Urticaria is an immune-mediated allergic disease. This study explored the effect of Jingfang Mixture on spleen T lymphocyte subsets of urticaria mice. A total of 50 Kunming mice were randomized into normal group(C), model group(V), and low-(JF-L, 0.5 g·kg~(-1)), medium-(JF-M, 1 g·kg~(-1)) and high-dose(JF-H, 2 g·kg~(-1)) Jingfang Mixture groups, with 10 mice in each group. The mixture of ovalbumin and aluminum hydroxide(0.1 mg + 0.1 mL) was used(intraperitoneal injection) to induce urticaria in mice. The administration began 6 days after the first immunization, and the second immunization was carried out 10 days after the first immunization. The pruritus index was detected within 30 min after the second immunization. The administration lasted 21 days. After 21 days, the serum was taken to detect the total IgE level. Based on hematoxylin and eosin(HE) staining, the pathological changes of skin tissue were observed, and Western blot was used to detect the levels of p-Janus kinase 2(JAK2)/JAK2 and p-signal transducer and activator of transcription 3(STAT3)/STAT3 in skin tissue. The spleen was taken to detect the spleen index, and flow cytometry was employed to determine the expression of lymphocyte subsets. The results showed that group V had obvious pathological changes in skin tissue compared with group C. Moreover, group V showed more scratches, higher spleen index, and higher level of total serum IgE than group C. In addition, higher levels of p-JAK2 and p-STAT3, lower proportions of CD4~+T, Th1, and Treg, higher proportions of CD8~+T, Th2, and Th17, and lower ratios of CD4~+/CD8~+, Th1/Th2, and Terg/Th17 were observed in group V than in group C. Compared with group V, each administration group showed alleviation of the pathological morphology of skin tissue, obvious epidermal thickening, relatively intact collagen fiber structure of dermal reticular layer, alleviated edema, and relief of vasodilation and peripheral inflammatory cell infiltration. Moreover, less scratching, lower spleen index, lower p-JAK2/JAK2 and p-STAT3/STAT3 were observed in the administration groups than in group V. JF-M group and JF-H group demonstrated lower levels of total IgE, larger proportions of CD4~+T, Th1, and Treg, smaller proportions of CD8~+ T, Th2, and Th17, and higher ratios of CD4~+/CD8~+, Th1/Th2, and Terg/Th17. In conclusion, Jingfang Mixture may improve the symptoms of urticaria mice by regulating the balance of spleen T lymphocyte subsets through JAK2-STAT3 signaling pathway.
Mice ; Animals ; Janus Kinase 2/pharmacology* ; Spleen ; T-Lymphocyte Subsets/metabolism* ; Signal Transduction ; Urticaria ; Immunoglobulin E

This study was aimed to explore the effect of homoharringtonine in combination with AG490 on JAK2-STAT5 associated signal pathway in HEL cells, and analyze its mechanism so as to provide theoretical basis for therapy of chronic myeloproliferative neoplasma by new program. The cell survival rates were tested by MTT, apoptosis was tested by flow cytometry after HEL cells were treated by 20 ng/ml HHT, 100 µmol/L AG490 and 20 ng/ml HHT in combination with 100 µmol/L AG490, while the signal proteins such as P-JAK2, P-STAT5 and BCL-xL activated by abnormal activated JAK2 were tested by Western blot. The results showed that both HHT and AG490 could inhabit the HEL cell proliferation after being treated for 24 hours, and Annexin V-PI double staining confirmed early apoptosis while HHT effect was more obvious, Western blot showed that the expressions of P-JAK2 and P-STAT5 were down-regulated, while the total protein levels of JAK2 and STAT5 were stable. It is concluded that HHT combined with AG490 can obviously inhibit the proliferation and induce early apoptosis of HEL cells, and there is synergistic effect between the two drugs. HHT possibly acts as a broad-spectrum PTK inhibitor and synergistically with AG490 inhibits the phosphorylation of signal proteins caused by JAK2V617F, thus down-regulating the transcription of STAT5.
Cell Line, Tumor ; Harringtonines ; pharmacology ; Humans ; Janus Kinase 2 ; metabolism ; STAT5 Transcription Factor ; metabolism ; Signal Transduction ; drug effects ; Tyrphostins ; pharmacology

