Research The Effects Of The Regulation Of Autophagy To Silicosis Fibrosis Of Rats

Objects 1 Wistar rats were given routine silica building, randomly divided into three gro ups, two groups were given three methyladenine and rapamycin, three groups of rats put to death in 7 days, 14 days, 28 days, 60 days and 90 days respectively. Through making HE staining and Masson staining of the lung tissue of rat, determination the collagen fiber in the area of lung tissue, we analyzed silicosis fibrosis development in different stages of the process. 2 We gave 3-MA inhibitting autophagy activity and rapamycin promoting autophagy activity of rats, we observated pulmonary macrophages(Alveolar macrophage, AM) the strength of the autophagy activity, analyzed the relationship between autophagy and silicosis fibrosis cells through the electron microscopeslices and the expression of protein levels in the lung tissue. 3 We measured its early apoptosis rate and analyzed the connection between autophagy and apoptosis cells by purified cells lavage in pulmonary macrophages.Methods SPF health male Wistar rats were randomly divided into three groups: model group, trimethyladenine group(3-methlyadenine, 3-MA) and rapamycin groups, each gro up were only 40. Erevy group perfused silicosis conventional building. Rats in each group were dusted in 7 days, 14 days, 28 days, 60 days and 90 days and respectively executed only 8. We collected the left lung lavage fluid, extraction of pulmonary macrophages, used flow cytometry instrument to detect early pulmonary macrophage apoptosis rate; Heart blood, centrifugal obtaining serum, used enzyme-linked immunoassay determina- tion of cytokines in the serum TNF-ɑ, TGF-beta, and the content of IL-10, observed the development of pulmonary fibrosis; Taking 100 mg of left lung tissue, real-time fluoresce nt quantitative polymerase chain reaction(Real-time PCR)analysisⅠand Ⅲ before collagen m RNA expression level; Apply adequate amount to the lung tissue homogenate, organization after cracking, cracking fluid using western blot method in the lung tissue LC3, Beclin1, AKT and PDK1 protein expression level; Taking120 mg of lung tissue, according to the kit instructions to determine the hydroxyproline content; Put the size of soybean on the right side of the lung tissue in paraformaldehyde solution, the lung tissue of HE staining and Masson staining, observed the pathological changes of the experimental group rats lung tissue and tissue changes of collagen fibers,and used of IPP(the Image Pro Plus) Image analysis software was developed for thedetermination of collagen fiber area; Taked the corn grain size lung tissue fixed by 2.5%glutaraldehyde solution, preparing section, observed the strength of the pulmonary macrophages autophagy activity.Results 1 Each experimental rat lung tissue pathological changes: the model group rats lung tissue with inflammatory cells infiltration, alveolar walls were obvioued thickening,alveolar cavity obviously decreases, and the lung tissue into fiber cells increased, with the increase of dye dust of time, a large number of cells within the lung nodules and typical silicon nodules; 3-MA group of rats in the early dye dust lung tissue inflammation was not obvioued, but still had a small amount of inflammatory cells infiltration in lung tissue,alveolar cavity with different degree of broadening, the lung tissue of rats in the late dye dust in a large amount of inflammatory exudate, and cell nodule; Rapamycin group of rats appeared at the beginning of the dye dust obvious inflammatory reaction and alveolar cavity with a large number of inflammatory exudate, significantly broadening alveolar walls, mid dye dust appear a large number of fibroblasts, and a typical silicon nodules,dye dust late lung fibrosis degree was serious, the lung tissue was completely destroyed.2 Different treatment group rats lung tissue fibrosis degree were different at a different time.We maked Masson staining to observe the changes of the collagen fibers in the lung tissue, and used morphology quantitative analysis system to measure collagen area, we found model group rats of collagen fiber area were increasing time in the whole process with dust time increased; And 3-MA group rats collagen fiber area have increased in 7and 14 days, and collagen fiber area of 28 days and 60 days were lower than it of 7 days and 14 days, collagen area again increased in 90 days. Compared with the model group in different periods, the collagen fiber area were lower than the model group. Rapamycin group of five different periods of collagen area were increasing in constant, compared with model group, collagen area were more than the model group, there were differences in both experimental group compared with model group and the difference was statistically significant(P<0.05). Because the hydroxyproline content can be used as important indicators of collagen metabolism, this study further determinated hydroxyproline content in the lung tissue, and found that the change trend of each hydroxyproline and the change trend of collagen fiber area were consistent. 