The Effects And Mechanisms Of Lendvirus-mediated LXRs Overexpression On High Glucose-induced Inflammatory Response In H9C2Cells

Objective：1.To construct lentiviral vector with overexpression LXRs, to further transfectH9C2cells.2.To observe the effect of lentiviral overexpressing LXRs on highglucose-induced inflammatory response in the H9C2cells.3.To investigate the mechanisms of LXRs attenuate the inflammation of H9C2cells.Methods:1. The H9C2cells were cultured with low sugar medium in vitro, and thenintervende by high sugar and lentivirus respectively.2. The gene fragments of Nr1h2and Nr1h3were obtained from the plasmidharboring the target gene and cloned into the lentivirus vector with green fluorescentprotein (GFP) by enzyme digestion method with the result of constructingrecombinant lentiviral plasmids (GV287-Nr1h2and GV287-Nr1h3). After sequenceidentification pHelper1.0and pHelper2.0were co-transfected into293T cells and thenthe recombinant lentivirus carrying Nr1h2and Nr1h3genes was obtained.3.1~4days after infection, the fluorescence of H9C2cells was observed byfluorescence microscopeto determine the appropriate MOI.4. In the experiment, five groups were divided:①Control group (Normalcontrol): The H9C2cells were cultured in low sugar medium;②High glucose group(The positive control): The H9C2cells were cultured in33mmol/L glucose medium;③LXRα group: The H9C2cells were infected with lentiviral of GV287-Nr1h3;④LXRβ group: The H9C2cells were infected with lentiviral of GV287-Nr1h2;⑤GFPgroup: The H9C2cells were infected with lentiviral of empty vector.5.72hours after lentivirus infection, the proliferation inhibition rate of H9C2cells was determined by CCK-8. The mRNA expression of LXRα, LXRβ,TNF-α,MCP-1and IL-6were detected by qRT-PCR;The western blot and immunofluo-rescence were employed to detect the expression of NF-κB phosphorylated protein. Results:1. H9C2cells derived fromcloned cell lines of rat embryonic heart tissue. TheH9C2cells proliferated adhesively with fusiform shape.2. Gene sequencing indicated that the lentiviral vector of LXRs overexpressionwas successfully constructed.The titer of concentrated virus was2×108TU/ml.3. The lentivirus of LXRs overexpression infected H9C2cells, when the MOI is40, the weak green fluorescence was observed after24hours and culminated after72h. Administration of8μg/ml Polybrene synergistically increased the infectionefficiency.4. Compared with Control group, the mRNA levels of LXRα and LXRβ weresignificantly elevated in Group LXRα and LXRβ (P<0.01); There was no significantdifference between Control group and GFP group (P>0.05).5. Compared with control group, the inhibition rate of H9C2cells was highest inhigh glucose group(P<0.01); In LXRα and LXRβ group, the inhibition rate werelower compared with high glucose group. The inhibition rate was more elevated inLXRβ group than in LXRα group (P<0.01). No significance were seen between GFPgroup and high glucose group (P>0.05).6. Compared with control group, the mRNA levels of MCP-1, IL-6, TNF-α werehigher in high glucose group (P<0.01) and lower in group LXRα and LXRβcompared with high glucose group. The mRNA levels were lower in LXRα groupthan LXRβ group (P<0.01). No significance were seen between GFP group and highglucose group (P>0.05).7. Compared with extremely weak fluorescence in control group,we got intensered fluorescence of NF-κB p65protein in both high glucose group and GFP groupcell nucleus,which accounts for about75%in each group; The average rate of redfluorescence of NF-κB p65protein in LXRα group than LXRβ group weredifferent:40%and68%of all nucleus,respectirely.8. The NF-κB phosphorylated protein expression of high glucose group washigher than that of control group (P<0.01);In LXRα and LXRβ group, NF-κBprotein level were lower compared with high glucose group. The NF-κB proteinlevel was lower in LXRα group than in LXRβ group (P<0.01). No significance were seen between GFP group and high glucose group (P>0.05).Conclusions:1. The lentiviral vector of LXRs overexpression was successfully constructed,and infected H9C2cells effectively.2. LXRs overexpression can attenuate high glucose-induced inflammatoryreaction in myocardial cells. The LXRα could be more potent to inhibit theinflammation reaction.3. The potency of LXRs overexpression could inhibition of NF-κB activation inmyocardial cells. and may attenuate the inflammation in myocardial cells by thiseffect, These results suggest that inhibition of NF-κB activation could be one of theway of LXRs to attenuate myocardial inflammation.