Autoimmune Hepatitis In Mice Research Articles

Inhibition of miR-431-5p attenuated liver apoptosis through KLF15/p53 signal pathway in S100 induced autoimmune hepatitis mice

AimsThe purpose of this study was to investigate the effects of miR-431-5p on hepatocyte apoptosis in AIH. Materials and methodsWe used intraperitoneal injection of S100 to establish AIH mouse model and injected AAV into tail vein on day 14 of modeling to regulate miR-431-5p expression. The expression of ALT, AST, IgG and apoptosis-related proteins Bax, Bcl-2 and cleaved caspase 3 were measured in each group. Cellular experiments were performed using miR-431-5p mimics or inhibitors to transfect LPS-stimulated AML12 cells, and apoptosis was verified using Western blot and Hoechst 33342/PI Double Staining. The target of miR-431-5p, KLF15, was screened using databases and verified by the luciferase reporter assay. The relationship between KLF15 and p53 was verified by si-KLF15 and PFTβ (a p53-specific inhibitor). Key findingsHere, we observed that the increase in the level of miR-431-5p was accompanied by a decrease in the expression of Krüppel-like zinc finger transcription factor 15 (KLF15). In addition, the deletion of miR-431-5p significantly reduced hepatocyte apoptosis in AIH mice induced by liver S100 and apoptosis of AML12 cells induced by LPS stimulation, accompanied by decreased expression of Bax and cleaved caspase-3 as well as increased expression of Bcl-2. Moreover, KLF15 was the direct and functional target of miR-431-5p. Furthermore, miR-431-5p negatively regulated the expression of KLF15, and KLF15 deletion partially abolished the inhibitory effect of miR-431-5p deletion on apoptosis by activating p53 signaling. SignificanceIn summary, miR-431-5p may be a potential therapeutic target for AIH.

The effects of oral administration of Aureobasidium pullulans-cultured fluid containing β-glucan on concanavalin A injected mice

A black yeast, Aureobasidium pullulans, extracellularly produces β-(1,3), (1,6)-D-glucan (β-glucan) under certain conditions. The β-glucan is known to be an immunomodulatory agent, and β-glucan enriched A. pullulans cultured fluid (AP-CF) is used in supplements to maintain human health. Concanavalin A (ConA) is a lectin, and when injected it is known to cause T cell mediated autoimmune hepatitis in mice. The present study investigated the effects of oral administration of AP-CF on ConA injection in mice. The results demonstrated that increases in serum alanine transaminase (ALT) levels after ConA injection were significantly suppressed in an AP-CF administered group of mice. To understand the mechanism of the ALT lowering effects of AP-CF, we used Foxp3 (forkhead box P3) knock-in mice which express the green fluorescent protein (GFP) in Foxp3 induced cells, and the effects of AP-CF on the regulatory T cell (Treg) populations were investigated. The results show that the basal level of Foxp3+ Treg populations in peripheral blood lymphocytes, liver infiltrating lymphocytes, and splenocytes was decreased after 7 days of administration of AP-CF. These findings suggest that oral administration of AP-CF suppresses the basal level of inflammation, and that it may be postulated to be involved in the ALT lowering effects of AP-CF.

C/EBPα/miR-7 Controls CD4+ T-Cell Activation and Function and Orchestrates Experimental Autoimmune Hepatitis in Mice.

Increasing evidence in recent years has suggested that microRNA-7 (miR-7) is an important gene implicated in the development of various diseases including HCC. However, the role of miR-7 in autoimmune hepatitis (AIH) is unknown. Herein, we showed that miR-7 deficiency led to exacerbated pathology in Concanavalin-A-induced murine acute autoimmune liver injury (ALI) model, accompanied by hyperactivation state of CD4+ T cells. Depletion of CD4+ T cells reduced the effect of miR-7 deficiency on the pathology of ALI. Interestingly, miR-7 deficiency elevated CD4+ T-cell activation, proliferation, and cytokine production in vitro. Adoptive cell transfer experiments showed that miR-7def CD4+ T cells could exacerbate the pathology of ALI. Further analysis showed that miR-7 expression was up-regulated in activated CD4+ T cells. Importantly, the transcription of pre-miR-7b, a major resource of mature miR-7 in CD4+ T cells, was dominantly dependent on transcription factor CCAAT enhancer binding protein alpha (C/EBPα), which binds to the core promoter region of the miR-7b gene. Global gene analysis showed that mitogen-activated protein kinase 4 (MAPK4) is a target of miR-7 in CD4+ T cells. Finally, the loss of MAPK4 could ameliorate the activation state of CD4+ T cells with or without miR-7 deficiency. Our studies document the important role of miR-7 in the setting of AIH induced by Concanavalin-A. Specifically, we provide evidence that the C/EBPα/miR-7 axis negatively controls CD4+ T-cell activation and function through MAPK4, thereby orchestrating experimental AIH in mice. This study expands on the important role of miR-7 in liver-related diseases and reveals the value of the C/EBPα/miR-7 axis in CD4+ T-cell biological function for the pathogenesis of immune-mediated liver diseases.

Formononetin protects against concanavalin-A-induced autoimmune hepatitis in mice through its anti-apoptotic and anti-inflammatory properties.

Autoimmune hepatitis (AIH) is a chronic inflammatory liver disease that seriously threatens the health of humans globally. Formononetin (FMN) is a natural herb extract with multiple biological functions. In this study, an experimental model of AIH was established in mice through the use of concanavalin A (ConA). To investigate the effects of FMN on ConA-induced hepatitis, the mice were pretreated with 50 or 100 mg/kg body mass of FMN. The results show that FMN alleviated ConA-induced liver injury of mice in a dose-dependent manner. Moreover, pretreatment with FMN inhibited the apoptosis of hepatocytes in the ConA-treated mice through downregulating the expression of pro-apoptotic proteins (Bax, cleaved caspase 9, and cleaved caspase 3) and upregulating the expression of anti-apoptotic protein (Bcl-2). It was also found that the levels of proinflammatory cytokines were greatly reduced in the serum and liver tissues of mice pretreated with FMN. Further studies showed that FMN reduced the level of phosphorylated nuclear factor kappa B (p-NF-κB) p65 and enhanced the level of IκBα (inhibitor of NF-κB), suggesting that FMN inhibits the activation of the NF-κB signaling pathway. In addition, FMN inhibited activation of the NOD-like receptor protein 3 (NLRP3) inflammasome. Therefore, FMN could be a promising agent for the treatment of AIH.

Integrated bioinformatic analysis revealed biological processes and immune cells implicated in autoimmune hepatitis.

Autoimmune hepatitis (AIH) is an immune-mediated inflammatory liver disease for which the pathogenesis remains incompletely understood. The current study aimed to reveal key biological processes and immune cells implicated in AIH by integrated bioinformatic analysis. The global gene expression in livers from wild-type BALB/c mice, mice with Tgfb1 deficiency, and mice with both Tgfb1 and Ifng deficiency was assessed by microarray data analysis. Differentially expressed genes were identified and subjected to functional enrichment analysis. AIH mice with Tgfb1 deletion showed significantly enhanced immune responses but impaired metabolic processes, whereas increased T cell activation and cytokine production, but weakened organic acid and lipid metabolic processes were observed in mice with deletion of both Tgfb1 and Ifng. In addition, infiltration of immune cells was evaluated by CIBERSORT. Increased infiltration of T cells, macrophages, and natural killer cells, and decreased infiltration of neutrophils, eosinophils, plasma cells, and B cells were observed in AIH mice. In conclusion, we identified potential biological processes and immune cells that contributed to AIH; further investigations are needed to confirm these findings and thus provide a potential novel therapeutic target for AIH treatment.

BMSCs attenuate hepatic fibrosis in autoimmune hepatitis through regulation of LMO7-AP1-TGFβ signaling pathway.

In a previous study, we reported that transplantation of bone mesenchymal stem cells (BMSCs) significantly attenuated liver damage in a mouse autoimmune hepatitis (AIH) model. Moreover, expression of the LIM domain protein, LMO7, correlated positively with the invasive capacity of hepatoma cells. However, whether LMO7 plays a role in inflammation and fibrosis of AIH remains unknown. This investigation aimed to explore the effect of BMSC transplantation on LMO7 and the role of LMO7 in hepatic fibrosis. S100-induced murine AIH and LPS-induced hepatocyte injury models were successfully established. Three doses of BMSCs were injected into AIH mice via the tail vein. LPS-treated AML12 cells were co-cultured with BMSCs in vitro. Small interfering (si) LMO7 RNA and T5224 (a specific inhibitor of AP-1) were used to demonstrate the relationship between LMO7-AP1-transforming growth factor (TGF)-β. Pathological examination and serum alanine and aspartate aminotransferase levels indicated that liver damage was notably ameliorated in the BMSC-treated mice. LMO7 level was upregulated, while AP-1 and TGF-β levels were downregulated upon intervention with BMSCs. AP-1 expression was upregulated in the siLMO7 group, whereas TGF-β level was downregulated in the T5224 group when compared to those in the control group. BMSC transplantation significantly limits liver fibrosis and upregulates the expression of LMO7. LMO7 inhibits the TGF-β pathway by inhibiting AP-1. This implies that BMSCs are a potential means of treating liver fibrosis. This approach has important implications for the treatment of AIH and other fibrotic diseases.

RIP3 Blockade Drives Myeloid-Derived Suppressor Cell-Mediated Immune Tolerance in Immune-Mediated Hepatitis

Background: Excessive and uncontrolled immune activation plays a deleterious role in liver damage of autoimmune hepatitis (AIH), thus, it`s of great significance to identify the regulation of immune tolerance. Receptor-interacting protein kinase 3 (RIP3) has been found to be down-regulated in tumor tissues and mediates tumor immune evasion. However, the role of RIP3 in AIH remains elusive. We aimed to investigate the role of RIP3 in the regulation of inflammation and immune tolerance in immune-mediated hepatitis (IMH). Methods: RIP3 expression was evaluated in the livers of AIH patients and mice models. Mice were treated with GSK872, a RIP3 specific inhibitor, or glucocorticoid dexamethasone before concanavalin A (ConA)-induced IMH. Real-time PCR, western blotting, and flow cytometry served to explore the molecular mechanisms involved. The depleting anti-mouse Gr-1 antibody and isotype control were used for further determine the role of MDSCs in GSK872 treatment. Findings: AIH patients and mice model displayed pronounced RIP3 activation in the liver tissues. RIP3 blockade by GSK872 prevented ConA-induced IMH by reduced hepatic proinflammatory cytokines and immune cells including Th17 cells and macrophages. Further experiments revealed that RIP3 inhibition resulted in an increase in CD11b+ Gr-1+ MDSCs with immunoregulatory properties in the livers, spleen, and peripheral blood. Moreover, the depletion of Gr-1+ MDSCs abrogated the protective effect and immune suppression function of GSK872 in ConA-induced IMH. Interpretation: RIP3 blockade prevents ConA-induced IMH through a MDSC-dependent mechanism. Inhibition of RIP3 kinase may be a novel therapeutic avenue for AIH treatment. Funding Statement: The study was supported by the National Natural Science Foundation of China (81860109). Declaration of Interests: The authors declare no conflicts of interest. Ethics Approval Statement: This study was approved by the Ethics Committee of Tianjin Medical University General Hospital, and all participants provided signed informed consents. The animal use protocol has been reviewed and approved by the Animal Ethical and Welfare Committee of Tianjin Medical University, Approval No. IRB2020-WZ-175.

GPX4-Regulated Ferroptosis Mediates S100-Induced Experimental Autoimmune Hepatitis Associated with the Nrf2/HO-1 Signaling Pathway.

Autoimmune hepatitis (AIH) is an inflammatory autoimmune disease of the liver. Oxidative stress triggered by reactive oxygen radicals is a common pathophysiological basis for the pathogenesis of many liver diseases, and ferroptosis is associated with the toxic accumulation of reactive oxygen species. The signaling transduction pathways responsible for iron processing and lipid-peroxidation mechanisms are believed to drive ferroptosis. However, the specific mechanisms regulating ferroptosis remain unclear. The aims of this investigation were to identify the possible effector functions of ferroptosis, based on glutathione peroxidase 4 (GPX4) regulation in an S100-induced autoimmune hepatitis mouse model and hepatocyte injury models. The S100 liver antigen-induced AIH mouse model was used to detect ferroptotic biomarkers using western blotting. Upregulated levels of cyclooxygenase2 (COX2) and Acyl-Coenzyme A synthase long-chain family member 4 (ACSL4) were observed in the S100-induced AIH model group, while levels of GPX4 and ferritin heavy chain 1 (FTH1) were downregulated (P < 0.05). The expression profiles of COX2, ACSL4, GPX4, and FTH1 were restored following the administration of ferrostatin-1. In addition, Nrf2 and HO-1 levels in the S100-induced AIH model mice after treatment with ferrostatin-1 were downregulated compared to the nonferrostatin-1-treated S100-induced AIH model mice (P < 0.05). Moreover, COX2 and ACSL4 levels were significantly upregulated, with significant FTH1 downregulation, in the AIH model mice when liver-specific GPX4 was silenced using AAV8 constructs. These data indicate that inhibition of ferroptosis significantly ameliorated the influence of AIH on the Nuclear factor E2-related factor 2 (Nrf2)/Heme oxygenase-1 (HO-1) signaling pathway, and that ferroptosis may act as an initiator or intermediate mediator leading to AIH.

Ubiquitously specific protease 4 inhibitor-Vialinin A attenuates inflammation and fibrosis in S100-induced hepatitis mice through Rheb/mTOR signalling.

Inflammation and fibrosis are major consequences of autoimmune hepatitis, however, the therapeutic mechanism remains to be investigated. USP4 is a deubiquitinating enzyme and plays an important role in tissue fibrosis and immune disease. Vialinin A is an extract from mushroom and is a specific USP4 inhibitor. However, there is lack of evidences that Vialinin A plays a role in autoimmune hepatitis. By employing S100‐induced autoimmune hepatitis in mice and AML12 cell line, therapeutic mechanism of Vialinin A was examined. Inflammation was documented by liver histological staining and inflammatory cytokines. Fibrosis was demonstrated by Masson, Sirius red staining and fibrotic cytokines with western blot and real‐time RT‐PCR. In experimental animal, there were increases in inflammation and fibrosis as well as USP4, and which were reduced after treatment of Vialinin A. Vialinin A also reduced Rheb and phosphorylated mTOR. Moreover, in LPS‐treated AML12 cells, LPS‐induced USP4, inflammatory and fibrotic cytokines, phosphorylated mTOR and Rheb. Specific inhibitory siRNA of USP4 reduced USP4 level and the parameters mentioned above. In conclusion, USP4 was significantly elevated in autoimmune hepatitis mice and Vialinin A reduced USP4 level and attenuate inflammation and fibrosis in the liver. The mechanism may be related to regulation of Rheb/mTOR signalling.

Circular RNAs associated with a mouse model of concanavalin A-induced autoimmune hepatitis: preliminary screening and comprehensive functional analysis.

Without treatment, autoimmune hepatitis (AIH) often leads to cirrhosis, liver failure and, in some cases, death. However, the pathogenesis of AIH remains incompletely understood. Here, we explored the relationship between differentially expressed circular RNAs (DECs) and development of AIH by obtaining an expression profile of DECs in a concanavalin A-induced AIH mouse model by microarray. In total, we identified 27 DECs; the host genes of these DECs were annotated with 140 Gene Ontology terms and 19 pathways, revealing potential roles in the metabolism of cellular ions and regulation of protein expression, as well as possible involvement in endocytosis and apoptosis. We constructed a circular RNA-microRNA network that was used to infer that a mmu_circ_0001520/mmu-miR-193b-3p/MAPK10 network may be associated with the occurrence of AIH. These findings may help lay the foundation for validation of the potential roles of circular RNAs in AIH.

Regulatory T Cells in Autoimmune Hepatitis: Unveiling Their Roles in Mouse Models and Patients.

Autoimmune hepatitis (AIH) is a severe and chronic liver disease, and its incidence has increased worldwide in recent years. Research into the pathogenesis of AIH remains limited largely owing to the lack of suitable mouse models. The concanavalin A (ConA) mouse model is a typical and well-established model used to investigate T cell-dependent liver injury. However, ConA-induced hepatitis is acute and usually disappears after 48 h; thus, it does not mimic the pathogenesis of AIH in the human body. Several studies have explored various AIH mouse models, but as yet there is no widely accepted and valid mouse model for AIH. Immunosuppression is the standard clinical therapy for AIH, but patient side effects and recurrence limit its use. Regulatory T cells (Tregs) play critical roles in the maintenance of immune homeostasis and in the prevention of autoimmune diseases, which may provide a potential therapeutic target for AIH therapy. However, the role of Tregs in AIH has not yet been clarified, partly because of difficulties in diagnosing AIH and in collecting patient samples. In this review, we discuss the studies related to Treg in various AIH mouse models and patients with AIH and provide some novel insights for this research area.

Microarray-based transcriptional profiling of a mouse model of autoimmune hepatitis.

Long noncoding RNAs (lncRNAs) are RNA molecules longer than 200 nucleotides that do not typically code for a protein. lncRNAs have regulatory roles in many physiological processes, and their dysregulation can contribute to cancer, cardiovascular and neurodegenerative diseases, as well as the onset of autoimmune diseases, including systemic lupus erythematosus and rheumatoid arthritis. However, lncRNA expression changes in autoimmune hepatitis (AIH), a form of inflammation induced by immunological tolerance disorders, are poorly understood. Here, for the first time to our knowledge, we used microarrays to profile 1161 differentially expressed lncRNAs (DELs; 608 up- and 553 down-regulated) and 11 512 differentially expressed mRNAs (DEMs; 5189 up- and 6323 down- regulated) in a concanavalin A-induced AIH mouse model. We used quantitative real-time PCR to confirm the expression of eight DELs and DEMs, and analyzed the coexpression relationship between them. Potential biological functions of screened DELs and DEMs were predicted with Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis. DEL-DEM interaction networks were also constructed. Our study revealed the roles of DELs and DEMs in the pathogenesis of AIH. We also provided potential candidate biomarkers that may have potential for future development into possible diagnostics or as a treatment for this disorder.

THU121 - MSCs attenuate cona-induced autoimmune hepatitis in mice through Nrf2/HO-1 pathway by altering macrophage polarization

THU121 - MSCs attenuate cona-induced autoimmune hepatitis in mice through Nrf2/HO-1 pathway by altering macrophage polarization

THU124 - Insufficient deletion of autoreactive CD4 T cells and plasticity of Tregs drive spontaneous autoimmune hepatitis in mice

THU124 - Insufficient deletion of autoreactive CD4 T cells and plasticity of Tregs drive spontaneous autoimmune hepatitis in mice

IL-37 increases liver inflammation in Con A-induced hepatitis by increasing IFN-γ secretion of infiltrated NK Cells

Abstract Liver is an immunologically tolerogenic organ, while, autoreactive immune cells mediated autoimmune hepatitis (AIH) is observed. Suppression of the hyper-reactive immune responses may restore the tolerance of liver. IL-37, an emerging IL-1 family cytokine with anti-inflammatory effects on innate and adaptive immunity, shows benefit on many autoimmune diseases. We proposed IL-37 could inhibit immune responses and improve AIH. IL-37 expressing adeno-associated virus (AAV-IL-37) successfully dampened the activation of macrophages in vitro. We next analyzed the effect of IL-37 in Con A-induced autoimmune hepatitis mouse model. Surprisingly, the liver histopathology showed more necrotic hepatocytes and infiltrating immune cells in AAV-IL-37 treated Con A mice than in control mice. Higher levels of serum IFN-γ in IL-37-expressing Con A mice were observed. Moreover, expression of inflammation-related mRNA in the liver were also highly induced with AAV-IL-37 treatment. Among hepatic lymphoid cells, NK and NKT cells were particularly increased in IL-37 treated Con A mice. The frequency of IFN-γ-secreting NK cells was also significantly increased. Further, CCL5, a chemoattractant for NK cells, was significantly elevated in the serum of AAV-IL-37 injected Con A mice. These results suggest that IL-37 worsen liver inflammation instead of relieving the disease. Of note, there was no obvious inflammation in AAV-IL-37 treated naïve mice, suggesting the proinflammatory effect of IL-37 in Con A treated mice resulted from the inflamed liver microenvironment. In conclusion, IL-37 aggravated liver inflammation in Con A induced hepatitis through recruiting NK cells and elevating the IFN-γ secretion of NK cells.

Montelukast ameliorates Concanavalin A-induced autoimmune hepatitis in mice via inhibiting TNF-α/JNK signaling pathway

BackgroundConcanavalin A (ConA) is a well-established model to induce autoimmune hepatitis (AIH) in mice which mimics pathological alterations that occur in human. The pathogenesis of ConA-induced AIH involves many signaling pathways. Montelukast is a leukotriene receptor antagonist that is mainly used in the management of asthma. The antioxidant, anti-inflammatory and anti-apoptotic effects of montelukast have been reported in previous studies. Lately, montelukast has been documented to confer protection against various inflammatory diseases. Up to date, no study has explored the effect of montelukast on AIH induced by ConA. Aim and methodThis study aims to detect the protective effects of montelukast (10 mg/kg) on ConA (20 mg/kg)- induced AIH in mice and to demonstrate its hepatoprotective mechanisms. Hepatic function, histological changes, oxidative stress, inflammation, autophagy, and apoptotic markers were investigated. ResultsHepatic function and histological data revealed that treatment with montelukast significantly attenuated ConA-induced hepatic damage. Montelukast significantly reduced JNK level and NFκB p65 expression, and inhibited proinflammatory cytokines (TNF-α and IL-6) as well as oxidative stress (MDA, NO, and GSH). Moreover, inflammatory cells (CD4+ infiltration and the levels of apoptotic markers (Bax and caspase-3) besides autophagy biomarkers (Beclin1 and LC3) were reduced. ConclusionOur results suggest that montelukast could be a potential therapeutic drug against the ConA-induced AIH through its anti-oxidant, anti-inflammatory, anti- autophagy as well as anti-apoptotic properties.

Deficiency of O-linked-glycosylation regulates activation of T cells and aggravates Concanavalin A-induced liver injury

ObjectiveProtein glycosylation is involved in immunological recognition and immune cell activation. The role of O-glycosylation in Concanavalin A (Con A)-induced autoimmune hepatitis (AIH) was elucidated in the present study. MethodsMice were intravenously injected with Con A (10 mg/kg) to establish an AIH mouse model. Here, 24 h prior to administration of Con A, experimental mice were intragastrically administrated with O-glycosylation inhibitor (benzyl-α-GalNAc) at doses of 1 and 5 mg/kg, respectively, while control mice were administrated with the same volume of saline. Before and after administration of Con A for 6 and 12 h, mice were sacrificed and their plasma and livers were collected to score liver injury. Peripheral blood, spleen, and thymus were collected for flow cytometry analysis. The expression levels of neutrophilic alkaline phosphatase-3 (NALP3) and NALP6 in liver were evaluated as well. ResultsPre-treatment with benzyl-α-GalNAc increased the serum transaminase levels and induced more infiltration and necrosis in livers of Con A administrated mice. The levels of some pro-inflammation cytokines also increased in administrated mice. In addition, pretreatment with benzyl-α-GalNAc up-regulated the expression levels of NALP3 and NALP6. And benzyl-α-GalNAc inhibited the levels of apoptosis of thymus cells and influenced activation of T cells in peripheral blood and spleen of Con A administrated mice, especially that accelerated the physiological progression of CD4+CD25−CD69+ subset. ConclusionThe present research demonstrated that benzyl-α-GalNAc aggravated Con A-induced AIH, and the role of the O-glycosylation inhibitor as the aggravation may be related to regulation of the levels of cytokines, as well as influencing proliferation of T cells.

Demethyleneberberine attenuates concanavalin A-induced autoimmune hepatitis in mice through inhibition of NF-κB and MAPK signaling

Demethyleneberberine (DMB) is a natural product which has been reported to possess mitochondria-targeting anti-oxidative and anti-inflammatory effect. However, the pharmacological action and molecular mechanism of DMB on autoimmune hepatitis (AIH) have not been explored. In this study, AIH was induced by intravenously injecting Con A (20 mg/kg) in mice for 8 h, and DMB protected against Con A-induced AIH, evidenced by obvious reduction of hepatic enzymes in serum and histological lesion. DMB significantly inhibited the infiltration of CD4+ T cell and Kupffer cell as well as the expression of inflammatory cytokines, such as TNF-α, IL-6, IL-1β and IFN-γ by ELISA and qPCR analysis. Western blotting analysis illustrated that DMB remarkably inhibited Con A-induced phosphorylation of IKK, IκB, NF-κB p65, ERK, JNK, p38 MAPK and STAT3 induced by Con A. Moreover, DMB also effectively suppressed hepatic oxidative stress with reduction of MDA and elevation of GSH. Taken together, our findings indicated that DMB could prevent Con A-induced AIH by regulating NF-κB and MAPK signaling, suggesting that DMB can serve as a promising candidate for therapy of AIH.

