Essential role of the adhesion receptor LFA-1 for T cell-dependent fulminant hepatitis

Viral hepatitis affects more than 2 billion people worldwide. In particular, no effective treatment exists to abrogate death and liver damage in fulminant hepatitis. Activation of T cells is an initial and critical event in the pathogenesis of liver damage in autoimmune and viral hepatitis. The precise molecular mechanisms that induce T cell-mediated hepatocyte injury remain largely unclear. In mice, T cell-dependent hepatitis and acute liver damage can be modeled using ConA. In this study, we examined the role of the adhesion receptor LFA-1 in ConA-induced acute hepatic damage using LFA-1(-/-) (CD11a) mice. Massive liver cell apoptosis and metabolic liver damage were observed in LFA-1(+/+) mice following ConA injection. By contrast, LFA-1(-/-) mice were completely resistant to ConA-induced hepatitis and none of the LFA-1(-/-) mice showed any hepatic damage. Whereas activated hepatic T cells remained in the liver in LFA-1(+/+) mice, activated T cells were rapidly cleared from the livers of LFA-1(-/-) mice. Mechanistically, T cells from LFA-1(-/-) mice showed markedly reduced cytotoxicity toward liver cells as a result of impaired, activation-dependent adhesion. Importantly, adoptive transfer of hepatic T cells from LFA-1(+/+) mice, but not from LFA-1(-/-) mice, sensitized LFA-1(-/-) mice to ConA-induced hepatitis. Thus, LFA-1 expression on T cells is necessary and sufficient for T cell-mediated liver damage in vivo. These results provide the first genetic evidence on an adhesion receptor, LFA-1, that has a crucial role in fulminant hepatitis. These genetic data identify LFA-1 as a potential key target for the treatment of T cell-mediated hepatitis and the prevention of liver damage.     

Protective Effects of Astaxanthin on ConA-Induced Autoimmune Hepatitis by the JNK/p-JNK Pathway-Mediated Inhibition of Autophagy and Apoptosis

Objective

Astaxanthin, a potent antioxidant, exhibits a wide range of biological activities, including antioxidant, atherosclerosis and antitumor activities. However, its effect on concanavalin A (ConA)-induced autoimmune hepatitis remains unclear. The aim of this study was to investigate the protective effects of astaxanthin on ConA-induced hepatitis in mice, and to elucidate the mechanisms of regulation.

Materials and Methods

Autoimmune hepatitis was induced in in Balb/C mice using ConA (25 mg/kg), and astaxanthin was orally administered daily at two doses (20 mg/kg and 40 mg/kg) for 14 days before ConA injection. Levels of serum liver enzymes and the histopathology of inflammatory cytokines and other maker proteins were determined at three time points (2, 8 and 24 h). Primary hepatocytes were pretreated with astaxanthin (80 μM) in vitro 24 h before stimulation with TNF-α (10 ng/ml). The apoptosis rate and related protein expression were determined 24 h after the administration of TNF-α.

Results

Astaxanthin attenuated serum liver enzymes and pathological damage by reducing the release of inflammatory factors. It performed anti-apoptotic effects via the descending phosphorylation of Bcl-2 through the down-regulation of the JNK/p-JNK pathway.

Conclusion

This research firstly expounded that astaxanthin reduced immune liver injury in ConA-induced autoimmune hepatitis. The mode of action appears to be downregulation of JNK/p-JNK-mediated apoptosis and autophagy.     

Hydrogen from intestinal bacteria is protective for Concanavalin A-induced hepatitis

It is well known that some intestinal bacteria, such as Escherichia coli, can produce a remarkable amount of molecular hydrogen (H₂). Although the antioxidant effects of H₂ are well documented, the present study examined whether H₂ released from intestinally colonized bacteria could affect Concanavalin A (ConA)-induced mouse hepatitis. Systemic antibiotics significantly decreased the level of H₂ in both liver and intestines along with suppression of intestinal bacteria. As determined by the levels of AST, ALT, TNF-α and IFN-γ in serum, suppression of intestinal bacterial flora by antibiotics increased the severity of ConA-induced hepatitis, while reconstitution of intestinal flora with H₂-producing E. coli, but not H₂-deficient mutant E. coli, down-regulated the ConA-induced liver inflammation. Furthermore, in vitro production of both TNF-α and IFN-γ by ConA-stimulated spleen lymphocytes was significantly inhibited by the introduction of H₂. These results indicate that H₂ released from intestinal bacteria can suppress inflammation induced in liver by ConA.     

