Epigenetical Regulation Of Lanthanum On LPS-induced NF-?B-Jmjd3 Downstream Inflammatory Genes In HUVECs

ObjectiveHuman umbilical vein endothelial cells?HUVECs?activated by endotoxin?LPS?were set as an endothelial injure model in the current study.By this model,we try to explore the inhibitory effects of lanthanum chloride?LaCl₃?on NF-?B-Jmjd3signaling induced by LPS and the underlying epigenetic mechanisms,to provide the experimental basis for efficient protect of vascular from inflammation end to develop medicinal value of rare earth element.Methods1.Cell culture and grouping:Primary HUVECs were thawed from frozen and were cultured routinely.The HUVECs in logarithmic growth phase were randomly divided into control group?incubated in serum-free medium for 2 hours?,LPS group?incubated in serum-free medium containing 1?g/ml LPS for 2 hours?,LaCl₃ group?incubated in serum-free medium containing 2.5?M/ml LaCl₃ for 2 hours?and LPS+LaCl₃ group?first incubated in serum-free medium containing 2.5?M/ml LaCl₃ for 30min,then transferred into serum-free medium containing 1?g/ml LPS for 2 hours?.During the incubation,the cells were cultured in a humidified incubator set at 37?with 5%CO₂?2.The effect of LaCl₃ on the viability of HUVECs was detected by MTT method.3.Cytoplasmic and nuclear proteins in HUVECs were separated,and the expression levels of NF-?B pathway related proteins were detected by Western blot.4.The binding activity between NF-?B/p65 protein and target sequence in nuclear extracts was detected by Millipore?Transcription Factor Assay Kit.5.The mRNA and protein expression level of Jmjd3 was detected by real-time quantitative PCR and Western blot.6.NF-?B/p65,Jmjd3 and H3K27me3 associated DNA libraries were setup by theMillipore?chromatinimmunoprecipitation?ChIP?kit,and ChIP-qPCR?quantitative real-time PCR?was applied to analyze of the accumulation of NF-?B/p65,Jmjd3 and H3K27me3 at the promoter region of the target genes such as TNF-?,MMP-9,IL-6,IL-1?,COX-2 and ICAM-1.7.The gene or protein levels of inflammatory factor TNF-?,MMP-9,IL-6,IL-1?,and ICAM-1 in cell or culture supernatant were detected by real-time quantitative PCR assay or ELISA,respectively.Results1.The influence of LaCl₃ on the viability of HUVECs:the growth of HUVECs had no significant difference under the treatment of 0,2.5,5.0,25,50 and 100?mol/L LaCl₃,however,the growth of HUVECs with LaCl₃ at the dosages of200?mol/L was inhibited?P<0.05,when compared with the control group?.It showed that the dose of LaCl₃ used in this experiment has no toxic effects.2.LaCl₃ inhibited LPS induced NF-?B-Jmjd3 up-regulation in HUVECs:?1?The nuclear NF-?B/p65 expression in LPS group was significantly increased when compared with that of the control group,as proved by western blot assay.While in the LPS+LaCl₃ group,the level goes down remarkably.There was no significant difference between LPS group and LaCl₃ group.The expression of I?B?decreased significantly in LPS group and LPS+LaCl₃ group,maintain at the same level in the LaCl₃ group,when compared to that of the control group.?2?Compared with control group,Jmjd3 expression was much higher in LPS group and went down significantly LPS+LaCl₃ group?P<0.01 vs.LPS group?.Jmjd3 expression has little change in LaCl₃ group without significant difference to control group.RT-qPCR and Western blot assays were employed to detect either the gene or protein expression respectively.3.The effect of LaCl₃ on the binding activity between nuclear NF-?B/p65 and target sequence:NF-?B/p65 binding activity was significantly higher in LPS group than that in the control group?P<0.05?,and it went down sharply in the LPS+LaCl₃group?P<0.01 vs.LPS group?.However,no significant change was found in LaCl₃group.4.Recruiment of NF-?B/p65 to inflammatory gene promoters:?1?Recruitment of NF-?B/p65on the gene promoter region of TNF-?,MMP-9,IL-6,IL-1?,COX-2and ICAM-1 increased remarkably in LPS group?P<0.05 vs.control group?;?2?The NF-?B/p65 accumulated to the above gene promoters were all lower in LPS+LaCl₃group than that in LPS group?P<0.05?;when compared with that of the control group,the recruitment of NF-?B/p65 in TNF-?,IL-6,IL-1?and ICAM-1 promoter region significantly decreased?P<0.05?,while in MMP-9 and COX-2 gene promoter region,no significant change was found;?3?Compared with the control group,the accumulation of NF-?B/p65 of LaCl₃ group was found significantly up-regulated in TNF-?,ICAM-1 gene promoter region?P<0.05?and the accumulation of NF-?B/p65 in MMP-9 gene promoter region down-regulated significantly?P<0.05?,meanwhile,the accumulation level in IL-6,IL-1?,and COX-2 gene