Molecular Mechnism Of Artesunate Interacts With Vitamin D Receptor To Reverse Sepsis Immunosuppression

BackgroundSepsis immunosuppression is the leading cause of death in sepsis patients because of the decreasing of the body’s resistance to secondary infection after the cytokine storm phase.Decreased reactivity of innate immune cells to pathogen stimuli is the main charateristic of immunosuppression,among which the LPS-tolerance state of macrophages is the most typical one.LPS-tolerance is a special cellular state formed after long-term exposure to LPS.Under this state,the reactivity of macrophages to the secondary LPS stimulation is reduced,which is manifested by the significantly reduced release of pro-inflammatory cytokines（TNF-α,IL-6）.Autophagy,a highly conserved self-protection mechanism in eukaryotes,is closely related to the immune system and plays an important role in regulating the release of proinflammatory factors and the clearance of pathogens.Calcium(Ca²⁺),an important regulator that controls various stages of autophagic flux,can either promote autophagy or inhibit autophagy.Thus,Ca²⁺signal may be a feasible strategy to up-regulating autophagy,which contributes to restore the ability of releasing pro-inflammatory factors and killing pathogens in LPS-tolerant macrophages.Our previous studies have found that intravenous injection of artesunate in cecal ligation puncture（CLP）-induced sepsis immunosuppression mouse model significantly increased the levels of the pro-inflammatory cytokines（TNF-α,IL-6）in the blood,spleen and lungs,reduced the number of bacteria in the blood,spleen and lungs,and improved the survival rate of immunosuppressive mice.The mechanism of artesunate reversing sepsis immunosuppression is closely related to the regulation of autophagy through the genetic effect of nuclear vitamin D receptor（VDR）.VDR exists in a variety of cells in two forms:membrane VDR（m VDR）and nuclear VDR（n VDR）,mediating the rapid and genetic effects of vitamin D respectively,and participating in the regulation of calcium and phosphorus metabolism,cell apoptosis,immune response and autophagy.Previous studies have shown that mVDR can mediate the rapid effect of vitamin D,regulate Ca²⁺/calmodulin protein kinaseⅡ（Ca MKⅡ）activation and downstream calcium channels,and then affect the autophagy signaling pathway related to cellular immune regulation.Therefore,this study focused on the rapid effect of mVDR,and explored the molecular mechanism of artesunate reversing sepsis immunosuppression by using LPS-tolerant peritoneal macrophages as an immunosuppression model.ObjectiveTo establish the model of LPS-tolerant mouse peritoneal macrophages,and investigate the molecular mechanism of artesunate reversing LPS tolerance based on the rapid effect of mVDR.Methods1.Establishment and autophagy characterization of LPS-tolerance model of mouse peritoneal macrophagesMouse peritoneal macrophages（PMs）were isolated from BALB/c mice;the adherent PMs were treated with LPS（5 ng/m L）for 4 h to induce LPS-tolerance,then the culture supernatants were replaced and added with LPS（100 ng/m L）for 4 h to verify the LPS-tolerance model via detecting TNF-αand IL-6 by ELISA;changes of phagocytosis and bactericidal abilities in LPS-tolerant PMs were detected by CFU assay;autophagy levels of LPS-tolerant PMs were detected by Western blotting and immunofluorescence assay;Fluo-4 AM（calcium ion probe）and calcium-free DMEM were used to detect the changes and sources of calcium ions in LPS-tolerant PMs.2.The effect of artesunate on regulating autophagy of LPS-tolerant PMsThe effect of artesunate and autophagy inhibitor 3-MA on TNF-αand IL-6 release in LPS-tolerant PMs was detected by ELISA;the effect of artesunate and autophagy inhibitor3-MA on bactericidal ability in LPS-tolerant PMs was detected by CFU assay;the effect of artesunate on autophagy level（autophagy flux was evaluated using lysosome inhibitor Bafilomycin A1）,Ca MKⅡactivation in LPS-tolerant PMs was detected by Western blotting and immunofluorescence assay;the effect of artesunate on intracellular calcium ion in