Regulation Of Resolvin D1 On Monocyte Gene Expression Under LPS Stimulation

Objective: Systemic inflammatory response syndrome(SIRS)is a systemic waterfall-like inflammatory response caused by infection or non-infectious factors acting on the body,over-activating inflammatory cells and releasing excess inflammatory mediators.When the body is in a state of inflammation,monocytes respond rapidly to inflammatory stimuli and participate in immune regulation.Resolvin is a new type of polyunsaturated fatty acid derivatives that promote inflammation resolution.Its biosynthesis is derived from the omega-3 essential fatty acids docosahexaenoic acid(DHA)in vivo,which is an endogenous anti-inflammatory mediator.Purpose: This paper focuses on the regulation of Resolvin D1(RvD1)derived from DHA on the gene expression of monocytes stimulated by LPS.Methods: This study is divided into three parts.First,we used CCK-8 method to detect the concentration of LPS that affects cell proliferation.Next,we used enzyme-linked immunosorbent method to detect the protein levels of TNF-α and IL-1β in the supernatant of THP-1 cells in the Control group,LPS group,LPS and RvD1 group.m RNA sequencing experiments were used to detect the differential gene expression among the Control group,LPS group,LPS group and RvD1 group.PCR experiments and Western Blot experiments were used to verify the effects of apoptosis-related genes and MAPK signaling pathway on the anti-inflammatory effect of RvD1.Results: Part One: LPS(100 ng/m L)significantly reduced the THP-1 activity of monocytes after stimulation for 24 hours.ELISA results of the cell supernatant of the Control group,the LPS group(100 ng/m L,24 h),the RvD1 group(100 ng/m L,24 h),the LPS+RvD1 group(100 ng/m L,24 h)showed that: after LPS treatment,the protein levels of TNF-α in the LPS group were significantly increased(P<0.05),the protein level of IL-1β in the LPS group showed an increasing trend but not statistically significant.Under the co-treatment of LPS and RvD1,the protein levels of TNF-α and IL-1β in the LPS+RvD1 group were significantly decreased(P<0.01)(P<0.05).Part Two: The results of m RNA sequencing showed that: among the LPS group versus the Control group,three hundred and seventy-three differential genes were up-regulated and one hundred and seventy-four differential genes were down-regulated in the LPS group.The LPS+RvD1 group versus the LPS group,one hundred and sixteen differential genes were up-regulated and two hundred and ninety-two differential genes were down-regulated in the LPS+RvD1 group.According to differential Gene Ontology(GO)enrichment analysis and hierarchical clustering analysis,the following inflammation-related genes were screened: c AMP response element binding protein-1(CREB1)and BCL-2(BCL-2L12)were down-regulated in LPS group vs.Control group,which were up-regulated in LPS+RvD1 group vs.LPS group.In addition,interleukin 1β(IL1B),TNF-α-inducible protein 6(TNFAIP6),caspase 3(CASP3)and nuclear factor κB subunit 2(NFKB2)were up-regulated in LPS group vs.Control group,but blocked by RvD1 treatment in LPS+RvD1 vs.Control group.According to the distribution of the above-mentioned key differential genes in the KEGG-enriched metabolic pathways,the next part of the experiment focuses on the verification of the secretion levels of these differentially expressed genes and the levels of proteins related to the MAPK signaling pathway.Part Three: PCR results showed that: among the LPS group versus the Control group,the expression of BCL-2m RNA showed a decreased trend but not statistically significant,and the expression of Caspase-3 m RNA significantly increased in the LPS group(P<0.001),while the LPS+RvD1 group versus the LPS group,the expression of BCL-2 m RNA in the LPS+RvD1 group significantly increased(P<0.01),the expression of Casepase-3m RNA significantly decreased(P<0.001).Western Blot results showed that compared with the Control group,LPS group could significantly enhance NF-κB(p65),PI3 K,BCL-2,Caspase-3,My D88,AKT,p38 protein level(P<0.01).Compared with the LPS group,the LPS+RvD1 group could significantly inhibit NF-κB(p65),PI3 K,Caspase-3,Myd88,p38 protein levels after RvD1 treatment(P<0.01)(Myd88,P<0.05),while it could significantly enhance BCL-2(P<0.01),AKT protein level(P<0.05).Conclusion: Under the stimulation of LPS,RvD1 can mediate monocytes to increase the activity of protein kinase B(AKT),as well as the expression of BCL-2and the activity of inhibiting apoptosis cysteine protease-3(Caspase-3).It implies that RvD1 can play an anti-apoptotic effect to a certain extent.RvD1 may play an anti-inflammatory role by regulating the anti-apoptotic gene BCL-2 and Caspase-3,reducing the damage of monocytes and activating the NF-κB(p65),PI3 K,Myd88,p38 pathways in the MAPK signaling pathway.