This study was aimed to investigate the therapeutic effect of two molecular targeted therapeutic drugs, tyrosine kinase inhibitors gefitinib and lapatinib, on JAK2 V617F positive myeloproliferative disorders (MPD). The human leukemia cell line (HEL cell line) carrying JAK2 V617F mutation was treated with gefitinib (0.5, 1, 5, 10, 25 µmol/L) and lapatinib (0.5, 1, 2, 4, 8, 16 µmol/L) respectively. MTT method was used to detect HEL cell proliferation. The apoptotic rate and cell cycle were measured by flow cytometry. The results showed that gefitinib could significantly inhibit the proliferation of HEL cells in a dose-dependent manner, it's correlation coefficients for 24 and 48 h were 0.991 and 0.895 respectively. IC(50) at 48 h was 5.4 µmol/L. Gefitinib could effectively induce apoptosis of HEL cells in a dose-dependent manner (r = 0.896). Otherwise, gefitinib could arrest HEL cells at G(0)/G(1) phase. The inhibitory effect of lapatinib was less than gefitinib, it's IC(50) of inhibiting proliferation of HEL cells was 19.6 µmol/L. It is concluded that both gefitinib and lapatinib can inhibit the proliferation of HEL cells. These two tyrosine kinase inhibitors can be used for researching of targeted therapy of JAK2 V617 positive MPD.
Antineoplastic Agents ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Janus Kinase 2 ; genetics ; Mutation ; Myeloproliferative Disorders ; metabolism ; pathology ; Protein Kinase Inhibitors ; pharmacology ; Quinazolines ; pharmacology

OBJECTIVETo investigate the regulatory role of JAK2/STAT3/vimentin signaling pathway on the proliferation and migration of human colon cancer cells.

METHODSThe human colon cancer cell Lovo was treated with Janus kinase inhibitor AG490. The proliferation of Lovo cells was quantified by MTT assay. The migration of Lovo cells was measured with scratch assay. The intracellular phosphorylation STAT3 (P-STAT3) and vimentin protein was detected by Western blot and immunofluorescence.

RESULTSAfter AG490 treatment, the proliferative ability of Lovo cells decreased as compared with control group (P<0.05). This suppression was dose-independent and time-independent. At 24 hrs after scratching, scratch width in the AG490 group recovered to 20%, lower than that in the control group (60%, P<0.05). After AG490 treatment, the expression of P-STAT3 and vimentin in Lovo cells decreased significantly (P<0.05).

CONCLUSIONJAK2/STAT3/vimentin signaling pathway participates in regulating the proliferation and migration of human colon cancer cells.

Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Colonic Neoplasms ; metabolism ; pathology ; Humans ; Janus Kinase 2 ; metabolism ; STAT3 Transcription Factor ; metabolism ; Signal Transduction ; Vimentin ; metabolism

OBJECTIVETo investigate the role of Janus kinase-signal transducer and transcription activator (JAK-STAT) pathway in the regulation of synthesis and release of lipopolysaccharide-induced high mobility group box-1 protein (HMGB1).

METHODSPeritoneal macrophages harvested from male Wistar rats were incubated for 3 days before the experiment. The activation of Janus kinase-2 (JAK2), signal transducer and activator of transcription-1 (STAT1) and STAT3 was observed before and 10, 30, 60 and 120 mins after LPS stimulation (4 determinations at each time point) and it was expressed as A value (absorption). In addition, the cells were divided into normal control, LPS stimulation, JAK2 inhibition (with AG490 treatment 2 hours before LPS stimulation), STAT1 inhibition (with fludarabine treatment 2 hours before LPS stimulation) and STAT3 inhibition (with rapamycin treatment 2 hours before LPS stimulation) groups. The cells in all groups except control group were stimulated with LPS 3 days after culture. The expression of HMGB1 gene and its protein release in each group were determined for 4 times and were expressed as A value.

RESULTSLPS could activate JAK2, STAT1 and STAT3 within 2 hours, especially the activation of STAT3 appeared more quickly, peaking at 10 minutes after LPS stimulation (7.47 +/- 0.56). Pretreatment with the inhibitors of JAK-STAT pathway could markedly reduce the expression of HMGB1 mRNA (P < 0.01), but exerted no effect on HMGB1 release.

CONCLUSIONJAK-STAT pathway can be activated early during endotoxin challenge, and it may play a role in the regulation of HMGB1 synthesis.