Pretreatment with Fucoidan from Fucus vesiculosus Protected against ConA-Induced Acute Liver Injury by Inhibiting Both Intrinsic and Extrinsic Apoptosis

This study aimed to explore the effects of fucoidan from Fucus vesiculosus on concanavalin A (ConA)-induced acute liver injury in mice. Pretreatment with fucoidan protected liver function indicated by ALT, AST and histopathological changes by suppressing inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ). In addition, intrinsic and extrinsic apoptosis mediated by Bax, Bid, Bcl-2, Bcl-xL and Caspase 3, 8, and 9 were inhibited by fucoidan and the action was associated with the TRADD/TRAF2 and JAK2/STAT1 signal pathways. Our results demonstrated that fucoidan from Fucus vesiculosus alleviated ConA-induced acute liver injury via the inhibition of intrinsic and extrinsic apoptosis mediated by the TRADD/TRAF2 and JAK2/STAT1 pathways which were activated by TNF-α and IFN-γ. These findings could provide a potential powerful therapy for T cell-related hepatitis.     

Divergent effects of RIP1 or RIP3 blockade in murine models of acute liver injury

Necroptosis is a recently described Caspase 8-independent method of cell death that denotes organized cellular necrosis. The roles of RIP1 and RIP3 in mediating hepatocyte death from acute liver injury are incompletely defined. Effects of necroptosis blockade were studied by separately targeting RIP1 and RIP3 in diverse murine models of acute liver injury. Blockade of necroptosis had disparate effects on disease outcome depending on the precise etiology of liver injury and component of the necrosome targeted. In ConA-induced autoimmune hepatitis, RIP3 deletion was protective, whereas RIP1 inhibition exacerbated disease, accelerated animal death, and was associated with increased hepatocyte apoptosis. Conversely, in acetaminophen-mediated liver injury, blockade of either RIP1 or RIP3 was protective and was associated with lower NLRP3 inflammasome activation. Our work highlights the fact that diverse modes of acute liver injury have differing requirements for RIP1 and RIP3; moreover, within a single injury model, RIP1 and RIP3 blockade can have diametrically opposite effects on tissue damage, suggesting that interference with distinct components of the necrosome must be considered separately.The etiologies of acute liver injury are diverse and its overall public health burden is considerable. Liver injury from acetaminophen (APAP) overdose is the most common cause of death from over-the-counter drugs and is the leading cause of acute liver failure in the developed world.^{1, 2, 3} Hepatic dysfunction from autoimmune hepatitis has a prevalence of 10–20/100 000.^{4, 5} Other etiologies of acute liver failure include idiosyncratic reaction to medications such as tetracycline, severe viral or alcoholic hepatitis, acute fatty liver of pregnancy, and idiopathic causes. Clinical complications resulting from liver failure include hepatic encephalopathy, impaired protein synthesis, and coagulopathies. Moreover, there are no effective means to reverse liver failure once advanced disease sets in – regardless of etiology – and transplantation frequently remains the only option for survival.⁶Concanavalin-A (ConA) is a lectin derived from the jack-bean plant with a unique ability to induce hepatitis in a well-described murine model of acute hepatic injury. ConA stimulates mouse CD4⁺ T-cell subsets to mediate insult to hepatocytes. The resulting cytokine release can further lead to recruitment and activation of innate inflammatory mediators, which perpetuate an insidious cycle of inflammation and hepatocellular injury.^{7, 8, 9}APAP is a widely used analgesic and antipyretic. Although usually considered safe at therapeutic doses, at higher doses APAP causes acute liver failure characterized by centrilobular hepatic necrosis.