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Objective To investigate the effect of atomization inhalation of budesonide and terbutaline sulfate on serum levels of cyclcoxygenase 2 (COX-2) and chemokine like factor-1 (CKLF-1) in children with bronchial asthma.Methods A total of 78 children with bronchial asthma in Hangzhou Children's Hospital from April 2016 to August 2017 were selected and divided into the control group(n =39) and the study group(n =39).The control group was treated with routine treatment,and the study group was treated with budesonide and terbutaline sulfate on the basis of the control group.The treatment was continued for 7 d.After t treatment,the clinical effects,clinical symptoms improvement,hospitalization time,the serum levels of COX-2 and CKLF-1 before and after treatment,and the incidence of adverse reaction of two groups were observed.Results The total effective rate of the study group(94.87％) was higher than that of the control group (74.36％)(x2 =6.303,P ＜ 0.05).The improvement time of chest tightness,wheezing,cough and hospitalization time of the study group were shorter than those of the control group (t1 =13.054,t2 =7.365,t3 =4.944,t4 =8.342,all P ＜ 0.05).After treatment,the levels of serum COX-2 and CKLF-1 in the two groups were lower than those before treatment,which of the study group were lower than those in the control group(t1 =4.934,t2 =4.660,all P ＜0.05).There was statistically significant difference in the incidence rate of adverse reactions between the study group(12.82％) and the control group(7.69％) (x2 =0.139,P ＞ 0.05).Conclusion Atomization inhalation of budesonide and terbutaline sulfate in the treatment of children with bronchial asthma can effectively alleviate the clinical symptoms,reduce serum levels of COX-2 and CKLF-1,improve therapeutic effect,promote children's recovery and shorten the hospitalization time.Besides,the incidence rate of adverse reactions is low,and it is safe.
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Objective To investigate the effects of chemokine like factor 1 (CKLF1) gene on the proliferative activities and osteogenic potentials of hip ligaments of ankylosing spondylitis (AS) in situ and in vitro. Methods Normal and AS hip ligament specimens were collected from 6 patients with femoral neck fracture and 4 AS patients with severe hip deformities. Ligament specimens were exposed to type Ⅱ colla-genase and obtained a single cell suspension, while phase contrast microscopy and anti-vimentin immuno- fluorescence staining (IFC) were applied to observe the cells. The specimens and fibroblasts were divided and cultured in situ and in vitro respectively, and the recombinant adeno-associated virus (rAAV)-lacZ (E. coli beta-galactosidase gene)and rAAV-hCKLF1 (human CKLF1 cDNA cloned in rAAV-lacZ in place of lacZ) were transduced for 21 days. Cell proliferation (cellularity), secretion of pro-inflammatory cytokines, expression of CKLF1 and CCR4 genes were detected by the water-soluble tetrazolium (WST-1) assay and Hoechst 33258 test (DNA content), enzyme-linked immunosorbent assay (ELISA), IFC test and fluorescent quantitative reverse transcription polymerase chain reaction (RT-PCR), respectively. Statistical analysis significance was conducted using the Student's t test and one-way analysis of variance (ANOVA) (LSD) test where appropriate. Results The second passage of normal and AS cells were positive for anti-vimentin, indicating that the cells were fibroblasts. After transducing with rAAV-hCKLF1 for 21 days, cellularity, WST-1 and Hoechst 33258 assays illustrated that CKLF1 gene transfer promoted cell proliferation (compared with the non-viral transduction and lacZ groups, F=6.98, 64.32, 115.91, P<0.05 or P<0.01). Overexpression of CKLF1 gene enhanced the secretion of pro-inflammatory cytokines (interleukin-6 and tumor necrosis factor-alpha) and the expression of bone-specific extracellular matrix proteins (osteopontin and osteocalcin) (F=34.57, 8.89, P<0.05 or P<0.01). Similar results were observed in fluorescent quantitative RT-PCR test. Conclusion Overexpression of CKLF1 promotes the proliferation of fibroblasts, the secretion of pro-inflammatory cytokines and the expression of osteogenic related target genes, suggesting that CKLF1 might be involved in the pathological ossification of AS.
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Objective To investigate the effects of chemokine like factor 1 (CKLF1) gene on the proliferative activities and osteogenic potentials of hip ligaments of ankylosing spondylitis (AS) in situ and in vitro. Methods Normal and AS hip ligament specimens were collected from 6 patients with femoral neck fracture and 4 AS patients with severe hip deformities. Ligament specimens were exposed to type Ⅱ colla-genase and obtained a single cell suspension, while phase contrast microscopy and anti-vimentin immuno- fluorescence staining (IFC) were applied to observe the cells. The specimens and fibroblasts were divided and cultured in situ and in vitro respectively, and the recombinant adeno-associated virus (rAAV)-lacZ (E. coli beta-galactosidase gene)and rAAV-hCKLF1 (human CKLF1 cDNA cloned in rAAV-lacZ in place of lacZ) were transduced for 21 days. Cell proliferation (cellularity), secretion of pro-inflammatory cytokines, expression of CKLF1 and CCR4 genes were detected by the water-soluble tetrazolium (WST-1) assay and Hoechst 33258 test (DNA content), enzyme-linked immunosorbent assay (ELISA), IFC test and fluorescent quantitative reverse transcription polymerase chain reaction (RT-PCR), respectively. Statistical analysis significance was conducted using the Student's t test and one-way analysis of variance (ANOVA) (LSD) test where appropriate. Results The second passage of normal and AS cells were positive for anti-vimentin, indicating that the cells were fibroblasts. After transducing with rAAV-hCKLF1 for 21 days, cellularity, WST-1 and Hoechst 33258 assays illustrated that CKLF1 gene transfer promoted cell proliferation (compared with the non-viral transduction and lacZ groups, F=6.98, 64.32, 115.91, P<0.05 or P<0.01). Overexpression of CKLF1 gene enhanced the secretion of pro-inflammatory cytokines (interleukin-6 and tumor necrosis factor-alpha) and the expression of bone-specific extracellular matrix proteins (osteopontin and osteocalcin) (F=34.57, 8.89, P<0.05 or P<0.01). Similar results were observed in fluorescent quantitative RT-PCR test. Conclusion Overexpression of CKLF1 promotes the proliferation of fibroblasts, the secretion of pro-inflammatory cytokines and the expression of osteogenic related target genes, suggesting that CKLF1 might be involved in the pathological ossification of AS.