Animals ; Cell Culture Techniques ; HMGB1 Protein ; biosynthesis ; Janus Kinase 2 ; metabolism ; Lipopolysaccharides ; Macrophages, Peritoneal ; metabolism ; Male ; Rats ; Rats, Wistar ; STAT1 Transcription Factor ; metabolism ; STAT3 Transcription Factor ; metabolism

Coexistence of chronic lymphocytic leukemia (CLL) and essential thrombocythemia (ET) in a patient is extremely rare, with only 10 cases reported thus far in literature. This paper describes a 94-year-old male having atypical B-CLL with CD5⁻ (CD5⁻) phenotype and ET. In this patient, we performed interphase fluorescence in situ hybridization (FISH) analysis which revealed 13q14.3 deletion in 31% of B-lymphocyte nuclei and RB1 deletion in 27% of B-lymphocyte nuclei, but not in neutrophils and T-lymphocytes. Furthermore, we identified JAK2 V617F mutation in the peripheral blood nucleated cells and neutrophils, but not in the B- and T-lymphocyte populations. Therefore, it was concluded that the occurrence of CD5− B-CLL and ET in this patient was pathogenically independent.
Aged, 80 and over ; CD5 Antigens ; metabolism ; Humans ; In Situ Hybridization ; Janus Kinase 2 ; genetics ; Leukemia, Lymphocytic, Chronic, B-Cell ; genetics ; metabolism ; Male ; Mutation ; Thrombocythemia, Essential ; genetics ; metabolism

OBJECTIVETo investigate the effects of cycle-dependent kinase (CDK) inhibitor SNS-032 on apoptosis in human acute myeloid leukemia (AML) HL-60 cells and its molecular mechanisms.

METHODSCultured AML HL-60 cells were treated with various concentrations of SNS-032. Cell apoptosis was determined with flow cytometry;cell viability was measured by MTT assay; the profiles of microRNA expression of HL-60 cells were analyzed by microRNA microarray;the protein expressions of JAK2/STAT3 pathway were detected by Western blotting.

RESULTSApoptosis of AML HL-60 cells was induced by SNS-032; the rate of apoptosis was (5.9±1.7)%, (12.1±3.1)% and (59.4±3.6)% when HL-60 cells were treated with 0,100 and 200 nmol/L SNS-032. MicroRNA microarray analysis revealed that the levels of miR-30a, miR-183, miR-20b, miR-26b, miR-20a, miR-589, miR-107, miR-181a, miR-106a, miR-17 and miR-378c were down-regulated by SNS-032,whereas the levels of miR-320a and miR-H7* were up-regulated. Western blotting showed that SNS-032 strongly inhibited phosphorylation of STAT3 and protein expression of JAK2,C-MYC and MCL-1.

CONCLUSIONCDK inhibitor SNS-032 can induce apoptosis of AML HL-60 cells, which is associated with the inhibition of MCL-1,C-MYC and JAK2/STAT3, and down-regulation of miR-17-92 family.

Apoptosis ; Cell Survival ; Down-Regulation ; Flow Cytometry ; HL-60 Cells ; Humans ; Janus Kinase 2 ; metabolism ; MicroRNAs ; metabolism ; Oxazoles ; pharmacology ; Phosphorylation ; STAT3 Transcription Factor ; metabolism ; Signal Transduction ; Thiazoles ; pharmacology

OBJECTIVETo investigate the frequency of JAK2 V617F mutation in 145 myeloproliferative disorders (MPDs) patients, analyze the correlation between JAK2 V617F mutation and clinical features.

METHODSThe JAK2 V617F mutation was detected by direct DNA sequencing of PCR product and allele-specific PCR respectively. The expression of JAK2, phospho-JAK2 and phospho-STAT5 proteins was determined by Western blot. The clinical data of MPDs patients with or without JAK2 V617F mutation was collected and analyzed for evaluating the clinical significance of JAK2 V617F mutation.

RESULTS1) The frequency of JAK2 V617F mutation for PV, IMF, ET was 92%, 58%, 50% respectively. Compared with conventional DNA sequencing (PV 84%, IMF 44%, ET 39%, respectively), allele-specific PCR exhibited a higher sensitivity in JAK2 V617F mutation detection. 2) The expression levels of phospho-JAK2 and phospho-STAT5 in peripheral blood mononuclear cells (PBMNCs) were upregulated significantly in JAK2 V617F-positive patients than in JAK2 V617F negative patients. 3) Compared with the patients with no JAK2 V617F mutation, the JAK2 V 617F-positive patients' features were as follows: older age of onset, higher mean leukocyte counts, lower platelet counts and smaller spleen volume. Frequency of thrombosis events in PT, ET, IMF was 17%, 32%, 16% respectively for JAK2 V617F positive group, and 0% (PV), 16% (ET), 5% (IMF) for JAK2 V617F negative group.

