The Influence And Mechanism Of Cilica To CC16 And SP-D In Patients With Silicosis And Silica-Treated Rats

Silicosis, an occupational disease with the characteristics of external cell matrix(ECM)deposition in lung interstitial, is due to the long-time inhalation of free SiO2 dust. It has reported in the 10th International Occupational Respiratory Disease meeting of China (10th ICORD) that patients with occupation diseases in china were 10467, and pneumoconiosis accounted for 80 % of all occupational disease in 2003, and silicosis resulted in more than nine billion RMB economy losses directly. It is one of the occupational disease with high morbidity in our country, and extensive harmful to people. There is no effective means for early diagnosis and health surveillance up to now. When patient was definitely diagnosed by chest roentgenogram, pulmonary fibrosis can not be reversed with medicines, chest high-resolution computed tomography (HRCT) can detect silicotic nodule earlier than roentgenogram, but it was not suitable as a routine method. So finding biomarker for early diagnosis of silicosis is very urgent. Recent several years, the investigations indicated that proteins with lung specificity, for instance, CC16 and SP-D play importance roles in the progression of pathogenesis of silicosis. So some researcher recommended that CC16 is a sensitive biomarker of Clara cell injury, which might improve our ability to detect exposure to silica harmful to the respiratory tract. And SP-D assay was given license by Japan government as biomarker of interstitial lung disease. The exact functions of CC16 are still unknown, but there is increasing evidence that CC16 plays important immunosuppressive, anti-inflammatory, anti-fibrosis and anti-tumor roles in the lung. SP-D acts as immunologic active material can reinforce the resistance, anti bacterial activity and dust invasion, and stabilize the lung immunity through recognition and prompt clearance of the necrotic and apoptoptic cells. The aim of this study is to explore the influence and mechanism of silica to CC16 and SP-D in patients with silicosis and silica-treated rats, and the potential value of discriminant equation set by SP-D and CC16 for early diagnosis of silicosis.ObjectiveTo explore the influence and mechanism of silica to CC16 and SP-D in patients with silicosis and silica-treated rats, and potential value of discriminant equation set by SP-D and CC16 for early diagnosis of silicosis.Methods1. Silica-treated rat model: A total of 80 Wistar rats were randomly divided into model group and control group (n=40 for each group). Model group was lightly anesthetized with pentobarbital and received a single intratracheal instillation of 1ml 50mg/ml silicon suspension. The control group was treated in the same way instead of 1 ml sterilized saline. On 7d, 14d, 21d, 28d and 60d after establishment of the animal model, rats were sacrificed and lung tissue and bronchoalveolar lavage fluid (BALF) were collected. I,III type collagen of lung tissue on tissue microarray were stained by Sirius red, and analized by a computerized morphometry system, Image-Pro Plus Version 4.5 for windowsTM. Expressions of SP-D and CC16 on tissue microarray were detected with immunohistochemistry and quantified by Image-Pro Plus Version 4.5 for WindowsTM; The SP-D and CC16 levels in BALF were detected with Western Blot and quantified by Quantity One Version 4.6.2.2. Silica-exposed workers: The subjects consisted of 30 healthy volunteers and 90 silica-exposed workers including the silica-exposed group (0), the silicosis suspects group(0+) and the silicosis phase I group, 30 subjects each group respectively. Subjects were selected strictly according to the National Diagnostic Standard (GBZ70-2002) of pneumoconiosis. The concentrations of CC16 and SP-D were measured in the serum by sandwich enzyme-linked immunosorbent assays, and discriminant equation set was established with SP-D and CC16 value.ResultsHistochemistry (HE staining) proved that the rat model of silicosis was successfully established by intratracheal infusion of silic dust suspension.Exposure to silica could increase the mass coefficient of organ to body of rats at each observed time point. In comparison with control group, I type collagen positive area percent of lung in model groups exhibited a sustained significant increase at all time points (P <0.05), but the increase of III collagen positive area percent of lung in model group was just significant at 14d and 21d (P <0.05).There were very few SP-D expressions in alveolar typeâ…¡and Clara cell intracytoplasm of control group while it markedly increased in lung tissue of silica group (P <0.05). SP-D positive cells reached the peak on the 14d. There was significant difference of SP-D integrated optical density between silica group and control gtoup (P <0.01). The SP-D levels of BALF in silica-treated group were significant higher than control group on 7d and 28d (P <0.01), and comparing with SP-D levels in BALF of silica-treated group on 7d, the 28d was still significantly elevated((P <0.01). CC16 expressed strongly in the Clara cell intracytoplasm of control group while it markedly decreased in lung tissue of silica-treated group((P <0.01). The integrated optical density of CC16 positive cells correlated negatively with the development of silicosis stage(rs= -0.967, P <0.01). The CC16 levels of BALF in silica-treated group were significant lower than those of control group on 7d and 28d(P <0.01), and comparing with CC16 levels of 7d BALF in silica-treated group, the 28d was still significant reduction (P <0.01).The concentration of CC16 in the serum was significantly decreased in silica-exposed workers compared with controls(P< 0.01); The concentration of CC16 in the serum was higher in lifelong nonsmokers than the current smokers in control subjects(P< 0.05), but there were no differences between lifelong nonsmokers and current smokers of 90 silica-exposed workers . Compared with control subjects, the levels of SP-D in the serum of silicosis suspects (0+) and silicosis phase I groups were significantly elevated (P< 0.01, respectively), which were also higher than the silica-exposed group (P <0.05 and P <0.01, respectively). Discriminant equation set by CC16 and SP-D were used in diagnosis of silicosis, and the rate of accuracy in healthy volunteers, the silica-exposed group and the silicosis phase I group were 86.7%, 89.2% and 76.7%, respectively. The total rate of correct classification was 84.2%.ConclusionTreatment with silica can reduce CC16 expression in rat lung tissue and BALF, and serum CC16 is decreased in patient with silicosis,So detection of CC16 may reflect early Clara cell injury.After silica instillation, There is a parabolic changing trend in the expression of lung tissue SP-D during 7~60 day exposure to silica, and increase SP-D level of BALF,and serum SP-D concentration is increased gradually with the progress of silica-induced lung injury. So detection of SP-D may reflect the injury of aveolus typeâ…¡cells and alveolar membrane permeability.Detection of CC16 and SP-D may be useful biomarker of silicosis early diagnosis, and reflecting Clara cell, ATâ…¡, integrality of alveolar membrane injury. The determination of SP-D can evaluate the progress of silica-induced lung injury. Discriminant equations set by CC16 and SP-D were useful for diagnosis of silicosis.