Immune Effect Of Thymosin-α1 On CD4 ~+ CD25~ + Regulatory T Cell In Septic Mice

ObjectiveTo investigate the changes of CD4+ CD25+ regulatory T cells (Treg) in a mouse sepsis model produced by cecal ligation and puncture and to explore the relationship between Treg and severity of sepsis. Effects of Thymosin al on variations of Treg in blood of septic mice would be observed to discuss the possible role of Thymosin al in the immunomodulation of immune dysfunction in sepsis, thus, interaction of Thymosinal and Treg might be clarified by a new approach.MethodsSepsis mouse model reproduced by cecal ligation and puncture in order to maintain a stable mortality, a pre-experiment was undertaken. By regulating the ligation length of remote cecal, the severity of sepsis was controlled, while the perforation time remained twice. Events after operation were recorded, such as consumption of water and food, clinical manifestation, and survival time, etc. Correction were carried based on the pre-experimental results, and a 50% survival rate of the model after producing 72h was maintained, and the stability of sepsis model was ensured. Specific pathogen free (SPF) grade Kunming mice, weight 25-35g, were randomly divided into Sham-operated control group (Sham), CLP group (CLP) and Thymosin al treated group (THI). Then, each group were randomly divided into 6 subgroups according to specimens harvest (2h,6h,12h,24h,48h and 72h), there were 10-20 mice in each subgroups. The mice were sacrificed and their blood, liver, lung and terminal ileum obtained for detecting. The quantities and relative ratio of CD4+CD25+T cells, CD4+ CD25+Foxp3+ T cells, and the apoptosis of CD4+CD25+ T cells in blood were detected by flow cytometry, IL-2, IL-6, IL-10, TNF-a and TGF-βin serum were examined by enzyme linked immunosorbent assay, the function indicators of liver and kidneys were checked by full-automatic biochemistry analyzer, and the he pathological changes in liver, lung and distal ileum stained Hematoxylin-Eosin (HE) were observed by optical microscopy. The survival rates of 72h were recorded. Data were processed by Excel2003 and statistical analysis software SPSS 14.0 for Windows. ResultsThe 72 hours survival rate of THI group was higher than CLP group (x2=5.558, P=0.0184). According to the flow cytometry detecting, there was little change of Treg in Sham group; However, the blood Treg accounted for the proportion of CD4+T cells in THI group were significantly lower than in CLP group at the same time points (P<0.05 or P<0.01); Contrarily, apoptosis rate of CD4+CD25+T cell in THI group was remarkable higher than CLP group (P<0.01); Apoptosis rate of CD4+CD25+T in CLP group was even lower than Sham group during the period of 6-24h after operation (P<0.05 or P<0.01). Serum cytokines IL-2, TNF-a, IL-10 and TGF-p varied notably among the three groups (P<0.01), and the change was smallest in Sham group. Compared with CLP group, the increase of IL-2 and TNF-a was lower in early phase and the decline was lower also in late phase in THI group (P<0.01); Although IL-10 and TGF-βshowed a trend of continuous increase in THI group, but the increase degree was obviously lower than CLP group (P<0.01). Histological analysis showed that the pathological damage of the distal ileum in THI group reduced evidently at the same time points comparing to CLP group.ConclusionRegulatory T cells in blood changes in characteristics were observed and analyzed on the basis of establishing a stable model of septic mice model in the experiment, and impact of intervention of immunomodulator, thymosin al, were understood. Thus, the effect of Thymosin al on the pathophysiology of development in sepsis could be discussed.It was shown that the increase of percentage of Treg in blood related to the severity of illness under sepsis pathological condition, higher proportion of Treg indicated more serious sepsis, on the other hand, Treg apoptotic rate got lower with the disease progresses. Treg increase suppressed and apoptotic rate promoted significantly were observed when the model treated with Thymosin al, pro-inflammatory factor TNF-a, IL-2 in THI group did not exceed CLP group in the early time but dropped slower than the latter, inflammatory response inhibitory factor TGF-β, IL-10 had been restrained in the late course of sepsis. In brief, Thymosin al can improve survival in septic mice, and promote Treg apoptosis in sepsis. The new finding may become evidence of the pharmacological mechanism for application to sepsis immunomodulation.