| AIM To observe the role of activated LXRαon the expression of IRAK-4 and NF-kappaB in the inflammatory response which induced by LPS in the Kupffer Cells in vitro and to explore the possible mechanisms of LXR negative regulation of inflammatory response.MEHTODS Male Kun-Ming mice, isolation and culture the Kupffer Cells by method of Collagenase perfusion in situ. And these cells were divided into four groups:○1 Normal Control group,○2 LPS treatment group,○3 LXR agonist T0901317 treatment group,○4 LPS and T0901317 combined treatment group. The LPS-treated group with a final concentration of 1μg/ml LPS in RPMI 1640 cultured for 6 h, the T0901317-treated group with a final concentration of 5μg/ml in RPMI 1640 cultured for 24 h, the Combined-treated group pre-cultured for 24 h with a final concentration of 1μg/ml T0901317 in RPMI 1640 and then cultured for 6 h with a final concentration of 5μg/ml LPS in RPMI 1640. All groups were cultured to 30 h. The expression of LXR, IRAK-4 and NF-kappaB gene and protein level were detected by Real Time-PCR, immunocytochemical and Western blotting, the activation of NF-kappaB was detected by electrophoretic mobility shift assay, the TNF-αand IL-1βlevel were detected by ELISA.RESULTS○1 The level of LXR mRNA and protein were highest in T0901317-treated group (mRNA 0.9912±0.0098, protein 1.740±0.034), and lowest in LPS-treated group (mRNA 0.2722±0.0038, protein 0.534±0.014). In Combined-treated group the level of LXR mRNA and protein (mRNA 0.7963±0.0075, protein 1.224±0.027) were higher than that in LPS group but lower than that in LXR agonist T0901317-treated group, the difference were significant between each other (p<0.05).○2 The level of IRAK-4 mRNA (2.0132±0.0406) was evidently lower in the Combined-treated group than that in LPS group (3.9238±0.0594), and the level was lowest in LXR agonist T0901317 group (0.8756±0.0093), but no difference between the Combined-treated group and Control group (p>0.05). The level of NF-κB mRNA (25.2142±0.3501) was highest in LPS group, and the difference was significant compared with other groups (p<0.05), but no differences among Control group (9.6881±0.1272), T0901317 group (8.9615±0.1723), and Combined-treated group (11.0173±0.2508).○3 The level of IRAK-4 protein expression was highest in LPS-treated group (0.710±0.018), and lowest in LXR agonist T0901317 group (0.102±0.004), the difference was significant (p<0.05). In the Control group (0.271±0.008) and the Combined-treated group (0.257±0.008) the protein level were lower than that in LPS-treated group but higher than that in LXR agonist T0901317-treated group, the difference was no significant between LPS-treated group and LXR agonist T0901317-treated group (p>0.05), but had evidently difference compared with LPS-treated group and T0901317 group (p<0.05). The level of NF-κB protein was highest in LPS-treated group (0.693±0.017), the difference was significant than other groups (p<0.05), but in other groups the level of NF-κB protein was lower, and no difference was observed (p>0.05).○4 The phosphorylation level of IRAK-4 were low in Control group (0.242±0.008) and LXR agonist T0901317-treated group (0.201±0.007), and no difference between the two groups (p>0.05). And the level were high in LPS treated group (0.893±0.019) and Combined-treated group (0.865±0.018), and no significant difference between the two groups (p<0.05), but the difference were notable compared with Control group and LXR agonist T0901317-treated group (p<0.05). The relative activation of NF-κB were low in Control group (24%±1%) and LXR agonist T0901317-treated group (28%±1%), but no significant difference was observed between the two groups (p>0.05), the activation of NF-κB were higher in Combined-treated group (75%±2%) than that in Control group and T0901317 group, the difference was significant (p<0.05), the highest activation of NF-κB was in LPS-treated group (100%±0%), and the difference was notable compared with other groups (p<0.05).○5 And the level of TNF-α(15.45±0.59μg/ml) and IL-1β(20.23±0.64μg/ml) were observed highest in LPS group (p<0.05), but no difference among the Control group, T0901317 group and Combined-treated group (p>0.05).CONCLUSION This date suggests that the LXR agonists can effectively up-regulate the level of LXR gene and protein and inhibit the IRAK-4 and NF-κB mRNA and protein level in TLR4 signaling pathway, and also inhibit the activity of NF-κB, but no significantly impacted on the IRAK-4 phosphorylation level. This may be one of mechanism that LXR agonist can down-regulate the inflammatory response. |
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