4Ⅰ,Ⅲtype collagen m RNA of rat lung tissue cells were determinated in different treatment groups at different periods:Ⅰtype collagen of control group was high in 7 days,Ⅰtype collagen of14 and 28 days decreases,Ⅰtype collagen was again rising trend in 60 days; the tendency ofⅠtype collagen 3-MA was the same with the model group, in addition,Ⅰtype collagen of 7 days in 3-MA group is higher than the model group, other times lower than the model group; Contents ofⅠtype collagen in rapamycin group was rise-reduce-rise-again again the change trend of rise, in additionⅠtype collagen of 60 days was lower than the model group, and every other period were higher than the model group. Model group Ⅲtype collagen content had been rising trends in addition it of 28 days had smaller amplitude to reduce; 3-MA groupⅢ type collagen content was higher at the beginning of the drug, and Ⅲ type collagen content had been lower trends in the delivery period of14 days and 28 days and 60 days after discontinuation, 90 days after drug Ⅲ type collagent rise again, and Ⅲ type collagen of 3-MA group were lower than the model group in different historical periods. Rapamyc the whole change process of Ⅲ type collagen content was the same as the change of this group of Ⅰtype collagen, and in addition to 7 and 14 days, other times lower than model group in different periodsⅠ,Ⅲtype collagen m RNA of two groups are differences compared with model group, and the difference was statistically significant(P < 0.05). 4 The change of serum cytokine levels in different group: based on the determination of the cytokines level of rats and we found cytokine TNF-a of the model group was increasing in the whole process with the of dye dust of time had been rised; Contents of cytokine TNF-a in 3-MA group was lower in the7 day of dosing, the level of 14 days increased, in stop drug dosing 28 days and 60 days content was reduced with varying degree, and 90 days the content rised again, but the period of cytokine levels was lower than the model group; Rapamycin group content had been rised in drug delivery period, 60 days content decreased obviously because the drug was stopped, 90 days content rises again. 5 Autophagy activity of rat pulmonary macrophage during different periods. Electron microscope found that different treatment group of autophagy activity was different during different periods. Model group rats appeared autophagy precursor and autophagy-lysosome in the early days of dye dust, in middle days of dye dust autophagy appeared, autophagy body gradually disappear in the late days of dye dust, autophagy body quantity rat pulmonary macrophage was little in 3-MA group and rat pulmonary macrophage autophagy body of rapamycin group was higher than the model group. In order to further illustrate the strength of the autophagy activity, we measured the autophagy marker protein LC3 and beclin1, we found that 3-MA group LC3 Ⅱ /LC3 Ⅰ of 7 days was higher than model group, but lower than rapamycin group; in 3- MA group LC3Ⅱ/LC3Ⅰof 14 and 28 days was low and lower than the model group and rapamycin group; 3-MA group after discontinuation LC3 Ⅱ/LC3Ⅰincreased again and 60 days was higher than the model group, and there was no significant difference between three groups data in 90 days; Rapamycin group LC3 Ⅱ/LC3 Ⅰ have been showing a tendency of increase, and rapamycin groups of various periods of LC3Ⅱ/LC3Ⅰwere higher than model group in each period. The change of beclin1 protein expression was the same as this group of LC3. 6 Analysis the protein expression level of rat lung tissue in different groups: research had shown that PI3K/Akt pathway was involved in a variety of cytokines to fibrosis process and played an important role in the cell proliferation, differentiation and apoptosis. In this article,through western blotting method, we determinated AKT and PDK1 of protein content in lung tissue of rats to reflect the degree of pulmonary fibrosis in rats. The experimental results showed that the lung tissue fibrosis degree of the model group rats was aggravating with the extension of dye dust time, AKT protein content in the lung tissue had been rise; 3-MA group and rapamycin AKT protein content also showed the trend of increase in the lung tissue of rats during the entire study, the difference was protein content of 3-MA group were lower than the model group in different historical periods, and the protein content of rapamycin group were higher than model group, and the two groups was compared with the control group were statistically significant. 7 Rapamycin group LC3 protein expression were increased and higher than the model group in different time;Rapamycin group early apoptosis rate of rats pulmonary macrophage( 1.74±0.73)、(1.25±0.35)、(1.44±1.24)、(3.70±1.51)(36.98 ±11.91)was lower than the model group( 5.63±1.01) 、( 4.95±0.63) 、( 4.10± 0.43) 、( 6.75±4.19) 、( 30.23±11.05) out of 90 days; 3-MA group LC3 protein expression was lower than model group during the dosing, protein expression was higher when the drug is stopped, protein expression level was similar as the model group in the 90 day; 3-MA group early apoptosis rate of rats pulmonary macrophage( 2.86±1.13) 、( 8.80±0.28)、(6.84±1.29)、(2.54±1.65)、(37.22±16.22),3-MA group apoptosis rate was low in 7 days; apoptosis rate increased significantly and higher than the model group in 14 and 28 days; apoptosis rate was down again in 60 days; apoptosis rate was no obvioued difference with the control group in 90 days.Conclusions 1 Rapamycin promotes the occurrence of autophagy activity, silicotic rats lung tissue fibrosis degree was aggravating, and was subjected to the regulation of autophagy, protein AKT, PDK1 presented a tendency of increasing as the dust of time. 23-MA can delay or partially inhibit autophagy activity, silicotic rats lung tissue fibrosis degree reduced, and protein AKT, PDK1 presented a tendency of increasing as the dust of time, but content were lower than the normal silicotic model rats. 3 3-MA and rapamycin inhibit and promote autophagy activity, cell early apoptosis rate reduced with the increase of autophagy activity at the beginning of the dye dust, but late in the rats with dust, cell early apoptosis rate with enhanced autophagy activity also showed a trend of rising.