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Objective:To explore the influences of neutrophilic granule protein (NGP) on nitric oxide (NO) production in lipopolysaccharide (LPS)-induced macrophages and the regulatory mechanism.Methods:NGP highexpression RAW264.7 cells (NGP/RAW) and negative control empty vector cells (NC/RAW), NGP knockout RAW264.7 cells (NGP KO/RAW) and wild-type cells (WT/RAW) were cultured in vitro. Cells in logarithmic phase were stimulated with 10 mg/L LPS (LPS group) or phosphate buffered saline (PBS group) respectively. The content of NO in the supernatant was detected by Griess method. The mRNA expression of inducible nitric oxide synthase (iNOS) was detected by quantitative reverse transcription-polymerase chain reaction (RT-qPCR). The protein expressions of iNOS and phosphorylated signal transducer and activator of transcription 1 (p-STAT1) were detected by Western blotting.Results:Compared with PBS group, iNOS mRNA and NO expression were significantly increased at different time after LPS stimulation, the mRNA expression of iNOS peaked at 12 hours after LPS stimulation (2 -ΔΔCt: 38.45±1.34 vs. 1.00±0.00 in NC/RAW cells, 56.24±2.41 vs. 1.45±0.30 in NGP/RAW cells, 37.84±1.52 vs. 1.00±0.00 in WT/RAW cells, 5.47±0.62 vs. 0.98±0.40 in NGP KO/RAW cells, all P < 0.05), and the production of NO peaked at 24 hours after LPS stimulation (μmol/L: 24.15±1.26 vs. 0.15±0.04 in NC/RAW cells, 58.80±2.11 vs. 0.18±0.02 in NGP/RAW cells, 25.04±1.80 vs. 0.16±0.02 in WT/RAW cells, 2.42±0.38 vs. 0.12±0.03 in NGP KO/RAW cells, all P < 0.05). After being stimulated by LPS, the expression of iNOS mRNA and NO in NGP/RAW cells were increased significantly compared with NC/RAW cells [iNOS mRNA (2 -ΔΔCt): 8.42±0.59 vs. 4.63±0.37 at 2 hours, 27.16±1.60 vs. 14.25±1.02 at 6 hours, 56.24±2.41 vs. 38.45±1.34 at 12 hours; NO (μmol/L): 4.12±0.25 vs. 2.23±0.17 at 6 hours, 16.50±1.52 vs. 6.35±0.39 at 12 hours, 58.80±2.11 vs. 24.15±1.26 at 24 hours, all P < 0.05]. At the same time, the protein expressions of p-STAT1 and iNOS were also significantly enhanced (p-STAT1/GAPDH: 4.26±1.84 vs. 1.00±0.32 at 0 hours, 20.59±4.97 vs. 0.93±0.21 at 2 hours, 141.99±10.99 vs. 11.17±2.11 at 6 hours; iNOS/GAPDH: 1.27±0.86 vs. 1.00±0.22 at 0 hours, 7.94±1.94 vs. 2.01±0.92 at 2 hours, 24.24±4.88 vs. 3.72±1.11 at 6 hours, all P < 0.05), indicating that NGP might increase the expression of iNOS by promoting the phosphorylation of the signal transducer and activator of transcription 1 (STAT1) pathway, thereby increasing the production of NO. After being stimulated by LPS, the expression of iNOS mRNA and NO in NGP KO/RAW cells were significantly lower than that of WT/RAW cells [iNOS mRNA (2 -ΔΔCt): 2.46±0.31 vs. 4.22±0.18 at 2 hours, 3.61±0.44 vs. 13.02±1.34 at 6 hours, 5.47±0.62 vs. 37.84±1.52 at 12 hours; NO (μmol/L): 1.22±0.19 vs. 2.01±0.12 at 6 hours, 1.60±0.44 vs. 5.15±0.62 at 12 hours, 2.42±0.38 vs. 25.04±1.80 at 24 hours, all P < 0.05]. It showed that iNOS activation was reduced after NGP knockout, which in turn reduced NO production. Conclusion:NGP can positively regulate NO production in activated macrophages by activating the STAT1/iNOS pathway.
