| Sulfur mustard (2,2-dichlorodiethyl sulfide, SM) is a powerful vesication chemical warfare agent that is still the main chemical warfare agent in many countries, and has very important strategic position. With its complex toxic mechanism is unclear, there are several injury pathway of SM and they can influence each other. Therefore studying on a certain genes or proteins change is difficult to clarify the toxic mechanism of SM. As important parts of systems biology, proteomics study on all the proteins that expressed by one cell or one organization, and metabonomics study on the changes of metabolites of organism by exterior stimulation. These two technologies can lead a technology platform and a new break point for SM toxic mechanism research at the level of systems biology.This study is based on previous researches, and guided by systems biology conception, and applied methods combined with traditional pharmacology and proteomics and metabonomics, and study on SM toxic mechanism and potential drug target preliminary on the macro and micro level. This study observed dynamic changes of hematopoietic system and immune system of SM-exposed mice. With MALDI-TOF-MS and NMR technology, the results showed that there were plenty of proteins and metabolites which were significantly different from control group in plasma of SM-exposed mice, and had dynamically changed through experiments. These materials maybe closely related to SM exposure. So this study provides evidences to discover SM toxic mechanisms.1. Establishment of sulfur mustard systemic intoxication model in mice1.1 Effects of various doses of sulfur mustard on general conditions of miceActivities and diets of each SM-exposed group were reduced. The average weight of each SM-exposed group began to fall at the 1st day, and reached the lowest level at the 3rd day (72 h), and was still significantly lower than propanediol (PF) group at the 7th day. Compared with PF group, weight loss of high doses (20 mg/kg) SM-exposed group was most obvious (P<0.01), and in later period the high-dosage groups had a part of animals dead. 1.2 Effects of various doses of sulfur mustard on blood routine of miceThe blood routine of SM-exposed group was measured by routine blood detector. Compared with PF group, the results showed that the dynamic changes of red, white, platelets of three SM-exposed groups began to fall at the 1st day, and reached the lowest level at 3rd day. Compared with PF group, 20mg/kg group had change most obviously (P<0.01), and it is still significantly lower than PF group at the end of experiment (168 h). The results were consistent to the weigh changes.1.3 Effects of various doses of sulfur mustard on DNA damage of bone marrow cells of miceThe dynamic changes of bone marrow cell DNA damage were measured by SCGE technology. The results showed that the bone marrow cells of high dose(20mg/kg) group had most injured(comet cell)than other groups at the 3rd day, and still had many injury cells while low and mediate doses of SM-exposed groups are no different from control group.Through these researches, the study determined 20 mg/kg dose to setup systemic intoxication model of SM.2. Effects of systemic exposure on hematopoietic and immune systems of sulfur mustard-exposed mice 2.1 Effects of systemic exposure on hematopoietic system of sulfur mustard-exposed miceSCGE results showed that the migration degrees and migration rates of SM groups had significantly higher than PF group (P<0.01) at 24 h and reach the peak (P<0.01) at 72 h and reduced a little at 168 h, but still significant higher than PF group (P<0.01). Routine blood test results showed that white, platelet counts of SM group were significantly lower than PF group (P<0.05) at 24 h after SM exposure. At 72 h, red white and platelet counts of SM group reached the lowest level that significantly lower than PF group (P<0.01). At 168 h, red white and platelet counts of SM group rised up a little, but still significantly lower than PF group (P<0.05).2.2 Effects of systemic exposure on immune system of sulfur mustard-exposed mice2.2.1 Dynamic changes of spleen weight and coefficient of sulfur mustard-exposed mice The dynamic observation results showed that the spleen weight and coefficient of SM group is significantly lower than PF group at 24 h (P<0.01) and reach the lowest level at 72 h (P<0.01) and recovered