Study On The Mechanism Of Apoptosis In Lymphocytes Induced By Sulfur Mustard

Sulfur mustard, as blister agent, can directly impair tissue and cell, leading local inflammation and general intoxication after absorption. One of the most threatening effects of sulfur mustard is a severe suppression of the immune system, which can lead, in the case of lesions and blisters, to opportunistic infections, septicemia and death. The suppression of the immune system induced by sulfur mustard happens earlier and recovers more rapidly. Lymphocyte is the major immunocyte and performs immunological function. It has been found to be sensitive to sulfur mustard. Lymphocytopenia has been thought to be the major cause, which leads to the suppression of the immune system after sulfur mustard exposure. However, the mechanism of sulfur mustard-induced lymphocytopenia has not been completely clarified. Therefore, the effective measures of early-diagnosis, prevention and cure have not been found. Objective: Apoptosis and necrosis are two different types of cell death induced by sulfur mustard. The model of lymphocyte exposure to sufur mustard was established to observe apoptosis and the role of caspase-3 on it. The experiment is aimed to offer more understanding on the rule of immunological impairment induced by sulfur mustard and to explore Caspase-3 protease as therapeutic targets for the control of inappropriate apoptosis induced by sulfur mustard in lymphocyte.Method:(1) Lymphocyte in vitro intoxication model: lymphocyte was separated and cultured from spleen of Wistar rat. Relative fluorescence intensity of EB combined with DNA, DNA ladder, apoptotic index and cell cycle were detected to make them as the markers of cell damage induced by sulfur mustard; RT-PCR, Western blot and fluorescent quantitative assay were explored to detect mRNA expression, protein expression and the activity of caspase-3. Fluorescent probe labeled with Rhodamine 123 was used to study mitochondrial potential; Meantime, the intervention of tetrapeptide (Ac-DEVD-CHO) on cytotoxic action induced by sulfur mustard was evaluated. (2) Animal in vivo intoxication model: Wistar rats were randomly divided into control group and experimental group. The spleen in the experimental group was collected after 1d, 3d, 5d intoxication. Themorphology of spleen with HE staining and Caspase-3 mRNA expression with RT-PCR were investigated.Results: (1) Apoptotic index increased with intoxication time extention,keeping high levels till 24h. However, apoptotic index of inhibitor group decreased markedly compared with sulfur mustard intoxication group accordingly. (2) DNA fragment analysis displayed DNA ladder more obviously with time extention in contrast with DNA ladder disappearing or being obscure in the inhibitor group. (3) Sulfur mustard interfered with cell cycle, S and G2/M cell cycle decreasing and G1/G0 cell cycle increasing. (4) Caspase-3 mRNA expression increased significantly at 3h compared with control group. (5) Caspase-3 was activated by sulfur mustard. The protein expression of pro-caspase-3 was increased at 9h and the fragment of P20 was detected earlier at 3h. (6) The activity of Caspase-3 increased significantly at 3h after intoxication analyzed with fluorescent quantitative assay. (7) Bcl-2 protein expression dropped significantly at 9h and continued to drop in the following time. (8) The mitochodrial potential also decreased at 3h after intoxication. (9) The morphology and Caspase-3 mRNA expression of spleen were changed after sulfur mustard intoxication.Conclusions: 1.Apoptosis, Caspase-3 and Bcl-2 are involved in lymphocyte cytotoxic action induced by sulfur mustard. Apoptosis induced by sulfur mustard is regulated by polygene and their abnormal changes determine the cytotoxic effect. 2.Sulfur mustard induced-apoptosis is mediated by Caspase-3 in various forms, such as gene expression, protein expression and activity regulation. This suggestes the process of apoptosis induced by sulfur mustard may be dependent on Caspase-3. Caspase-3 protease as therapeutic targets to regulate sulfur mustard intoxication may b...