The Preliminary Study On The Mechanism Of Cadmium-induced Pulmonary Fibrosis

BackgroundCadmium (Cd) is a kind of accumulation poison, it is normally found at a low concentration in the soil, and it is not biodegradable in the environment through the aggravation of industry pollution. Humans are exposed to Cd in the work place or through ingestion Cd-contaminated foods or water, the half life of Cd lasts for sixteen to thirty years in human body, it can accumulate in various organs and make damages and it is even carcinogenic. Cd has been classified as the sixth toxic substance harmful to human health by United Nations Environment Programme (UNEP) and the Committee of International Public Health of Heavy Metals, second only to aflatoxin and arsenic. In1987,International Agency for Research on Cancer(IARC) ranked Cd as ⅡA Carcinogens, and amended it to ⅠA in1993.Cd was extensively used in electroplate, paints, battery and photograph industry production. With the development of industry, the environmental pollution caused by toxic substance containing Cd becomes more and more severe. The American Committee of Agriculture has ranked Cd as first-line Agriculture environment pollutant; and the environment pollution brought by cadmium has attached great importance around world. Pulmonary fibrosis is a suite of pulmonary mesenchymal disease characteristic as progressive fibration of pulmonary mesenchymal and the fall-off of the respiratory function caused by varies factors, and usually harmful to human body. After final diagnosis, the survival time of it is only12-5years, five years survival rate is20%, and there is no effective prevention and treatment. Statistics shows that, with intensify of encironmental pollution, the incidence rate of pulmonary fibrosis is increased. With the development of modern research technics, scholars want to find out new method based on the mechanism study of pulmonary fibrosis. Although there has been some progress but no breakthrough has been achieved because of the complicated mechanism and numerous influencing factors. Besides the occupational expose to Cd and ingestion the polluted water or plants, smoking is another main route of non-occupational expose to Cd.In present, most of the studies about the damage caused by Cd to human body focus on the kidney injurious while there are insufficient studies reporting the lung injury. After the finding of extensive lung fibration in an autopsy dead from Cd poisoning, we tested this phenomenon through animal experiment. In our experiment, the animal exposed to Cd was found with widespread lung fibration, based on finding of this autopsy and animal experiment, we carried out a series of animal experiments and extro cell culture in order to interpret the molecule mechanism of Cd induced lung fibration. Epithelium-mesenchymal translation (EMT) has intimate relationship with fibroblast cell aggregation in pulmonary fibrosi. EMT is a complicated multi-step progress, the key feature is epithelium lost cell polarity and epithelial specificity maker E-cadhering(E-cad), got mesenchymal specificity maker α-SMA and Vimentin, and the shape of cell changed from cube to spindle-shaped, there is several signal pathway can have relationship with EMT, of the total epithelium growth factor receptors (EGFR) pathway play an important role. ObjectiveFrom the molecule mechanism study of Cd-induced lung fibration, we try to provide a new method for the prevention and therapy of the pulmonary fibrosis and lesion caused by Cd, and we also try to offer new thought in the study of lung fibration caused by other reasons. Methods1. Animal experiment:The rat is exposed to Cd through inhalation from respiratory and peritoneal injection method, we observe the gross anatomy of lung tissue, and the micro morphology change after HE dyeing.2. Followed with the immunohistochemisty dyeing to determination the localization and expression of E-cad and a-SMA in lung tissues. 3. Cell culture test in vitro, the cultered type Ⅱ penumonocyte (A549) was treated with10mM/LCdCl2, observe the cell morphology change after24h,48h and72h.4. Fluorescent quantitation testing the expression of a-SMA mRNA and E-cad mRNA in cultered type Ⅱ penumonocyte (A549) which have been treated with10mM/LCdCl2for24h、48h、72h.5. Western blot testing the change of a-SMA and E-cad expression in cultured cell be treated with10mM/LCdCl2for24h、48h、72h.6. Western blot testing the p-EGFR, which represents the activation status of EGFR signaling pathway. Results1. Lung injuries in gross autophy is found in both the rat exposed to CdCl2through respiratory inhalation or peritoneal injection, and the micro observation after HE dyeing shows lung inflammation, lung congestion, lung hydrops and pulmonary fibrosis, and in the group exposed to CdCl2through respiratory is more grievous.2. Observation after immunohistostaining in both two groups mesenchymal specificity maker a-SMA is high positive expression and epithelial specificity maker E-cadhering (E-cad) is positive expression in type Ⅱ penumonocyte, epithelial specificity maker E-cadhering (E-cad) is positive expression in part of the fibroblast in interstitial tissue, this phenomenon gives the hint that this part of fibroblast comes from type Ⅱ penumonocyte.3. Observation of the cultured type Ⅱ penumonocyte cell treated by lOmM/L CdCl2after24h,48h and72h under inverted phase contrast microscope, the shape of type Ⅱ penumonocyte is changed from rhombus to spindle-shaped same as fibrocyte and the intercellular space is widen, cells are separated from each other.4. Fluorescent quantitation testing shows that the expression of a-SMA mRNA is up-regulation and E-cad mRNA is down-regulation in cultered type II penumonocyte (A549) which have been treated with10mM/LCdCl2for24h,48h and72h.5. Western blot testing shows that with the increase of time exposs to Cd, epithelial specificity maker E-cadhering (E-cad) expression is wind-down and mesenchymal specificity maker a-SMA expression is increased gradually in cultured type Ⅱ penumonocyte cell.6. Western blot testing shows following with the increase of time exposed to Cd, p-EGFR is increased gradually, which represents the activation status of EGFR signaling pathway. Conclusion1. Both animal experiment and dead case of cadmium poisoning support the view that cadmium exposure can induce lung fibration, and in the group exposed to CdCl2through respiratory is more grievous.2. Animal experiment indicates that Cd can induce type Ⅱ penumonocyte translation into fibrolast.3. In vitro cell culture experiment indicates that Cd can induce the type Ⅱ penumonocyte translation into fibrolast.4. In the process of EMT epithelial specificity maker E-cadhering (E-cad) expression is wind-down and mesenchymal specificity maker a-SMA expression is increased gradually.5. All of the results support the view that EMT plays an important part in the process of cadmium-induced pulmonary fibrosis.6. The activation of EGFR signal transduction pathway is related to to cadmium induced EMT and lung fibration.