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Objective@#To quantify the serum levels of apoptosis-related molecules M30 and M65 in the patients with primary biliary cholangitis (PBC) and analyze their clinical significance for PBC. @*Methods@#A total of 79 patients with PBC and 40 healthy individuals were involved in this study from January 2017 to April 2019. The levels of serum M30 and M65 were measured by enzyme-linked immunosorbent assay (ELISA) and the ratio of M30/M65 was calculated. The parameters of liver function were tested by automatic biochemical analyzer. ALT, AST and GGT were determined by rate method, ALP was assayed by the method of NPP substrate-AMP buffer and T-Bil was determined by oxidation method. Autoantibodies including AMA-M2, anti-GP210 and anti-SP100 were detected by automated multiplexed bead-based and immunoblotting assays. The correlation of M30, M65 and M30/M65 ratio with liver function items were analyzed by spearman assay. The levels of M30, M65 and M30/M65 ratio were analyzed by ROC curve to evaluate their values in PBC screening. @*Results@#M65 level in PBC group was significantly higher than that in control group (P＜0.001) with statistically significant difference and M30/M65 ratio was significantly lower than that in control group with statistically significant difference (P＜0.001). There was no significant difference for the level of M30 between the two groups (P=0.641). Among the PBC patients, 18 were anti-GP210-positive and 17 were anti-SP100-positive. In anti-GP210 positive group the levels of M30 and M65 were significantly higher than those in the negative group (P values were 0.002 and 0.001 respectively). In anti-SP100 positive group the level of M65 was significantly higher than that in the negative group (P=0.027) and M30/M65 ratio was significantly lower than that in negative group with statistically significant difference (P=0.005). Spearman correlation analysis showed that the levels of M30 and M65 were positively correlated with ALT, AST and T-Bil (P＜0.05). The cut off values of M30 and M65 were 63.62 U/L and 155.37 U/L and M30/M65 was 0.32, respectively. The screening sensitivities were 63.30%, 89.10% and 93.30%, and the specificities were 51.60%, 90.00% and 75.00%, respectively. @*Conclusion@#The level of M65 in the serum of PBC patients significantly increased and M30/M65 ratio significantly decreased, which could be used as a promising serum marker of laboratory for PBC screening.
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Objective@#To investigate the IL-10+CD19+ regulatory B cells (Breg) in peripheral blood of patients with rheumatoid arthritis (RA), and analyze the correlation between the expression of IL-10+CD19+ Breg cells and disease severity. @*Methods@#A total of 40 patients with active RA and 30 healthy individuals (healthy controls, HC) were involved in this study. The peripheral blood mononuclear cells (PBMC) were isolated. The isolated PBMC were co-cultured with CpG oligodeoxynucleotide 2006 (CpG ODN 2006) and phorbol myristate acetate (PMA) in vitro. Flow cytometry was employed to analyze the expression of Breg before and after stimulation. The correlations of Breg expression with ESR, CRP and DAS28 were analyzed. @*Results@#Before stimulation in vitro, the expression of Breg in PBMC of RA group and HC group were 1.92%(1.58%, 2.56%) and 2.04%(1.73%, 2.93%) respectively. There was no significant difference between the two groups (P=0.258). After induction in vitro, the expression of Breg of RA group [13.00%(9.75%, 14.85%)] was significantly higher than that of HC group [9.12%(6.83%, 10.22%)], P＜0.001. Spearman correlation analysis showed that Breg expression was negatively correlated with DAS28 (P=0.002), but not with ESR and CRP (P＞0.05). @*Conclusion@#The expression of Breg was significantly increased after stimulation in vitro and negatively correlated with DAS28, which indicated Breg may play important regulatory roles in pathogenesis and development of RA.
