| Diabetes mellitus (DM) patients showed many immunological abnormalities. Recently,many researchers focused on immune therapy to prevent 1 type diabetes mellitus.Furthermore, they have gotten satisfactory results of immune tolerance, immune inhibitionand immune stimulation in diabetic animal model. Low dose radiation (LDR) could induceradiation hormesis and adaptation reaction. Research work results showed that singleradiation 100 mGy could strengthen rat's immune function and increase lymphocytereactivity, while 500 mGy radiation could inhibit rat's immune function. However, there'sno report on repeated LDR on diabetic immune function. The present study planned tostudy the effects of repeated LDR on diabetic rat's immune function, which will furnishexperimental basis on diabetic immune intervention therapy.The present experiment detected the diabetic rat's immune function and its possiblemechanism of LDR with immune and molecular biolog ical techniques. Spleen cellsapoptosis percentage decreased significantly after the rats were irradiated with LDR(especially 50 and 75 mGy), which mean LDR inhibited spleen cells apoptosis andincreased immune function. The results showed that LDR protected the diabetic rat'simmune functions, but the detailed mechanisms need to be explored further.1. Rat diabetes mellitus modelDiabetic rat's model was made with streptozotocin (STZ). STZ was a kind of broadspectrum antibiot ics, which has anti-bacteria, anti-tumor and causing diabetes associate effects. STZ could destroyβcells of pancreatic islet selectively. There's a high activeglucose side chain in STZ molecular structure, which madeβcells of pancreatic isletrecognize it incorrectly and enter into the cells, and the other section of STZ was the toxicgroup, which has toxic effects toβcells of pancreatic islet. And this model imitated the 1 typediabetes mellitus of human, which could be applied to study I type diabetes and itscomplications.Diabetic rats were raised routinely, and they were irradiated with X-ray every other dayfor one month. The rats in DM+NAC group was given NAC (150 mg/kg) by garbage andlasted for 4 weeks. And then the rats were raised fro another 4 weeks. Urine glucose wasdetected every week and blood glucose was measured every other day. All the rats werekilled at the end of 8th week after the rats suffered with DM. Serum was collected and storedat -70℃for further examination and some parts of spleens were made into single cellsuspension and other parts of spleen were made into paraffin package for cell apoptosisindexes research.2. Protective effects of LDR on diabetic rat's oxidative damage in serumOxidative-antioxidant balance was destroyed in diabetic rats, which showed increasedROS content, decreased antioxidant enzyme (SOD and CAT) activity and lead to diabeticrat's lipid peroxidation in serum. MDA, NO content and NOS activities increased, whichcaused mitochondra membrane structure and function abnormalities and increased normalcells apoptosis percentage. That is ROS is the important factor in DM rat's oxidative damage.It interacted with AGES, which was the important agent in diabetic complications. LDRrelieved the oxidative stress in diabetic rats through increasing antioxidant substance andantioxidant enzymes and protected the diabetic rats to some extent.LDR, especially 50 mGy could increase antioxidant enzyme contents in diabetic rat'sserum, which has antagonism effects to diabetic rat's oxidative damage, and at the same time, it decreased lipid peroxidation as MDA content in diabetic rat's serum. LDR,especially 25 and 50 mGy decreased NO and NOS content in diabetic rat's serum, whichtook protective effects to DM rats caused by NO in diabetic rats.3. Effects of LDR on spleen lymphocytes subgroup and its relative factors3.1 Effects of LDR on spleen lymphocytes1 type diabetes mellitus is a kind of autoimmune disease. Many immune cells wereinvolved in its development, and CD4+ T cell takes critical effects in its development. Itmainly showed that the percentage of CD4+ T cells increased and the percentage of CD 8+ Tcells decreased. That means T lymphocyte subgroup showed unbalance in diabetic rats andabnormal activation and proliferation. The ratio of CD4+ T cells to CD8+ T cells lost balanceand the decrease of CD 8+ cells made immune function out of control and it enhance thedamage effects of CD 4+ T cells toβcells and hasten the diabetic mellitus development.The change of CD4+ and CD8+ T cells percentage in spleen cells were detected withflow cytometry methods. CD4+ T cells content in 8 and 12 week diabetic rats spleenincreased significantly, and CD8+ T cells decreased significantly and the ratio ofCD4+/CD8+ increased significantly in 8 week diabetic rats, CD8+ T cells percentage in 12week diabetic rats increased significantly and the ratio of CD4+/CD8+ decreased to thenormal level. The results of 8 week diabetic rats were in accordance with the above reports,and CD4+T cell percentage was high still, and the ratio of CD4+/CD8+ T cell recovered to thenormal level.CD4+ T cells percentage in diabetic rats of NAC and LDR+DM groups was lower thanthat in diabetic rats, while CD8+T percentage increased compared with the diabetic rats.The ratio of CD4+/CD8+ T cell increased significantly nearly to the normal level. CD 4+ Tcell percentage was in 12 week LDR+DM group was lower than that in DM group, whileCD8+ T cell percentage was lower than that in