Objective To study the expression of NLRP3 inflammasome and its downstream inflammatory factors in patients with chronic obstructive pulmonary disease (COPD) and healthy controls, and to reveal the effect and significance of NLRP3 inflammasome in the pathogenesis of COPD. Methods Forty patients with acute exacerbation COPD (AECOPD) who were hospitalized from November 2016 to May 2017 were recruited in the AECOPD group, and recruited in the stable COPD group when they entered the stable stage. Forty healthy individuals were recruited in the control group. General information and peripheral blood were collected from each subject. The levels of NLRP3 mRNA and caspase-1 mRNA in peripheral blood mononuclear cells were measured by real-time PCR. The levels of IL-18 and IL-1β were measured by enzyme-linked immunosorbent assay. Results The levels of NLRP3 mRNA, IL-18 and IL-1β in the AECOPD patients were significantly higher than those in the stable COPD group [2.11±0.77, 12.79 (7.10, 43.13) pg/ml, 17.02 (8.36, 52.21) pg/ml vs. 1.60±0.44, 10.66 (6.32, 18.59) pg/ml, 13.34 (7.07, 16.89) pg/ml, all P<0.05] . The levels of NLRP3 mRNA, IL-18 and IL-1β in the AECOPD patients were significantly higher than those in the control group [2.11±0.77, 12.79 (7.10, 43.13) pg/ml, 17.02 (8.36, 52.21) pg/mlvs. 1.00±0.49, 6.29 (4.73, 7.93) pg/ml, 5.93 (4.81, 9.67) pg/ml, all P<0.05]. The levels of NLRP3 mRNA, IL-18 and IL-1β were significantly higher in the stable COPD group than the control group [1.60±0.44, 10.66 (6.32, 18.59) pg/ml, 13.34 (7.07, 16.89) pg/mlvs. (1.00±0.49, 6.29 (4.73, 7.93) pg/ml, 5.93 (4.81, 9.67) pg/ml, all P<0.05]. Correlation analysis showed that the plasma IL-18 level was positive correlated with leukocyte count and neutrophil percentage in the AECOPD group (r=0.372, P<0.05;r=0.386, P<0.05). The expression of NLRP3 mRNA in the AECOPD group and stable COPD group were positively correlated with the CAT score (r=0.387, P<0.05;r=0.399, P<0.05) . Conclusion NLRP3 inflammasome is involved in the inflammatory response in COPD patients.
ObjectiveTo investigate the association between IL-1β gene-511C/T polymorphisms and the risk of chronic obstructive pulmonary disease (COPD). MethodsSuch databases as PubMed, EMbase, CNKI, CBM, VIP and WanFang Data were searched for the studies on the association between IL-1β gene-511C/T polymorphisms and the risk of COPD up to May 2014. According to inclusion and exclusion criteria, two reviewers independently screened literature, extracted data, and assessed methodological quality of included studies. Then meta-analysis was performed using RevMan 5.0 software. ResultsA total of 10 case-control studies from 9 articles involving 1 171 cases and 1 268 controls were included. The results of meta-analysis showed that, no significant association was found between IL-1β gene-511C/T polymorphisms and the risk of COPD:TT+CT vs. CC:OR=1.06, 95%CI 0.66 to 1.70, P=0.82; TT vs. CT+CC:OR=0.87, 95%CI 0.60 to 1.26, P=0.32; TT vs. CC:OR=0.95, 95%CI 0.51 to 1.75, P=0.86; CT vs. CC:OR=1.10, 95%CI 0.71 to 1.70, P=0.15; T vs. C:OR=0.97, 95%CI=0.72 to 1.30, P=0.84. The results of subgroup analysis by ethnicity showed that, no significant association was found between IL-1β gene-511C/T polymorphisms and the risk of COPD among Caucasians and Asians. ConclusionIL-1β gene-511C/T polymorphisms might not contribute to the risk of COPD.