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Objective To evaluate the changes of adenosine metabolism pathway related molecules and their contribution to inflammatory injury in primary biliary cholangitis (PBC).Methods The consecutive samples of 49 subjects with PBC from The First People's Hospital of Taicang and The Second People's Hospital of Changshu were recruited from October 2016 to October 2017,and 36 healthy controls were involved in this study.The expression of CD39 and CD73 on CD4+T cells and Foxp3 + regulatory T cells were assayed by flow cytometry and the concentration of adenosine triphosphate (ATP) in serum was analyzed by ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS).The correlations between Tregs,ATP and liver function were analyzed,i.e.,alanine aminotransferase (ALT),aspartate aminotransferase (AST),gamma-glutamyltransferase (GGT),alkaline phosphatase (ALP) and Mayo scores.Results In the patients with PBC,low proportions of CD4+CD39+T cells were noted compared with healthy controls [(5.28 ± 1.92) ％ vs (11.0l ± 3.19) ％,t =10.25,P ＜ 0.01].The patients with PBC also had significantly low proportion of CD4+CD25 + Foxp3+ CD39+ T cells compared with healthy controls [(23.75 ± 9.48) ％ vs (54.68 ± 5.18) ％,t =13.79,P ＜0.01].No significant difference of the proportion of CD4+CD73+T or CD4+CD25+ Foxp3+CD39+T cells was found between PBC and control groups (t values were 2.235 and 1.083,P ＞ 0.05).The level of serum ATP was higher in the patients with PBC than that of healthy controls [(200.28 ± 79.41) μg/L vs (89.20 ± 33.76) μg/L,t =8.367,P ＜ 0.01].A significant correlation was demonstrated between the proportion of CD39 + Treg in total Treg cells and the levels of ATP (r =-0.413,P =0.003),GGT (r=-0.378,P=0.007) and Mayo score (r=-0.382,P=0.007).Conclusion The low proportion of CD39+ Treg cells may contribute to the down-regulation of ATP hydrolysis in the patients with PBC.No significant change of CD73 + Treg cells was found in PBC patients.
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Background The mitochondrial Na+/Ca2+exchanger, NCLX, plays an important role in the balance between Ca2+influx and efflux across the mitochondrial inner membrane in endothelial cells. Mitochondrial metabolism is likely to be affected by the activity of NCLX because Ca2+activates several enzymes of the Krebs cycle. It is currently believed that mitochondria are not only centers of energy produc-tion but are also important sites of reactive oxygen species (ROS) generation and nucleotide-binding oligomerization domain receptor 3 (NLRP3) inflammasome activation. Methods&Results This study focused on NCLX function, in rat aortic endothelial cells (RAECs), induced by glucose. First, we detected an increase in NCLX expression in the endothelia of rats with diabetes mellitus, which was induced by an injection of streptozotocin. Next, colocalization of NCLX expression and mitochondria was detected using confocal analysis. Suppression of NCLX expression, using an siRNA construct (siNCLX), enhanced mitochondrial Ca2+influx and blocked efflux induced by glucose. Un-expectedly, silencing of NCLX expression induced increased ROS generation and NLRP3 inflammasome activation. Conclusions These findings suggest that NCLX affects glucose-dependent mitochondrial Ca2+signaling, thereby regulating ROS generation and NLRP3 in-flammasome activation in high glucose conditions. In the early stages of high glucose stimulation, NCLX expression increases to compensate in order to self-protect mitochondrial maintenance, stability, and function in endothelial cells.
