[Study on the expression of TLR4 on splenocytes and its relation with CD4(+) CD25(+) regulatory T cells in peripheral blood in severely burnt rats].
OBJECTIVE
To investigate the expression of Toll like receptor 4(TLR4) on splenocytes and TNF-alpha mRNA, the dynamic changes of CD4(+) CD25(+) regulatory T cell(Treg) and endotoxin(ET) in peripheral blood of burnt rats during the early phase, and to explore the mechanism against inflammatory reaction in intestine-derived infection after burn.


METHODS
64 SD male rats were randomly separated into control group and burn model groups. Rats of burn model group were burnt with vapor under 3 mPa pressure and 108 degrees Celsius temperature for 8 seconds to achieve deep partial-thickness burn, and a 30% total body surface area (TBSA) burn model was made. Rats were sacrificed before and 2, 5, 8, 12, 24, 48 and 72 h after burn, and the TLR4 and TNF-alpha mRNA expression in splenocytes were measured at various intervals by RT-PCR. The expression of TLR4 protein was measured by Western blot, and the percentage of Treg cells in CD4(+) T cells was detected by flow cytometry (FCM), and the LPS concentration of plasma was detected by limulus lysate test.


RESULTS
The expression of TLR4 mRNA, TNF-alpha mRNA, TLR4 protein and the levels of Treg, ET were significantly increased at some times points after burn. The expression of TLR4 mRNA and protein reached the peak at 8 h, whereas the TNF-alpha mRNA at 12 h, and Treg and ET at 8 h. The peak values of them were 3.66+/-0.51, 2.27+/-0.19, 1.65+/-0.23, 63.19+/-12.65% and 11.68+/-1.71 Eu/mL respectively, which were of significant difference when compared with the control group (P<0.01). The expression of TLR4 mRNA showed a positive correlation with that of Treg, ET and TNF-alpha mRNA (r=0.898, 0.811, 0.462, P<0.01).


CONCLUSIONS
Treg might play a major role in the process of immune regulation in rats after burn, the mechanism of which may be correlated with the increase of LPS-TLR4 signal transduced by intestine-derived infection.
Fu
Ying-jun
YJ
Department of Pharmacology, Nanchang University Medical College, Nanchang 330006, China. fuyingjun123@126.com
Xie
Yong
Y
Shi
Jun-qiang
JQ
Zhou
Nan-jin
NJ
Guo
Guang-hua
GH
D000818:Animals; D053264:B-Cell Activating Factor / Q000378:metabolism*; D015153:Blotting, Western; D002056:Burns / Q000097:blood / Q000276:immunology* / Q000378:metabolism* / Q000503:physiopathology; D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism; D005434:Flow Cytometry; D008070:Lipopolysaccharides / Q000097:blood; D008297:Male; D011897:Random Allocation; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013154:Spleen / Q000166:cytology / Q000378:metabolism*; D050378:T-Lymphocytes, Regulatory / Q000276:immunology* / Q000378:metabolism*; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
D053264:B-Cell Activating Factor; D008070:Lipopolysaccharides; C495347:Tlr4 protein, rat; D051197:Toll-Like Receptor 4; D014409:Tumor Necrosis Factor-alpha