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Objective@#To investigate the potential effects of cytochrome P450 1A1 (CYP1A1) in regulating macrophages polarize to M2 type and explore the molecular mechanism.@*Methods@#All trials were completely randomized. ① Experiment 1: 6-8 weeks old healthy male C57BL/6J mice were collected, and primary peritoneal cells were extracted, then the cells were divided into phosphate buffered saline (PBS) group and interleukin-4 (IL-4) group. The cells in the IL-4 group were stimulated with 10 mg/L IL-4 (M2 macrophage inducer); and those in the PBS group were given with an equal amount of PBS. The mRNA expressions of intracellular M2 type polarized marker molecules including arginase-1 (Arg-1) and chitinase 3 like protein 1 (YM1) at 2, 4, 6 hours after IL-4 challenge were determined by quantitative reverse transcription-polymerase chain reaction (RT-qPCR). The phosphorylation of tyrosine protein kinase 1/signaling transcriptional and transduced activator 6 (JAK1/STAT6) signaling pathway and protein expressions of CYP1A1 and Arg-1 at 6, 12, 24 hours after IL-4 challenge were determined by Western Blot. ② Experiment 2: RAW264.7 cells with high expression CYP1A1 (CYP1A1/RAW) and their negative control cells (NC/RAW) were cultured in vitro. The cells in logarithmic growth phase were collected, and then they were divided into PBS control group and IL-4 group. The treatment method was the same as experiment 1. The phosphorylations of intracellular JAK1/STAT6 and phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathways in different cells at 1 hour and 2 hours after IL-4 challenge were determined by Western Blot. The mRNA and protein expressions of Arg-1 in different cells at 2 hours and 4 hours after IL-4 challenge were determined by RT-qPCR and Western Blot, respectively.@*Results@#① Experiment 1: after IL-4 challenge for 2 hours, the mRNA expressions of Arg-1 and YM1 in the primary peritoneal macrophages of mice were significantly increased as compared with the PBS control group [Arg-1 mRNA (2-ΔΔCt): 1.75±0.82 vs. 1.00±0.21; YM1 mRNA (2-ΔΔCt): 2.58±0.53 vs. 1.00±0.20, both P < 0.05] which indicated that IL-4 induced macrophage to M2 type polarization successfully. Meanwhile, the mRNA expression of CYP1A1 in polarized mouse peritoneal primary macrophages was also elevated as compared with the PBS control group, and peaked at 4 hours [CYP1A1 mRNA (2-ΔΔCt): 2.25±0.69 vs. 1.00±0.17, P < 0.01]. The results indicated that CYP1A1 expression was enhanced when macrophages polarized to M2 type. Compared with the PBS control group, the bands of phosphorylated JAK1 (p-JAK1), phosphorylated STAT6 (p-STAT6), Arg-1 and CYP1A1 were enhanced in primary peritoneal macrophages of mice in the IL-4 group, and reached the peak value at 12 hours then gradually decreased. This result indicated that the phosphorylation of JAK1/STAT6 pathway was enhanced in M2 macrophages with high expression of CYP1A1, and the pathway was activated. ② Experiment 2: after IL-4 challenge for 2 hours, the expression of Arg-1 mRNA in CYP1A1/RAW cells was significantly higher than that in NC/RAW cells (2-ΔΔCt: 3.02±0.60 vs. 1.47±0.43, P < 0.05), and the protein band signal was also stronger, and both peaked at 4 hours. This indicated that CYP1A1 could promote the polarization of macrophages to M2. After IL-4 challenge for 2 hours, the expression of p-JAK1 and p-JAK3 protein bands in both cells were significantly enhanced as compared with the PBS control group, but the enhancement of p-STAT6 band in CYP1A1/RAW cells was stronger than that of NC/RAW cells, indicating that CYP1A1 promoted macrophage polarization by promoting phosphorylation of the JAK1/STAT6 pathway. In the meantime, the protein band of Akt, a downstream protein of the PI3K/Akt pathway, in CYP1A1/RAW cells was significantly lower than that of NC/RAW cells, indicating that CYP1A1 did not promote macrophage polarization through this pathway.@*Conclusions@#CYP1A1 promotes the polarization of macrophage to M2 type. The mechanism is related to promoting the phosphorylation of JAK1/STAT6 pathway.
