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Earthworm Extract Attenuates Silica Induced Pulmonary Fibrosis In Mouse

Posted on:2016-03-01Degree:MasterType:Thesis
Country:ChinaCandidate:T WangFull Text:PDF
GTID:2284330461493307Subject:Occupational and Environmental Health
Abstract/Summary:PDF Full Text Request
Pneumoconiosis is due to long-term inhalation of dust in working environment,which is a systemic disease mainly characterized by irreversible progressive pulmonary fibrosis. Pneumoconiosis is the most serious occupational disease in China,more than 85% of the newly diagnosed occupational disease every year were attributed to pneumoconiosis and over 90% of them are coal workers’ pneumoconiosis(CWP) and silicosis. CWP contains of coal pneumoconiosis, silicosis and anthraco-silicosis, their pathogenesis are basically the same, which are strongly linked to free silica content of coal dust / silica dust. At present, massive whole lung lavage(MWLL) for cleaning dust in lungs is very effective, suitable for early-stage patients. However, MWLL is not easy to accept by the patients. Drug is the most likely accepted therapy for patients, but there are few treatment strategies available that specifically target the pathogenesis of lung fibrosis. China already has more than700000 diagnosed pneumoconiosis patients, with about 15000 new cases annually, so it is urgent to find effecive medicine for treating these kinds of diseases.Earthworm, also known as lumbricus, is a common terrestrial annelids that live in soil and rich in protein, mineral and fatty acid. Earthworm is one of the important Chinese herbal medicine and belongs to the 67 kinds of animal medicine collected in the earliest pharmacy monograph named " Shen Nong’s herbal classic", always used to treat a variety of diseases. In 1995, China Food and Drug Adminisration approved the production of earthworm extract(EE), which is widely used in the treatment of cardiovascular and cerebrovascular diseases. The active ingredients of EE are Lumbrokinase and plasmin. Animal experiments and clinical observations indicatedthat EE has many pharmacological functions, such as anticoagulant, fibrinolysis,anti-inflammatory and antioxidant. These konwn functions are able to inhibit the silica induced lung inflammatory reaction and fibrosis theoretically, but there is no related report. Therefore, our hypotheses is EE can inhibit or treat silica induced pulmonary fibrosis. Through the observation of pathology, pulmonary fibrosis markers and μCT, we determine its proventive and therapeutic effects. This study aims to provide scientific basis for the exploitation of effective therapy of silicosis and also give new clues for expansing EE treatment field.Objective:Establish the animal model of silica-induced pulmonary fibrosis and design three kinds of experimental scheme namely earthworm extract prevention,early fibrotic stage treatment and fibrotic stage treatment of silicosis to explore whether the EE could prevent / treat silica induced pulmonary fibrosis, in order to provide scientific basis for the exploitation of effective drug of silicosis.Methods:(1) Design three kinds of experimental scheme namely EE prevention,early fibrotic stage treatment and fibrotic stage treatment of silicosis; ①Prevention program: mice were randomly divided into 4 groups, control group, silica group, EE low dose group and EE high dose group. High, low dose group were treated with purified EE(unit of enzyme activity were respectively 24U/g and 240U/g), 1 time per day, for 5 days. Then the pulmonary fibrosis models were conducted intratrachally with SiO2 dust suspension disposable and continue EE treatment by intraperitoneal injection, while the control and silica group were given normal saline until different time end points(7 days, 14 days, 28 days). ② Early fibrotic stage treatment program:Grouping as previously, add a pirfenidone group. Then control group received instillation of normal saline, and other groups were undergoing disposable infusion of SiO2 dust suspension; Starting EE/ pirfenidone treatment from the fourth week, we totally give intraperitoneal administration for 5 weeks then observe the curative effects. ③ Fibrotic stage treatment program: Grouping as previously, starting EE treatment from the fifth week, we totally give intraperitoneal administration for 4weeks then observe the curative effects.(2) BALF cell count, HE staining, μCT andlung fibrosis score were used to observe the dynamic changes of SiO2 dust induced mouse silicosis development.(3) We detect the dynamic expression of collagen fiber,α-SMA, E-cad, TGF-β1, Smad2 by Masson staining, ICH and Western blot. The changes of silicon content of mice serum were detected by ICP-MS.Results:1 EE having preventive effect against silica induced pulmonary fibrosis in mice(1) BALF cell count: Variation tendency of changes in cell number, macrophage and neutrophil is nearly the same. Silica could significantly increase the number of cells in the alveoli, but cell number was gradually decreased over time. EE could inhibit the infiltration of inflammatory cells caused by silica within a certain extent. The number of cells in the BALF was higher than that of control group while lower than those of silica group. At the beginning of treatment, EE could increase the number of lymphocytes in BALF, then the lymphocytes of high dose EE group reduced largely with time, on the 28 th day, the number of lymphocytes was lower than that of silica dust group, higher than control group. The above description shows that EE could inhibit the silica induced inflammatory cell exudation.