The Effect And Mechanism Of Artesunate To Reverse LPS Tolerance In Macrophages Via Inhibiting MVDR Internalization

Objective:To clarify the role and molecular mechanism of membrane vitamin D receptor（mVDR）in artesunate（AS）to reverse LPS tolerance in macrophages and to lay a foundation for further elucidate the effect and its molecular mechanism of artesunate to treat sepsis.Methods:1.Effect of artesunate on inflammatory cytokines before release from LPS-tolerant cellsMouse peritoneal macrophages were pretreated with low dose of LPS（5 ng/m L）for 4 h,and then treated with high dose of LPS（100 ng/m L）for a second time to establish LPS tolerance model.The levels of TNF-α,IL-6,IL-1βand IFN-γin the supernatant were detected4 h after the addition of AS2.The effect of artesunate on the distribution of VDR on cell membrane and intracellular2.1 The effects of AS on VDR m RNA and protein expression in LPS-tolerant cells were detected.2.2 The effect of AS on the intracellular distribution of VDR was observed by inverted fluorescence microscopy,laser confocal microscopy and ELISA.2.3 The cytoplasmic protein and nucleoprotein of the cells were extracted,and the concentration of VDR in the cytoplasm and nucleus of the cells was determined by ELISA to determine the influence of AS on the distribution of VDR in the cells.3.The importance of mVDR in the immunomodulatory effects of artesunate3.1 After the cells were treated with anti-VDR antibody,the formation of LPS-tolerantmacrophage model and the influence of AS on the release level of pro-inflammatory cytokines,m RNA expression level and expression level of autophagy-related molecular proteins in the LPS-tolerant macrophage model were detected.3.2.The effect of 1α,25（OH）₂D₃on the release level of TNF-αwas detected after the cellswere treated with anti-VDR antibody.3.3 The lipid raft inhibitor methyl-β-cyclodextrin（MβCD）was added to observe the formation of LPS tolerant macrophage model after lipid raft destruction.3.4.After caveolin-1 si RNA was interfered with the key lipid raft molecule,the effects of AS on VDR internalization,ATG16L1 protein expression and TNF-αrelease level of LPS-tolerant cells were detected.3.5 After incubation with wild VDR peptides H305,H397 and mutant peptides H305A,H397A and AS,the effects of AS on the release level of TNF-αin LPS-tolerant macrophages were detected.4.Effect of transcriptional inhibitors and protein synthesis inhibitors on reverse LPStolerance in macrophages by artesunateThe effect of AS on the level of TNF-αfrom LPS-tolerant macrophages treated withTranscriptional inhibitors（mifepristone）and Inhibitors of protein synthesis（actinomycin D）were detected.Results:1.Artesunate can increase the level of inflammatory cytokines before the release ofLPS-tolerant cellsAfter pretreatment with low dose of LPS（5 ng/m L）for 4 h,and then treated with high dose of LPS（100 ng/m L）for a second time,the release level of pre-inflammatory cytokines（TNF-α,IL-6,IL-1β,IFN-γ）was significantly reduced compared with that of cells stimulated by LPS alone（100 ng/m L）.The model of LPS tolerance was established successfully.After the addition of AS,the release level of pro-inflammatory cytokines was significantly increased compared with that of LPS-tolerant cells,suggesting that AS can improve the level of pro-inflammatory cytokines in LPS-tolerant cells.2.Artesunate can significantly alter the intracellular distribution of VDR2.1.VDR was highly expressed in LPS-tolerant cell models,AS can reduce the level of VDR in LPS-tolerant cells.2.2.As can reduce the level of VDR in the cytoplasm and nucleus of LPS-tolerant cells.3.mVDR is closely related to the effect of artesunate on the increase the level of pro-inflammatory cytokines in LPS-tolerant cells3.1.After treatment with anti-VDR antibody,AS did not increase the release level of pro-inflammatory cytokines and m RNA expression,as well as m RNA and protein expression of autophagy-related molecules in LPS-tolerant cells.3.2.1α,25（OH）₂D₃did not decrease the release level of TNF-αafter treatment with anti-VDR antibody.3.3.Lipid raft is a key pathway for mVDR internalization into cells,the LPS tolerancemodel could not be formed after the lipid raft was destroyed.3.4.After interfering with the expression of key molecules in lipid rafts,intracytoplasmic VDR expression was decreased,and the effect of AS on increasing the expression of autophagy-related molecules in LPS-tolerant cells and the release level of TNF-αdisappeared.3.5 VDR wild peptides H305 and H397 incubated with AS did not increase the release level of TNF-αin LPS-tolerant cells.4.Transcription inhibitors and protein synthesis inhibitors had no effect on artesunate to reverse LPS tolerance in macrophagesTranscriptional inhibitors（mifepristone）and protein synthesis inhibitors（actinomycin D）had no effect on AS to increase the level of TNF-αin LPS-tolerant cells.Conclusions:1.Artesunate significantly increased the release level of pro-inflammatory cytokines in LPS-tolerant cells;2.Artesunate can alter the distribution of VDR on the cell membrane and intracellular,and inhibit mVDR internalization and nuclear translocation.3.The addition of anti-VDR antibodies,wild VDR peptides containing H305 and H397together with artesunate inhibited the effects of artesunate,H305A mutant peptide had no such effects.4.The formation of LPS tolerance model was significantly inhibited by lipid raft interference,leading to the disappearance of artesunate effect;5.Transcription inhibitors and protein synthesis inhibitors had no significant effect on the effects of artesunate;6.The effect of artesunate on the reversal of LPS tolerance is closely related to its inhibition of mVDR internalization..