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To investigate the potential effects of cytochrome P450 1A1 (CYP1A1) in regulating macrophages polarize to M2 type and explore the molecular mechanism. Methods All trials were completely randomized. ① Experiment 1: 6-8 weeks old healthy male C57BL/6J mice were collected, and primary peritoneal cells were extracted, then the cells were divided into phosphate buffered saline (PBS) group and interleukin-4 (IL-4) group. The cells in the IL-4 group were stimulated with 10 mg/L IL-4 (M2 macrophage inducer); and those in the PBS group were given with an equal amount of PBS. The mRNA expressions of intracellular M2 type polarized marker molecules including arginase-1 (Arg-1) and chitinase 3 like protein 1 (YM1) at 2, 4, 6 hours after IL-4 challenge were determined by quantitative reverse transcription-polymerase chain reaction (RT-qPCR). The phosphorylation of tyrosine protein kinase 1/signaling transcriptional and transduced activator 6 (JAK1/STAT6) signaling pathway and protein expressions of CYP1A1 and Arg-1 at 6, 12, 24 hours after IL-4 challenge were determined by Western Blot. ② Experiment 2: RAW264.7 cells with high expression CYP1A1 (CYP1A1/RAW) and their negative control cells (NC/RAW) were cultured in vitro. The cells in logarithmic growth phase were collected, and then they were divided into PBS control group and IL-4 group. The treatment method was the same as experiment 1. The phosphorylations of intracellular JAK1/STAT6 and phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathways in different cells at 1 hour and 2 hours after IL-4 challenge were determined by Western Blot. The mRNA and protein expressions of Arg-1 in different cells at 2 hours and 4 hours after IL-4 challenge were determined by RT-qPCR and Western Blot, respectively. Results ① Experiment 1: after IL-4 challenge for 2 hours, the mRNA expressions of Arg-1 and YM1 in the primary peritoneal macrophages of mice were significantly increased as compared with the PBS control group [Arg-1 mRNA (2-ΔΔCt): 1.75±0.82 vs. 1.00±0.21; YM1 mRNA (2-ΔΔCt): 2.58±0.53 vs. 1.00±0.20, both P < 0.05] which indicated that IL-4 induced macrophage to M2 type polarization successfully. Meanwhile, the mRNA expression of CYP1A1 in polarized mouse peritoneal primary macrophages was also elevated as compared with the PBS control group, and peaked at 4 hours [CYP1A1 mRNA (2-ΔΔCt): 2.25±0.69 vs. 1.00±0.17, P < 0.01]. The results indicated that CYP1A1 expression was enhanced when macrophages polarized to M2 type. Compared with the PBS control group, the bands of phosphorylated JAK1 (p-JAK1), phosphorylated STAT6 (p-STAT6), Arg-1 and CYP1A1 were enhanced in primary peritoneal macrophages of mice in the IL-4 group, and reached the peak value at 12 hours then gradually decreased. This result indicated that the phosphorylation of JAK1/STAT6 pathway was enhanced in M2 macrophages with high expression of CYP1A1, and the pathway was activated. ② Experiment 2: after IL-4 challenge for 2 hours, the expression of Arg-1 mRNA in CYP1A1/RAW cells was significantly higher than that in NC/RAW cells (2-ΔΔCt:3.02±0.60 vs. 1.47±0.43, P < 0.05), and the protein band signal was also stronger, and both peaked at 4 hours. This indicated that CYP1A1 could promote the polarization of macrophages to M2. After IL-4 challenge for 2 hours, the expression of p-JAK1 and p-JAK3 protein bands in both cells were significantly enhanced as compared with the PBS control group, but the enhancement of p-STAT6 band in CYP1A1/RAW cells was stronger than that of NC/RAW cells, indicating that CYP1A1 promoted macrophage polarization by promoting phosphorylation of the JAK1/STAT6 pathway. In the meantime, the protein band of Akt, a downstream protein of the PI3K/Akt pathway, in CYP1A1/RAW cells was significantly lower than that of NC/RAW cells, indicating that CYP1A1 did not promote macrophage polarization through this pathway. Conclusions CYP1A1 promotes the polarization of macrophage to M2 type. The mechanism is related to promoting the phosphorylation of JAK1/STAT6 pathway.