(2) HE staining and fibrosis scoring: After intervention for 7 days, 14 days, 28 days respectively, mice were harvest. Compared with silica group, which demonstrated typical development process of pulmonary fibrosis, high and low dose EE treatment group exerted the same trend, alveolitis and pulmonary fibrosis was significantly alleviated. Separately, lung tissue inflammatory cell infiltration area was significantly reduced after intervention for 7 days. On 14 days, not only the lung tissue structure seems more complete, but also the proliferation of fibroblasts and cell aggregation decreased compared with the silica group. Although the fibrotic nodules emerged at28 day, the size and the number were all less than those of silica group. The fibrosis scoring provide a quantitative view to further confirm that EE could significantly inhibit development of fibrosis induced by silica dust.(3) μCT: 28 days after intervention, The voxels number of aerated lung volume of silica group calculated by software are significantly less than that of saline group while high. Low dose EE treatment group is slightly higher than that of silica group,especially the different between low dose EE group and silica group was statistically significant. That indicate EE might partially inhibit a lung volume reduction of silica dust.(4) Fibrosis markers: Masson staining showed that the collagen deposition was significantly increased in silica group than that of high and low dose EE treatment group. The expression level of α-SMA in silica group increased significantly and those of high, low dose EE group were close to control;however, the expression levels of E-cad showed the opposite trend. TGF-β1 and Smad2, which are two important molecular of TGF-β signaling pathway, were up-regulated in silica group while down regulated in EE treatment groups. All these suggested EE could inhibit the deposition of extracellular matrix, activation of fibroblasts and epithelial mesenchymal transition.The mechanism may be partly through the influence of TGF-β signaling pathway.(5) Silicon content in serum: After intervention for 7 days and 14 days, compared with control, serum content of silicon were increased in the silica group, the high and low does of EE treatment groups. At the day 28, the silicon content level of silica group is also higher than control and lower than that of high and low doses of EE groups, and the differences among these four groups are statistically significant.These results suggested that EE could increase the silicon absorbed into the blood,thus reducing the deposition of silicon in lung tissues.2 EE having theropeutic effect against silica induced pulmonary fibrosis in mice(1) Early fibrotic stage treatment: The treatments with EE and pirfenidone started from the fourth week after silica exposure and treated for five weeks. Ten mice of the control group showed no fibrosis lesions; among silica group, average fibrosis score was 3.0 and average distribution grade was 2.3. The index of severity and distribution range among EE and pirfenidone treatment group were obviously lower, and there was statistical difference compared with the silica group. These indicated that EE may be an effective therapeutic drug for silica induced lung fibrosis.(2) Fibrotic stage treatment: The treatment with EE started from the fifth week aftersilica exposure and treated for four weeks. Five mice of the control group showed no fibrosis lesions; the average fibrosis score was 2.8 and the average distribution grade was 2.6 in silica group. There was no statistical difference between the severity of fibrosis in EE treatment group and silica group(P>0.05) but the average lesion distribution of EE high dose treatment group was obviously lower than that of silica group(P<0.05). Pathology results also showed that EE was effective on reducing lesion distribution of silicosis. μCT analysis showed that EE treatment group had larger lung volume voxel value, but without statistically significant differences comparing with the silica group. μCT illustrated that EE could increase lung volume in the treatment of pulmonary fibrosis, but the efficiency was limited.Conclusion:(1) EE could inhibit the occurrence and development of silicosis both in inflammation and fibrosis stages, and the effect of EE low dose was better than that of high dose.(2) EE and pirfenidone could truly effect on slowing down fibrosis development at the early fibrotic stage induced by silica and the effect of EE at least was no less than that of pirfenidone.(3) EE played a certain function at the firotic stage, however, the therapeutic effect was poor and required higher doses.
Keywords/Search Tags:silica dust, pulmonary fibrosis, earthworm extract, pirfenidone
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