CONCLUSIONSMPDs patients display higher frequency of JAK2 V617F mutation. JAK2 V617F mutation positive patients predispose to a thrombosis tendency.

Female ; Humans ; Janus Kinase 2 ; genetics ; metabolism ; Male ; Mutation ; Myeloproliferative Disorders ; genetics ; metabolism ; Phosphorylation ; STAT5 Transcription Factor ; metabolism ; Sequence Analysis, DNA

Objective: To investigate the role and mechanism of tumor-derived mesenchymal stem cells in regulating the M2 polarization of macrophages within gastric cancer microenvironment. Methods: Gastric cancer tissues and the adjacent non-cancerous tissues were collected from patients underwent gastric cancer resection in the First People's Hospital of Lianyungang during 2018. In our study, THP-1-differentiated macrophages were co-cultured with gastric cancer-derived mesenchymal stem cells (GC-MSCs). Then, the M2 subtype-related gene, the markers expressed on cell surface and the cytokine profile were analyzed by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), flow cytometry and Luminex liquid chip, respectively. The key cytokines mediating the inducing effect of GC-MSCs on macrophage polarization into the M2 subtype were detected and screened by Luminex liquid chip, which were further confirmed by the neutralizing antibody test. The expressions of macrophage proteins involved in M2 polarization-related signaling pathways under the different co-culture conditions of GC-MSCs were detected by western blot. Results: In Mac+ GC-MSC-culture medium (CM) group, the expression levels of Ym-1 and Fizz-1 (1.53±0.32 and 13.22±1.05, respectively), which are markers for M2 subtype, were both significantly higher than those of Mac group (1.00±0.05 and 1.21±0.38, respectively, P<0.05). The level of iNOS in Mac+ GC-MSC-CM group (0.60±0.41) was significantly lower than that of Mac group (1.06±0.38, P=0.023). In Mac+ GC-MSC-Transwell (TW) group, the expression levels of Ym-1 and Fizz-1 (1.47±0.09 and 13.16±2.77, respectively) were both significantly higher than those of Mac group (1.00±0.05 and 1.21±0.38, respectively, P<0.05). The level of iNOS in Mac+ GC-MSC-CM group (0.56±0.03) was significantly lower than that of Mac group (1.06±0.38, P=0.026). The ratios of CD163(+) /CD204(+) cells in Mac+ GC-MSC-CM and Mac+ GC-MSC-TW groups (3.80% and 4.40%, respectively) were both remarkably higher than that of Mac group (0.60%, P<0.05). The expression levels of IL-10, IL-6, MCP-1 and VEGF in Mac+ GC-MSC-CM group were (592.60±87.52), (1 346.80±64.70), (11 256.00±29.03) and (1 463.90±66.67) pg/ml, respectively, which were significantly higher than those of Mac group [(41.03±2.59), (17.35±1.79), (5 213.30±523.71) and (267.12±12.06) pg/ml, respectively, P<0.05]. The levels of TNF-α, IP-10, RANTES and MIP-1α were (95.57±9.34), (410.48±40.68), (6 967.30±1.29) and (1 538.70±283.04) pg/ml, which were significantly lower than those of Mac group [(138.01±24.31, (1 298.60±310.50), (14 631.00±4.21) and (6 633.20±1.47) pg/ml, respectively, P<0.05]. The levels of IL-6 and IL-8 in GC-MSCs [(11 185.02±2.82) and (12 718.03±370.17) pg/ml, respectively] were both strikingly higher than those of MSCs from adjacent non-cancerous gastric cancer tissues [(270.71±59.38) and (106.04±32.84) pg/ml, repectively, P<0.05]. The ratios of CD86(+) cells in Mac+ IL-6-blocked-GC-MSC-CM and Mac+ IL-8-blocked-GC-MSC-CM groups (28.80% and 31.40%, respectively) were both higher than that of Mac+ GC-MSC-CM group (24.70%). Compared to Mac+ GC-MSC-CM group (13.70%), the ratios of CD204(+) cells in Mac+ IL-6-blocked-GC-MSC-CM and Mac+ IL-8-blocked-GC-MSC-CM groups (9.90% and 8.70%, separately) were reduced. The expression levels of p-JAK2 and p-STAT3, which are proteins of macrophage M2 polarization-related signaling pathway, in Mac+ GC-MSC-CM group (0.86±0.01 and 1.08±0.01, respectively) were significantly higher than those of Mac group (0.50±0.01 and 0.82±0.01, respectively, P<0.05). The expression levels of p-JAK2 in Mac+ IL-6-blocked-GC-MSC-CM group (0.47±0.02) were significantly lower those that of Mac+ GC-MSC-CM group (0.86±0.01, P<0.05). The expression levels of p-JAK2 and p-STAT3 in Mac+ IL-8-blocked-GC-MSC-CM group (0.50±0.01 and 0.85±0.01, respectively) were both significantly lower than those of Mac+ GC-MSC-CM group (0.86±0.01 and 1.08±0.01, P<0.05). The expression levels of p-JAK2 and p-STAT3 in Mac+ IL-6/IL-8-blocked-GC-MSC-CM group (0.37±0.01 and 0.65±0.01, respectively) were both significantly lower than those of Mac+ GC-MSC-CM group (0.86±0.01 and 1.08±0.01, P<0.05). Conclusion: GC-MSCs promote the activation of JAK2/STAT3 signaling pathway in macrophages via high secretions of IL-6 and IL-8, which subsequently induce the macrophage polarization into a pro-tumor M2 subtype within gastric cancer microenvironment.
Humans ; Interleukin-6/genetics* ; Interleukin-8/pharmacology* ; Janus Kinase 2/metabolism* ; Macrophages/metabolism* ; Mesenchymal Stem Cells ; STAT3 Transcription Factor/metabolism* ; Signal Transduction ; Stomach Neoplasms/pathology* ; Tumor Microenvironment

