[An experimental study of the inhibitory effects on the activation of endotoxin-induced Kupffer cells through short hairpin RNA targeting interleukin-1 receptor associated kinase-4 gene].
OBJECTIVE
To explore the inhibitory effects on the activation of endotoxin-induced Kupffer cells (KCs) through short hairpin RNA (shRNA) targeting interleukin-1 receptor associated kinase-4 (IRAK-4) gene.


METHODS
Two effective transfection shRNA plasmid (pSIIRAK-4-A, pSIIRAK-4-B) and one invalidated plasmids (pSIIRAK-4-C) targeting IRAK-4 gene were constructed. The isolated mouse KCs were divided into three groups: the normal control group, the RNAi control group (pSIIRAK-4-C) and the RNAi effective group (pSIIRAK-4-A, pSIIRAK-4-B). Then KCs were stimulated with 0.1 microg/ml lipopolysaccharide (LPS) after 24 h transfection with the constructed plasmid. The expression of IRAK-4 gene and protein level were determined by RT-PCR and Western blot at 6 h after LPS stimulation, and the activities of NF-kappaB in KCs and the TNFalpha level were estimated by ELISA at 0 h, 1 h, 3 h, 6 h and 12 h.


RESULTS
The level of IRAK-4, the activities of NF-kappaB and the TNF-alpha level in the RNAi effective group were evidently lower than those in normal and RNAi control groups (P < 0.01) at 1 h, 3 h, and 6 h. Especially, the pSIIRAK-4-A group in which the changes of the above indices were of no difference (P > 0.05), had better inhibited effects than that of the pSIIRAK-4-B group (P < 0.01).


CONCLUSIONS
The shRNA targeting IRAK-4 gene could effectively inhibit the activation of endotoxin-induced KCs.
Liu
Zuo-jin
ZJ
Department of Hepatobiliary Surgery, Second Affiliated Hospital of Chongqing University of Medical Sciences, Chongqing 400010, China.
Li
Sheng-wei
SW
Liu
Chang-an
CA
You
Hai-bo
HB
Peng
Yong
Y
Li
Xu-hong
XH
Chen
Xian-feng
XF
Gong
Jian-ping
JP
D000818:Animals; D004731:Endotoxins; D053592:Interleukin-1 Receptor-Associated Kinases / Q000235:genetics* / Q000378:metabolism; D007728:Kupffer Cells / Q000378:metabolism*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D015398:Signal Transduction / Q000502:physiology
D004731:Endotoxins; D034741:RNA, Small Interfering; C507773:IRAK4 protein, human; D053592:Interleukin-1 Receptor-Associated Kinases