diabetes group . The ratio of CD4+/CD8+ T cells in 50 mGy radiation group was higher than that in normal group, but there's nostatistic significance. The ratio of CD4+/CD8+ T cells increased significantly compared withthe normal group. The present study showed that repeated LDR radiation could regulate theimmune unbalance in diabetic rats and modified the immune function abnormalities.3.2 Effects of LDR to T cell receptorsT cell receptor (TCR) consists of many subunits. TCRαβis the unit that specificlybinds with antigen peptide MHC molecular, and then T cell was stimulated by TCR and cellactivation, proliferation and cytokines production or offering signal and inducing cellapoptosis.TCRαβcontent in spleen cells was measured with flow cytometry in this experiment,and TCRαβpercentage in 8 and 12 week DM rat's spleen cells increased significantly, whileTCRαβpercentage in NAC and radiation group was lower than that in the DM group. Somestudies showed that LDR could increase the content of TCRαβand facilitate T cellproliferation, differentiation and maturation. The present results showed that TCRαβpercentage in repeated LDR radiation group decreased significantly, and this result showedthat the reaction in diabetes mellitus rats was not accordance with the normal body, and theexact mechanism need to be explored further.3.3 Effects of LDR on IL-2 factorIL-2 is the lymphocyte factor that secreted by activated D cells and it could facilitate celldifferentiation and proliferation and it is involved in many immune diseases development.Some studies showed that IL-2 took important effects in diabetes development. The presentstudy showed that IL-2 in 8 week diabetes rats serum increased significantly and, while IL-2content in 8 week DM rats spleen cells supernatant and 12 week DM rats serum and spleencell supernatant decreased significantly. The increase of IL-2 content in 8 week DM ratsmight by induced by the increase of lymphocyte in peripheral blood, while they were not damaged still at that time. The decrease of IL-2 content in spleen cells supernatant might byinduced by long time high blood glucose leading T lymphocyte function defect. Thisphenomenean occurred just because the ability to induce IL-2 decreased or the T lymphocytein the body was stimulated continuously and secreted IL-2 excessively, and it seemed like ina"depletion"false imagination. The decrease of IL-2 in 12 week DM rat's serum and spleencells supernatant might be the result of the above phenomena developed. IL-2 content in 8week NAC and LDR rat's serum recovered to the normal level. IL-2 content in spleen cellsupernatant was still lower than that in the normal control group. IL-2 content in NAC andLDR group rats serum and spleen cells supernatant recovered to the normal level, while IL-2content in 25 mGy and 75 mGy radiation group was higher than that in the normal controlgroup significantly.4. Effects of LDR on diabetic rat's spleen cell apoptosisI type diabetes was the autoimmune disease that mediated by T cell. The mechanism ofit was the loss of auto-tolerance to theβcell normal factors in pancreatic islet, which causedβcell destruction and quantity loss progressively. The correlated factors in this diseaseincluded auto-antigens (such as concealed antigen discharge, the change of auto-antigen,molecular imitation and antigen epitope expansion), abnormal immune modulation,abnormal cell apoptosis and genetic agents, et al.Recent studies showed that there's some relationship between cell apoptosis and 1 typediabetes development. The cell apoptosis adjustment lost might cause many kinds of disease,for example, excessive apoptosis might cause AIDS, autoimmune diabetes (some researchersregarded it as the result ofβcell auto-antibody because the T cell apoptosis was inhibited),Alzheimer's and Parkinsons diseases.The present study detected the spleen cell apoptosis with Annexin V and PI double dyeand TUNEL methods. The results showed that the percentage of apoptotic spleen cell increased in 8 and 12 week diabetic rats, while low dose ionizing radiation decreased theapoptotic spleen cells percentage significanly.The previous studies showed that LDR could decrease the apoptotic spleen cellspercentage. The related mechanism might involve some protective proteins production. Andsome proteins increased in thymus, spleen and lymph nodes after irradiated, in which therelative molecular quality as 10×10~3 protein could stimulate thymocyte cells and spleen cellsproferalition.The present study showed that LDR (especially 50 and 75 mGy radiation) decreased thediabetic rat's spleen cell apoptosis and increased the diabetic rats'immune function andprotected the diabetic rats to some extent. But the detailed mechanisms need to be exploredfurther.In summary, LDR decreased lipid peroxidation and NO and NOS level in diabetic rat'sserum and increased the content of anti-oxidative enzymes and protected the diabetic rats tosome extent. Meantime, CD4+T content, the ration of CD4+/CD8+ Tcell and TCRαβinirradiated diabetic rats decreased, while CD8+ T cell content increased significantly. Theresults indicated that LDR regulated the immune unbalance by inhibiting the CD4+T andfacilitating CD8+ T proliferation and modified the immune function disorder and protectedthe diabetic rats. This study offered the experimental basis for the further study on theimmune interference therapy in diabetes with LDR.
|
PDF Full Text Request