| Objective: Chronic obstructive pulmonary disease (COPD) is a commonbut serious disease which harms to human health, and not fully reversibleairflow limitation, it is one of the main reason for the increased morbidity andmortality around the world, caused serious economic and social burden, Thissituation getting worse[1]. Now it is still lack of clinical timely and accurateassessment of the AECOPD (acute exacerbation of COPD) occurrence,development and prognosis indicators.Researchers are searching reasons whyacute exacerbation of COPD occurred. To investigate a sensitive indicator ofthe development and prognosis has much significance. Infection plays animportant role on the occurrence and development of COPD.Lipopolysaccharide (LPS) could produce large amounts of inflammatorymediators by stimulating the body’s immune cells, which would causes aseries of inflammatory reactions on the body.P38mitogen-activated proteinkinase (MAPK) was involved in intracellular signal transductionkinase-mediated inflammatory response, playing an important role in theinflammatory cascade caused by LPS[2]. SB203580is a specific inhibitor ofp38MAPK which inhibits LPS-induced inflammatory response to the lungs. Inthis experiment, serum and peripheral blood mononuclear cells from healthypeoples and patients with acute exacerbation of COPD were studied. Toobserve the levels of TNF-a, IL-10, RANTES in serum which from healthycontrol group and different levels of AECOPD patients,the effect ofSB203580on production of TNF-a, IL-10, RANTES from PBMCs stimulatedby LPS. The correlation analysis of the severity of AECOPD patients andcytokines TNF-a, IL-10, RANTES.These may supply a new method forclinical prevention and treatment of AECOPD. Methods:1Objects and settings：Select subjects AECOPD patients hospitalizedfrom March2012to January2013in our hospital respiratory and outpatientmedical examination that for confirmed healthy volunteers in the same period.According to the Chinese Medical Association in2011respiratory branch toAEOPD and diagnostic criteria, all objected peoples are divided into:(1)AECOPD group (2) healthy control group. Lung function results will beobject of study (AECOPD patients), moderate group (50%≤FEV1%<80%)and severe group (50%<FEV1%≤30%), very severe group (FEV1%<30%). Experimental groups of four groups:①healthy control group;②The moderate group of AECOPD;③The severe group of AECOPD;④Thevery severe group of AECOPD.The levels of TNF-a, IL-10, RANTES in serum and cell supernatantswere measured1.1Specimen collection: All objected peoples were selected for the controlgroup and the experimental group (AECOPD patients) fasting mining cubitalvein8ml3ml injected dry vacuum non-anticoagulant tube,4℃3000r/mincentrifugal15min, the supernatant the solution As regards the-80°Crefrigerator be measured TNF-a, IL-10, RANTES levels. Another5ml bloodwas injected into EDTA anticoagulant tube for the separation of PBMCs.1.2Isolation of mononuclear cells: Each blood mononuclear cells isolatedusing lymphocyte separation medium.0.4%trypan blue staining, excludinginactive cells, the number of active cells>95%may be used. Containing10%fetal calf serum in and RPMI1640culture fluid regulating cell concentrationare1×106/ml, spare.1.3Assaying the levels of TNF-a, IL-10, RANTES in serum by ELISA.1.4Detecting the levels of TNF-a, IL-10, RANTES in cell supernatantscontent of PBMC by ELISA.Experimental groups: first will be adjusted after the group separation ofPBMCs count cell concentration to1×10~6/ml, incubated in24-well plates(37℃CO2incubator) were divided into five groups. Results:1(1) AECOPD patients the presence of serum TNF-a levels between thethree groups a significant difference (P <0.05); the AECOPD patients serumTNF-a levels were significantly higher than that in the control group, astatistically significant (P <0.05).(2) AECOPD between the three groupsRANTES there is a significant difference (P <0.05); AECOPD patients withmoderate group, the group of severe, very severe serum RANTES water wassignificantly higher than the moderate group was statistically significant (P <0.05).(3) AECOPD patients with moderate group, the severe group, levels ofserum IL-10were significantly lower than the control group (P <0.05); theAECOPD patients between serum IL-10level was not statistically differences(P>0.05).(4) AECOPD serum TNF-a, of RANTES in levels of FEV1%wasnegatively correlated (r=-0.957, P=0.00; r=-0.964, P=0.00); IL-10levelsand FEV1%were positively correlated (r=0.656, P=0.00).2(1) PBMCs from healthy group and AECOPD moderate group, severegroup, very severe group in vitro in the same conditions (adding serum RPMI1640),comparing levels of TNF-a, RANTES and IL-10between differentgroups:Comparing levels of cell supernatant TNFa, IL-10were significantlydifference between the healthy group and three groups of AECOPD (P <0.05);The levels of cell supernatant TNFa, IL-10were significant differencesbetween the three AECOPD groups (P <0.05), the health group AECOPDmoderate group was statistically significant (P <0.05); The very severe groupcompared with the healthy group, the moderate group, severe group ofRANTES content in the cell supernatant: RANTES contented in cellsupernatant of AECOPD, the extreme group is higher than the healthy group,the moderate group and severe group ((P <0.05), RANTES in cell supernatantcontent has any obvious difference between health and moderate group orsevere group of AECOPD（P＞0.05）.There is no significant differencebetween the moderate group and severe group of AECOPD（P＞0.05）.(2) LPSstimulation of PBMCs, health groups and AECOPD three groups of cells inthe supernatant of TNF-a, of RANTES in content, and reduced IL-10levels,with statistically significant by univariate analysis of variance (P <0.05); significant difference (P <0.05) in the control group, LPS group and SB+LPSgroup, among the three groups of TNF-a, RANTES, IL-10content. TheSB203580(5μΜ,10μΜ,15μΜ) pretreatment significantly inhibitedLPS-induced TNF-a, RANTES, content increased but the levels of IL-10increased with SB203580pretreatment concentration of AECOPD betweenthe three groups showed no significant difference; AECOPD group PBMCsecretion of TNF-a, RANTES was significantly higher than that in healthycontrol group (P <0.05), IL-10secretion was significantly lower than thecontrol group (P <0.05).Data are presented as mean±standard deviation（(x|-)±s）. Each group wasused one-way ANOVA (One way ANOVA) for comparison. If any significantStudent-Newman-Keuls (SNK-q) test, P <0.05was considered statisticallysignificant.Conclusions:1The levels of TNF-a, RANTES in serum and the severityof AECOPD negative correlation with lung function;There is negativecorrelation between levels of IL-10and the severity of AECOPD, but there isa positive correlation with lung function. TNF-a and RANTES may take partin occurrence and development of COPD in airway inflammation, that may bethe important index of AECOPD, while IL-10, play a certainanti-inflammatory effects.2TNF-a and RANTES is secreted increased of peripheral bloodmononuclear cells by LPS stimulation from the control group and theexperimental group; p38MAPK inhibitors SB203580after the intervention,TNF-a, RANTES reduced suggest that SB203580may inhibit TNF-a,RANTES secretion. The levels of IL-10increased slightly.There may bereduce inflammation reaction and consumption of anti-inflammatory factorsby inhibiting p38MAPK pathways. |
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