LPS-tolerant PMs was detected by Fluo-4 AM;the relationship between artesunate’s effect and CaMKⅡ-IP3R-Ca²⁺pathway was determined by using KN-93（CaMKⅡinhibitor）,2-APB（IP3R blocker）and BAPTA(Ca²⁺chelator);the relationship between artesunate’s effect and m VDR in LPS-tolerant PMs was determined by using VDR antibodyand agonist 1α,25（OH）₂D₃.3.Mechanism of artesunate on restoring autophagy of LPS tolerant PMsULK1 inhibitor ULK-101 was used to determine the relationship between artesunate’s effect and ULK1 in LPS-tolerant PMs;the relationship between artesunate’s effect and different phosphorylation sites of ULK1 was determined by using ULK1（Ser317,Ser637,Ser757）phosphorylation antibodies;ULK1-related calcium pathway inhibitors（m TOR inhibitor Rapamycin,AMPK inhibitor Compound C,CaMKKβinhibitor STO-609）were used to further clarify the molecular mechanism of artesunate restoring autophagy of LPS tolerant PMs through the intervention of corresponding kinase activation.Results1.The results of establishment and autophagy characterization of LPS-tolerance model of mouse peritoneal macrophagesAfter LPS 5 ng/m L induction for 4 h and LPS 100 ng/m L secondary stimulation,the pro-inflammatory cytokines（TNF-α,IL-6）release compared with normal PMs stimulated by LPS 100 ng/m L alone was significantly reduced,suggesting that the LPS-tolerance model was successfully established;the characteristics of LPS tolerant PMs include:significantly reduced in bactericidal ability;autophagy level significantly decreased,and autophagy function could not be induced by LPS;calcium ion level was significantly increased,and calcium ion was mainly released from endoplasmic reticulum（ER）IP3channel.2.The results of the effect of artesunate on regulating autophagy of LPS-tolerant PMsArtesunate enhanced the ability of LPS-tolerant PMs to release pro-inflammatory cytokines（TNF-α,IL-6）and kill bacteria under the secondary stimulation of LPS,but this effect was antagonized by autophagy inhibitor 3-MA;artesunate enhanced the ability of TNF-αand IL-6 release and bacteria clearance by restoring the autophagy induction of LPS-tolerant PMs;artesunate restored autophagy of LPS-tolerant PMs by suppressing CaMKⅡhyperphosphorylation,then reducing the calcium release of endoplasmic reticulum IP3 channel;the effect of artesunate on regulating autophagy in LPS-tolerant PMs could be blocked by VDR antibody and agonist 1α,25（OH）₂D₃.3.The results of mechanism of artesunate on restoring autophagy of LPS-tolerant PMsULK1 inhibitor ULK-101 could significantly inhibit the effect of artesunate on restoring autophagy and TNF-αrelease in LPS-tolerant PMs;arteunate inhibited phosphorylation of ULK1-Ser637（autophagic inhibitory site,activated by AMPK hyperphosphorylation）,thereby restored the initiation of autophagy by LPS-induced ULK1-Ser317（autophagy promoter site,activated by AMPK normal phosphorylation）;arteunate inhibited phosphorylation of ULK1-Ser637 by suppressing AMPK phosphorylation incompletely（from hyperphosphorylation to normal phosphorylation）via Ca²⁺-Ca MKKβ-AMPK pathway.Conclusions1.LPS 5 ng/m L induction for 4 h is an effective method to establish the model of LPS-tolerance in PMs;in addition to the decreased release of pro-inflammatory cytokines,the characteristics of LPS-tolerant PMs include the weakened ability to kill bacteria,the decreased level of autophagy,and the increased level of intracellular calcium ions,which are mainly from the endoplasmic reticulum IP3 channel.2.Artesunate can inhibit CaMKⅡ-IP3R-Ca²⁺pathway via m VDR,reduce the release of calcium ions from the endoplasmic reticulum IP3 channel,and then restore the autophagy of LPS-tolerant PMs,thereby increasing pro-inflammatory cytokines release and enhancing the bactericidal ability.3.Artesunate inhibits AMPK hyperphosphorylation incompletely via Ca²⁺-Ca MKKβ-AMPK pathway,which results in dephosphorylation of ULK1-Ser637（autophagic inhibitory site,activated by AMPK hyperphosphorylation）,and then recovers the initiation of autophagy by LPS-induced ULK1-ser317（autophagy promoter site,activated by AMPK normal phosphorylation）.