[Effect of apoptosis of CD4+ CD25+ regulatory T lymphocytes on polarization of helper T lymphocytes and potential interventional influence of Xuebijing injection in septic rats].
OBJECTIVE
To evaluate the influence of apoptosis of CD4(+)CD25(+) regulatory T lymphocyte (Treg) on polarization of helper T lymphocyte (Th) and effect of Xuebijing injection in septic rats.


METHODS
A sepsis model was reproduced by cecal ligation and puncture (CLP). Wistar rats were randomly divided into the normal group (n=8), sham operation group (n=8), model group (n=8) and Xuebijing injection treatment group (n=8). CD4(+)CD25(+)Tregs in each group were separated by immunomagnetic beads isolation system on day 3, the apoptotic rates, expression of forkhead/winged helix transcription factor p3 (Foxp3) as well as cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) of Treg were analyzed by flow cytometry, and the secretion level of interleukin-10 (IL-10) of Tregs was determined by enzyme linked immunosorbent assay (ELISA) after culturing Tregs for 12 hours. Interferon-gamma (IFN-gamma), IL-4, and IL-17 levels, which were respectively secreted by Th1, Th2 and Th17, were measured by ELISA in the supernatant after CD4(+)CD25(+)Tregs were co-cultured with CD4(+)CD25(-)T lymphocytes for 68 hours.


RESULTS
The apoptosis rate of the normal group was (12.03+/-0.89)%, which was not significantly different compared with the sham operation group [(9.48+/-2.17)%]. The apoptosis rate of model group [(5.87+/-0.44)%] was much lower than that of the normal and sham operation groups, while the Xuebijing injection treatment group [(27.29+/-2.48)%] had the highest apoptosis rate compared to others (all P<0.01) . The expression of Foxp3, CTLA-4, and the secretion of IL-10 of Treg were negatively correlated with the apoptosis rates, correlation coefficients (r) respectively were -0.878 (P=0.042), -0.877 (P=0.042), and -0.743 (P=0.010). The secretion of IFN-gamma, IL-4 and ratio of IFN-gamma/IL-4 in model group were significantly elevated compared with the normal group [IFN-gamma: (254.70+/-44.88) ng/L vs. (0.68+/-0.78) ng/L , IL-4: (8.82+/-0.61) ng/L vs. (3.48+/-0.98) ng/L, ratio of IFN-gamma/IL-4: 30.28+/- 4.87 vs. 0.23+/-0.30, all P<0.01], and secretion of IFN-gamma as well as ratio of IFN-gamma/IL-4 were markedly higher in Xuebijing injection treatment group [(491.54+/-84.28) ng/L, 45.31+/-8.01, respectively] than in model group (P<0.01 and P<0.05). No marked change in IL-17 levels was noted in various groups (all P>0.05) .


CONCLUSIONS
Due to the apoptosis of Treg, the suppression function of CD4(+)CD25(+)Tregs on CD4(+)T lymphocyte appears to be abated, and treatment with Xuebijing injection could effectively enhance the apoptosis of Tregs, mediating the response of shifting Th2 to Th1.
Dai
Xin-gui
XG
Intensive Care Unit, Xiangya Hospital of Central South University, Changsha 410008, Hunan, China.
Yao
Yong-ming
YM
Ai
Yu-hang
YH
D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects; D060908:CTLA-4 Antigen; D002478:Cells, Cultured; D018920:Coculture Techniques; D004195:Disease Models, Animal; D004365:Drugs, Chinese Herbal / Q000494:pharmacology; D051858:Forkhead Transcription Factors / Q000378:metabolism; D007371:Interferon-gamma / Q000378:metabolism; D007378:Interleukins / Q000378:metabolism; D008297:Male; D011897:Random Allocation; D051381:Rats; D017208:Rats, Wistar; D018805:Sepsis / Q000188:drug therapy / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D006377:T-Lymphocytes, Helper-Inducer / Q000276:immunology*; D050378:T-Lymphocytes, Regulatory / Q000276:immunology* / Q000378:metabolism / Q000473:pathology
D015703:Antigens, CD; D060908:CTLA-4 Antigen; C556708:Ctla4 protein, rat; D004365:Drugs, Chinese Herbal; D051858:Forkhead Transcription Factors; C500522:Foxp3 protein, rat; D007378:Interleukins; D007371:Interferon-gamma