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. 2004 Oct 1;10(19):2890-3.
doi: 10.3748/wjg.v10.i19.2890.

Expression of toll-like receptor 4 and MD-2 gene and protein in Kupffer cells after ischemia-reperfusion in rat liver graft

Yong Peng  1 , Jian-Ping Gong, Chang-An Liu, Xu-Hong Li, Ling Gan, Shou-Bai Li
Affiliations

Expression of toll-like receptor 4 and MD-2 gene and protein in Kupffer cells after ischemia-reperfusion in rat liver graft

Yong Peng et al. World J Gastroenterol. .

Abstract

Aim: 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).

Conclusion: 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.

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Figures

Figure 1
Figure 1
Expression of TLR4 and MD-2 mRNA by RT-PCR analysis. A: PCR products were electrophoresed on agarose gels and photographed. B: Quantitative data of mRNA levels were shown as the ratio of relative absorbance and expressed as mean ± SD. Expression of TLR4 and MD-2 mRNA were sig-nificantly increased in the IR group compared with the con-trol group (P < 0.01).
Figure 2
Figure 2
Percentage of TLR4 and MD-2 positive cells. The percentage of TLR4 and MD-2 positive cells was significantly increased after IR compared with the control group.
Figure 3
Figure 3
Comparison of TNF-α production in supernatant of KCs. In IR group, the TNF-α level in supernatant was in-creased with time, and reached the maximum (529.1 ± 30.9 ng/mL) at 12 h. But in anti-TLR4 group, the production of TNF-α was obviously inhibited by Ab against TLR4 compared with the IR group (P < 0.01).
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