| AIM:To isolate, culture and identificate rat Kupffer cells (KCs), and to investigatethe effects of urotensin II (UII) and its receptor UT on the expressions of proinflammation cytokinesin in lipopolysaccharide (LPS)-stimulated cells.METHODS:Rat KCs are isolated and purified by means of in situ perfusion, density gradient centrifugation and early changing medium. Isolated cells are identified by ink phagocytosis and ED2 staining test. The cells were then divided into four groups: group A:normal control; group B:urantide; group C:LPS; group D:urantide plus LPS. The levels of UII and UT mRNA expression were detected by real-time PCR. The level of UII secretion was detected by enzyme-linked immunosorbent (ELISA) in supernatant. The levels of TNF-α and IL-1β mRNA and protein were detected by RT-PCR and ELISA.RESULTS:Rat KCs were successfully isolated and purified, and confirmed by ink phagocytosis and ED2 staining test. Therelative levels of the primary KC UII mRNA expression in group C were significantly higher than those in group A, B and D [(14.785±1.441) vs 1, (0.926±0.263), (4.069±0.575), all P<0.01], and the levels in group D were higher than in group A and B (all P<0.01), but no significant difference was found between group A and group B (P>0.05). The relative levels of UT mRNA expression in group C were significantly higher than those in group A, B and D [(18.046±0.56) vs 1, (0.811±0.09), (2.485±0.532), all P< 0.01], and the levels in group D were higher than in group A and B (all P<0.01),but no significant difference was found between group A and group B (P> 0.05). The levels of UII secretion in supernatant in group C were significantly higher than group A, B and D [(354.485±10.658) pg/mL vs (202.24±5.426) pg/mL, (178.175±17.837) pg/mL, (281.4±6.104) pg/mL, all P<0.01], and the levels in group D were higher than those in group A and B (all P<0.01), but no significant difference was found between group A and group B (P>0.05).The relative levels of KC TNF-α mRNA expression in group C were significantly higher than those in group A, B and D [(1.073±0.02) vs (0.087±0.006), (0.076±0.006), (0.357±0.021), all P<0.01], the levels in group D were higher than those in group A and group B (all P<0.01), but no significant difference was found between group A and group B (P>0.05). The relative levels of primary KC IL-1β mRNA expression in group C were significantly higher than that in group A, B and D [(0.523±0.021) vs (0.13±0.01), (0.12±0.01), (0.193±0.012), all P <0.01], the levels in group D were higher than those in group A and group B (all P <0.01), but no significant difference was found between group A and group B (P> 0.05). The levels of TNF-α secretion in supernatant in group C were significantly higher than those in group A, B and D [(435.10±18.30) vs (39.41±15.30), (48.51±15.77), (265.29±14.12), all P<0.01], the levels in group D were higher than those in group A and group B (P<0.01), but no significant difference was found between group A and group B (P>0.05). The levels of IL-1β secretion in supernatant in group C were significantly higher than those in group A, B and D [(1385.20±66.58) pg/mL vs (88.51±49.10) pg/mL, (49.82±30.99) pg/mL, (629.12±146.09) pg/mL, all P<0.01], the levels in group D were higher than those in group A and group B (P< 0.01), but no significant difference was found between group A and group B (P> 0.05). In addition, the KCs were treated with different doses of UⅡ polypeptide. It was shown that the relative levels of KC TNF-α mRNA expression in the 500 nM dose was higher than those in 250 nM.100 nM and 0 nM [(1.11±0.002) vs (0.79±0.001), (0.74±0.002), (0.66±0.003), all P<0.01], and the levels in 250 nM and 100 nM doses were higher than in 0 nM dose (all P<0.01), no significant difference was found between 250 nM and 100 nM dose (P>0.05).CONCLUSION:Rat KCs can be effectively isolated and purified by means of the in situ perfusion and density gradient centrifugation; The expressions of UⅡ/UT system and proinflammation cytokines can be induced by LPS challenge, but inhibited by urantide pretreatment in the primary KCs; The UⅡ polypeptide can stimulate the expression of TNF-α gene. This suggests that the UⅡ/UT system may play a crucial role in inflamedly liver injury. Such a study may provide the foundation for novel pharmacological approaches to reduce hepatic inflammation. UTR inhibitor is expected to be a new candidate for treatment of cytokine-related liver diseases. |
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