promoter region changed little.The above results suggested that LaCl₃ can block LPS induced NF-?B/p65 recruitment in pro-inflammatory genes'promoter,while LaCl₃ alone had various effects on the recruitment of NF-?B/p65 to each pro-inflammatory gene promoter respectively.Compared to control group,NF-?B/p65 accumulation increased in TNF-?and ICAM-1 gene promoter region,remained unchanged in IL-6,IL-1 and COX-2 gene promoter region,and significantly decreased in the MMP-9gene promoter region.All the results were obtained by NF-?B/p65 ChIP-qPCR analysis.5.Enrichment activity of Jmjd3 to inflammatory gene promoters:?1?Compared with the control group,the enrichment of Jmjd3 to the above pro-inflammatory gene promoters was significantly higher?P<0.05?in both LPS group and LaCl₃ group;?2?The recruitment of Jmjd3 to above gene promoters in LPS+LaCl₃ group were lower in the group than that in LPS group,and the difference was statistically significant?P<0.05?.When compared with the control group,Jmjd3 accumulation in the IL-1?and COX-2 gene promoter region decreased significantly.And in IL-10 and ICAM-1 gene promoter region,the Jmjd3 accumulation increased significantly?P<0.05?.There was no significant change in TNF-?,MMP-9 and IL-6 promoter region.It suggested that LaCl₃ can inhibit LPS induced Jmjd3 recruitment in TNF-?,MMP-9,IL-6,IL-1?,COX-2 and ICAM-1 gene promoter region,and however,when HUVECs treated with LaCl₃ alone,the recruitment of Jmjd3 in pro-inflammatory genes promoter region increased significantly compared with that of control group.6.The level of H3K27me3 in inflammatory gene promoter region:?1?The level of H3K27me3 in the above gene promoters went down significantly in LPS group?P<0.05 vs.control group?;?2?H3K27me3 levelin the above gene promoter region was higher in LPS+LaCl₃ group than that of LPS group,but lower than that of the control group?both P<0.05?;?3?In LaCl₃ group,H3K27me3 level in TNF-?,IL-6,COX-2 and ICAM-1 gene promoter region significantly increased?P<0.05 vs.control group?.H3K27me3 level in MMP-9 gene promoter region was lower compared to?P<0.05 vs.the control group?,and H3K27me3 level in the IL-1?gene promoter did not change.The results indicated that LaCl₃ can inhibit the demethylation of H3K27me3 in all the above inflammatory gene promoter region.When the HUVECs treated with LaCl₃ alone,H3K27me3 level remain increased in TNF-?,IL-6,COX-2 and ICAM-1 promoter region or remain the same in IL-1?despite of the increased recruitment of Jmjd3.However,in the MMP-9 gene promoter region,the H3K27me3 level decreased with a decrease of NF-?B/p65 recruitment.7.The inhibiting effect of lanthanum chloride on LPS induced up-regulation of inflammatory factors in endothelial cells:as evaluated by RT-qPCR and ELISA,TNF-?,IL-6,IL-1?,ICAM-1 and MMP-9 were up-regulated at both gene and protein level by LPS induction while LaCl₃ was able to repress the effects?P<0.05?.Treatment with LaCl₃ alone showed no obvious difference.Totally,the results proved that LaCl₃ can inhibit the secretion of TNF-?,MMP-9,IL-6,IL-1 and ICAM-1effectively.Conclusion1.Jmjd3 was recruited to pro-inflammatory genes promoter regions by LPS induction and worked as a demethylase on H3K27me3,which resulted in rapid demethylation of H3K27me3 and exposure of transcription start sequence of targeted genes.Then LPS promotes the up-regulation and recruitment of NF-?B and Jmjd3 to gene promoter region of TNF-?,MMP-9,IL-6,IL-1?,COX-2 and ICAM-1 and finally activated TNF-?,MMP-9,IL-6,IL-1?,COX-2 and ICAM-1 gene expression.2.LaCl₃ blocked LPS induced up-regulation of inflammatory factors such as TNF-?,MMP-9,IL-6,IL-1?and ICAM-1 in HUVECs,via NF-?B-Jmjd3 signaling.By depressing LPS-activated nuclear expression and recruitment of NF-?B/p65 and Jmjd3 into pro-inflammatory genes promoter region,and inhibiting the demethylation of H3K27me3 in aforementioned pro-inflammatory gene promoter region,these genes were silenced.3.We found that while HUVECs were treated with LaCl₃ alone,which was set as a LaCl₃ control group,the enrichment of Jmjd3 in aforementioned pro-inflammatory gene promoter region increased significantly.However,this phenomenon did not lead to up-regulation of pro-inflammatory genes.A further analysis demonstrated that the expression and activity of NF-?B/p65-Jmjd3 was suppressed,the accumulation of NF-?B/p65 and H3K27me3 in targeted gene promoters was regulated variously.Essentially,both the recruitment of NF-?B/p65 and demethylation of H3K27me3were indispensable.All these suggested LaCl₃ regulate inflammatory genes through precise and comprehensive regulatory mechanisms.