[Effect of apoptosis of CD4+ CD25+ regulatory T cells on proliferation as well as secretion of effector T cells and interventional activity of Xuebijing injection in septic rats]

Abstract

Objective: To investigate the effect of apoptosis of CD4+ CD25+ regulatory T cells (Tregs) on proliferation as well as secretory function of effector T cells (Teff) and potential influence of Xuebijing injection on them in septic rats.

Methods: A sepsis model was reproduced by cecal ligation puncture (CLP), and Wistar rats were randomly divided into the control group (n = 8), sham-operated group (n = 8), CLP group (n = 8), and Xuebijing injection treatment group (n = 8). CD4+ CD25+ Tregs in each group were separated by immunomagnetic beads isolate system on day 3, the apoptosis rate, expression of forkhead/winged helix transcription factor p3 (Foxp3) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) on Tregs were analyzed by flow cytometry, and secretion levels of interleukin (IL)-10 from Tregs were measured by ELISA. Following co-culture of CD4+ CD25+ Treg with CD4+ CD25- T cells (1:1) for 68 hours, proliferative activity of Teff was determined by MTT, and IL-2/sIL-2R alpha levels were measured by ELISA.

Results: The apoptosis rate of Tregs in control group was 12.03% +/- 0.89%, which was not significantly different from sham-operated group 9.48% +/- 2.17%. The apoptosis rate of Tregs in CLP group 5.87% +/- 0.44% was lower than that in control group (P < 0.01), and treatment with Xuebijing injection markedly enhanced the apoptosis of Tregs 27.29% +/- 2.48%. Compared to CLP group, expression of Foxp3, CTLA-4, and the secretion of IL-10 of Treg were significantly lowered in Xuebijing injection group (all P < 0.01). The Teff proliferative activity in response to ConA, and IL-2 levels of Teff in CLP group were significantly suppressed compared with control group (P < 0.01), and secretion of sIL-2R alpha in the supernatants was much higher than that of the control group. In comparison to the CLP group, inhibitory rate of Teff proliferative activity and the sIL-2R alpha levels were significantly decreased, while the secretion of IL-2 was increased in Xuebijing injection group (P <0.01).

Conclusion: CD4+ CD25+ Tregs could markedly upregulate the suppressive function on Teff in sepsis, and treatment with Xuebijing injection effectively enhanced apoptosis of Tregs, thereby down-regulating the suppression on Teff.