Regulatory Network Of Bioactivity Medium Of Silica-reduced Pulmonary Fibrosis

Silicosis, one of most severe occupation diseases, is an gradually aggravated and irreversiblely pulmonary interstitial fibrosis disease, induced by long-term inhalation of free silica powder dust, such as the dust of mining excavation in the gold and hydropower armed police force, civil mine exploitation, raw material quassation in the procedure of metallurgy and ceramics, which can cause momentous lose of economy directly and indirectly, and induce substantially aggravated problems in the fields of society and public health. Up to now, the study of the single cytokine effectiveness of silicosis fibrosis had got some achievements, but the mechanism of systematic regulatory network is still not being demonstrated.Objective To ascertain related bioactivity mediums of silica-reduced pulmonary fibrosis in rats, to construct the regulator network of the material bioactivity and its genes as well as both integration, to evaluate network regulatory mechanism of silicosis fibrosis systematically.Method The regulatory network of the bioactivity mediums was constructed by the method of systems biology in this paper. 1. Identification of the bioactivity mediums. The mediums of biological activity were identified by the meta-analysis software RevMan 4.2. The investigative biases were also evaluated by analytical methods such as funnel plot, heterology and susceptibility. 2. Construction of initial regulatory network with meta-analysis database. The potential regulatory network pathway was constructed and analyzed by meta–analysis, cubic spline interpolation, differential model and weighting matrix obtained with least square method. 3. Silicosis model in rats and construction of initial regulatory network with animal experiment database. The rats models in silicosis or control group were established with intratracheal infusion of silica dust suspension or physiological saline by trachea exposure method. Wistar rats were divided randomly into two groups including control group and silicosis group. At each time point such as day 1, 3, 7, 14, 21, and 28 after establishment of the rat model, eight rats from each group were sacrificed. After the serum and pulmonary tissue of the model were required, the related bioactivity mediums were detected systematically. The abnormal syntheses of collagenⅠandⅢin pulmonary tissue with Sirius red staining were detected by polarization microscopy. The area percentages of collagensⅠandⅢwere quantitatively analyzed by image analysis systems. The concentration of NF-κB, IL-1β, IL-10, TNF-α, INF-γ, TGF-β1, and GM-CSF protein in serum was measured by ELISA method. And NO content was determined by nitric acid reeducates method. The regulatory network was constructed with correlation coefficient and differential equation model by systematical detection database. 4. Revise of regulatory network by intervention experiment. Silicosis model in rats was established by the same trachea exposure method. 32 rats were divided into two groups randomly, i.e. TNF-αintervention group and silica group, which were observed at the 7th day and 14th day after establishment of the animal model, respectively. Eight rats from each group at each time point were sacrificed, and the same bioactivity mediums as above-mentioned were detected systematically. The regulatory network was reconstructed and revised by comparing the difference of each bioactivity mediums in the two groups. 5. The experiment ascertainment and expansion of the gene regulatory network. The total RNA of pulmonary tissue in silicosis and control group was abstracted by Trizol method at the 14th day after model construction. The different express genes were screened with diffscore value by illumina gene expression microarray chip. The cluster of the related cytokines gene was analysised by the software including Cluster and TreeView and by the database platform such as KEGG, GO and DAVID. Then the gene regulatory network was established by correlation coefficient matrix and was also integrated with regulatory network of bioactivity medium for the purpose of expanding and improving the network.Result 1. The related bioactivity mediums of silica-reduced lung fibrosis were ascertained to be TNF-α,TGF-β1,IFN-γ,MCP-1, IL-4, IL-6,IL-18,MMP-9, IL-10,IL-1β,IL-5,IL-8, IGF-1, GM-CSF, MMP-2 and EGF by meta-analysis. Of the mediums, TNF-αcontent in lung tissue increased not on a straight line but on a wave line in a time-dependent manner. The medium level wasn't coincident in lung tissue, macrophage and serum. Specially, the medium content in macrophage was more than the others'at the 1st day after model constructing, and the medium of pulmonary tissue in silica group was more significant statistically than that in control group except on the 1st day, and the mediums content in the serum was lower than that in the lung tissues. 