Experimental Study On Model Rat Of Heat Stroke Treated With Ulinastain By CD4~+ CD25~+ Foxp3~+ Treg

Background:Heat stroke (HS) is a life-threatening illness characterized by a rapid increase in core temperature to more than 40℃ and central nervous system (CNS) abnormalities during people exposure to high-temperature and high-humidity ambient. Research shows that systemic inflammatory response syndrome (SIRS) will appear rapidly after HS, leading to muliple organ dysfunction syndrome(MODS). As a consequence, its mortality is up to 60%-90%. CD4+CD25+Foxp3+ regulatory T cells (Tregs) is a kind of important immunosuppressive cells, which can regulate the immune-mediated systemic inflammatory response, but whether Tregs could regulate SIRS of HS is unknown. As a protease inhibitor, Ulinastatin (UTI), has the function of anti-inflammatory and immune regulation, and it can decrease the number of Tregs in peripheral blood of SIRS patients and improve the immune function. However, whether UTI could treat HS and its therapeutic mechanism remain unclear.Objective:This study is aimed at determine whether Tregs could regulate the systemic inflammatory response of HS, and to explore the therapeutic effects and mechanism of UTI in HS rats.Methods:In the fist part of our study, we establish the rat model of HS. And we then (1) performed laboratory tests on serum AST, ALT and cytokines include TNF-α, IL-2, IL-6, TGF-β, IL-10 of 5 groups with different time (6h、12h、24h、48h and 72 h after HS); (2) performed HE staining to observe and analyse the morphological changes of liver tissue at each time of 6h,24h and 72h after onset of HS; (3) Record the 72h survival rate.In the second part of our study, to clarify whether Tregs was involved in the pathophysiological process of HS, we performed flow cytometry to enumerate the percentage of Tregs in spleen and peripheral blood, and assayed the apoptosis rate of Tregs in spleen.In the last part of our study, in order to demonstrate the efficacy of using UTI to treat HS and explore its possible therapeutic mechanism, we inject UTI via abdomen after onset of HS. We then (1) performed laboratory tests on serum AST, ALT and cytokines include TNF-a, IL-2 and IL-6 ,TGF-β,IL-10,(2) performed HE staining to observe and analyse the morphological changes of liver tissue; (3) Record the 72h survival rate; (4) performed flow cytometry to enumerate the percentage of Tregs in spleen and peripheral blood, and assayed the apoptosis rate of Tregs in spleen.Results:In the first part of our study, we constructed stable HS model. Our data showed that the serum cytokines, liver function, hepatopathy changes and 72h survival rate are in accord with the clinical features of HS, and well consistency with the seriousness of the disease. The serum cytokines of HS group at different time were significantly higher than control group. TNF-α, IL-2 and IL-6 levels significantly rised before 24h, and then began to decline. TGF-β, IL-10 increased over time. The liver function index of AST and ALT markedly increased in HS group at each time, and the pathological of liver tissues damaged severly, and aggravated with the advance of disease.In the second part of our study, our data showed that the percentage of Tregs in spleen and peripheral blood decreased firstly and increased subsequently, which kept rising with the progression of disease. The apoptosis rate of Tregs in spleen increased at first, and then with time progress it significantly reduced after 24h after onset of HS, which was negative correlative with the changes of Tregs.In the third part of our study, we found that the cytokines, liver function, liver tissue injury and the 72h survival rate in HS-UTI group, as compared with HS group, was significantly improved. Flow cytometry showed that the apoptosis of Tregs in spleen was decreased at early stage, the percentage of Tregs in spleen and peripheral blood was increased after treat with UTI compared to HS group.24h after onset of the disease the apoptosis of Tregs in spleen was increased and the percentage of Tregs was significantly reduced in rats treated with UTI.Conclusion:(1) The severity of HS could be reflected by related cytokines, indexes of liver function, pathological changes of liver tissue and the survival rate of rats at 72h after HS.24h can be regarded as the time dividing ponit between pro-inflammatory and anti-inflammatory reaction. (2) Tregs was involved in the pathogenesis of the inflammatory reaction in HS. (3) UTI alleviate the inflammatory reaction in HS and improved the prognosis by inhibiting the apoptosis of Tregs to control the excessive reduce of Tregs at early stage, and inducing Tregs apoptosis to inhibit the continue increasing of Tregs in the middle and later stage.