Objective To construct a regulatable plasmid containing single chain fusion gene of murine interleukin-12 (mIL-12) which was regulated with mifepristone (RU486) and explore its expression in vitro. Methods The p40 and p35 subunit sequence of mIL-20 were respectively obtained from the plasmid GCp35Ep40PN by polymerase chain reaction (PCR) and they were cloned into pCA14 plasmid after introducing a linker by overlap PCR. The single chain mIL-12 gene was comfirmed by sequencing and subcloned into pRS-17 vector which contains RU486 regulator cassette. The positive clone named pRS-RUmIL-12 was identified by restriction endonuclease digestion and PCR. Lipofectamine 2000 was used to transfect the pRS-RUmIL-12 to HEK293 cells followed by manufacturer’s recommendations. The protein concentration of mIL-12 induced with RU486 in supernatant of the transfected HEK293 cells was measured by ELISA. Results The sequence of single chain mIL-12 what we obtained was the same as the expected result. The results of restriction endonuclease digestion and PCR showed that the RU486-inducible regulatory vector (pRS-RUmIL-12) was successfully constructed. No significant mIL-12 protein concentration in supernatant of HEK293 cells activation was measured without the inducer RU486, whereas higher concentration of the mIL-12 protein was observed in the presence of RU486. The relationship of concentration of the mIL-12 protein and RU486 was positive correlated under definite range. Conclusion A regulatable eukaryotic expression plasmid of mIL-12 single chain fusion gene was constructed, which could be used in the further research of gene regulation and gene therapy.
Objective To investigate the impacts of cytokines （interleukin-4，IL-4；tumor necrosis factor-α，TNF-α） and medications of bronchial asthma （dexamethasone，aminophylline，salbutamol） on the activity of histamine N-methyltransferase（HMT） in tracheal epithelial cells.Methods BEAS-2B bronchial epithelial cells were cultured and treated with different concentration of TNF-α, IL-4, dexamethasone, salbutamol and aminophylline respectively. The activity of HMT in BEAS-2B cells was determined by high performance liquid chromatography.Results The activity of HMT in tracheal epithelial cells was (50±7) pmol?min-1?mg pro-1.TNF-α and IL-4 lowered the activity of HMT significantly at the concentration equal to or higher than 1 ng/mL and 5 ng/mL respectively，and reached the maximum inhibitory effect at the level of 10 ng/mL.Dexamethasone and aminophylline could ameliorate distinctly the inhibitory effect of TNF-α on the activity of HMT, while salbutamol had no significant inhibitory effect.Conclusions TNF-α and IL-4 exert the lowering effect on the activity of HMT，which would be one important cause of airway hyperreactivity.Glucocorticoids and theophyllines are administered to treat asthma partly due to its relieving mechanism of TNF-α negative effects on HMT.
Objective To study the regulative effect of angelica sinensis on cellular immune function in perioperative patients with obstructive jaundice. Methods Fourteen patients with obstructive jaundice were injected with angelica before and after operation for 14 days. The activity of IL-2 and the expression of IL-2R in lymphocytes in peripheral blood were measured, respectively. Results The activity of IL-2 and the expression of IL-2R decreased significantly in patients with obstructive jaundice (P＜0.01). The activity of IL-2 and the expression of IL-2R in peripheral blood lymphocyte increased significantly before and after operations (after treatment using angelica) (P＜0.01), though there was a little decrease after operation but they were still higher than that befor using angelica.Conclusion It maybe useful to use angelica to improve the cellular immune function in patients with obstructive jaundice.
Objective
To observe the effect of celastrol on the secretion of interleukin (IL)-17 in peripheral blood mononuclear cells in patients with sympathetic ophthalmia (SO), and its possible mechanisms.
Methods
Venous blood samples were extracted from 10 cases of sympathetic ophthalmia patients and 10 health objectives. The peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation and then were divided into 4 groups. Group A (control group): PBMCs of health objectives; Group B: PBMCs of SO patients; Group C: PBMCs of SO patients with 0.5 μmol/L celastrol in the medium; Group D: PBMCs of SO patients with 1 μmol/L celastrol in the medium. After culturing the cells for 3 days, the supernatant of 4 groups was collected, and the levels of IL-23 and IL-17 were detected by enzyme-linked immuno sorbent assay (ELISA). Then, the 50 ng/ml rIL-23 was added into the medium of group A which was the group A1; the 50ng/ml rIL-23 and 1 μmol/L Cela were added into to the medium of group A which was the group A2. For the medium of group B, the 50 ng/ml rIL-23 was added into the medium which was the group B1; the 50 ng/ml rIL-23 and 1 μmol/L celastrol were added into to the medium of group B which was the group B2. After culturing for 3 days, the supernatant of cells of these 4 groups was collected, and the levels of IL-17 were detected by ELISA.
