights and blood pressure of TT group were decreased (P < 0.05). The levels of serum TG, TC, and LDL-C of TT group were decreased significantly (P < 0.05). Kidney morphology of TT group was improved obviously and the size of lipocyte decreased. The levels of serum Ang II, Lep, and β2-MG of TT group decreased significantly (P < 0.05). The density of LepR in kidney of TT group decreased significantly (P < 0.05). The mRNA and protein expression of JAK2 and STAT3 in kidney of TT group was decreased significantly (P < 0.05). Conclusion: TT improves the leptin resistance of the obesity-related hypertensive rats mainly through JAK2/STAT3 pathway.
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Aim Based on the effect of total flavonoids extracted from Polygonum perfoliatum L.(TFP) against dimethylnitrosamine(DMN)-induced hepatic fibrosis(HF), to investigate the anti-fibrotic mechanism of TFP.Methods Ninety SD rats were divided into normal group, model group, colchicines(0.1 mg·kg-1) group, and TFP(200, 100, 50 mg·kg-1) group.Except the rats of normal group, other rats were injected intraperitoneally with volume fraction 0.5% DMN solution(2 mL·kg-1) for eight weeks, once every two days.From the first day of modeling, each administration group was given the corresponding dose of drugs to intervene, and the normal group and model group were given an equal volume of solvent, once a day.At the end of the eighth week, the blood and liver tissues were collected.Liver tissue was taken at a fixed position, and the degree of liver tissue was observed by HE staining.The contents of serum ALT, AST, SOD and MDA were measured using colorimetric method;the levels of serum HA, LN, PCⅢand Ⅳ-C were detected using enzyme-linked immunosorbent assay(ELISA);the levels of TNF-α, IL-1β and IL-6 in liver tissues were detected by ELISA;the expression of α-SMA, TGF-β1, p-JAK2 and p-STAT3 were detected by Western blot.Results Compared with the model group, TFP(200, 100, 50 mg·kg-1) could improve the liver tissue lesions, reduce the expression of ALT, AST, HA, LN, PCⅢ, IV-C and MDA, increase SOD activity, reduce the levels of TNF-α, IL-1β and IL-6, and inhibit the expression of α-SMA, TGF-β1, JAK2, STAT3, p-JAK2 and p-STAT3.Conclusion TFP could inhibit DMN-induced HF of rats, which may be involved with antioxidant and inhibiting expression of TGF-β1, JAK2/STAT3 signaling pathway and inflammatory response.
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AIM:To investigate the effect of piceatannol on the viability, and the abilities of migration and invasion in the prostate cancer cells.METHODS:DU145 cells were treated with piceatannol at different doses (0, 5, 10, 20, 40 and 80 μmol/L) for different time (12, 24, 36 and 48 h) as indicated.The cell viability was assessed by CCK-8 assay.The migration and invasion abilities of the cells were analyzed by wound healing assay and Transwell assay, respectively.The protein levels of p-JAK2 and p-STAT3 were detected by Western blot.RESULTS:Piceatannol dose-dependently decreased the cell viability.After treatment with piceatannol, the abilities of migration and invasion of the cells were significantly inhibited.Moreover, treatment with piceatannol resulted in marked decreases in the protein levels of p-JAK2 and p-STAT3.CONCLUSION:Piceatannol inhibits the viability, migration and invasion of the prostate cancer cells via regulating the JAK2/STAT3 signaling pathway.