^{1, 10} At therapeutic doses, >90% of APAP is metabolized by glucuronidation and sulphation and its metabolites are excreted via the renal system. Of the remaining APAP, roughly 2% is excreted intact in the urine, and approximately 8% is metabolized by the cytochrome P450 system to N-acetyl-p-benzo-quinone imine (NAPQI), which is highly reactive.^{11, 12} Hepatic glutathione (GSH) then induces the formation of a safely excretable APAP-protein adduct. However, at toxic doses of APAP, GSH becomes depleted and NAPQI is able to exert harmful effects by forming covalent bonds with mitochondrial proteins, inhibiting the Ca²⁺-Mg²⁺-ATPase and inducing mitochondrial dysfunction.^{1, 2} This disturbance leads to a decrease in ATP synthesis, disruption of cellular membrane, and eventually hepatocyte death.¹³Although GSH depletion and the resulting toxic metabolites are prerequisites for APAP hepatotoxicity, there is evidence that the severity of liver injury may depend on subsequent participation of innate immunity.^{10, 14, 15, 16} In particular, APAP-induced injury has been reported to be contingent on activation of the NLRP3 inflammasome via DAMPs released from injured hepatocytes. Inflammasome activation cleaves Caspase 1 inducing IL-1β release and galvanizing intrahepatic neutrophils and inflammatory monocytes, which exacerbate injury.¹⁷ However, alternate studies using transgenic mice suggest that NLRP3 inflammasome is largely dispensable for APAP toxicity.¹⁸ Thus the role of inflammasome activation in APAP toxicity is controversial and may be dependent on the precise experimental conditions or strain of mice employed.Apoptosis and necrosis are classically understood processes of cell death that denote either organized Caspase 8-dependent programmed cell death or non-programmed disorganized death, respectively. In contrast to necrosis, which leads to the release of DAMPs and sustains inflammation, apoptosis produces cell fragments called apoptotic bodies, which phagocytic cells are able to engulf before the contents of the cell can spill out onto the surrounding space and activate innate immunity. ‘Necroptosis'' is a recently described Caspase 8-independent method of cell death that denotes organized cellular necrosis. Necroptosis requires the co-activation of RIP1 and RIP3 kinases. Both in vitro and in vivo investigations have suggested that APAP can induce cellular demise via necrosis or Caspase 8-dependent apoptosis, which is determined, in part, by ATP availability from glycolysis.¹⁹ Zhang et al.²⁰ recently confirmed that RIP1 is necessary in APAP-induced necroptosis. Similarly, Takemoto et al.²¹ showed that RIP1 inhibition protects against reactive oxygen species (ROS)-induced hepatotoxicity in APAP-induced acute liver injury. Further, a recent report suggested that selective inhibition of RIP3 using the anticancer drug Dabrafenib alleviates APAP injury.²²In the ConA model of acute liver injury, experiments using apoptosis-resistant mice expressing mutant FADD revealed that ConA alone induced primarily necrotic cell death, whereas ConA combined with d-galactosamine induced apoptosis and necrotic cell death.²³ Zhou et al.²⁴ reported that Necrostatin-1 (Nec-1) prevents autoimmune hepatitis in mice via RIP1- and autophagy-related pathways. Another recent report investigated the role of RIP1, RIP3, and PARP-1 in murine autoimmune hepatitis. This study found that in cases where death of mouse hepatocytes is dependent on TRAIL and NKT cells, PARP-1 activity was positively correlated with liver injury and hepatitis was prevented both by RIP1 or PARP-1 inhibitors.²⁵ Our goal in the current study was to investigate, in parallel, the effects of RIP1 and RIP3 blockade in diverse models of acute liver injury. Our work suggests that modulating necroptosis may have divergent effects, depending on the etiology of hepatic injury and the specific component of the necrosome being targeted.     

Constitutive expression of cyclo-oxygenase 2 transgene in hepatocytes protects against liver injury