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Objective:To explore the effect of humanistic care in patients with hematopoietic stem cell transplantation.Methods:Totally 51 patients with hematopoietic stem cell transplantation were recruited from the First Affiliated Hospital of Xi'an Jiaotong University,between September 2014 and July 2016,and were randomly divided into two groups.Patients in the intervention group (n =25) received humanistic care,while those in the control group (n =26) received routine nursing.The incidence of adverse psychological status and nursing satisfaction were compared between the two groups.Results:The incidence rate of adverse psychological state in the intervention group (16％) was significantly lower than that in the control group (53％),while the nursing satisfaction in the interventiongroup (92％) was significantly higher than that in the control group (69％),P ＜0.05.Conclusion:Application of humanistic care in the patients with hematopoietic stem cell transplantation can significantly improve nursing satisfaction and reduce the incidence of adverse psychological state,which will improve the confidence of the patients and the quality of nursing satisfaction.Consequently,it will promote the relationship between the nurses and patients.
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Objective To evaluate the therapeutic effect of Maxing-Shigan decoction combined with salmeterol/fluticasone inhalation for acute exacerbation of chronic obstructive pulmonary disease (COPD). Methods A total of 86 patients with acute exacerbation of COPD were enrolled and randomly divided into a salmeterol/fluticasone group (41 patients) and a combined treatment group (45 patients). The salmeterol/fluticasone group was treated by salmeterol/fluticasone inhalation, and the combined treatment group by Maxing-Shigan decoction combined with salmeterol/fluticasone inhalation. Serum C-reactive protein (CRP) was detected by a immunonephelometric assay, and Toll-like receptor 9 (TLR9) in hemocytes was detected by flow cytometry. The score of the syndromes in traditional Chinese medicine (TCM), such as cough, sputum, gasping and shortness of breath, as well as pulmonary function and therapeutic effect were evaluateds. Results After the treatment, the serum C-reactive protein in the combined treatment group was significantly lower than that in the salmeterol/fluticasone group (4.3 ± 1.2 mg/L vs. 8.4 ± 2.5 mg/L;t=5.417, P<0.01), and the TLR9 expression was significantly higher (1.9 ± 0.7 vs. 1.6 ± 0.4;t=3.418, P<0.05). The scores of the syndromes in TCM, such as cough (1.7 ± 0.6 vs. 3.8 ± 1.1;t=2.859, P<0.05), sputum (1.6 ± 0.4 vs. 3.9 ± 1.2;t=3.027, P<0.05), gasping (1.2 ± 0.5 vs. 3.4 ± 1.3;t=3.416, P<0.05) and shortness of breath (1.5 ± 0.7 vs. 3.7 ± 1.6;t=3.468, P<0.05) in the combined treatment group were significantly lower than those in the salmeterol/fluticasone group. The forced expiratory volume in first second (75.4 ± 5.8 L vs. 62.8 ± 6.9 L;t=3.526, P<0.05) and the percentage of forced expiratory volume in first second to forced vital capacity (85.7%± 10.3%vs. 71.9%± 15.4%;t=5.648, P<0.01) in the combined treatment group were significantly higher than those in the salmeterol/fluticasone group. The time to symptoms alleviated (3.4 ± 0.7 d vs. 5.6 ± 1.2 d; t=3.256, P<0.05) and the use dose was (1.8 ± 0.2) ×103μg vs. (5.3 ± 0.4)×103μg, and use times of salmeterol/fluticasone (7.4 ± 1.3 vs. 16.5 ± 3.4;t=4.574, P<0.05) in the combined treatment group were significantly decreased than those in the salmeterol/fluticasone group. The total effective rate in in the combined treatment group were significantly decreased than those in the salmeterol/fluticasone group (84.4% vs. 73.2%; χ2=4.519, P<0.05). Conclusion Maxing-Shigan decoction combined with salmeterol/fluticasone inhalation can improve the pulmonary function in patients with acute exacerbation of COPD, its effiency is suppior to salmeterol/fluticasone inhalation alone.