Objective Melatonin (MLT) can increase the expression of cartilage-derived growth factor and stimulate the synthesis of cartilage matrix. To investigate the prevention and treatment effects of MLT on damaged cartilage through observing the expressions of bone morphogenetic protein 2 (BMP-2) and interleukin 1β (IL-1β) in articular cartilage of the rats with osteoarthritis (OA). Methods Forty SPF 4-week-old male SD rats (weighing 120-150 g) were randomly divided into 4 groups (n=10): normal control group (group A), OA group (group B), OA/pinealectomy group (group C), and OA/ pinealectomy/MLT group (group D). The rats of group A served as a control without treatment. The rats of groups B, C, andD underwent left knee joint injection of 0.2 mL 4% papain solution 1 time every other day for 2 weeks for establ ishing OAmodel. Two weeks after papain injection, the rats of groups C and D were exposed to continuous l ight for 24 hours (intensity of illumination: 500 lx) for creating pinealectomy models. And at the next day after pinealectomy model establ ishing, the rats of group D were treated with intra-articular injections of 0.2 mL 20 mg/mL MLT solution 4 times a week for 4 weeks. At 1 week after last MLT injection, the venous blood samples were taken in groups A, B, and C to test the level of serum MLT by ELISA, respectively, and then the specimens of left cartilage of femoral condyle were harvested for macroscopic, histological, and immunohistochemical examinations in 4 groups. Results The OA and pinealectomy models of rats were successfully establ ished, and all rats survived. There were significant differences in the serum MLT level among groups A, B, and C, and among different time points at the same group (P lt; 0.05). In group A, articular cartilage surface was smooth and elastic, and chondrocytes arranged regularly. In groups B and C, articular cartilage surface was rough, cartilage defects and subchondral bone exposure were observed in some areas, and chondrocytes arranged irregularly. In group D, cartilage surface was more smooth than that in groups B and C, and the degrees of cartilage defect and subchondral bone exposure decreased with regular arrangment of chondrocytes. There were significant differences in Mankin scores and integral absorbance values among 4 groups (P lt; 0.05). Conclusion Exposure to continuous l ight can accelerate degeneration process of articular cartilage of OA rats. Injections of 0.2 mL MLT solution (20 mg/mL) by intra-articular for 4 weeks can inhibit the progress of cartilage defects. Upregulationof anabol ic factor of BMP-2 as well as down-regulation of catabol ic factors of IL-1β is associated with cartilage repairin the pathological features of OA.
Objective To investigate interleukin-1β (IL-1β), IL-6, and IL-17 levels in both synovial fluid and serum of patients with primary knee medial osteoarthritis (OA) after high tbial osteotomy (HTO). Methods Twenty-six patients with primary knee medial OA undergoing HTO between January 2011 and June 2014 (experimental group) and 30 healthy individuals (control group) were recruited into the study. There was no significant difference in gender, age, and body mass index between 2 groups (P>0.05). The X-ray film was taken to record healing time at osteotomy site, to measure the tibiofemoral angle, and to assess limb alignment after HTO. Visual analogue scale (VAS) pain score and knee society score (KSS) were used to evaluate pain level and function of the knee. The IL-1β, IL-6, and IL-17 concentrations in both plasma and synovial fluid were measured before operation and at 6, 12, and 18 months after operation in the experimental group using ELISA method; the levels in plasma were measured in control group. Results Primary healing of incisions was achieved in patients. All patients were followed up 18-24 months (mean, 21 months). The X-ray film showed osseous healing at osteotomy site at 9-14 weeks (mean, 11.5 weeks). The average tibiofemoral angle was 167.5° (range, 165-170°) after bone healing. Satisfactory limb alignment was obtained in all patients. The postoperative VAS pain score was significantly decreased and KSS score was significantly improved when compared with preoperative scores (P<0.05), but no significant difference was found between different time points after operation (P>0.05). The preoperative plasma and synovial fluid IL-1β, IL-6, and IL-17 concentrations were significantly higher in patients than controls (P<0.05). The postoperative IL-1β, IL-6, and IL-17 concentrations in plasma and synovial fluid were significantly lower than preoperative ones in patients (P<0.05), but the concentrations were significantly higher than those in controls (P<0.05). The postoperative plasma and synovial fluid IL-1β, IL-6, and IL-17 concentrations were significantly declined in patients, but there was no significant difference between different time points after operation (P>0.05). Conclusion HTO can significantly improve the pain symptom and joint function and reduce IL-1β, IL-6, and IL-17 levels in both plasma and synovial fluid of patients with medial compartment knee OA, but these cytokines can not return to normal level.
