A Preliminary Study On Recovery Of Mice Lung Damage And Mechanism Of Inflammation Induced By Ricin

Ricin toxin(RT) is potent protein toxin which is extracted from castor beans(Ricinus communis), the holotoxin consists of two polypeptide chains(A and B) linked by disulfide bond with the molecular weight of 65 kDa. RT is one of typeⅡribosome inactivating protein(RIPⅡ)which could inhibit protein synthesis in mammalian cells. Ricin’s toxic effect is obviously dose dependent, high dose of ricin’s main effect is inhibiting the synthesis of protein, low dose could induce cell to produce cytokines and could cause lipid peroxidation in vivo. Various patterns of ricin’s exposure like ingestion, intramuscular injection, inhalation could present different toxic effect. Compared with other exposure pattern, aerosolized ricin exposure could produce strongest toxic effect, cause severe pulmonary injury, compressive diffuse alveolar edema, acute alveolitis which is induced by abundant number of activated macrophage. Aerosolized ricin exposure could cause severe capillary hyperemia and osmosis in pulmonary interstitial tissue. Once ricin enters into the pulmonary tissue, it could combine with variety types of cells to inhibit the activity of ribosome and finally induce the cell death. Ricin toxin B chain is one of lectins which could bind with galactose residues on most cell surface to transport the A-B toxin complex into cytoplasm. Macrophage plays the key role in innate immunity and adapted immunity, has an ability of phagocytosis. Mannose receptors expressed on the macrophage cell surface could bind with mannose or fucose residues of glycoproteins, such as ricin A1 and A2 chain. Innate immunity is the first defend line against pathogenic microorganism, cells could recognize pathogen through pattern recognition receptors expressed on cell surfaces and trigger the innate immune system to eliminate the invasion of pathogens. TLR4 is a member of the Toll-like receptor(TLR) family, is the most well-kown receptor which is associated with pathogen-associated molecular patterns TLR4 plays a fundamental role in activation of innate immunity which caused by pathogenic microorganisms and inflammatory response in tissue damage. Cell injury induces degradation and secretion of cell substance, TLR4 mediates series of inflammatory response, activation and maturation of macrophages and dendritic cells, and enhances the expression of inflammatory cytokines. TLR4 signaling pathway is divided into MyD88 dependent pathway and TRIF dependent pathway.At present, the studies on ricin were focused on inflammatory response and tissue pathological changes after superalethal and sublethal dose of ricin exposure, there are few studies on low dose ricin exposure. Our results include three parts below:(1) Pathological changes of pulmonary tissue in mice exposed to low dose aerosol ricinThe experimental measurement of LD50 in mice with aerosol ricin was about 4.95 ± 0.14μg/kg, when exposure at a concentration of 164ng/kg which is corresponding to 1/30 LD50, mice pulmonary tissue damage could be self-recovered within a certain time. Through histopathological observation, we found that after the exposure of low dose of aerosol ricin, the pulmonary tissue showed obvious pathological changes in 12 hours with symptoms of alveolar structural damage, bronchial edema, smooth muscle hyperplasia. The symptoms were aggravated and most serious in day 3-4, and were relieved in day 5-6. Part of edema around the blood vessels were eliminated, the pulmonary peribronchial edema were disappeared. Compared with the control group, the alveolar structure had not been recovered to the normal state in day 7. Immunohistochemistry results showed that the expression levels of MMP-2 and MMP-9 were weakly positive in normal tissues. The expression of MMP-2 and MMP-9 in pulmonary tissue were increased slightly in 12 hours after aerosol ricin exposure. Compared with the control group, expression of MMP-2 was highest in third days and in experimental group, and the highest expression of MMP-9 was in the fourth day. Then the expression of MMP-2 and MMP-9 were decreased and returned to the normal level in day 7.(2) Detection of pulmonary alveolar lavage inflammatory cytokines and TLR4 and its downstream signaling pathway proteins in alveolar macrophage.The extracted mice alveolar macrophages were round, ranging in size. In a cultivation time of 2-3 hours, most of cells were adherent and after 1-2 days of cultivation the shapes were changed. We took both of Wright-Giemsa staining and Diff-Quik staining method for the identification of alveolar macrophages and determination of its purity, results showed that: Diff-Quik staining method could replace the Wright-Giemsa method to be an alternative with its advantages of clean background with no impurity and short terms of staining process. Pulmonary alveolar macrophage activity assay showed that the alveolar macrophage phagocytic rate and phagocytic index were mainly dependent on the ratio between FITC labeled bacteria and alveolar macrophages. At a fixed ratio of 20:1, phagocytic rate and phagocytic index reached the peak, and under the condition of prolonging of incubation time, phagocytic rate and phagocytic index represented a tendency increased first and then decreased subsequently.After aerosol ricin exposure at a concentration of 33ng/kg which is corresponding to 1/150 LD50, We took the ELISA method to determine the concentration of inflammatory cytokines in supernatant of mice alveolar lavage at each time points, We found that expression level of TNF-α and IL-1β of experimental group were higher than control group, and the expression level reached the peak in 12 hour then decreased. The differences between two groups were significant. And the expression of IL-6 also presented same tendency, IL-6 reached the peak in 48 h. The expression level of the total protein in the supernatant of alveolar lavage increased first and then decreased. The total protein concentration was highest at 60 h. We took RT-PCR method to determine the expression level of TLR4 and its downstream signaling pathway in mice pulmonary alveolar macrophages at each time points. The results showed that the expression of TLR4, MyD88, TRAF6, IRAK-1 and IRAK-4 was highest in 12 h which is compared with other time points, showed a tendency of increasing first and then decreased, while the expression of TRAM and TRIF had no significant difference at each time point, indicating that low dose of ricin exposure could mediate the TLR4 signaling pathway which is dependent on MyD88, we achieved the same result compared with our previous study. The conclusions drawn from this study:Mice pulmonary inflammation which was induced by 1/30 LD50 dose of aerosol ricin could be identified through the detection of MMP-2 and MMP-9,and could be self-recovered in 7 days; Diff-Quik staining method could replace the Wright-Giemsa staining method, to be a novel alternative to consider the purity of alveolar macrophage; The ratio between FITC labeled E.coli and macrophages of 20:1, incubation time of 25 min are the best cultivation condition to determine the phagocytosis of alveolar macrophage; After aerosol ricin exposure at a dose of 1/150 LD50 in mice, ricin could mediate the expression of TLR4 and its downstream signaling pathway protein MyD88,TRAF6,IRAK-1,IRAK-4 in alveolar macrophages to induce pulmonary inflammatory response. Our research lays a foundation for clarify the mechanism on ricin induced pulmonary inflammatory injury and its self recovery.