Results
In group A, the levels of IL-23 and IL-17 were (228.43±17.27) pg/ml and (220.55±31.15) pg/ml respectively. In group B, the levels of IL-23 and IL-17 were (513.85±36.46) pg/ml and (866.77±72.92) pg/ml respectively. In group C, the levels of IL-23 and IL-17 were (381.07±20.93) pg/ml and (517.43±54.87) pg/ml respectively. In group D, the levels of IL-23 and IL-17 were (237.14±17.97) pg/ml and (242.89±34.09) pg/ml respectively. Between group A and D, there was no statistically significant difference in IL-23 or IL-17 level (P＞0.05); but when comparing other groups, the differences were statistically significant (P＜0.05). The levels of IL-17 in group A1 and group A2 were (428.43±24.53) pg/ml and (229.15±23.28) pg/ml and the difference was statistically significant (P＜0.05). The levels of IL-17 in group B1 and group B2 were (1373.39±89.51) pg/ml and (571.01±94.88) pg/ml and the difference was statistically significant (P＜0.05).
Conclusion
Celastrol can inhibit the secretion of IL-17 by PBMCs in SO patients via inhibiting the secretion of IL-23.
ObjectiveTo investigate the effects and clinical significance of edaravone on serum tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and intercellular adhesion molecule-1 (ICAM-1) in elderly patients with obstructive sleep apnea hypopnea syndrome (OSAHS).MethodsA total of 90 elderly patients with moderate to severe OSAHS confirmed by polysomnography were recruited from North China University of Science and Technology Affiliated Hospital in February 2016 to October 2017. According to random number table method the OSAHS patients were randomly divided into group A (n=30), group B (n=30) and group C (n=30). Group A received continuous positive airway pressure treatment for six months, group B received edaravone therapy and continuous positive airway pressure treatment for six months, and group C only received edaravone therapy for six months. The changes of serum TNF-α, IL-6 and ICAM-1 were detected by enzyme-linked immunosorbent assay before and after treatment.ResultsThe differences of serum TNF-α, IL-6 and ICAM-1 before treatment in the three groups were not statistically significant (P>0.05). Compared with before treatment, the levels of serum TNF-α, IL-6 and ICAM-1 decreased in the three groups (P<0.05). After six months of treatment, the levels of serum TNF-α, IL-6 and ICAM-1 decreased in group A and group B compared with group C (P<0.05), and decreased significantly in group B compared with group A (P<0.05).ConclusionEdaravone can inhibit the expressions of serum TNF-α, IL-6 and ICAM-1 in elderly patients with moderate to severe OSAHS, and thereby reduce vascular endothelial dysfunction and injury.
Objective To investigate the effects of ginkgo biloba extract （GBE） on expressions of IL-1β, IL-6,and TNF-α in the pancreas and brain tissues of rats with severe acute pancreatitis （SAP）, and further to explore the pathogenesis of SAP and the efficacy of GBE on brain injury. Methods Fifty-four Winstar rats were randomly divided into normal control group, model group, and treatment group, with 18 rats for each group. For rats in the normal control group, only conversion of pancreas was performed by abdomen opening , followed by wound closure immediately. For rats in the model group and treatment group, 5% sodium taurocholate hydrate were injected under pancreatic capsule to establish SAP model, and then GBE and normal saline were infected into intra-abdomen repeatedly every 8 hours, respectively. At 6 h, 12 h, and 24 h after the model establishment, experimental samples were extracted and serum amylase was detected. Pathogenic scoring for pancreas tissues was performed under light microscopy, and immunohistochemistry method was employed to detect the expression levels of IL-1β, IL-6, and TNF-α in pancreas and brain tissues. Results For the treatment group, both serum amylase and pancreas scoring were significantly lower than those of the model group （P＜0.01）. At 24 h after model establishment, the expressions of IL-1β, IL-6, and TNF-α of pancreas tissues in model group were significantly higher than those at 6 h and 12 h （P＜0.05 or P＜0.01）, but no significant differences wereobserved in treatment group （P＞0.05）. The expressions of IL-1β, IL-6, and TNF-α of brain tissues in model group were significantly higher than those at 6 h and 12 h （P＜0.05 or P＜0.01）, but in treatment group decreased （P＜0.05 or P＜0.01）. The expressions of IL-1β, IL-6, and TNF-α in the treatment group were significantly lower than those of the model group at same time （P＜0.01）. Conclusions During SAP, the expressions of IL-1β, IL-6 and TNF-α in pancreas and brain tissues increased obviously. GBE showed suppressing and scavenging effects on IL-1β, IL-6 and TNF-α in pancreas and brain tissues.
