The Impact Of Tobacco Smoke On The Ultrastructure Of Human Macrophages And The Role Of Erythromycin

Objective:To study the effects of tobacco smoke on the ultrastructure of human macrophages and the role of erythromycin(EM).Methods:U937 cells were gained by serial subcultivation in vitro, and induced by PMA(10ng/ml). Human macrophages were treated with cigarettes smoke extract(CSE) and pre-treated with EM.1.Cells were divided into groups of four:(1) blank control group; (2) CSE group(incubated with 1%CSE for 24 hours); (3) CSE+EM (24h) group(incubated with 1μg/ml EM 24 hours,and incubated with 1%CSE for 24 hours).;(4)CSE+EM(48h) group(incubated with 1μg/ml EM 48 hours,and incubated with 1%CSE for 24 hours).2.The pre-incubated macrophages were processed through pre-embedded, fixed, rinsing, dewatering, osmosis, polymerization, trimming, sliced. Use the electron microscopy to study the ultrastructure of each treatment group after the uranyl acetate and lead citrate staining.3.After the supernatants of each group were centrifuged precipitate.The 2’,7’-dichlorofluorescin-diacetate was added under the conditions of protection from light. Incubated 30 minutes on the same conditions. Finally, the macrophages were examined the fluorescence intensity with the fluorescence microplate.Results:1.Observation of the three groups of ultrastructure The blank control group:Most of cells were round or oval, a few of mitochondria, lysosome and endoplasmic can be visibled. The vast majority of surface had many microvilli-like projections, and the nuclear was eccentric, circular or oval. A small number were irregular shape. The chromatin was accumulated in the nuclear membrane. The CSE group:Compared to the blank control group, in CSE group, not only the numbers of lysosome, mitochondria, endoplasmic reticulum increased, but also the cristaes were disarrangenment, fractured gradually(Called mitochondria vacuolation). The matrix of mitochondria with electron density increased.The primary lysosome and secondary lysosome existed at the same time, even showed multiple lysosome gathered. Endoplasmic reticulum expanded. The intracellular matrix were concentraton, which was called the degranulation of rough endoplasmic reticulum. The chromatin gathered more in the mitochondria. The CSE+EM group:Compared to the CSE group, the numbers of mitochondria reduced more and mitochondria did not vacuolate. The secondary lysosomes were invisible, and the numbers decreased. Endoplasmic reticulum were not expanding. And the degranulation of rough endoplasmic reticulum were not obvious.2. The effect of CSE and EM on ROS relative in human macrophages:In CSE group, the release of ROS was increased obviously compared with control group (P<0.05). In CSE+EM(24h) group,the release of ROS was lower than CSE group (P<0.05). And in CSE+EM(48h) group,the release of ROS was obviously decreased compared with the CSE group (P<0.05).Conclusion:1.Cigarette smoking caused changes of the ultrastructure, including mitochondria swelled and vacuolated, lysosome gathered, endoplasmic reticulum expanded and the degranulation of rough endoplasmic reticulum. EM could improve the phenomenon, such as the numbers of mitochondria, endoplasmic reticulum reduced more.Mitochondria did not vacuolate. Endoplasmic reticulum were not expanding. And the degranulation of rough endoplasmic reticulum were not obvious 2.CSE stimulated the macrophages to release ROS more. But EM could inhibit macrophage from secreting ROS.It can point out that EM may be inhibit the release of ROS by protecting the function of mithochondria, which may be the mechanism of oxidative stress.