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Objective:To establish an animal model of chronic systemic inflammation with long-term high expression of circulating IL-6 by introducing exogenous IL-6 gene transfer vector.Methods:Recombinant murine IL-6-encoding adeno-associated virus (AAV-IL-6) was constructed. Twenty-one 24-week-old male C57BL/6J mice were randomly divided into three groups with seven in each group: AAV-IL-6 group, vector control (AAV-ctrl) group and blank control group. At 0, 8 and 16 weeks of intervention, the mice in the three groups were injected with AAV-IL-6 (100 μl 0.5×10 10 vp/ml), unloaded AAV (100 μl 0.5×10 10 vp/ml) and the same volume of saline in the tail vein, respectively. IL-6 levels in mouse serum were measured by ELISA. The general condition of mice was observed and blood routine tests were performed. Changes in blood biochemical parameters and C-reactive protein (CRP) levels were detected. At the end of 24-week intervention, the mice were sacrificed and the myocardium, liver, spleen, quadriceps femoris, knee joint and middle femur were taken for HE staining. Results:At 4, 8, 16 and 24 weeks after intervention, serum IL-6 levels were (75.41-169.28) pg/ml in the AAV-IL-6 group, while in the two control groups, the levels were below the lower limit of detection (7.8 pg/ml). At 24 weeks after intervention, the body weight of mice in the AAV-IL-6 group was significantly lower than that of mice in the two control groups; the neutrophil counts and CRP level in the AAV-IL-6 group were higher than those in the two control groups, while the levels of albumin, creatinine, triglyceride and cholesterol were lower than those in the two control groups. There were no differences in the aforementioned parameters between the two control groups. Compared with the blank control group, both AAV-IL-6 and AAV-ctrl groups showed increased lymphocyte counts. All mice had normal liver and kidney functions at the end of intervention. Histopathological findings indicated that the mice in the AAV-IL-6 group had focal infiltration of lymphocytes in the central venous area of the liver and around the myocardial and the skeletal muscle fibers, diffuse infiltration of multinucleated giant cells in the spleen, atrophic skeletal muscle, disorganized growth plate, reduced chondrocyte hypertrophic zone, thinner bone cortex and trabecular, and reduced osteoid. There were no histopathological changes in mice of the two control groups.Conclusions:Repeated tail vein injection of AAV-IL-6 could achieve long-term high expression of circulating IL-6 in mice, which manifested the phenotype of chronic systemic inflammation in preliminary detection and provided a safe, effective and simply accessible animal model for related studies.
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Objective:To explore early-onset gout and related risk factors in Shandong Province, and provide decision-making information on prevention.Methods:Data from electronic medical records and face-to face interview were collected from 8 393 patients with gout who first visited the gout clinic of the Affiliated Hospital of Qingdao University from September 2016 to December 2021. Data included demographics, comorbidity and biochemical examinations. The dynamic changes of onset age from 2002 to 2021 were statistically analyzed. The clinical characteristics and related risk factors of patients with early-onset and late-onset gout were statistically analyzed.Results:The age of onset of gout decreased significantly from 2002 to 2021. Compared with 2002, the average age of onset in 2021 decreased by 2.3 years [(41.9±10.6 vs 39.6±14.0) years]. The median age of onset decreased by 3 years in 2012-2021 compared with 2002-2011(37 vs 40 years, P<0.001). The proportion of gout patients with onset age<40 years old increased significantly, from 45.1% in 2002 to 57.8%, and increased by 12.7% in 20 years( P<0.001). The constituent ratios of 20-29 years old group( Ptrend<0.001) and≤19 years old group( Ptrend=0.011) increased by 9.3%( P<0.001) and 4.2%( P=0.002) over 20 years, which was the highest increase among all age groups with onset age<40 years old. Multivariate stepwise linear regression analysis showed that positive family history, blood uric acid level, metabolic syndrome and smoking were independent risk factors for early onset of gout. Conclusion:The age of gout onset in tends to be younger. The increase of the proportion of patients younger than 30 years old is probably the key factor leading to the early-onset gout in Shandong Province. Early and effective intervention on the risk factors related to early-onset gout is essential to prevent the early-onset gout as well as to reduce the prevalence of gout and complications.
