| Silicosis, a systematic disease characterized with lung interstitial fibrosis caused by inhalation of silica dust, has been listed as occupational disease. From 1991 to 2002, a survey focusing on the morbidity of occupational diseases in China revealed that a remarkable concave-shape rebounding tendency of occupational diseases had been shown. A decrease trend was shown from the early 1990s and its number of 10 thousand, reached it bottom then rose to 15 thousand in 2002. Therefore, the incidence status of silicosis still needs concern. Up to 2006, a total of 616,000 accumulative number cases of pneumoconiosis had been reported nationwide. Among them, 146,195 cases died and the prevalence number of pneumoconiosis is 470,247. About one thousand new cases per year were reported in recent 15 years and the loss caused by this disease per year is about 90 billion RMB yuan directly or indirectly. Almost 80% of pneumoconiosis was silicosis cases. These data indicated that silicosis was one of the most dangerous occupational diseases.Though in some condition, silica exposure was stopped, silicosis still develops. With high concentration silica dust exposing, more and more younger cases were reported with more serious disease condition and higher mortality rate. What's more, with more and more farmer workers taking part in non-protective exposure of silica, the prevention condition becomes more and more serious. Facing such condition, a plan aimed at decreasing and finally eliminating silicosis in 2010 and in 2030 had been put forward by ILO and WHO. However, up to date, the mechanism of silicosis was still obscure which resulted in no specific methods for early diagnosing and treating. Once diagnosed by traditional back-chest position X-ray method, no treatment could reverse silicosis. Till now, though there were several drugs could be used in clinics, yet these drugs with high toxicity. So, it is urgent to develop new skills and tracing new treatments to interfere with this disease.ObjectiveThe aim of this study was to explore the molecular mechanism of silicosis and the role of Chinese traditional medicine on this disease. Differential gene expression profiling in pulmonary tissue of rats exposed to silica in early period was carried out and the effects of Gymnadenia conopsea alcohol extract (GcAE) were evaluated on silicosis. According to this study, molecular mechanism for silicosis and basic information for studying new more effective and low toxic drugs could be offered.Methods 2.1 Grouping and treatmentsGrouping A total of 160 wistar rats were divided into four experimental groups randomly: drug-treated group (GcAE-treated) and positive drug tetrandrine (TT) treated (TT-treated) group, silicosis group (silica-instilled group) and the control group (n=40 for each group).Silicosis rat models After lightly anesthetized with pentobarbital, rats were received a single intratracheal instillation of 1ml 50mg/ml silica suspension in GcAE-treated, TT-treated and silica-instilled groups. In control groups, rats were teated in the same way but instillation of 1 ml sterilized saline instead.Treating Rats in drug-treated groups were administered intragastrically GcAE (8g/kg body weight per day, orally) or TT (50mg/kg body weight, 3 times/wk, orally) 24 hours after the establishment of the silicosis model. The silica-instilled and control groups were administered intragastrically 2 ml sterilized saline per day orally.Sacrifice of animals and specimen collection At the 7th d, 14th d, 21th d, 28th d and 60th d after establishment of animal models, eight rats in each group were sacrificed, and lungs were removed and stored immediately in liquid nitrogen. Then specimens were maintained at -80℃until RNA extraction. I, III type collagens of lung tissue were stained by sirius red, and determined by a computerized morphometry system, Image-Pro Plus Version 4.5 for windows TM. 2.2 Lung gene expression analysis on 14th dayA gene chip system (Illumina Company, USA) was used in studying gene profile of lung total RNA in rats exposed to silica. Following scanning and standardizing (Illumina Company, USA), diffscore was calculated and thus differential expressed gene could be got. Bio-information system(http://david.abcc.ncifcrf.gov/)was used in gene functional annotation and classification. Also, real-time PCR method was used in verifying the discovered genes.Results3.1 Pathological results of silicosis model Staining with normal HE method, obvious pathological changes of lungs could be observed in silicosis animal model, such as granuloma, proliferation of fibroblasts and smooth muscle cells, and thicken of alveolar wall in pulmonary alveolus. Also, accumulate of collagen, destruction of lung cell structure and dotted fibrosis could also be seen. However, the changes of lung in GcAE treated group were slighter than that of the silicosis groups.3.2 Differential expression genes in early period of silicosis model Compared with the control group, 1 567 genes were differential expressed including up-regulated 762 genes and down-regulated 799 genes in silica exposed rats among a total of 22 107 available genes. Thirty-five and twenty clusters could be classified according to their function in up-regulated and down-regulated genes, respectively. The clusters involved genes: (1) cell bio-chemical reaction related genes for cell cycle, differentiation and proliferation, apoptosis, cytoskeleton, signal transduction, transfer and stress reaction, DNA damage and heat shock protein; (2) metabolism and transcription related genes of DNA, RNA and protein; (3) enzyme related genes including oxidoreductase, phosphatase, hydrolase, glutathione transferase and protein kinase; (4) cytokines, chemokines and immunological reaction related genes.Also, KEGG pathways analysis showed that genes related with biosynthesis of terpenoid, steroid and ATP and riboflavin were up-regulated. However, genes related with signal pathway of Wnt, TGF-βsignaling pathway and lymphocyte transendothelial migration were down-regulated. The chip results were verified using real-time PCR for SOD2 and HMOX1 gene with the same trend.3.3 Differential expression genes in early period of silicosis model treated with GcAEThree hundred and eight and 231 were up-regulated and down-regulated genes among a total of 539 available genes compared with the GcAE vs Silica group, respectively.Up-regulated pathways might associate with CAMS, notch signaling pathway, and leukocyte transendothelial migration and cell communication. Down regulated pathways might relate with genes for linkage of complement and coagulatlon cascades, hematopoietic cell lineage, urea cycle and Alzheimer's disease. The chip results were verified using real-time PCR for SOD2 and HMOX1 gene with the same trend. ConclusionsA complex gene regultion network exists in the development of silicosis; this may offer basic information and guild for further research for regulation network of silicosis, treatment target gene and biomarkers in the early period of this disease.Also, our results have showed that GcAE may inhibit the progress of silicosis in the early period and could attenuate airsacculitis and fibrosis of the lung. The GcAE may be one potential drug for silicosis with almost the same effect as TT.
|
PDF Full Text Request