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Objective To investigate the population and role of IL-10+ CD19+ regulatory B cell (Breg) in patients with chronic hepatitis B.Methods Patients with acute hepatitis B (AHB) (n =28),chronic hepatitis B (CHB) (n =31) and normal subjects (n =25) were collected from Changshu No.2 People's Hospital between 2011 June and 2012 October.Peripheral blood mononuclear cells (PBMC) were isolated and stimulated with CpG ODN 2006 and PMA.Flow cytometry was used to analyze the population of IL-10-CD19 + Breg,CD4 + CD25high Treg,and ELISA was used to analyze the concentration of IL-10 in culture supernatant.Results The population of Breg in Peripheral blood of the CHB group [1.28％ (1.05％-2.20％)] was higher than that in the AHB group [0.87％(0.55％-1.22％)] and the HCs group [0.89％ (0.51％-1.37％)] (P =0.001,0.006),and the difference between the AHB group and the HCs group was not statistically significant (P=0.669).Breg in the CHB group [14.30％ (10.70％-16.70％)] was higher than that in the AHB group [10.30％ (7.05％-13.30％)] and the HCs group [10.40％(6.85％-12.60％)] (P =0.003,0.001),treg in the CHB group [5.80％ (4.20％-9.10％)] was also higher than that in the AHB group [4.05％ (2.53％-5.40％)] and the HCs group [4.50％ (2.55％-5.50％)] (P ＜0.001,P =0.005),and there was no significantly difference between the AHB group and the HCs group (Breg:P =0.796 ; Treg:P =0.227).Spearman correlation analysis showed that Breg was positively correlated with Treg in the CHB group (r =0.50,P =0.004),however there was no significantly correlation in the AHB group and the HCs group (r =-0.15,P =0.462; r =0.09,P =0.669).The concentration of IL-10 in the CHB group was higher than that in the AHB group and the HCs group (P ＜ 0.001),and the difference between the AHB group and the HCs group was not statistically significant (P=0.341).Spearman correlation analysis showed that IL-10 were positively correlated with the population of Breg in the CHB group (r =0.409,P =0.022).Conclusion The poluations of regulatory B cell and regulatory T cell increased in patients with chronic hepatitis B,and Breg cell might play the immune regulation role through secreting IL-10 in chronic HBV infection.
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Objective To investigate the role of interleukin-27 (IL-27) in proliferation and differentiation of CD4+ T cells in ankylosing spondylitis(AS).Methods CD4+ T cells were separated from peripheral blood which collected from AS patients and health controls(HCs).Cells proliferation was detected by CCK-8 kit,and cytokines were analyzed by ELISA.Real-time PCR was used to determine the mRNA expression of T-bet and GATA3.Results Serum IL-27 level in patients with AS was obviously higher than that in HCs(P ＜0.01).The proliferation rate of CD4+ T cells and the level of IFN-γin cultured medium in AS were higher than those in HCs group after IL-27 stimulation (P ＜ 0.01).IL-27 could induce T-bet mRNA expression in CD4+ T cells in AS (t =14.3,P ＜ 0.01),but no change was found in GATA3 mRNA expression.Conclusions IL-27 could induce the proliferation of CD4+ T cells and the activation of T-bet pathway in AS.Furthermore,Th1 immune response and related cytokines could be induced by IL-27 in AS.These implicate that IL-27 may play an important role in AS related inflammation.
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Objective To analyze the features of bone minimal density and bone quantitative ultrasound in men with different osteoporotic risk graded by osteoporosis self-assessment tool for Asian (OSTA).Methods After exclude the secondary osteoporosis,724 subjects over 50 years old were involved.The parameters of hight,weight,quantitative ultrasound index (QUI),QUS-T score were examined.The bone density (BMD) were measured by dual-energy X-ray absorptiometry (DXA)in 120 elderly men.All subjects were grouped into low (osteoporotic) risk group,moderate risk group and high risk group by OSTA index.120 subjects measured BMD were grouped into normal bone mass group,osteopenia group and osteoporosis group by WHO standard.The differences and correlation analysis in BMD,QUST,and QUI between these groups were analysed.Results The percents of low risk people,moderate risk people and high risk people were 56.4％ (408 cases),28.2％ (204cases),15.5％ (112 cases),respectively.There were 30.0％ (36 cases) normal bone mass people,58.3％ (70 cases) osteopenia people and 11.7％ (14 cases) osteoporosis people in groups measured BMD.QUS-T score,QUI were gradually decreased in groups of low risk,moderate risk and high risk (-0.56±1.09,-0.88±-1.28,-1.21±1.40; 98.47±19.04,92.62±22.49,87.68±24.43; all P ＜0.05) and had statistical significant differences between low risk and moderate risk,high risk groups,while had no differences between moderate risk and high risk groups.The femoral neck BMD and total BMD were gradually decreased in all the three groups (0.89±0.12,0.85±0.10,0.77± 0.10; 1.0±0.15,0.93 ± 0.11,0.83±0.1; all P＜0.01).Osteoporosis in the three groups were 3.4％ (2 cases),13.0％ (6 cases),37.5％ (6 cases),respectively and osteoporosis percents in moderate risk group and high risk group were higher compared with low risk group (x2=11.77,P＜0.01).QUS-T score and QUI decreased gradually in groups of normal mass,osteopenia and osteoporosis (0.99±0.08,-0.70±1.07,-1.96±0.73; 109.26±17.05,96.15±18.20,72.54±10.00; F=10.47,11.73,all P＜ 0.01).Except for lumbar BMD,a positive linear correlation emerged between OSTA and QUS-T score,QUI,hip BMD(all P＜0.01).The values of R with femoral neck BMD,torch BMD and total hip BMD were 0.45,0.38,0.39,respectively.And the same value with QUS-T score and QUI was 0.23.Conclusions With the decreasing of OSTA index,risk of osteoporosis is increased and QUS-T score,QUI and BMD are decreased gradually.There are positive linear correlation between OSTA index and QUS-T score,QUI,hip BMD.