2. The mediums, IFN-γ,IL-10,IL-1β,IL-18,IL-6,IGF-1, GM-CSF, TGF-β1 and TNF-αwere the key nodal points in regulatory network of the pulmonary fibrosis, but IL-4, IL-5,IL-8,MCP-1, MMP-2, MMP-9 and EGF were not in the regulatory, if threshold value in weighting matrix exceeded absolute value 2σ. The mediums, IFN-γ,IL-10,IL-1β, GM-CSF, TGF-β1 and TNF-αwere the key nodal points in regulatory network of the pulmonary fibrosis, but IL-18, IL-6,IGF-1, IL-4, IL-5, IL-8,MCP-1, MMP-2, MMP-9 and EGF were not in the regulatory, if threshold value in weighting matrix exceed absolute value 3σ. 3. The lung tissue with sirius red staining were observed by microscopy and image analysator, with the result that the rat model of silicosis was established successfully at day 21, 28. Compared with that in the control group, the content of NF-κB and NO in serum increased significantly at all the time points in silicosis group except for NO at day 21, and dynamic state express ratio tendency of both mediums was coincident. At the 21st day and 28th day, the express ratio of TGF-β1 in silicosis group was higher, but that of IFN-γwas lower than one in the control group. TNF-αprotein level decreased in earlier period and increased in the later period, but had no statistical significance during the whole experiment in both comparisons. The dynamic express ratio of the content of IL-1β, IL-10, and GM-CSF exceed 1 at various time point. Of the total mediums, the tendency of the former two protein level was elevated in fibrosis prophase and degraded in fibrosis anaphase, but the tendency of the later GM-CSF was raised by waved method. 4. The mediums in regulatory network in which the degree weren't less than 5 had IFN-γ, GM-CSF, TGF-β1, TNF-α, and the mediums of which the degree was less than 5 had IL-10, IL-1β, NF-κB, NO, COLⅠ, COLⅢ. The soluble TNF-αreceptor which was feasible to the intervention of pulmonary fibrosis could suppress formation of collagenⅠandⅢin lung tissues, and play role of anti-fibrosis through peripheral serum mediums such as NO, NF-κB, TGF-β1, and IL-1βin cytokine network in silicotic process. In the above regulatory network, the mediums in the pathway related to TNF-αshould include COL I, GM-CSF. 5. 29 genes related to cytokines function categorization in serum were divided to the regulatory network in silicosis fibrosis. The key genes with degree exceeding 5 in regulatory network were CCL7, GRN, IL-18, SFTPD, SPN, SPP1, TNF-αand TNFRSF.Conclusion The pulmonary fibrosis was regulated by the network method of silica-induced changes of bioactivity mediums. The regulatory factors which involve in pulmonary fibrosis consist of 16 bioactive mediums including TGF-β1,IFN-γ,MCP-1, IL-4, IL-6,IL-18,MMP-9, TNF-α,IL-10,IL-1β,IL-5,IL-8, IGF-1, GM-CSF, MMP-2, EGF with space-time transmutation rule. In the fibrosis process, the mediums'levels which weren't consistent in various time points increased or decreased not by straight line method, but by wave line method. In the various specimens related pumomary fibrosis, the detection of the mediums'variation in the related cells was the earliest than that in the others, but the one in serums was the latest than that in the others. The mediums levels in lung tissue were the maximum, but the one in serums was the minimum. The mediums, IFN-γ, IL-10, IL-1β, GM-CSF, NF-κB, NO, TGF-β1 and TNF-α, were the key nodal points in regulatory network of the pulmonary fibrosis, but IL-18, IL-6,IGF-1, IL-4, IL-5, IL-8,MCP-1, MMP-2, MMP-9 and EGF weren't in the regulatory. NO and NF-κB were a prime center in the regulatory network of pulmonary fibrosis, and TNF-α, TGF-β1 not only acted as the promoter, but also held the post of intermediate transferring factor in regulatory network which caused lung fibrosis. The changed content of GM-CSF,IL-1β,IFN-γamplified and inhibited cascade network effective, respectively. Then network effective of these bioactivity mediums resulted in lung fibrosis under the condition of imbalance of promoting agent and suppressing agent.