Lxr-α Activation Promoted Nor-1 Expression And Suppressed Lps-mediated Inflammation In Kupffer Cells

Objective: To explore the relationship between Neuron-derived orphan nuclear receptor-1(NOR-1) and Liver X receptor-α(LXRα) expression and their anti-inflammatory reaction in Kupffer cells (KCs).Methods: KCs isolated from Male KM mice by gradient centrifugation combined with selective adherence, and the KCs were seeded in 6-well plates at 1×106 cells/well in RPMI1640 containing 20% FBS for 24h, and were randomly divided to four groups: (1) control group: Cultured in RPMI1640 for 30h; (2) T0901317 treated group: Cultured in RPMI1640 containing T09601317(1μM) for 24h, and then treated with RPMI1640 containing LPS (10μg/ml) for 6h; (3) LPS treated group: Cultured in RPMI1640 for 24h, and then treaded with RPMI1640 containing LPS (10μg/ml) for 6h; (4) LPS+T0901317 treated group (combination group): Cultured in RPMI1640 containing T09601317(1μM) for 24h, and then treaded with RPMI1640 containing LPS (10μg/ml) for 6h. The expression of NOR-1 and LXR-αgene and protein level were determined by Real Time-PCR, Western blot and immunofluorescent assay. The concentrations of TNF-αand IL-10 in supernate were evaluated by enzyme linked immunosorbent assay (ELISA).Results: (1) Real-Time fluorescence quantitative RT-PCR results showed that the expression levels of LXRαmRNA in the combination group and T0901317 treated group were significanctly higher than that in LPS treated group, and 2-△△CT>2, the differences were significant; and the expression levels of LXRαmRNA in the control group was higher than that in LPS treated group, and 2-△△CT>2, the difference was significant. the expression levels of NOR-1 mRNA in the combination group and T0901317 treated group were significanctly higher than that in LPS treated group, and 2-△△CT>2, the differences were significant; and the expression levels of NOR-1 mRNA in the control group was higher than that in LPS treated group, and 2-△△CT>2, the difference was significant.(2)Western blot analysis of LXR-αand NOR-1 protein in KCs: The protein levels of LXRαand NOR-1 in LPS treated group (0.569±0.045 and 0.335±0.042, respectively) was significant lower than the other groups (control group, 1.013±0.049 and 0.541±0.167; T0901317 treated group, 1.736±0.038 and 1.031±0.040; combination group 1.232±0.027 and 0.819±0.022, respectively), the difference was significant (P<0.05). The protein levels of LXR of LXRαand NOR-1 in the T0901317 treated group was the highest of all the four groups, the difference was significant (P<0.05); The protein levels of LXRαand NOR-1 in the combination group were significant lower than T0901317 treated group, but, higher than the control group and LPS treated group, the differences were significant (P<0.05).(3) Protein level of LXRαand NOR-1 in immunofluorescence: LXR-αin KCs was determined by immunofluorescence analysis, slides were viewed on laser scanning confocal microscope×800. The results showed that LXRαand NOR-1 protein were found in the cytoplasm and nuclear of KCs, The mean MII levels of LXR-αin control, T0901317, LPS and combination group were 97.52±10.39; 110.04±14.54; 56.98±7.95; 105.58±14.03, respectively. Values are expressed as the mean±standard deviation, T0901317 group was the highest versus the other groups (P<0.05). The mean MII levels of NOR-1 in control, T0901317, LPS and combination group were 80.15±4.88; 115.65±8.47; 65.50±5.76; 94.14±4.48, respectively. Values are expressed as the mean±standard deviation, T0901317 group was the highest versus the other groups (P<0.05).(4) ELISA results showed that: The levels of supernatant Tumor necrosis factor-α(TNF-α) in LPS group (450.89±78.52) was higher than in control group (5.84±2.35) and the T0901317 treated group (6.73±1.90), the differences were significant (P<0.05). The levels of supernatant TNF-αin the combination group (61.21±17.45) was lower than in LPS treated group, the differences were significant (P<0.05). The levels of supernatant Interleukin-10 (IL-10) in the combination group (537.41±36.41) was significant higher than in control group (10.67±3.48) and the T0901317 treated group (10.17±1.82), the differences were significant (P<0.05). The levels of supernatant IL-10 in the combination group was higher than in the LPS treated group (61.85±12.41), the differences were significant (P<0.05).Conclusions: Using T0901317, a synthetic ligand of LXRs, we observed that it can induce LXRαexpression. KCs, After T0901317 challenge, the mRNA and protein expression of both LXR-αand NOR-1 in T0901317 group were higher than the control group. On the other hand, the mRNA and protein expression of the two nuclear receptors in the combination group were higher than in the LPS group, and the inflammatory factor-TNF-αwas inhibited in the combination group, the expression level of anti-inflammatory factor-IL-10 was significant elevated in the combination group. After LPS pretreatment, the mRNA and protein expression of both LXR-αand NOR-1 in LPS group were lower than in the control group. Then, we can hypothesis that LXR-αcan regulate NOR-1 expression in KCs. one way of LXR-αrealize the anti-inflammatory mechanism is regulating NOR-1 expression.