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ObjectiveTo investigate mechanisms for IL-27 induced proliferation and differentiation of peripheral blood CD4+ T cells in primary biliary cirrhosis (PBC).MethodsPeriperal blood CD4+ T cells were isolated from patients with PBC,chonic hepatitis B (CHB) and health controls (HCs).After IL-27 stimulation,proliferation ability of CD4+ T cells was evaluated by CCK-8 kit,and cytokines were analyzed by ELISA.Real-time PCR was employed to assay mRNA expression of T-bet and GATA3 in CD4+ T cells.p-STAT-1 and pSTAT-3 expression in CD4+ T cells were detected by Western blot.ResultsEnhanced proliferation of CD4+ T cells was found in all subjects after IL-27 stimulation.However,the proliferation ability in patients with PBC was greater than that in CHB and HCs ( P＜0.001 ).Levels of IL-2 and IFN-γ in supernatant from IL-27-incubated PBC blood CD4+ T cells were higher than that from CHB and HCs (P＜0.001 ).In normal situation,T-bet mRNA of CD4+ T cells in PBC group was higher than that in CHB group (P=0.007).Furthermore,after IL-27 stimulation,elevated T-bet mRNA expression and GATA3 inhibition were found in patients with PBC.High expression of p-STAT-1 and p-STAT-3 in blood CD4+ T cells were found in PBC,CHB and HCs after stimulation by IL-27.But their expression in patients with PBC were higher than those in patients with CHB and HCs.ConclusionProliferation of blood CD4+ T cells could be induced by IL-27 in patients with PBC.The signaling pathways of p-STAT-1,p-STAT-3 were involved to induce Th1 immune response and related cytokines expression.This study implicated that IL-27 may play important roles in early inflammation damage in PBC.
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Objective To investigate a new therapeutic pathway for primary biliary cirrhosis (PBC) by immune tolerance reestablishment in a PBC mouse model. Methods Spleenic cells from naive mice were incubated with M2 in the presence of ECDI and the ceils were injected into caudal vein of the mice which would be used for development of PBC model. Spleenic cells incubated with bovine serum albumin (BSA) were injected as controls. 16 weeks later, anti-mitoehondrial antibody (AMA) , alkaline phosphatase(AKP) and portal inflammation were assayed for evaluating the prevention effect. Results AMA positive rate in tolerance group was lower than that in BSA and PBC groups ( P = 0. 007, P = 0. 003 ). The difference between BSA and PBC was not significantly. Serum AKP levels in tolerance, BSA and PBC group were (80.5 ±9.8) U/L, (93.8 ±15.7) U/L and (92.5 ±17.7) U/L, separately. The level in tolerance group was lower than that in BSA and PBC groups (P =0.0095, P =0.029). The rates of portal areas with cell infiltration were 42. 67% ± 12. 30% , 57. 07% ± 11. 35% and 51. 53% ± 9. 96% , separately. The number of infiltrated portal tracts in tolerance group was less than that in PBC group (P = 0.039) and BSA group (P = 0. 0024). Conclusion PBC was prevented to some extent by reestablishing immune tolerance to M2 autoantigen. This provides clues for finding a better treatment proposal.