at 168 h.2.2.2 Dynamic changes of apoptosis and necrosis of spleen lymphocytes of sulfur mustard-exposed miceFlow cytometry analysis results showed that the percentage of apoptosis of spleen lymphocytes of SM group had no difference from PF group at 4h, and reached the peak at 24 h (P<0.01), and little reduced at 72 h but still higher than PF group (P<0.01), and recovered at 168 h. The percentage of necrosis was significantly higher than PF group at all the four time points (P<0.01) but continuously reduced since 4 h and reached the lowest level at 168 h.2.2.3 Dynamic changes of splenocyte proliferation of sulfur mustard-exposed miceThe results showed that the splenocyte original proliferation of SM group was significantly lower than PF group at all the four time points (P<0.01). The proliferation of T-cells of SM group was significantly lower at 4 h 72 h and 168 h(P<0.01) but significantly higher at 24 h(P<0.01).The proliferation of B cells of SM group was significantly lower than PF group(P<0.01) at 4 h and 72 h but significantly higher than PF group at 24 h and 168 h(P<0.05).2.2.4 Dynamic change of CD4~+/CD8~+ T cells of sulfur mustard-exposed miceThe results showed that the percentage of CD4~+T-cells of SM group was significantly higher than PF group at 24 h 72 h and 168 h (P<0.01).The percentage of CD8~+ T cells of SM group was significantly higher than PF group at 72 h (P<0.01) and no different from PF group at other three time points. The proportion of CD4~+/CD8~+ of SM group was significantly higher than PF group at 24 h and 168 h (P<0.01).2.2.5 Dynamic changes of plasma levels of IL-1β, IL-6, IL-10 and TNF-αof sulfur mustard-exposed miceThe dynamic changes of plasma cytokine levels, including IL-1β, IL-6, IL-10 and TNF-αwere measured by Luminex technology. The results showed that the concentration of IL-1βin plasma of SM group was significantly higher than PF group at 24 h and 72 h (P<0.05). The concentration of IL-6 of SM group was significantly higher than PF group at 4 h, 24 h and 72h (P<0.05). The concentration of IL-10 of SM was significantly lower than PF group at 24 h and 72 h (P<0.05). The concentration of TNF-αhad a gradual rise, and was significantly higher than PF group (P<0.05) at 72 h and 168 h.3. Study on metabolites and proteins in plasma of sulfur mustard-exposed mice3.1 Effects of systemic exposure on plasma metabolites of sulfur mustard-exposed miceThe dynamic changes of plasma metabolites were measured by nuclear magnetic resonance(NMR)technology. The results showed that, in the four time points, the map of metabolites of SM group were different from control group. Among these metabolites, PC, GPC, 3-HB, glycerin, lactic acid, lipid, VLDL, LDL, HDL and VLDL/LDL had significantly dynamic changes.3.2 Effects of systemic exposure on plasma proteins of sulfur mustard-exposed miceThe dynamic changes of plasma proteins were measured by plasma polypeptide spectrum technology (weakly positive magnetic beads and MALDI-TOF-MS). The results showed that, compared with PF group, SM systemic exposed mice had 17 proteins significantly changed in different time points, and there are 6 proteins had significantly dynamic changes. The proteins of M/Z 2000.40, 1960.83, 1969.07, 1980.52, 2794.32 and 8120.80 were related to SM exposure.In summary, there was hematopoietic system damage in SM systemic exposed mice.The damage of bone marrow cell DNA and hematopoietic system began to rise at 24h, and reached the highest level at 72h after SM exposure. There were immune abnormality and cytokine network imbalance in SM systemic exposed mice. Spleen lymphocytes apoptosis and necrosis scale, T, B cells proliferation capacity and CD4~+, CD8~+ T-cells proportion were significantly different from PF group. There were 15 metabolites which referred to sugar metabolism, energy metabolism, lipid metabolism and Oxidative stress etc. significantly changed in plasma of SM systemic exposed mice. There were 17 proteins significantly changed in plasma of SM systemic exposed mice, and 6 proteins had significantly dynamic changes. These metabolites and proteins may be closely related to SM exposure. This study provided new clues for discovering SM toxic mechanism and made up a certain experimental basis for the study of damage-related molecules and anti-toxin targets of SM. |
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