| Objective: Chronic obstructive pulmonary disease (COPD) is aninflammatory lung diseases, characterized by airflow limitation, and theairflow limitation that is not fully reversible and progressive, activation ofinflammatory cells release a variety of mediators in the pathogenesis ofprocess, is because of infection, immunity, poisoning and other factors causelung and bronchus large numbers of macrophages, neutrophils and otherinflammatory cell infiltration, release a lot of inflammatory mediatorswaterfalls cascade, which cause the lung disease caused by pulmonaryinflammatory mediators and inflammatory medium imbalance.Lipopolysaccharide (LPS) can stimulate the body neutrophil cells andmacrophages to the release of a large number of inflammatory mediators,causing inflammatory lung disease (ILD) such as COPD.Alveolarmacrophages (AMs) is the main effector cells of LPS, lung is the first line ofdefense, and AMs can release a variety of inflammatory factors causingpulmonary inflammatory mediators and inflammatory medium imbalance isthe result of inflammation. Inhibition of the mitogen-activated protein kinase(MAPK) signal transduction pathway can reduce the release ofpro-inflammatory cytokines. These cytokines involved may become a newtarget for the treatment of COPD. In this experiment, the mouse AM wasstudied, to observe the effect of AECOPD serum and p38MAPK specificinhibitor SB203580secretion of RANTES, IL-10of the cells stimulated byLPS, and further to investigate the expression of RANTES, providing atheoretical basis for the clinical application of p38MAPK inhibitors for thetreatment of COPD. Methods:1The concentration of RANTES in cell supernatant detectedby ELISACultured MH-S cell line, adjusting the cell concentration to5x10~6/ml, intwenty-four well polystyrene tissue culture plates and incubatedovernight.Groups were randomly divided as follows:(1) Control group: eachof1%,5%,10%of healthy persons and patients with COPD serum+RPMI1640and10%fetal calf serum+RPMI1640culture medium andincubated MH-S cells;②LPS group: respectively1%,5%,10%of healthypeople and patients with COPD serum+RPMI1640and10%fetal calf serum+RPMI1640culture medium incubated MH-S cells after6h, LPS stimulation,a final concentration of100ng/ml;③LPS+SB203580group:1%,5%,10%of healthy people and patients with COPD serum+RPMI1640and10%fetalcalf serum+RPMI1640culture MH-S cells6h incubation, the finalconcentration of10μmol/L SB203580pre-incubated for20min after LPSstimulation, a final concentration of100ng/ml.All groups were stimulated for6h, cell supernatant were collected, frozenand stored at-80℃refrigerator, to detect the RANTES concentration byELISA.2The concentration of IL-10in cell supernatant detected by ELISAMethods and groups were the same as determination of RANTES.3RANTES expression in MH-S cell was examined byImmunocytochemistry (ICC).Adjusting cell concentration of1x10~6/ml,on the twenty-four wellpolystyrene tissue culture plates overnight, based upon the above grouping ofMH-S cells after stimulation,expression of RANTES were detected byImmunocytochemistry method.Results:1The change of RANTES levels in MH-S cell supernatant:①After LPS-stimulation,the level of RANTES in supernatant of MH-S cells wasincreased,and with the increased serum concentration in the supernatant,theRANTES levels increased.In addition,the content of RANTES in MH-S cellsupernatant incubated with COPD patient serum was more than in healthy individuals. There was statistically significant difference compared with thecontrol group in serum of the same concentration and the same type(P<0.05).With p38MAPK inhibitor SB203580+LPS intervention,RANTES content hasbeen reduced,but still higher than the control group.Compared with the controlgroup,the difference was statistically significant in Serum of the sameconcentration and the same type(P<0.05),while LPS group andLPS+SB203580group were also considered statistically significant in Serumof the same concentration and the same type(P<0.05).②With the increasedserum concentrations of patients with COPD, RANTES content in the controlgroup, LPS group and LPS+SB group showed a significant positivecorrelation （r=0.966,P<0.01; r=0.974, P<0.01; r=0.945, P<0.01）； Withincreased serum concentrations of healthy people of RANTES in content inthe control group, LPS group and LPS+SB group was also found asignificant positive correlation（r=0.968,P<0.01; r=0.979, P<0.01; r=0.965,P<0.01）.③The serum of COPD patients and LPS have synergic effect on theproduction to further increase RANTES content.Compared with the sameserum concentration and the healthy control group, the difference wasstatistically significant(P<0.05).2The change of IL-10levels in MH-S cell supernatant:①IL-10levelexhibited increase after LPS-stimulation,the content of IL-10in supernatant ofMH-S cells was increased,and with the increased serum concentration in thesupernatant was increased in IL-10levels.In addition,the content of IL-10inMH-S cell supernatant incubated with COPD patient serum was less than inhealthy individuals.There was statistically significant difference comparedwith the control group in serum of the same concentration and the sametype(P<0.05).With p38MAPK inhibitor SB203580+LPS intervention, IL-10content has been reduced, but still higher than the control group.Comparedwith the control group,the difference was statistically significant in serum ofthe same concentration and the same type(P<0.05),while LPS group andLPS+SB203580group were also considered statistically significant in serumof the same concentration and the same type(P<0.05).②With the increased serum concentrations of patients with COPD, IL-10content in the controlgroup, LPS group and LPS+SB group showed a significant positivecorrelation （r=0.920,P<0.01; r=0.923, P<0.01; r=0.765, P<0.01）； Withincreased serum concentrations of healthy people of IL-10in content in thecontrol group, LPS group and LPS+SB group was also found a significantpositive correlation（r=0.983,P<0.01; r=0.878, P<0.01; r=0.985, P<0.01）.③The synergy effect is not significant while cells treated with both COPDpatient serum and LPS.3Immunocytochemistry results showed that: the cytoplasm of the controlgroup, only a faint yellow dye；the cytoplasm stained yellow significantly afterLPS stimulation of cell; using SB203580pretreatment, LPS stimulationcytoplasm stained yellow weakening but still stronger than the control group;The cytoplasm stained yellow Enhanced With COPD patients with serumconcentration increased significantly compared with healthy people and calfserum by yellow dye, RANTES expression in each group and the controlgroup were significantly increased(P <0.05).Conclusions:1In the MH-S cell supernatant,the levels of RANTES and IL-10increased after LPS stimulation, however the levels of RANTES and IL-10decreased after intervention by SB203580respectively,but it was still higherthan the control group.The results suggest that SB203580may inhibit thesecretion of RANTES and IL-10.2With incresaed serum concentrations in the same COPD patients, thelevel of RANTES and IL-10in the MH-S cell supernatant both were increased,and the content of RANTES was higher than healthy people, the content ofIL-10was lower than that of the healthy people. Serum in patients with COPDon MH-S cells may be an inflammatory stimulus,and there were Synergiesbetween COPD patient serum and LPS.3Inactive RANTES existed in the cytoplasm. LPS stimulation of MH-Scells, RANTES is activated to become the active RANTES. Application ofSB203580intervention, the cytoplasm stained yellow weakened, shows that the SB203580can reduce the expression by p38MAPK. |
PDF Full Text Request