ObjectiveTo isolate and identify the cartilage progenitor cells (CPCs) from normal cartilage, and to explore the influence of interleukin 1β (IL-1β) in different concentrations on its chondrogenesis. MethodsCPCs were isolated from normal cartilage of adult New Zealand white rabbit with the fibronectin adhesion assay;the cell phenotype was identified;and the cloning and differentiation of CPCs were observed. CPCs were incubated with H-DMEM in group A, with chondrogenic induced medium in group B, with chondrogenic induced medium+0.1 ng/mL IL-1β in group C and chondrogenic induced medium+1.0 ng/mL IL-1β in group D for 3 weeks. The histology, biochemistry, and real-time fluorescence quantitative PCR were performed to observe the effect of IL-1β on the chondrgenic differentiation. ResultsThe CPCs from normal cartilage expressed positively stem cell phenotype, which have similar ability of cloning and differentiation to stem cells. The cell pellets in groups C and D were significantly smaller than those in group B, and cell showed hypertrophic morphology change. There were more expressions of collagen type Ⅱ and collagen type X in group B than in group A, in group B than in groups C and D, and in group C than group D with Safranin O staining. The biochemistry results showed that collagen type Ⅱ content, glycosaminoglycan (GAG) content, and the ratio of GAG/DNA were significantly lower in groups C and D than in group B (P<0.05), and in group D than in group C (P<0.05);but the DNA content was significantly higher in groups C and D than in group B (P<0.05), and no significant difference between groups C and D (P>0.05). The real-time fluorescence quantitative PCR results showed that the relative mRNA expressions of collagen type Ⅱ, collagen type X, and Sox-9 were significantly lower in groups C and D than in group B (P<0.05), and in group D than in group C (P<0.05), but the relative mRNA expressions of Runx-2 and matrix metalloproteinase 13 were significantly higher in groups C and D than in group B (P<0.05), and in group D than in group C (P<0.05). ConclusionThere are CPCs having the character of stem cells in normal cartilage, and they have the capability of cloning and potential differentiation. IL-1β can inhibit the chondrogenesis of CPCs, and possibly promote the osteogenic differentiation.
Objective To access the protective effects of glucosamine hydrochloride capsules (OTL) on articular cartilage in osteoarthritis of rabbit. Methods Thirty-six New Zealand white rabbits were divided randomly into three groups (n=12): sham group (group A), anterior cruciate l igament transection (ACLT)/normal sal ine group (group B), and ACLT/ OTL group (group C). Rabbits in groups B, C received ACLT on the right knee. Rabbits in group A were not given ACLT ascontrol. Group C received a daily administration of OTL at a dose of 150 mg/kg of body weight for 12 weeks; in contrast, group B received normal sal ine at the same dose. All rabbits were sacrificed after 12 weeks. The right femoral condyle were removed and observed at pathologic changes with HE staining and graded by Mankin’s scale, the expression level of transforming growth factor β1 (TGF-β1) and interleukin 1β (IL-1β) were detected by immunohistochemical staining. Results All rabbits survived at the end of experiment and incision healed well. The gross observation showed that joint synovia increased and articular surface was smooth and integrity in group A; that ulcer was observed on the articular surface of group B; and that articular surface was smooth and integrity in group C. There were sigificant differences in articular cartilage scores between 3 groups (P lt; 0.05). The histological observation showed that the articular cartilage had normal structure and the cells arranged regularly in group A; that the articular cartilage became thin and the cells arranged irregularly in group B; and that the cells arranged with a clear layer and had regular shape in group C. The Mankin scores were 1.04 ± 0.13, 7.97 ± 0.12, and 2.81 ± 0.36 in groups A, B, and C, respectively; showing significant difference between 3 groups (P lt; 0.05). The result of immunohistochemistry showed that the expressions of TGF-β1 were 50.62 ± 1.51, 24.81 ± 1.28, and 41.57 ± 1.69 and the expressions of IL-1β were 13.12 ± 1.21, 62.53 ±2.37, and 30.67 ± 1.28; showing significant differences between 3 groups (P lt; 0.05). Conclusion A daily administration ofOTL at a dose of 150 mg/kg for 12 weeks can partially decrease the expression levels of IL-1β and increase the expression levels of TGF-β1, which delays the development of osteoarthritis.
ObjectiveTo investigate the relationship between alumina ceramic particles and aseptic loosening of the joint prosthesis and the effect of lanthanum chloride on the secretion of interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) of macrophage RAW264.7 induced by alumina ceramic particles. MethodsRAW264.7 cells were cultured in vitro and divided randomly into 4 groups according to different culture solutions:blank control group (group A),1 mg/mL alumina ceramic particles (group B),1 mg/mL alumina ceramic particles and 10 μmol/L lanthanum chloride (group C),and 10 μmol/L lanthanum chloride (group D).The cell growth was detected by MTT,and ELISA,RT-PCR,and Western blot were used to test the expressions of IL-1β,TNF-α,and nuclear factor κB (NF-κB). ResultsThere was no significant difference in cell growth among all groups by MTT (F=2.180,P=0.142).RT-PCR results showed that the expressions of IL-1β,TNF-α,and NF-κB mRNA in group B were significantly higher than those in the other 3 groups (P<0.05); the expressions in group D were significantly lower than those in group A (P<0.05).ELISA results showed that the contents of IL-1β and TNF-α in group B were significantly higher than those in the other 3 groups (P<0.05); the contents in group D were significantly lower than those in group A (P<0.05).Western blot analysis revealed that the expression of NF-κB protein in group B was significantly higher than that in the other 3 groups (P<0.05). ConclusionAlumina ceramic particles can stimulate the secretion of IL-1β and TNF-α of macrophage,and lanthanum chloride can inhibit the secretion of IL-1β and TNF-α of macrophage.