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Objective:To explore the trans-membrane signaling mechanism of interleukin-6 (IL-6)-induced osteogenic differentiation and calcification of human umbilical artery smooth muscle cells (HUASMCs).Methods:HUASMCs were primarily cultured in vitro and were stimulated with IL-6, IL-6+solutable IL-6 receptor (sIL-6R), IL-6+sIL-6R+solutable gp130 (sgp130), or vehicle (blank control). Alizarin red and Von Kossa staining were used for detecting cell calcification, Western blot was used to test the protein expression of tissue-nonspecific alkaline phosphatase (TNAP), osteopontin (OPN), bone morphogenetic protein-2 (BMP-2) and Runt related transcription factor 2 (Runx2), and immunofluorescence was used to examine the mIL-6R expression of HUASMCs. The comparison of measurement date between the two groups was conducted by t-test. The comparison of measurement date between multiple groups was conducted by one-way analysis of variance (ANOVA). Results:The intensity severity of calcification stain was IL-6+sIL-6R group >IL-6+sIL-6R+sgp130 group>IL-6 group=blank control. After stimulated for 12 hours, the TNAP expression in blank control, IL-6 group, IL-6+sIL-6R group, IL-6+sIL-6R+sgp130 group were (0.44±0.08), (0.52±0.14), (0.84±0.16) and (0.55±0.10) respectively ( F=290.96, P<0.001). After stimulated for 3 days, the OPN expression in blank control, IL-6 group, IL-6+sIL-6R group, IL-6+sIL-6R+sgp130 group were (0.61±0.84), (0.95±0.16), (1.65±0.24) and (0.99±0.10) respectively ( F=507.72, P<0.001). After stimulated for 12 hours, the BMP-2 expression in blank control, IL-6 group, IL-6+sIL-6R group, IL-6+sIL-6R+sgp130 group were (0.77±0.05), (1.69±0.16), (2.81±0.26) and (0.57±0.12) respectively ( F=959.09, P<0.001). After stimulated for 3 days, the Runx2 expression in blank control, IL-6 group, IL-6+sIL-6R group,IL-6+sIL-6R+sgp130 group were (0.57±0.03) , (0.92±0.10), (1.31±0.13) and (0.66±0.06) respectively ( F=1141.27, P<0.001). Comparing with Jurkat cells (positive control) and CEM cells (negative control), HUASMCs limited expressed mIL-6R. Conclusion:IL-6 may induce HUASMCs osteogenic differentiation and calcification mainly via the sIL-6R-mediated trans-signaling pathway.
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Objective To observe the aortic calcification level in patients with rheumatoid arthritis (RA),and to analyze the relationships between aortic calcification and some RA disease related presentations.Methods RA patients (RA group) were all in-patients consecutively recruited from the Department of Rheumatology in one single tertiary hospital,and healthy subjects (control group) were individuals for check-up from the same hospital at the same time.Subjects with long-term smoking and drinking history,diabetes,hypertension,coronary heart disease,cancer,active or chronic infection,other autoimmune diseases and liver or kidney dysfunction were excluded in both groups.The aortic calcification scores (including ascending aorta,arcus aorta and aorta thoracica) were obtained automatically by 256-slice spiral CT scanner using the Heart Beat-CS program.Statistical package from Soci-science (SPSS) 17.0 software was used for data analysis.Student's t test,Mann-Whitney U test,Spearman test and x2 test were used.Results One hundred RA patients and 60 healthy subjects were selected,and there were no differences of age [(53±10) vs (51 ±8),t=1.031,P=0.304) and gender compositions [male 40(40％) vs 25(41％),x2=0.430,P=0.869) between the two groups.The aortic calcification score in the RA group was higher than that in the control group [19.4(3.3,190.0) vs 2.1 (1.9,18.0),U=1 579.5,P＜0.01].In RA group,the calcification score was positively correlated with age (r=0.729,P＜0.01),course of disease (r=0.227,P=0.023),C-reactive protein (CRP) (r=0.229,P=0.022),total cholesterol (TC) (r=0.220,P=0.028) and low density lipoprotein cholesterol (LDL-C) (r=0.224,P=0.014),but not related with treatment duration,number of tender joints and swollen joints,erythrocyte sedimentation rate,rheumatoid factor,anti-CCP antibody,DAS-28 (CRP),DAS-28 (ESR),triglyceride (TG) and high density lipoprotein cholesterol (HDL-C).The aortic calcification was also positively correlated with age in control group (r=0.465,P＜0.01),but not related with TC,TG,HDL-C,LDL-C.Conclusion RA patients have more severe aortic calcification than the matched general population.Aortic calcification degree is related to disease course,CRP,TC and LDL-C,which indicates that chronic systemic inflammation is essential to aortic calcification in RA.
