Effects And Underlying Mechanisms Of Hyperglycemia-triggered ATF6-CHOP Pathway Of Kupffer Cell On Liver Ischemia/Reperfusion Injury

Hyperglycemia is an adverse risk factor for hepatic ischemia reperfusion injury(IRI)during liver transplantation or hepatectomy;however,the underlying mechanism remains unclear.Our previous studies implicated endoplasmic reticulum(ER)stress in the pathophysiology of hepatic IRI.In this study,we addressed the question of whether and how hyperglycemia triggered ER stress and affected liver IRI.Diabetic patients and streptozotocin(STZ)-induced diabetic mice were involved in vivo.Bone marrow-derived macrophages(BMDMs)were used in vitro.The ER stress-ATF6-CHOP signaling pathway was specifically activated in liver tissues and Kuppfer cells(KCs)from diabetic patients and STZ-induced diabetic mice.The ER stress inhibitor,4-phenylbutyrate(PBA),attenuated hyperglycemia-related liver IRI based on biochemistry and histology,and decreased TLR4-related pro-inflammatory responses based on inflammatory cytokines and macrophage/neutrophil trafficking.Furthermore,CHOP-knockout(CHOP-KO)had markedly reduced hyperglycemia-related liver IRI and inflammatory responses.Importantly,expression of β-catenin,as a negative regulator of inflammatory responses,was effectively inhibited,accompanied by activation of ATF6-CHOP signaling in diabetic mice,and was almost restored in liver tissues from PBA-treated or CHOP-KO diabetic mice.Furthermore,β-catenin small interfering RNA(si RNA)targeting KCs abrogated the CHOP-KO-related protective effects against hyperglycemia-related liver IRI.High glucose(HG)specifically activated ATF6-CHOP signaling,inhibited β-catenin expression,and enhanced TLR4-related inflammatory responses in BMDMs in vitro;these effects were partly reversed in PBA-or CHOP si RNA-treated/CHOP-KO BMDMs.However,the anti-inflammatory functions of CHOP-KO were almost abolished by β-catenin si RNA in BMDMs under HG conditions.In conclusion,this study revealed that hyperglycemia specifically triggers ER stress-ATF6-CHOP signaling,inhibits β-catenin activity,promotes inflammatory responses,and exacerbates liver IRI,which might provide the rationale for novel therapeutic strategies aimed at managing diabetic-related liver surgery.The First Part: Hyperglycemia Triggered ATF6-CHOP pathway regulates β-Catenin signaling during Liver Ischemia/Reperfusion Injury in vivoWild-type C57BL/6 mice were injected with multiple injections of low-dose STZ.Hyperglycemia was confirmed in the treated mice at day 14 post-STZ injection.Hyperglycemia specifically triggered ER-stress-related ATF6-CHOP signaling in liver tissues KCs.Compared with untreated diabetic mice,PBA significantly alleviated ischemia-induced liver injury at 0 and 6 h,but not at 24 h after reperfusion,as demonstrated by significantly decreased levels of s ALT and s AST,improved liver architecture,reduced the frequency of TUNEL-positive cells,and repressed pro-inflammatory gene expression and macrophage/neutrophil recruitment.CHOP-deficient and CHOP-proficient hyperglycemia mice were subjected to liver IRI model.CHOP deficiency markedly attenuated hyperglycemia-enhanced s ALT and s AST after I/R.CHOP-deficient relieved the damage of liver tissue structure,acted as an anti-apoptoic role,and significantly reduced pro-inflammatory responses.CHOP disruption affected macrophage and neutrophil trafficking after liver ischemia in diabetic mice.Hyperglycemia significantly decreased β-catenin expression accompanied by ATF6-CHOP activation.However,CHOP deficiency effectively restored hyperglycemia-inhibited β-catenin expression in control diabetic mice.We analyzed the role of β-catenin in this process by disrupting β-catenin expression in CHOP-deficient livers using a mannose-linked β-catenin si RNA in vivo.The mannose-linked fluorescence-labeled si RNA was efficiently transduced into KCs in the liver,followed by liver partial warm ischemia model.β-catenin si RNA significantly enhanced pro-inflammatory cytokines secretion,and exacerbated hepatic IRI.The Second part: How Hyperglycemia Triggered ATF6-CHOP pathway Regulates β-catenin Signaling Pathways in vitroBMDMs were differentiated in high-glucose(30 mM)or low-glucose(5 mM)conditions for 7 days.High-glucose specifically triggered m RNA and protein expression of ATF6 and CHOP,but not IRE1 a,XBP1,PERK,or ATF4 compared with low glucose.BMDMs exposed to high-glucose conditions for 7 days were pre-incubated with PBA for 12 h,and then stimulated with LPS for 24 h.BMDMs differentiated under high-glucose conditions produced significantly lower levels of pro-inflammatory TNF-α and IL-6(at 6,12,and 24 h),but higher levels of IL-10(6 and 12 h)in PBA-treated cells compared with untreated cells.Western Blots indicated β-catenin was rapidly inactivated(at both 2 and 6 h)and then gradually recovered(at both 12 and 24 h),but β-catenin expression was stabilized by PBA after LPS treatment.PBA markedly enhanced LPS-phosphorylated Akt(Ser473)between 6 and 24 h,as demonstrated by western Blot.In contrast,NF-κB p65 was decreased in BMDMs at different times after LPS treatment in the PBA group compared with the control group.CHOP si RNA successfully knocked down high-glucose-triggered CHOP gene expression,and effectively enhanced β-catenin gene expression(about 3-fold)under high-glucose conditions.High-glucose-stressed BMDMs produced lower levels of TNF-α and IL-6 and higher levels of IL-10 in response to LPS stimulation in the CHOP si RNA group compared with the NS si RNA group.BMDMs isolated from CHOPFL/FL and CHOP-/-mice were cultured in low/high-glucose conditions.High-glucose effectively inhibited β-catenin expression in CHOPFL/FL BMDMs,but CHOP deficiency markedly reversed the inhibition of β-catenin expression,which was then silenced by β-catenin si RNA.High-glucose-enhanced expression levels of TNF-α and IL-6 were decreased in CHOP-deficient BMDMs,while high-glucose-inhibited expression of IL-10 was increased after LPS treatment.β-catenin si RNA almost abolished the anti-inflammatory function of CHOP deficiency in the above conditions.Moreover,CHOP deficiency restored p-Akt expression,but this effect was neutralized by β-catenin si RNA after LPS treatment in high-glucose-derived BMDMs,as demonstrated by western Blot.The opposite effect was observed in relation to NF-κB protein expression.