Objective To investigate the effect and mechanism of miR-4287, a chondrogenesis associated microRNA, regulated the expression of aggrecanase-1 (a disintegrin and metalloproteinase with thrombospondin motif 4, ADAMTS4) in human chondrocytes. Methods First, the voluntarily donated normal and osteoarthritic knee articular cartilages were used to detect the expressions of miR-4287 and ADAMTS4 mRNA by real-time fluorescence quantitative PCR. Then, chondrocytes were isolated from knee articular cartilages. The effect of IL-1β on the expression of miR-4287 and ADAMTS4 mRNA was validated by the first generation of osteoarthritic chondrocytes. To confirm the influence of IL-1β signal pathways on the expression of miR-4287 and ADAMTS4 mRNA, osteoarthritic chondrocytes were pretreated with MAPK signal pathway inhibitor SP600125, NF-κB pathway inhibitor SN50, and finally stimulated with IL-1β. Chondro cytes were transfected with miR-4287 mimics and mimics negative control, inhibitors and inhibitors negative control respectively to value the effect of miR-4287 on ADAMTS4 expression. Luciferase reporter assay was used to verify the direct interaction between miR-4287 and putative site in the 3-untranslated region (3’UTR) of ADAMTS4 mRNA. Results Compared with normal knee articular cartilages, the miR-4287 expression was markedly diminished and conversely ADAMTS4 mRNA expression was raised in osteoarthritis cartilages (P<0.05). Stimulation with IL-1β led to a reduction in miR-4287 expression and upregulation in ADAMTS4 mRNA expression, showing significant difference when compared with the untreated groups (P<0.05). Pretreatment with IL-1β signal pathway inhibitors induced miR-4287 expression and attenuated ADAMTS4 mRNA expression in human chondrocytes, which were significantly different from that of unstimulated cells (P<0.05). ADAMTS4 mRNA and protein were suppressed by transfection with miR-4287 mimics (P<0.05) and elevated by transfection with miR-4287 inhibitors (P<0.05). As luciferase reporter assay showed, overexpression miR-4287 failed to alter the luciferase activity of a reporter construct containing either wild or mutant 3’UTR of ADAMTS4 mRNA (P>0.05). Conclusion miR-4287, a chondrogenesis associated microRNA, may play an important role in cartilage degeneration. miRNA-4287 is able to regulate ADAMTS4 expression in human chondrocytes, but not by means of directly targeted the ADAMTS4 mRNA 3’UTR. The exact mechanisms need to be further addressed.
ObjectiveTo explore the effects of interleukin 10 (IL-10) gene modified bone marrow mesenchymal stem cells (BMSCs) on the expression of inflammatory cytokines and neuronal apoptosis in rats after cerebral ischemia reperfusion injury.MethodsBMSCs were cultured by whole bone marrow adherence screening method. The properties of BMSCs were identified by immunocytochemical methods. BMSCs at passage 3 were transfected with recombinant adenovirus IL-10 gene (AdIL-10-BMSCs). The model of middle cerebral artery occlusion was made in 40 adult male Sprague Dawley rats by thread embolism method. The rats were randomly divided into 4 groups (n=10). At 3 hours after modelling, the rats of groups A, B, C, and D received tail intravenous injection of 1 mL L-DMEM medium containing 10% FBS, 61.78 ng IL-10, 1 mL BMSCs suspension (2×106 cells/mL), and 1 mL AdIL-10-BMSCs cell suspension (2×106 cells/mL), respectively. The cells were labelled with BrdU before cell transplantation in groups C and D. At 7 days after reperfusion, the brain tissue was harvested to detect the expression of OX42 by immunohistochemical assay, to determine the concentration of tumor necrosis factor α (TNF-α) and IL-1β by ELISA, and to detect the apoptosis by TUNEL assay. BrdU labelled cells were observed by immunofluorescence staining in groups C and D.ResultsBrdU labelled positive cells with green fluorescence were observed in the brain tissue of groups C and D, which mainly distributed in the striatum, cerebral cortex, and subcortex around the infarction area. The number of OX42 positive cells was significantly less in groups B, C, and D than group A (P<0.05), and in group D than groups B and C (P<0.05). Compared with the other 3 groups, the contents of TNF-α and IL-1β significantly decreased in group D (P<0.05). TUNEL assay showed that the apoptotic cells (TUNEL positive cells) were mainly seen in the striatum and fronto parietal subcortical tissues (equivalent to ischemic penumbra). The number of TUNEL positive cells in group D was significantly less than that in groups A, B, and C (P<0.05).ConclusionAdIL-10-BMSCs can inhibit secretion of TNF-α and IL-1β from microglial cells and inhibit the nerve cell apoptosis around infarct brain tissue, which might contribute to its protective role upon cerebral ischemia reperfusion injury.