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Chemokine-like factor 1 (CKLF1) is a newly cloned chemotactic cytokine with CCR4 being its functional receptor. Recent evidence demonstrates a role of CKLF1 in arthritis. The aim of this study was to quantify the expression of CKLF1 as well as assess the correlation between CKLF1 and plasma acute-phase markers. Synovium was obtained from 16 osteoarthritis (OA), 15 rheumatoid arthritis (RA) and 10 ankylosing spondylitis (AS) patients undergoing total joint arthroplasty, with other 11 patients treated for meniscal tears during sport accidents serving as normal controls. Levels of CKLF1 and CCR4 mRNA were detected by qRT-PCR, and the expression of CKLF1 was investigated by immunohistochemistry staining, subsequently analyzed with semiquantitative scores. Plasma acute-phase markers of inflammation were determined by ELISA. CKLF1 was found with a particularly up-regulated expression in synovim from AS and RA patients, and CCR4 mRNA levels increased in RA patients, not in OA or AS patients. Elevated levels of plasma markers of inflammation including CRP, ESR and D-dimer were observed in RA. Further, significantly positive correlations between relative expression levels of CKLF1 and CRP/ESR in RA patients and a positive correlation between CKLF1 and ESR in AS patients were found. There was no detectable correlation between CKLF1 and plasma D-dimer. This study confirms an increased but different level of CKLF1 in RA, OA and AS patients, all significantly higher than that in controls. Additionally, the significant positive correlations between CKLF1 levels and CRP/ESR in RA and between CKLF1 and ESR suggest that CKLF1 might contribute to the inflammation state and clinical symptoms in these rheumatic diseases. Further studies are required to investigate the utility of targeting specific CKLF1 for symptom control or disease modification in RA and AS.
Subject(s)
Adult , Female , Humans , Male , Middle Aged , Arthritis, Rheumatoid , Metabolism , Biomarkers , Metabolism , Case-Control Studies , Chemokines , Genetics , Metabolism , MARVEL Domain-Containing Proteins , Genetics , Metabolism , Osteoarthritis , Metabolism , RNA, Messenger , Genetics , Metabolism , Receptors, CCR4 , Genetics , Metabolism , Spondylitis, Ankylosing , Metabolism , Synovial Fluid , MetabolismABSTRACT
Objective To investigate the expression of chemokine-like factor 1(CKLF1) in the balloon injured aorta of Sprague-Dawley rats.Methods Balloon expansion induced aorta injury model was established in 80 male Sprague-Dawley rats.Model rats were randomly divided into 8 groups.Rats were sacrificed at the postoperative periods of 12 hours,1 day,3 days,1 week,2 weeks,4 weeks,6 weeks,and 8 weeks respectively.Sham injury operation was applied to 5 rats as control.The ratio of intimal area (IA) and medial area (MA) was calculated to determine the extent of neointimal hyperplasia.Expression of CKLF1 was examined at protein level with immunohistochemistry and at mRNA level with RT-PCR.Software IPP6.0 was used to examine the mean optical density of positive staining.With β-actin expression as an internal control,semi-quantity of CKLF1 expression was calculated by CKLF1/β-actin.Results Visible neointima was noticed at 1 week postoperation.Extend of intimal hyperplasia(IA/MA)was most remarkable at 4 weeks and receded afterwards. Immnohistochemistry study showed that expression of CKLF1 was stronger in the neointima than in the media(P=0.016).The expression was most obvious in the neotima at 1 week postoperation.RT-PCR showed peak expression at 3 days postoperation and declined gradually but still at a higher level than control(P<0.05).The extent of intimal hyperplasia(IA/MA)was positively correlated to the expression of CKLF1(R=0.70,P=0.188).Conclusion The expression of CKLF1 was up regulated in balloon injured rat aorta.The expression was more obvious in the neointima than in the media.CKLF1 may play a role in the development of intimal hyperplasia.
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Objective To express human chemokine-like factor 1 (CKLF1) in Drosophila S2 cells and study its function. Methods The pMT/V5-His-CKLF1 expression plasmid was constructed and transfected into Drosophila S2 cells. The positive clones were selected through PCR and RT-PCR. The culture medium was analyzed by Western blot with anti-CKLF1 polyclonal antibody. Chemotaxis and MTT assays on human peripheral blood and C2C12 cells, respectively, were then carried out with the medium. Results CKLF1 was transcribed efficiently in S2 cells. The expressed CKLF1 protein could be detected in the culture supernatant by Western blot, which showed weak chemotactic activity on both human peripheral blood neutrophils and lymphocytes as well as enhancing effect on the proliferation of C2C12 cells. Conclusion CKLF1 was expressed successfully in Drosophila S2 cells and secreted into the culture medium. The recombinant CKLF1 expressed in Drosophila cells can chemoattract leucocytes and promote the proliferation of C2C12 cells.