Analysis Of Components In Bronchoalveolar Lavage Fluid Of Rats Following Silica Exposure

Objectives Presently, pneumoconiosis is a frequent occupational lung disease Some specialists estimated that there are more than one million pneumoconiosis cases in China. And the number of the pneumoconiosis cases will increased within 10 to 15 years. The date reporter of pneumoconiosis cases in 2005 indicate that the pneumosilicosis and coal worker's pneumoconiosis(CWP) are dominant (90.8% in the pneumoconiosis cases). And a half of the pneumoconiosis cases is pneumosilicosis. It is one of the most serious occupational disease. Nowadays, the generally accepted mechanism of pneumosilicosis include toxcity of silica, lipid peroxidation, the cells interaction in lung, cytokine network and so on. But the detail mechanisms it was not clear yet, which has restricted the early diagnosis and treatment badly.BAL was involved in study of pulmonary disease has for 100 years. Due to the invention for bronchofiberoscope(BFS) ,as a relative safe method, BAL has been widely used for scientific research and clinic tracking. Components of BALF come from air tube and alveolar epithelium ecderon which can reflect process for affection of lung parenchyma. Analysis the the formed elements(cell) and some invisible elements of BALF will help us with, to exploring which kind of change play an criticality role in early affection for pneumonoconiosis, trying to find the biomarks for early diagnosis of pneumosilicosis and the process of pulmonary fibrosis. Further more all those researches will offer evidences for revealing the pathogenesy of pneumonoconiosis.Methods Forty male SD rats (weight is 200±16g) were divided into two groups randomly. The rats of the control group were injected with 1ml normal saline in trachea.The rats of the experiment group were injected with 1ml SiO2 suspension of 50mg/ml in trachea.Forty days later, the rats were killed and we collected the bronchoalveolar lavage fluid both of the two groups. Cell types and number in BALF were counted the content of protein and SOD activity, the content of Fe,Zn,Mn and the content of CC10 were measured in BALF by the method of Bradford, xanthinoxidase, ICP-AES and ELISA. To isolate the dissolubility protein in BALF by polyacrylamide gel electrophoresis.Misce bene the protein sample solution with protein of 200μg and hydration balanced solution which the total volume is 340μl (the length of IPG glue stick is 18cm, pH 3~10) with the method of two-dimensional gel electrophoresis (2-DE) for proteome, then progress the first ampholine electrophoresis.The second ampholine electrophoresis with 10% SDS-PAGE. Bolter the composition of the proteic substance in BALF. Paraffin-embedded lung sections with HE, and the pathology changes of the lung tissue r was observed under the light microscope .Results1 The quantity of the total cells is (1.21±0.44)×107/L , the white blood cells is (1.14±0.51)×106/L,the heterophil granulocyte is (0.53±0.08)×106/L, the macrophage is (9.97±3.91)×106/L,the total lymphocte is (0.94±0.42)×106/L of the dusted rats ; and the quantity of the total cells is (6.30±2.19)×106/L, the white blood cells (0.52±0.21)×106/L , the heterophil granulocyte is (0.21±0.09)×106/L , the macrophage is (5.04±1.52)×106/L, the total lymphocte is (0.31±0.15)×106/L of the control group.The results show that the quantity of the total cells and the inflammatory cells in BALF of dusted rats are higer than the control group (P <0.05).2 The content of protein is 1.06±0.56g/l, the SOD activity in supernatant is 14.24±8.01U/ml, the SOD activity in cells is 22.01±6.46U/mgprot in the BALF of dusted rats; and the content of protein is 0.62±0.27g/l, the SOD activity in supernatant liquid is 13.61±8.61U/ml, the SOD activity in cells is 14.01±7.38U/mgprot in the BALF of control group.The content of protein and the SOD activity in cells is higher than the control group (P <0.05).But the SOD activity in the supernatanttotal of the two groups has no statistically significant (P>0.05).3 The contents of Fe, Zn and Mn in the BALF of dusted rats is 1.84±1.10μg/ml, 6.03±2.54μg/ml and 0.16±0.05μg/ml.The contents of Fe, Zn and Mn in the control group is 4.76±1.70μg/ml, 2.51±1.77μg/ml, 0.09±0.04μg/ml. The content of Fe degraded in the BALF of dusts rats, but the contents of Zn and Mn steped up in the BALF of dusts rats (P <0.05).4 The content of CC10 is 0.77±0.22g/l in the BALF of dusted rats. The content of CC10 is 0.41±0.15g/l in the BALF of control group. The content of CC10 in BALF of dusted rats is lower than the control group (P <0.05).5 The limpid separating strips of the protein in the BALF were appeared in the electrophoregram by polyacrylamide gel electrophoresis. The separating strips have the difference between the quantity and the shade degree.6 Using the method of two-dimensional gel electrophoresis (2-DE) bolter protein, we gained the limpid proteinaceous dielectrophoresis patterns.To compare the 2-DE patten of BALF of the two groups, we found that there are evident difference between the two groups. The difference shows by the increase and decrease of proteinum blotch and the colored degree.7 The lung tissue of control group on routine pathological section is complete, uniformity and limpid; the lung tissue of Dusted Rats was destroyed at different level, and showed classical silicotic nodule, the collagen fibers(CF) is increased in silicotic nodule.The interstitial substance fibroplasia in lung was appeared.The lung tissue were damaged in dusted rats.Conclusions1 We detected that the quantity increased evidently of white blood cells, heterophil granulocyte, macrophage, lymphocte(P<0.05).The increasing quantity of inflammatory cells in the BALF of dusted rats state that silica can induce serous inflammatory reaction, and damage the lung tissue.2 With measuring the content of the protein, the activity of SOD and the contents of Fe,Zn,Mn, we detected that all those subjects has been evident changed during the period of injury and recovery of lungs(P<0.05).To analysis the mechanism of action, those date can be play an important roles to elucidate the nosogenesis of pneumonoconiosis.3 We detected that the content of CC10 express was reduced in BALF of dusted rats(P<0.05). To detect the content of CC10 can reflect the early damage of the Clara cell, accordingly that can get the state of the lung disease.The change of the content of CC10 in BALF can be a marker for the appearance of injury and recovery of lung tissue.4 With the method of two-dimensional gel electrophoresis (2-DE), we separated the dissolubility proteinum in BALF of two groups and gained the limpid protein dielectrophoresis patterns. And compared the control group, the difference of BALF 2-DE pattens revealed that the quality and quantity of the protein in BALF have been changed of the dusted rats.