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In 2013,the Chinese Society of Endocrinology published the'Management ofhyperuricemia and gout:Chinese experts consensus'.In the following five years,a large number of basic researches,clinical studies,and epidemiological results had emerged and need to be popularized in order to improve the understanding and standardizing management of these conditions in general physicians.On the other hand,the methodology of developing clinical guidelines has become more and more scientific,standardized and international.Therefore,based on the 2013 version of the consensus,Chinese Society of Endocrinology decided to develop a guideline for the diagnosis and management of hyperuricemia and gout under standardized methods and procedures of evidence-based clinical practice guidelines.This article will give a review of the status of clinical practice guideline development in China and explain the methods and procedures followed when developing this guideline.We hope this work may provide some useful recommendations for other developers to draft evidence-based clinical practice guidelines.
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Objective To explore whether different Epstein-Barr virus (EBV) infection status is involved in systemic lupus erythematosus (SLE) pathogenesis through type Ⅰ interferon pathway by observing the EBV antibodies,serum interferon α (IFN-α) level and four type Ⅰ interferon induced gene (ISGs;2'-5' oligoadenylate synthetase-like protein (OASL),myxovirous resistant 1 (MX1),interferon-stimulated gene 15 (ISG15) and lymphocyte antigen 6 complex,locus E (LY6E) expressions in SLE patients and healthy controls.Methods Forty-eight patients with SLE and 36 healthy controls were enrolled into this study.The serum antibodies of EBV capsid antigen-IgG/lgM and EBV nuclear antigen 1-IgG,and serum levels of IFN-α were measured by enzyme linked immunosorbent assay (ELISA) method.Real time reverse transcription polymerase chain reaction was used to test the mRNA levels of OASL,MX1,ISG15 and LY6E in the peripheral blood lymphocytes (2-△△Ct was used to indicate the gene expression).Statistical analysis was performed using t-test or Mann-Whitney U test,Chi square test and Spearman correlation test.Results ① The EBV lytic infection rate (VCA-IgM) in SLE patients was higher than that in healthy controls (40％ vs 11％,x2=5.381,P=0.027).② The serum levels of IFN-α in SLE patients were higher than those in the healthy controls [(206±151) ng/L vs (90± 76) ng/L,t=4.248,P＜0.05],as well as the mRNA levels of OASL,MX1,ISG15 and LY6E [1.8(0.6,5.1) vs 1.2 (0.5,1.4);1.9(1.0,4.4) vs 0.9(0.7,2.5);4.1(1.6,7.8) vs 0.8(0.5,1.7);1.6(0.7,3.3) vs 0.8(0.6,1.2),U=604,560,312,608;P＜0.05,respectively].The mRNA levels of OASL,MX1,LY6E and ISG15 were all positively related to SLE disease activity index (SLEDAI) scores (r=0.319,0.461,0.547,0.484,P＜0.05,respectively).Serum IFN-α levels were elevated in SLE patients with EBV lytic infection than in non-lytic infection patients [(282± 174) ng/L vs (157±114) ng/L,t=2.604,P＜0.05];The mRNA levels of OASL and ISG15 were also elevated in patients with lytic EBV infection [2.0(0.8,7.6) vs 1.2(0.6,3.1);6.2(2.4,15.5) vs 3.3(1.3,6.3),U=377,350,385,354;P＜0.05,respectively].The SLEDAI scores in patients with EBV lyric infection were higher than in patients with non-lyric infection (16±4 vs 12±8,P＜0.05).Conclusion EBV infection may be involved in the pathogenesis of SLE by activating the type Ⅰ interferon pathway.
