Expression of toll-like receptor 4 and MD-2 gene and protein in Kupffer cells after ischemia-reperfusion in rat liver graft.
OBJECTIVE
To investigate the expression of toll-like receptor 4 (TLR4) and MD-2 gene and protein in Kupffer cells (KCs) and their role in ischemia-reperfusion (IR) injury of rat liver graft.


METHODS
KCs were isolated at 0 (control group), 2, 12, 24 h (IR group) following IR in rat liver graft. mRNA expression of TLR4 and MD-2 was detected by RT-PCR analysis, protein expression of TLR4/MD-2 was detected by flow cytometric (FCM) analysis, and tumor necrosis factor-alpha (TNF-alpha) level in supernatant was measured by ELISA. Then isolated KCs were incubated with anti-TLR4 polyclonal antibody (anti-TLR4 group), and TNF-alpha level was measured again.


RESULTS
The mRNA and protein expression of TLR4/MD-2 and the level of TNF-alpha in IR group increased significantly at 2 h following IR, and reached the maximum at 12 h, and slightly decreased at 24 h, but were still significantly higher than those in the control group (P<0.01). The expression of these factors was markedly decreased after anti-TLR4 antibody treatment as compared with the IR group (P<0.01).


CONCLUSIONS
Lipopolysaccharide (LPS) following IR can up-regulate TLR4/MD-2 gene and protein expression in KCs, and synthesize cytokine TNF-alpha. Anti TLR4 antibody can inhibit the production of TNF-alpha induced by LPS. TLR4 and its partner molecule MD-2 may play an important role in Kupffer cell activation and IR injury.
Peng
Yong
Y
Department of Hepatobiliary Surgery, the Second Affiliated Hospital of Chongqing Medical University, 74 Linjiang Road, Chongqing 400010, China.
Gong
Jian-Ping
JP
Liu
Chang-An
CA
Li
Xu-Hong
XH
Gan
Ling
L
Li
Shou-Bai
SB
D000818:Animals; D000954:Antigens, Surface / Q000235:genetics*; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000235:genetics; D007728:Kupffer Cells / Q000473:pathology / Q000502:physiology*; D016031:Liver Transplantation / Q000473:pathology / Q000502:physiology*; D051218:Lymphocyte Antigen 96; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D011956:Receptors, Cell Surface / Q000235:genetics*; D015427:Reperfusion Injury / Q000473:pathology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D000954:Antigens, Surface; D018076:DNA, Complementary; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; C495347:Tlr4 protein, rat; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha