| Research BackgroundThe importance of the liver in the human body is self-evident.In addition to digestive functions,the liver plays an irreplaceable role in nutrient metabolism,immune defense,and blood volume regulation.However,the incidence of various liver diseases continues to rise worldwide,and a variety of acute and chronic liver diseases such as cirrhosis and viral hepatitis are seriously damaging human health.Acute liver injury is a key part of the development of many liver diseases,and it is both a direct cause of acute liver disease and an important aspect of the ongoing exacerbation of chronic liver disease.A variety of causes,including excessive alcohol consumption,drug abuse,and viral infections,can cause acute liver injury,resulting in a widespread and uncontrollable inflammatory response in the liver that impairs the normal function of liver cells.Severe acute liver injury can easily develop into acute liver failure,leading to severe destruction of liver cells and high mortality rate of patients,for which there is no effective treatment other than liver resection and liver transplantation.Therefore,it is important to study the pathogenesis of acute liver injury,to find new clinical treatment targets,and to provide a basis for early intervention.The pathogenesis of acute liver injury cannot be separated from the extensive and uncontrollable inflammatory response in the liver.When the liver is exposed to external stimuli,a variety of intrinsic immune cells in the liver quickly respond by secreting a large number of cytokines such as IL-1β and IL-6 to regulate hepatic homeostasis,however,when the stimuli are too strong,the inflammatory response in the liver will be out of balance,resulting in acute liver injury.Intrinsic immune cells in the liver include hepatic Kupffer cells,i.e.,hepatic intrinsic macrophages,natural killer cells,dendritic cells,etc.Among them,hepatic Kupffer cells are the largest population of intrinsic macrophages in the human body and are the most numerous and powerful intrinsic immune cells in the liver.In addition to immune cells,other cells in the liver are also involved in the regulation of the inflammatory response in acute liver injury.Hepatic stellate cells,also known as lipid storage cells,are rich in lipid droplets at rest,and the major components of lipid droplets are triglycerides and vitamin A esters.In fact,70%of the body’s vitamin A is stored in the liver,where it is mainly stored in the form of vitamin A esters in the lipid droplets of stellate cells.ATRA is an important immunomodulatory factor and has a wide range of roles in various systemic diseases.It has been clearly demonstrated that ATRA can regulate the secretion of multiple inflammatory factors by a variety of immune cells.For example,ATRA enhances the secretion of IL-1β in cells such as skin mast cells and alveolar macrophages.After acute liver injury,the concentration of ATRA increases rapidly and is accompanied by the loss of stellate cell lipid droplets.However,it is not clear whether ATRA is involved in the interaction between hepatic stellate cells and Kupffer cells to regulate the progression of acute liver injury.The pathogenesis of acute liver injury cannot be separated from the cellular interactions,and multiple cells in the liver interact with each other to regulate the disease process of liver injury.Hepatic stellate cells and Kupffer cells have been reported to play an integral role in regulating inflammation in various liver diseases.Meanwhile,our preliminary findings also revealed that the interaction between hepatic stellate cells and wither cells is involved in acute liver injury.On the one hand,hepatic stellate cells are a source of several soluble immunoreactive factors,including cytokines,chemokines,or exosomes,which can activate Kupffer cells under acute liver injury conditions.On the other hand,IL-1α/β secreted by Kupffer cells can enhance the survival of hepatic stellate cells in vitro and in vivo and enhance their pro-inflammatory effects.Therefore,the impact of the interaction between hepatic stellate cells and Kupffer cells on liver diseases should not be underestimated.However,the specific significance and mechanism of the interaction between hepatic stellate cells and Kupffer cells in acute liver injury is not clear.In summary,this study was divided into three main parts.In the first part,we used a mouse model of acute liver injury and a primary cell culture system in vitro to clarify the activation status of stellate cells and the level of ATRA secretion in acute liver injury.In the second part,we examined the effect of ATRA on the secretion level of inflammatory factors in Kupffer cells by combining transcriptome sequencing with various molecular biology tools.In the third part,we investigated the intracellular mechanism of ATRA regulation on the secretion of inflammatory factors in Kupffer cells through cellular experiments.This study reveals the intracellular mechanisms and interactions between stellate cells and Kupffer cells regulated by ATRA from a new perspective,providing new potential targets and corresponding theoretical basis for the treatment of acute liver injury.Part I.Activation and secretion of ATRA by hepatic stellate cells in acute liver injuryAimATRA of hepatic stellate cell origin has the function of regulating the secretion of cytokines by various immune cells,but the activation status of stellate cells and the level of ATRA secretion in acute liver injury have not been clearly reported.The aim of this study was to investigate whether high levels of activation of stellate cells and secretion of ATRA in stellate cells can occur in acute liver injury.Methods1.Detection of stellate cell activation in acute liver injury modelA mouse model of acute liver injury was established by intraperitoneal injection of CC14,and α-SMA was stained in the liver sections of the modeled mice and control mice by immunofluorescence staining technique.The serum of acute liver injury model mice and control mice were collected,and the serum levels of IL-1β secretion were detected by Elisa technique and statistically analyzed.2.Construction of stellate cell culture system in vitroStellate cells were extracted from normal mice at rest using primary cell extraction technology and cultured in vitro using a dedicated primary culture system.RT-qPCR and Western blot were used to monitor the relative expression of activation-related proteins in the in vitro culture of stellate cells,and statistical analysis was performed.3.Detection of stellate cell lipid droplet loss and ATRA secretion levelThe level of lipid droplet loss during activation of primary stellate cells was observed by oil red staining.The total RNA and total protein of primary stellate cells were extracted,and the expression of key enzymes of vitamin A ester catabolism was detected by qPCR and Western blot.The serum of acute liver injury model mice and the supernatant of in vitro cultured stellate cells were extracted,and the levels of ATRA were measured by liquid chromatography tandem mass spectrometry and statistically analyzed.Results1.Stellate cells were significantly activated in the mouse model of acute liver injuryThe expression of α-SMA was significantly increased in the liver of mice with acute liver injury,which means that stellate cells were significantly activated in the mouse model of acute liver injury.2.In vitro mock stellate cell activation model establishmentWe extracted mouse liver stellate cells and cultured them in vitro to establish an in vitro model of stellate cell activation.The primary cells were firstly stained by Desmin and it was confirmed that the proportion of stellate cells was more than 90%in the extracted cells.The expression of stellate cell activation markers such as α-SMA,Collagen I,and Fibronectin at the mRNA and protein levels also increased significantly with the increase of culture days,which implied the successful establishment of the stellate cell activation model in vitro.3.The activation of stellate cells was accompanied by the loss of lipid droplets and the release of ATRAOil red staining of primary stellate cells at different culture days revealed that a significant decrease of lipid droplets in stellate cells occurred with increasing culture days.The content of ATRA,a lipid droplet metabolite of stellate cells,increased significantly,which was consistent with the serum results in the acute liver injury model.Real-time fluorescence quantitative PCR with Western blot technique confirmed that the expression of Aldhla3 in the cells at both mRNA and protein levels increased significantly with the activation of stellate cells.Part Ⅱ ATRA promotes IL-1β secretion by Kupffer cells through RAR receptors to exacerbate acute liver injuryAimOur previous study demonstrated that stellate cells significantly activate and lose lipid droplets and secrete ATRA in acute liver injury,and the aim of this study was to investigate whether ATRA could affect acute liver injury by influencing the secretion of inflammatory factors by Kupffer cells,and to investigate its mode of action.Methods1.To detect the effect of ATRA on the synthesis and secretion of inflammatory factors in Kupffer cellsTranscriptome sequencing was used to detect the transcriptome expression of THP-1 cell line after ATRA stimulation.The relative expression of IL-1β at the mRNA and protein levels was measured by qPCR and Western blot.To detect the secretion of IL-1β in serum and cell culture supernatant using Elisa technique.2.To investigate the receptors of ATRA to promote IL-1β secretion in Kupffer cells and the effect on acute liver injuryThe expression of RAR receptor and PPARδ receptor in THP-1 cells was detected by Western blot technique.Stimulation of macrophages using agonists and inhibitors of RAR receptor or PPARδ receptor,and detection of IL-1β expression at mRNA level and protein level by qPCR and Western blot techniques.The secretion of IL-1β in cell culture supernatant and serum was detected by Elisa.Liver sections of mice after drug intervention were stained by HE staining to observe hepatocyte injury.The levels of ALT in serum were measured by ALT assay kit.Results1.Hepatic stellate cell-derived ATRA increased IL-1β production in vivo and in vitro in Kupffer cells1.1 Transcriptome sequencing suggests that ATRA can promote IL1β synthesis in macrophage cell linesATRA was stimulated using 5μM concentration for 24 hours,and whole transcriptome sequencing was performed after RNA collection.The results showed that 950 genes were significantly up-regulated and 1517 genes were significantly down-regulated in the ATRA-treated group compared to the control group,and among the genes with the most significant expression changes,IL1β expression was significantly up-regulated.1.2 ATRA can promote IL1β synthesis and secretion in vitro in Kupffer cellsMouse primary Kupffer cells were extracted and confirmed by F4/80 staining,and Kupffer cells accounted for more than 95%of the extracted primary cells.The mRNA and protein expression of IL1β were significantly increased after ATRA stimulation by real-time fluorescence PCR and Western blot assay.The expression of IL-1β in Kukui cells was increased by ATRA stimulation for 0,4,8,12,24,and 36 h.The Elisa technique confirmed that the secretion level of IL-1β in Kukui cells increased significantly with the increase of ATRA stimulation time,and it climbed more significantly after 24 h.The expression level of IL-1β in Kukui cells was also increased by ATRA stimulation time.The level of IL-1βsecretion in Kupffer cells increased significantly with the increase of ATRA stimulation time and climbed more significantly after 24 hours.The co-culture technique mimics the effect of stellate cell activation products on Kutcher cells in vitro,and the Elisa results confirm that the level of IL-1β in the co-culture supernatant increases significantly after 24 hours of co-culture.1.3 ATRA can promote the secretion of IL1β in vivoATRA gavage group showed a significant increase in serum IL1-β compared with the normal group,confirming that ATRA can promote the secretion of IL-1β in vivo.2.ATRA exerts pro-IL-1β effects through RAR receptors and exacerbates acute liver injury2.1 Agonists or inhibitors of RAR and PPARδ can agonize or inhibit the action of ATRAIn macrophages,both types of receptors are expressed at high levels,with RAR receptor expression dominated by the RARa subtype and PPARδ receptor expression levels comparable to those of RAR receptors.ATRA with the RAR agonist TTNPB can increase the expression of CYP26A,a recognized downstream target gene of the RAR receptor,while the RAR inhibitor AGN193109 can significantly inhibit the effect of ATRA on RAR receptor agonism.Similarly,ATRA with GW501516 could increase the expression of PDK4,a recognized downstream target gene of PPARδ receptor,while DG172 could significantly inhibit the agonistic effect of ATRA on PPARδ receptor.The upregulation of CYP26A was significantly greater than that of PDK4 in the ATRA group compared to the control group.2.2 In vitro,ATRA exerts pro-IL-1β effects mainly through RAR receptors,but not through PPARδ receptorsIn the presence of the RAR agonist TTNPB,IL1β expression was significantly elevated,while the RAR receptor inhibitor AGN193109 significantly inhibited the IL-1β-promoting effect of ATRA.In contrast,GW501516,an agonist of PPARδ,did not increase IL-1βexpression,and DG172,an inhibitor,did not reduce the increase of IL-1β expression under the effect of ATRA.protein expression levels of IL-1β were detected by Western blot technique,and the results were consistent with mRNA expression levels.in THP-1 and Kupffer cells,ATRA and Both ATRA and TTNPB could promote IL-1β secretion at the same level,while AGN193109 could also significantly inhibit the promotion of IL-1β secretion by ATRA.The expression of RARa receptor in macrophages was knocked down using viral transfection,and we found that the ability of ATRA to promote IL-1β synthesis and secretion was dramatically reduced in the RARα knockdown group of cells.2.3 In vivo,ATRA promotes IL-1β secretion and exacerbates acute liver injury through activation of RAR receptorsThe CC14+ATRA-treated group showed a more significant increase in IL-1β secretion compared with the CC14 group,while the CC14+AGN193109 group showed a significant decrease in IL-1β secretion compared with the CC14+AGN193109 group.The CC14+ATRA-treated group showed a more significant increase in ALT release compared to the CC14 group,while the CC14+AGN193109 group showed a significant decrease in ALT release,and the CC14+ATRA-treated group showed a more significant and extensive ballooning and lysis necrosis of hepatocytes compared to the CC14 group,while the CC14+AGN193109 group showed a significant decrease in hepatocyte injury.Part Ⅲ.ATRA-RAR exacerbates acute liver injury by activating NLRP3 in Kupffer cellsAimWe have demonstrated that in acute liver injury,hepatic stellate cells are activated and secrete ATRA,which further exacerbates liver injury by promoting IL-1β synthesis and secretion in Kupffer cells.This part of the study aims to reveal the specific mechanism of ATRA promotion of IL-1β secretion in Kupffer cells through the detection of inflammatory signaling pathways in Kupffer cells,and to provide potential targets for clinical intervention.Methods1.To investigate the effect of ATRA on NLRP3 in Kupffer cellsThe expression changes of NLRP3 and Caspase-1 at mRNA and protein levels were detected by qPCR and Western blot techniques.The secretion of IL-1β in cell supernatant was detected by Elisa.LDH assay kit was used to detect the amount of intracellular LDH,which represents the survival rate of the cells.Morphological features of macrophage death after drug stimulation were observed and photographed under light microscope.2.Probing the specific signaling pathway of ATRA activation of NLRP3 in Kupffer cellsTo detect the amount of ROS in macrophages after ATRA stimulation using ROS assay kit.To detect the secretion of IL-1β after ROS scavenger treatment using Elisa.To detect the relative expression of Akt and mTOR proteins and their phosphorylation levels by Western blot technique.THP-1 cells were transfected by LC3-GFP-RFP virus,and cellular autophagy was determined by observing fluorescence.Results1.ATRA activates macrophage NLRP3 inflammatory vesicles via RAR receptors,leading to cell scorching1.1 ATRA can drive the initiation process of NLRP3 in macrophagesATRA can increase the expression of IL-1β,NLRP3,and Caspase-1 at the mRNA level in macrophages as detected by real-time fluorescence quantitative PCR,while AGN193109 can block the effect of ATRA to some extent.1.2 ATRA promotes IL-1β secretion dependent on the activation of NLRP3 inflammatory vesiclesATRA can increase the expression of IL-1β,NLRP3,and Caspase-1 at the protein level,while AGN193109 can significantly block the effect of ATRA.There was also a significant increase in Cleaved Caspase-1,the active form of Caspase-1,in response to ATRA.Elisa assays confirmed that ATRA can significantly increase IL-1β secretion from macrophages,which can be inhibited by AGN193109.An inhibitor of NLRP3 activation,MCC950,was used,and it was found that MCC950 could completely block the IL-1β-promoting effect of ATRA,confirming that the promotion of IL-β secretion by ATRA is completely dependent on the NLRP3 inflammatory vesicle pathway.1.3A NLRP3 activation promotes macrophage focal death and burst release of IL-1βBy LDH assay,a significant death of macrophages was found to occur after 12 hours of ATRA stimulation.There was a significant increase in the expression of N-GSDMD at the protein level in response to ATRA stimulation,implying that macrophage death due to ATRA may be caused by focal death.VX765,an inhibitor of pyroptosis and caspase-1,was used to block the effect of ATRA,and it was confirmed that the macrophage death caused by ATRA was significantly reduced under the effect of VX765.Under light microscopy,it was clearly observed that ATRA could cause significant edema of macrophages,which is a hallmark of cell scorch death.Under the blocking effect of VX765,the secretion of IL-1β decreased dramatically.2.ATRA-RAR activates the AKT-mTOR pathway and inhibits cellular autophagy,leading to the accumulation of ROS,which activates NLRP3 inflammatory vesicles2.1 ATRA promoted the excessive accumulation of ROS in macrophagesExcessive ROS accumulation in macrophages occurred after 4 h of ATRA treatment.Statistical analysis of intra-image fluorescence intensity revealed that the intracellular ROS content after ATRA treatment was about 1.2 times higher than that of the control group.The effect of blocking ROS using Mito-Tempo,a scavenger of ROS,revealed that the promotion of IL-1β secretion by ATRA was completely blocked in the presence of Mito-Tempo.2.2 ATRA activated the Akt-mTOR pathway through the RAR receptorThe Akt-mTOR pathway was detected by Western blot,and it was found that the phosphorylation levels of Akt and mTOR were significantly increased in the presence of ATRA,implying that ATRA could activate the Akt-mTOR pathway.Using viral knockdown of RARa in macrophages and then giving cells ATRA stimulation,the results confirmed that ATRA activation of the Akt-mTOR pathway is dependent on the RAR receptor.2.3 ATRA blocked the autophagic flow in macrophagesAfter ATRA stimulation,the P62 protein clearly decreased in macrophages,while LC2BII had a significant increase,which means that early autophagy was normal,while late autophagy was inhibited and autophagic flow was blocked.LC3-GFP-RFP virus was transfected into macrophages and observed by confocal microscopy,and it was found that in the control group,the green fluorescence of LC3 protein disappeared and the intracellular autophagic flow was normal,while autophagic flow was blocked in the ATRA group.Conclusions1.Through the study of a mouse model of acute liver injury,it was confirmed that stellate cells undergo activation and loss of lipid droplets to release ATRA during acute liver injury.2.In acute liver injury,stellate cell-derived ATRA can activate NLRP3 inflammatory vesicles by activating RAR,activating the Akt-mTOR pathway,leading to disruption of cellular autophagic flow and excessive accumulation of ROS.3.Stellate cell-derived ATRA promotes the onset of pyroptosis and burst release of IL-1β by facilitating the initiation and activation of NLRP3.4.In hepatic blastocytes,ATRA exacerbates the inflammatory response to acute liver injury and exacerbates the development of liver injury through the above mechanisms. |
PDF Full Text Request