Plasma proteomic analysis of autoimmune hepatitis in an improved AIH mouse model

BackgroundThe prevalence of autoimmune hepatitis (AIH) is increasing, and its early clinical diagnosis is difficult. The pathogenesis of AIH remains unclear, and AIH-related studies are largely limited because of lack of suitable mouse models.MethodsTo obtain a good tool for research on AIH, we first established an improved immune-mediated mouse model that can mimic the pathological process of AIH as in the human body, through repeated injections of human cytochrome P450 2D6 (CYP2D6) plasmid. Next, a proteomic analysis based on isobaric tag (IBT) technology was performed to detect the differentially expressed proteins (DEPs), and related biological functions and pathways in the plasma of AIH and normal mice. Finally, we performed enzyme-linked immunosorbent assay (ELISA) to further confirm the most abundant DEP in the plasma of patients with AIH.ResultsAutoantibodies and the characteristic pathology of AIH were observed in our mouse model. Inflammatory infiltration also increased in the livers of AIH mice over time and plateaued by day 42 post the first injection. Chronic hepatitis was most severe on day 35 with the development of fibrosis as well, and the plasma of AIH mice were collected for proteomic analysis. A total of 176 DEPs were found in this experiment, of which 148 DEPs were up-regulated and 28 DEPs were down-regulated. Thirty significant Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (P < 0.05) were detected. Arginine biosynthesis was found to be the most significant pathway involved in the AIH process. During the Gene Ontology (GO) analysis, most DEPs were found to be involved in the binding, cellular, and metabolic processes. Using ELISA, the most overexpressed DEP, serum amyloid A 1 (SAA1), was confirmed to be increased specifically in the plasma of patients with AIH compared to other chronic hepatitis. Different plasma levels of SAA1 were also found related to different grades of inflammation and stages of fibrosis in the liver of patients with AIH.ConclusionsOur study is the first to describe the proteomics analysis of a true sense of AIH mouse model, which is beneficial for a better understanding of AIH pathogenesis and identifying potential biomarkers for its clinical diagnosis.

Characterization and functional prediction of the microRNAs differentially expressed in a mouse model of concanavalin A-induced autoimmune hepatitis.

In order to investigate the altered expression of microRNAs (miRNAs) in the development of autoimmune hepatitis (AIH), the aberrantly expressed miRNAs in the concanavalin A (Con A)-induced AIH mouse model were identified for the first time with microarray in this study. A total of 49 miRNAs (31 up- and 18 down-regulated) were screened out, and the qRT-PCR validation results of 12 chosen miRNAs were consistent with the microarray data. Combined with the profiling of differently expressed mRNAs in the same model (data not shown), 959 predicted target genes (601 for up- and 358 for down-regulated miRNAs) were obtained according to the intersection of databases miRWalk and miRDB, and several hub genes were obtained from the regulatory networks, including Cadm1 and Mier3. These target genes were significantly enriched in the Gene ontology (GO) terms of “transcription, DNA-templated”, and were annotated in 47 signaling pathways, comprising “Wnt signaling pathway”, “Hippo signaling pathway”, “Ferroptosis” and “mitogen-activated protein kinase (MAPK) signaling pathway”, according to the GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. In the miRNA-GO-network, mmu-miR-193b-3p were exhibited in 33 GO terms of biological processes (BP), and the most significantly regulated GO term in BP categories was “regulation of transcription, DNA-templated”. While in the miRNA-pathway-network, mmu-miR-7005-5p were enriched in 37 pathways, which was more than the other specifically expressed miRNAs, and the most significantly enriched pathways were “Endocytosis” and “MAPK signaling pathway”. In conclusion, these differently expressed miRNAs seemed to be associated with the onset of AIH, and have the potential to serve as the new targets on the treatment of this disease.