| ObjectivesCoal workers'Pneumoconiosis (CWP) is an occupational disease inducd by coal dust exposure, which hazard the health of workers. The pathogenesis hasn't been studied clearly yet, which make it difficult to prevent and cure. It is very significant to study the mechanism of pneumoconiosis for prevent and cure of pneumoconiosis, especially to research the earlier affairs and the regulation of occurrence. The data from in vitro and animal experiments showed that, apoptosis, especially the apoptosis of alveolar macrophages (AM), was the immunopathological base of pneumoconiosis. Apoptosis was induced by three signal transduction pathways: mitochondria pathway, endoplasmic reticulum pathway and membrane death receptor payway. Membrane death receptor payway have three basic death receptor signal pathways, included Fas/FasL pathway, Fas/FasL pathway and TRAILR/TRAIL pathway. Now, there were some reports about the role of lung cell apoptosis mediated by Fas/FasL and TNF/TNFR pathway triggering pneumoconiosis, but there was little research about the TRAILR/TRAIL pathway in this field. Therefore, our study focused on the contact between apoptosis induced by TRAILR/TRAIL pathway in CWP.The aim of the present study is to explore the apoptotic mechanism of AM mediated by TRAILR/TRAIL signal transduction pathway, and to provid scientific data for earlier prevention and cure of pneumoconiosis. First, the AM of subjects and the patients with CWP were separated from the massive whole lung lavage fluid and cultured in vitro, second, Fas/FasL and TNFR/TNF-αsignal transduction pathway and some important points of TRAIL/TRAILR signal transduction pathway were blocked, then apoptosis and apoptosis-related proteins were detect. Subjects and methodsObserved subjects and CWP cases without the other lung diseases were chosen as subjects in Beidaihe Coal Workers'Sanatorium. Cases were all men.Questionnaire was designed reasonably based on the study aims. The contents included the general information, history of employment, history exposed to dust and history of diseases, etc. The data of pneumoconiosis'diagnosis were collected from local hospital and the complication from the medical records of Beidaihe Coal Workers'Sanatorium Hospital. The standard of pneumoconiosis'diagnosis was chosen GBZ70-2009. All subjects were talked by face to face.Bronchoalveolar lavage fluid (BALF) was filtered, centrifuged and the supernatant was poured down, AM were cleaned 3 times and AMs suspension was made to count. Each subject's cells were cultivated in six-well plate: A (untreated group), B (SOD group), C(TRAIL/TRAILR group), D(anti-TRAIL antibody group), E(Caspase-8inhibitor group). F(NF-κB inhibitor group). AM were purified for 2 hours and cultivated in in plates for 24 hours, then collected for the following experiments.The apoptosis of AM were detected by ordinary optical microscope, laser scanning confocal microscope, flow cytometry and transmission electron microscope, signaling protein were detected by western blot.All the data were summarized in EXCEL and analyzed by SPSS13.0 statistical analysis software. Multiple comparision between the mean were analyzed by analysis of variance or rank sum test, comparision between the rates of multiple were analyzed by chi-square test.Results1 The expression level of signl protein in CWP and the effect of dust exposure factors on the apoptotic signaling proteins of AMThe relative expression of apoptotic signaling proteins(DR5,caspase-3 and caspase-8) in the observed objects were less than that in CWP, the difference had statistical significant(P<0.05). The relative expression of apoptotic signaling proteins in the group of dust exposure (≥20 yr) were more than that in the group of dust exposure(<20 yr), the difference had statistical significant(P < 0.05). The relative expression of apoptotic signaling proteins in group of incidence length of service (<10 yr) were more than that in group of incidence length of service (≥10 yr),the difference had statistical significant(P<0.05). The relative expression of caspase-3 in group of incidence age(<40 yr) was more than that in group (≥40 yr), the difference had statistical significant(P<0.05).2 The apoptosis of AM in CWP casesTypical morphology of apoptosis was observed under ordinary optical microscopy, laser scanning confocal microscope and transmission electron microscopy, such as chromatin marginalization, crescent formation and nucleus fragmentation into massive. Typical hypodiploid apoptotic peak were observed before the diploid peak (Go/G1) appearing in flow histogram in the detection of flow cytometry. The index of apoptosis of AM in CWP was higher than that in observed subjects, and the index of apoptosis of AM at stageⅡof CWP was higher than that at stageⅡ, the difference had statistical significant(P<0.05).The index of apoptosis was positively correlated with the expression of apoptotic protein, the difference had statistical significant(P<0.05).3 The comparison of index of apoptosis among the different treated groupsOur study found that in the CWP, the index of apoptosis in the group of SOD and the group of TRAILR/TRAIL was lower than that in the untreated group, the difference had statistical significant(P<0.05). The index of apoptosis in the group of anti-TRAIL, caspase-8 and NF-κB inhibitor were less than that of group of TRAILR/TRAIL, the difference had statistical significant(P<0.05).4 The change of DR5, Caspase-8, Caspase-3 in TRAIL/TRAILR pathwayWe found that in our research, the relative expression of DR5, caspase-8 and caspase-3 in SOD group were lower than that of in control and TRAIL/TRAILR groups, the difference had significant (P<0.05). The relative expression of caspase-8 and caspase-3 in anti-TRAIL group were less than that in untreated group and TRAIL/TRAILR group , The relative expression of DR5 in anti-TRAIL group were less that in untreated group, the difference had significant (P<0.05). The relative expression of caspase-8 and caspase-3 in caspase-8 inhibitor group were less than that in control group and TRAIL/TRAILR group, the difference was significant (P<0.05). The relative expression of DR5 in caspase-8 inhibitor group were less that in untreated group, the difference had significant (P<0.05). The relative expression of caspase-8 and caspase-3 in NF-ΚB inhibitor group were less that in control group and TRAIL/TRAILR group, The relative expression of DR5 in NF-ΚB inhibitor group was less that in untreated group , the difference had significant (P<0.05).Conclusions1 Apoptotic signaling proteins, included caspase-3, caspase-8 and DR5, had a certain extent expression in AM of CWP. The expression of apoptotic signaling proteins were increased with duration of dust exposure and incidence length of service and age at CWP onset.2 There was excessive apoptosis of AM in the CWP cases, the index of apoptosis of AM in CWP cases were higher than that of in the observed subjects, the index of apoptosis were inceased with the severty of fibrosis. The apoptosis of AM could be inhibited by SOD, anti-TRAIL antibody, caspase-8 inhibitor and NF-κB inhibitor.3 The relative expression of upstream protein (DR5) and downstream protein (caspase-3 and caspase-8) could be down regulated by SOD. The relative expression of downstream protein (caspase-3 and caspase-8) could be down regulated by anti-TRAIL antibody and caspase-8 inhibitor and NF-κB inhibitor. |
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