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Objective To investigate the immune tolerance in animal models of primary biliary cirrhosis (PBC) by determining the cell proliferation and activation induced cell death (AICD).Methods C57BL/6 mice were injected with 5 mg/kg of polyI:C to develope PBC models. The lymphocytes and CD4~+ T cells were separated from spleens and livers 16 weeks later and were stimulated by M2, conA and anti-CD3 for cell proliferation and AICD. Expression of apoptosis related genes and proteins were detected by real time polymerase chain reaction (PCR) and Western blotting, respectively. Results ① The lymphocyte proliferation was 0.1988 ± 0.0111 in blank controls and 0. 2068±0. 0115 in PBS treated mice with no significant difference (P>0.05). However, an abundant lymphocyte proliferation was found in PBC mice (0. 358 ± 0. 022), which was higher than that in controls and PBS treated mice. The proliferation of lymphocyte from liver was greater than that from spleen in PBC mice (P<0.01). ② The apoptotic rate in blank controls (74.70%±4.58%) and PBS treated mice (74.20%±4.44%) was higher than that in PBC mice (44.85%±6.47%,P<0.01),but no difference was found between blank controls and PBS treated mice (P>0.05). Furthermore, the apoptosis rate of T cells from livers were significantly lower than that from spleens in PBC mice (P<0.01). ③ The expressions of FasL and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in PBC mice were lower than those in PBS treated mice (P<0. 01), but there was no change in expression of Fas was found. ④ The expression of Fas-associated death domain-like interleukin-1-β-converting enzyme-inhibitory protein (FLIP_L) in PBC mice was higher than that in blank controls. Moreover, the expression of FLIP_L in livers was higher than that in spleens in PBC mice (P<0. 01). Conclusios The elevated expression of FLIP_L may inhibit AICD. Besides, the decreased expressions of FasL and TRAIL may also help in the enhancement of the anti-apoptotic ability in lymphocytes and in the aggravation of portal area inflammation.
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Objective To evaluate the safety of mefformin in the treatment of elderly type 2 diabetes mellitus(T2DM). Methods Two hundred and forty-three cases of elderly T2DM hospitalized from Jan.1996 to Dec. 2006 were reviewed; the changes of fasting blood glucose(FBG), postprandial blood glucose (PBG), glycosylated hemoglobin (HbA1c), liver and renal function and blood lactic acid were evaluate before and after treatment. Results The mean time of treatment with mefformin was (6.6±3.9) years (3 months-21 years)in these 243 cases. The levels of FBG, PBG and HbAlc significantly reduced after treatment with mefformin only in 43 cases (17.7%), mefformin combined with other oral hypoglycemic drugs in 124 cases (51.0%) and mefformin combined with insulin in 76 cases (31.3%). There was only 18.1% of the cases with normal range ( > 80 ml/min) of creatinine clearance rate (Ccr), and 25.8% of the cases with Ccr≤50 ml/min. The liver and renal function as well as the blood lactic acid had no significant change after treatment no matter in total cases or in different groups separated by Ccr.Conclusions Mefformin is safety in the treatment of elderly T2DM patients. Ageing is not the contraindication of mefformin. To the patients with high risk, we should monitoring the level of blood lactic acid.
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Objective To investigate the distribution and expression of adiponectin receptors in different tissues of normal Wistar rats. Methods The rats were euthanized and the total RNA was extracted from equal weight tissue samples of cardiac muscle, skeletal muscle, liver, kidney, testicle and fat. RT-PCR was employed to amplify the isolated cDNA, and the expression of adiponectin receptors in different tissues was semi-quantitatively analyzed. Results AdipoR1 was observed in rat cardiac muscle, skeletal muscle, liver, kidney, testicle and fat, with the highest expression in testicle and fat (P
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Primary hyperaldosteronism(PHA) is one of the most common reasons of secondary hypertension,which usually has long history,moderate hypertension and relative high incident of vascular complications.Its other complications include hypokalemia,alkalosis,hypocalcemia and IGT et al.Screening hypertension patients using plasma aldosteronism/renin rate will greatly increase the diagnosis and improve the treatment.Treatments of PHA include surgery,medicine and intervene.