OBJECTIVE: To investigate the effect of kaempferol on proliferation of acute myeloid leukemia (AML) KG1a cells and its mechanism. METHODS: Human AML KG1a cells in logarithmic growth stage were taken and set at 25, 50, 75 and 100 μg/ml kaempferol group, another normal control group (complete medium without drug) and solvent control group (add dimethyl sulfoxide) were also set. After 24 and 48 hours of intervention, the cell proliferation rate was detected by CCK-8 assay. In addition, interleukin-6 (IL-6) combined with kaempferol group (Plus 20 μg/l IL-6 and 75 μg/ml kaempferol) was set up, 48 hours after culture, the cell cycle and apoptosis of KG1a cells were detected by flow cytometry, the mitochondrial membrane potential (MMP) of KG1a cells was detected by MMP detection kit (JC-1 method), and the expression of Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway related proteins in KG1a cells were detected by Western blot. RESULTS: The cell proliferation rate of 25, 50, 75 and 100 μg/ml kaempferol group decreased significantly (P<0.05), and with the increase of kaempferol dose (r_{24 h}=-0.990, r_{48 h}= -0.999), the cell proliferation rate decreased gradually (P<0.05). The inhibitory effect of 75 μg/ml kaempferol on cell proliferation reached half of effective dose after 48 hours of intervention. Compared with normal control group, the G₀/G₁ phase cell proportion and apoptosis rate of cells in 25, 50 and 75 μg/ml kaempferol group increased, while the S phase cell proportion, MMP, phosphorylated JAK2 (p-JAK2)/JAK2 and phosphorylated STAT3 (p-STAT3)/STAT3 protein expression decreased in a dose-dependent manner (r=0.998, 0.994, -0.996, -0.981, -0.997, -0.930). Compared with 75 μg/ml kaempferol group, the G₀/G₁ phase cell proportion and apoptosis rate of cells in IL-6 combined with kaempferol group decreased, while the S phase cell proportion, MMP, p-JAK2/JAK2 and p-STAT3/STAT3 protein expression increased significantly (P<0.05). CONCLUSION Kaempferol can inhibit KG1a cell proliferation and induce KG1a cell apoptosis, its mechanism may be related to the inhibition of JAK2/STAT3 signal pathway.
Humans ; STAT3 Transcription Factor/metabolism* ; Interleukin-6/metabolism* ; Kaempferols/pharmacology* ; Signal Transduction ; Apoptosis ; Janus Kinase 2 ; Cell Proliferation ; Leukemia, Myeloid, Acute