ObjectiveTo investigate the effects of interleukin (IL)-26 on the late phase of lipopolysaccharides (LPS)-induced lung inflammation in mouse model.MethodsThirty-two mice were equally and randomly divided into four groups: blank control group, IL-26 control group, LPS model group, and IL-26 intervention group. The blank control group was given intranasal administration of phosphate buffered solution (PBS, 40 μl) and PBS (40 μl) 10 minutes apart. The IL-26 control group was given recombinant human interleukin-26 (rhIL-26; 50 μg/kg, dissolved in 40 μg PBS) and PBS successively. The LPS model group was given intranasal administration of PBS (40 μl) and LPS (10 mg/kg, dissolved in 40 μl PBS) at 10 minutes interval. The IL-26 intervention group was given intranasal administration of rhIL-26 and LPS at 10 minutes interval. Seventy-two hours later after treatment, bronchoalveolar lavage fluid (BALF) cell count, cytokine assay and pathological staining of lung tissue were performed in each group. The gene expression of inflammatory pathway in lung tissue was detected by RT-PCR. One-way ANOVA was used for comparison between groups. ResultsCompared with the blank control group, the expression of tumor necrosis factor-α and activating transcription factor 3 in IL-26 control group increased significantly (all P < 0.05). The number of peripheral blood mononuclear cells, total BALF cells, lymphocytes and neutrophils, and the content of macrophage inflammatory protein-1a in BALF were significantly increased in IL-26 intervention group comparing with LPS model group (all P < 0.05). IL-26 intervention group had more inflammatory subsidence in interstitial, perivascular, peribronchial and mean values than LPS model group (all P < 0.05). The expressions of Toll-like receptor 4, Toll-like receptor 2 and interferon γ induced protein 10 in IL-26 intervention group were significantly higher than those in LPS model group (all P < 0.05).ConclusionIL-26 can significantly alleviate the late inflammatory reaction of lung tissue in LPS-induced mouse inflammation model.
ObjectiveTo investigate the expression of programmed cell death-1 (PD-1), interferon-γ (IFN-γ), interleukin-4 (IL-4) and T help cells (Th) in peripheral blood of patients with asthma.MethodsForty-one asthma patients were divided into an acute group (20 cases) and a chronic group (21 cases) according to the disease state of asthma. Another 18 healthy subjects were recruited as a control group. Peripheral blood samples were collected from all the subjects at admission or inclusion. The concentrations of PD-1, IFN-γ and IL-4 were detected by enzyme-linked immunoassay method. The expressions of Th1 and Th2 were detected by flow cytometry method.ResultsThe PD-1 and IFN-γ concentrations, Th1 proportion and Th1/Th2 ratio in the two asthma groups were reduced compared with the healthy control group, and were significantly lower in the acute group compared with the chronic group (all P<0.05). The IL-4 level and Th2 proportion in the two asthma groups were increased compared with the healthy control group, and were significantly higher in the acute group than the chronic group (allP<0.05). In the acute group, the PD-1 level was positively correlated with IFN-γ and Th1 level (r values were 0.678 and 0.712, respectively), and negatively correlated with IL-4 and Th2 (r values were –0.745 and –0.700, respectively).ConclusionThe concentration of PD-1 in patients with asthma is reduced especially in acute asthma, and shows close correlation with conventional index of asthma.
ObjectiveTo study the effect of down-regulated leptin receptor by small interfering RNA (siRNA) in inhibiting the messenger RNA (mRNA) expressions of interleukin (IL)-1β and nitric oxide (NO) of human osteoarthritis chondrocytes, in order to provide reference for basic clinical research.
MethodsCartilage was harvested under sterile conditions from osteoarthritis knee joints in patients undergoing total knee arthroplasty. Human articular chondrocytes were isolated and the cells were cultured in vitro. The cells in the 3rd passage were transferred by siRNA Ob-Rb (experimental group) and blank Ob-Rb (control group), respectively. Then mRNA expressions of IL-1β and NO were tested by quantitative polymerase chain reaction at hour 24, 48 and 72 after successful transfection.
ResultsThe mRNA expressions of IL-1β increased slightly and that of NO declined slightly at hour 24, 48 and 72 after transfection in the treatment group, but they all were significantly lower than those in the control group (P ＜ 0.05) , and the differences became much larger as time went on.
ConclusionLeptin receptor under siRNA technology can significantly inhibit the mRNA expressions of IL-1β and NO in human osteoarthritis chondrocytes.
ObjectiveTo investigate the pathogenesis and treatment of obstructive sleep apnea hypopnea syndrome (OSAHS) by detecting the changes of serum interleukin-23 (IL-23) and C-reactive protein (CRP) levels of the OSAHS patients before and after treatment with continuous positive airway pressure (CPAP).MethodsFifty-eight patients with moderate to severe OSAHS diagnosed by polysomnography were recruited as an experimental group, 57 out-patient healthy subjects with matched age, sex and body mass index of the experimental group were enrolled as a control group. The serum concentrations of IL-23 and CRP in the experimental group were detected and compared before and after CPAP application for 3 months. The serum concentrations of IL-23 and CRP in the control group were also measured.ResultsThe serum levels of IL-23 and CRP in the OSAHS patients were significantly higher than those in the normal control subjects (P<0.05). The serum levels of IL-23 and CRP in the OSAHS patients after CPAP treatment were significantly lower than those before CPAP treatment (P<0.05). The serum concentrations of IL-23 and CRP were positively correlated with apnea hypopnea index (r=0.756, r=0.345, P<0.05, respectively), and negatively correlated with mean oxygen saturation (r=–0.715, r=–0.334, P<0.05, respectively).ConclusionsThe serum levels of IL-23 and CRP are positively correlated with the severity of OSAHS. After CPAP treatment, the levels of IL-23 and CRP decrease, which indicates that CPAP treatment may reduce the inflammatory reaction and correct anoxia of OSAHS patients.