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Objective To assess the safety and efficacy of adopting different vascular access during autologous peripheral blood stem cells collection.Methods 87 patients received autologous peripheral blood stem cells collection were divided into two groups:One was peripheral vein harvesting group (43 cases),which used the 16G disposable fistula needle for autolo-gous peripheral blood stem cell collection and the other central venous harvesting group (44 cases),which used double cavity of femoral vein catheter for autologous peripheral blood stem cell collection.The observation indicators included venous ac-cesses,collection efficiency,patient tolerance,the number of mononuclear cell and CD34 positive cells.Results The numbers of mononuclear cells and CD34 positive cells in two groups were all above the standard and there was no significant differ-ence (P >0.05).However,the success of venous accesses,the efficiency smooth of collection and-patient tolerance were bet-ter in double cavity of femoral vein catheter group (P <0.05).Conclusion Harvesting the autologous peripheral blood stem cell through central venous by using double cavity of femoral vein catheter had the advantages as follows:high success rate of puncture,acquisition smoothly and reducing the suffering extent of the patients,and also it did not affect the acquisition effi-ciency and effectiveness.
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non-myeloablative BuCy+fludarabine conditioning regimen,and another one was treated with TBI+VP-1 6 +CTX+CCNU conditioning regimen.Only one case received short-term MTX,cyclosporin A and ATG regimen for prevention of graft-versus-host disease (GVHD).The GVHD prevention regimens of the other patients were based on short-term MTX,cyclosporin A,ATG and mycophenolate mofetil regimen.The hematopoietic reconstitution, complications and prognosis were observed.Results One patient died of intracranial hemorrhage,and hematopoi-etic reconstitution was achieved in the other 20 patients.The median time for hematopoietic reconstitution shortened by one day in large-dose group compared with that in low-dose group.Adverse reactions included high fever, shivering,gastrointestinal tract adverse reaction,liver injury,oral mucositis and other rare side effects.GVHD occurred more frequently in patients with HLA mismatched transplantation.Nine patients with aGVHD and 9 patients with cGVHD recovered after effective treatment.Within 100 days after transplantation,18 patients had bacterial or fungal infection,mainly upper respiratory tract infection;7 patients had cytomegalovirus infection;2 had EB viremia,and one had urinary BK virus infection.Only one patient died of VOD.Hemorrhagic cystitis occurred in 5 patients and improved after treatment.The median survival time was 24 months (ranging from 136 days to 9 years).One-year and 3-year overall survival rates were 85.2% and 63.9%,the disease free survival rates were 81% and 23.8%,recurrence free survival rates were 71.4% and 14.3%,respectively.Conclusion URD-HSCT was an effective method to treat leukemia.Conditioning regimen of BuCy and modified BuCy2 were safe and effective,the adverse reactions were reversible and well tolerated.Hematopoietic reconstitution time shortened in large-dose MNC and CD34 + cell number groups compared with that in low-dose group.The occurrence rate of GVHD with HLA mismatched transplantation was more than that of HLA matched transplantation.Low-dose heparin,prostaglandin E1 and Danshen injection can effectively prevent VOD.
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Objective To establish a quantitative detection method for Mycobacterium tuberculosis by immunomagnetic capture combined with PCR-ELISA detection system with double internal standards(IMC-PCR-ELISA) .Methods The immunomagnetic (Dynabeads? ) which could specifically capture Mycobacterium tuberculosis were prepared .According to Mtp40 and IS6110 gene sequence of Mycobacterium Tuberculosis ,2 pairs of primers(upstream primer was modified with Biotin at 5′end) ,2 same-length mutant fragments with PCR amplified fragments ,and 3 capture probes(modified with digoxigenin at 3′end) were designed .Myco-bacterium tuberculosis were captured by immunomagnetic ,then detected by PCR-ELISA with double internal standards .Results The IMC-PCR-ELISA method could yield quantitative results in about 4 h with a detection limit at 5 × 103 copies/mL .There was a fine linear relationship between the copies of Mtp40(IS6110)in fact and in the calculation through formula when the concentrations of low internal standards were 30-70 copies/mL and the concentrations of high internal standards were 8 000-12 000 copies/mL (r2 =0 .998) .No nonspecific amplification was observed .Conclusion A rapid and quantitative method for the detection of Myco-bacterium tuberculosis was established successfully .The IMC-PCR-ELISA method was rapid ,sensitive ,secific and quantitative .
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Objective To investigate the clinical application of examination of specific IgG4 to food allergens.Methods Specific IgG4 as well as specific IgE to ten kinds of food allergens in sera of 51 patients with chronic eczema was examined by ELISA.Results Food allergen specific IgG4 was positive in 35 patients (68.6%) and food allergen specific IgE was positive in 45 patients(88.2%)of the 51 cases (P