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Objective To investigate the role of recombinant human interleukin 6 (rhlL-6) in calcification and osteogenic transition of cultured human umbilical artery smooth muscle cells (HUASMC), and the possible cell signal transduction way. Methods HUASMCs were isolated by the explant method. HUASMCs were treated with (treatment groups) or without (control group) rhIL-6. Alizarin Red S stain was applied for calcium deposition in extracellular matrix of control ceils and the cells treated with rhIL-6 50 μg/L at day 12. Calcium concentration in cell layer of control group and treatment group (treated with rhIL-6 10 μg/L and 50 μg/L, respectively) was determined calorimetrically by the o-cresolphthalein complexone method at day 3, 6, 9 and 12, and corrected by total cell proteins. The mRNA expressions of bone-specific alkaline phosphatase (BAP), osteopontin (OPN), bone morphogenetic protein-2 (BMP2) and osteoprotegerin (OPG) were estimated by real-time PCR in 12, 24 and 72 hours. OPN, BMP2 and OPG expressions were assessed by Western blotting and the BAP concentration at the same time was checked by fluorometry method . Electrophoretie mobility shift assays (EMSA) was used to detect the binding activity of transcription factor Cbfα1 with or without inhibitors of p38-MAPK (SB203580) and PKC (DHC) after 6 hours stimulation by rhIL-6 10 μg/L. Results rhIL-6 induced a positive Alizarin Red S stain and a time-dose-dependent increasing of cell layer calcium deposition.Compared with control group, rhIL-6 10 μg/L enhanced gene expression and protein levels of BAP and BMP2 at the early time (12 and 24 hours), and of OPN and OPG at later hours (24 and 72 hours). RhIL-6 still induced an increasing of binding activity of Cbfα1, which could be partially blocked by DHC but not SB203580. Conclusions rhIL-6 induces HUASMCs calcification and osteogenie transition in vitro, which may be one of the mechanism involved in IL-6 associated vascular calcification as observed in clinical studies. The role of IL-6 in HUASMCs may partially achieved through the PKC cell signal transduction way.
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ObjectiveTo investigate the effects of pravastation intervention on tumor necrosis factor (TNF)-α-indueed ossifie calcification in human umbilical artery smooth muscle cells (hUASMCs). MethodshUASMCs were cultured by tissue explant in vitro, hUASMC were treated with TNF-α 50 μg/L and pravastatin of three different concentrations. The calcium deposition was determined by O-cresolphthalein eomplexone method. The mRNA expression of BAP and OPN was determined by real time-PCR. The protein expression of BAP, OPN and BMP-2 was determined by Western blotting. ResultsPravastatin inhibited the proliferation of hUASMC (r=-0.946, P＜0.01) and decreased the cell calcium deposition (r=-0.973, P＜0.01) in a dosedependent manner. Pravastatin down-regulated the expression of BAP, OPN and BMP-2 induced by TNF-α in a dose-dependent manner (mRNA, r=-0.972, P＜0.01;BAP protein, r=-0.820, P＜0.01;OPN protein, r=-0.972, P＜0.01;BMP-2 protein, r=-0.928, P＜0.01). ConclusionPravastatin can inhibit the proliferation of hUASMC, decrease the cell calcium deposition and inhibit the ossifie calcification of hUASMC induced by TNF-α.
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Objective To study the effect of uremic serum on the calcification and osteogenic transition of cultured human umhilical artery smooth muscle cells(HUASMC).Methods Sera from 40 healthy controls(control group),40 nondialysis uremic patients(nondialysis group)and 45 uremic patients on dialysis(dialysis group)were detected fi)r biochemical indexes concerned and used to treat the cultured HUASMC.Alizarin red S stain was applied to examined calcium deposition in the cell layer.Calcium concentration was determined calorimetrically by the Ocresolphtha]ein complexone method,and corrected by total cell proteins.The mRNA expression of bone specific alkaline phosphatase(BAP),osteopontin(OPN)and bone morphogenelic protein 2(BMP2)was estimated by realtime PCR.OPN and BMP2 protein expression was assessed by Western blotting and fluorometry method was used to check the BAP concentration. Results Serum biochemical detection revealed thai both uremic groups had higher levels of phosphate,triglyseride,iPTH,C-reactive protein(CRP)and IL-6,and lower level of fetuin-A than healthy control(P＜0.05).Furthermore,dialysis serum had higher levels of triglyseride,CRP and IL-6 than nondialysis serum(P＜0.05).Compared with control group,both uremic scra induced more cell layer calcium deposition and higher mRNA and protein expression levels of BMP2,BAP and OPN(P＜0.05).Higher mRNA and protein expression levels of above factors were found in dialysis group as compared to nondialysis group(P＜0.05). Conclusions Uremic serum can induce HUASMC calcification and osteogenic transition in vitro,which may be one of the mechanisms involved in vascular calcification of ESRD patients.Microinflammatory state may promote the osteogenic transition and vascular calcification in dialysis patients.