Study On The Rivalry Influence Of OM To The TNF-α-induced Apoptosis Of Alveolar Macrophage

Objective:To study the molecular mechanism of the rivalry role of OM to the TNF-α-induced apoptosis of alveolar macrophage.Methods:male Wistar rats,adult and health,Macrophage collected by lung alveolus bronchial lavage was cultured with 100μg/ mlSiO₂ for 24h,then collect the supernatants.Devided to blank control,TNF-a(5,10,20μg /L)+AM-SiO₂ supernatant,OM(200,400,800mg/L)+10μg/L TNF-a+AM-SiO₂supernatant groups.Then stimulate the purified macrophages with the supernatants of all the groups.1)FCM was used to detect the apoptosis of of the alveolar macrophages;2)Expression of NF-κBP65 in pulmonary macrophages was measured with method of immunohistochemistry,and results were analyzed with MIAS-2000 image analytical system by means of positive unit(PU);3)Expression of G(Gαs)protein in cellular membrane were detected with method of Western-blot,and results were analyzed with gelatum imaging system.Results:1)Reasults of macrophages apoptosis:Apoptosis percentage of macrophages in TNF-a groups were significantly higher than that of the control group(P＜0.05).and the apoptosis percentage in TNF-a 10μg/L group was significantly lower than that of 5μg/L and 20μg/L group(P＜0.05);Apoptosis percentage in OM groups were significantly higher than that of the control group(P＜0.05).but that of 400mg /Lgroup was significantly lower than that of 200,800mg/L group(P＜0.05);2)NF-κBP65 immunohistochemistry reasults:MIAS-2000 image analytical system shows:Compared with the control group,the positive unit value of NF-κBP65 in TNF-a groups decreased,There weres tatistical significances between 5μg/L group and the control group(P＜0.05),5μg/L and 10μg /L group,5μg/L and 20μg/L group.The positive unit value of NF-κBP65 in OM groups were decreased than that of the control group,but only two statistical significances(P＜0.05)between 200mg/L group and the control group,800mg/L group and the control group.The positive unit value of NF-κBP65 negatively correlated with the apoptosis rate(r=-0.812,P＜0.001)and the expression of G(Gαs)protein(r=-0.538,P＜0.05).3)Reasults of Western-blot:G(Gαs) protein levels in the cellular membrane enhanced along with the increasing concentration of TNF-α,respectively,which in 5μg/L and 20μg/L TNF-a group were significantly higher than that in the control group(P＜0.05).and the expression of G(Gαs)protein in 10ug/L TNF-a was significantly lower than those in other two groups(P＜0.05).There was positive correlation between the apoptosis rate and the expression of G(Gαs)protein(r=0.740,P＜0.01);In OM groups,the expression of G(Gαs)protein were significantly higher than that in the control group (P＜0.05)along with the increasing concentration of OM and.there was a positive correlation between the apoptosis rate and the expression of G(Gαs)protein(r=0.831,P＜0.001); Conclusion:1)TNF-αcould induce macrophages apoptosis in form and structure,of which the mechanism though the upwards regulaotion of G protein and the downwards regulation of NF-κB.2)G protein and NF-κB play an important role during the course of macrophages apoptosis.3)OM may protect macrophages from apoptosis through the activation of G protein and NF-κB.