The effect of COX (cyclo-oxygenase)-2-dependent PGs (prostaglandins) in acute liver injury has been investigated in transgenic mice that express human COX-2 in hepatocytes. We have used three well-established models of liver injury: in LPS (lipopolysaccharide) injury in D-GalN (D-galactosamine)-preconditioned mice; in the hepatitis induced by ConA (concanavalin A); and in the proliferation of hepatocytes in regenerating liver after PH (partial hepatectomy). The results from the present study demonstrate that PG synthesis in hepatocytes decreases the susceptibility to LPS/D-GalN or ConA-induced liver injury as deduced by significantly lower levels of the pro-inflammatory profile and plasmatic aminotransferases in transgenic mice, an effect suppressed by COX-2-selective inhibitors. These Tg (transgenic) animals express higher levels of anti-apoptotic proteins and exhibit activation of proteins implicated in cell survival, such as Akt and AMP kinase after injury. The resistance to LPS/D-GalN-induced liver apoptosis involves an impairment of procaspase 3 and 8 activation. Protection against ConA-induced injury implies a significant reduction in necrosis. Moreover, hepatocyte commitment to start replication is anticipated in Tg mice after PH, due to the expression of PCNA (proliferating cell nuclear antigen), cyclin D1 and E. These results show, in a genetic model, that tissue-specific COX-2-dependent PGs exert an efficient protection against acute liver injury by an antiapoptotic/antinecrotic effect and by accelerated early hepatocyte proliferation.     

A randomized controlled trial to assess the safety and efficacy of silymarin on symptoms,signs and biomarkers of acute hepatitis

PurposeMilk thistle or its purified extract, silymarin (Silybum marianum), is widely used in treating acute or chronic hepatitis. Although silymarin is hepatoprotective in animal experiments and some human hepatotoxic exposures, its efficacy in ameliorating the symptoms of acute clinical hepatitis remains inconclusive. In this study, our purpose was to determine whether silymarin improves symptoms, signs and laboratory test results in patients with acute clinical hepatitis, regardless of etiology.MethodsThis is a randomized, placebo-controlled trial in which participants, treating physicians and data management staff were blinded to treatment group. The study was conducted at two fever hospitals in Tanta and Banha, Egypt where patients with symptoms compatible with acute clinical hepatitis and serum alanine aminotransferase (ALT) levels >2.5 times the upper limit of normal were enrolled. The intervention consisted of three times daily ingestion of either a standard recommended dose of 140 mg of silymarin (Legalon^®, MADAUS GmbH, Cologne, Germany), or a vitamin placebo for four weeks with an additional four-week follow-up. The primary outcomes were symptoms and signs of acute hepatitis and results of liver function tests on days 2, 4 and 7 and weeks 2, 4, and 8. Side-effects and adverse events were ascertained by self-report.ResultsFrom July 2003 through October 2005, 105 eligible patients were enrolled after providing informed consent. No adverse events were noted and both silymarin and placebo were well tolerated. Patients randomized to the silymarin group had quicker resolution of symptoms related to biliary retention: dark urine (p=0.013), jaundice (p=0.02) and scleral icterus (p=0.043). There was a reduction in indirect bilirubin among those assigned to silymarin (p=0.012), but other variables including direct bilirubin, ALT and aspartate aminotransferase (AST) were not significantly reduced.ConclusionsPatients receiving silymarin had earlier improvement in subjective and clinical markers of biliary excretion. Despite a modest sample size and multiple etiologies for acute clinical hepatitis, our results suggest that standard recommended doses of silymarin are safe and may be potentially effective in improving symptoms of acute clinical hepatitis despite lack of a detectable effect on biomarkers of the underlying hepatocellular inflammatory process.     

Glucocerebroside treatment ameliorates ConA hepatitis by inhibition of NKT lymphocytes

Concanavalin A (ConA) induces natural killer T (NKT) cell-mediated liver damage. Glucocerebroside (GC) is a naturally occurring glycolipid. Our aims were to determine the effect of GC in a murine model of ConA-induced hepatitis. Mice in groups A and B were treated with GC 2 h before and 2 h following administration of ConA, respectively; group C mice were treated with ConA; group D mice was treated with GC; group E mice did not receive any treatment. Liver damage was evaluated by serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels and liver histology. The immune effect of GC was determined by fluorescence-activated cell sorter analysis of intrahepatic and intrasplenic NKT lymphocytes, measurement of cytokine levels, and Western blot analysis for STAT 1, 4, 6, and NF-kappaB expression. The effect of GC on NKT cell proliferation was assessed in vitro. Serum AST and ALT levels were markedly reduced in GC-treated group A mice compared with nontreated group C animals, and histological damage was markedly attenuated in group A. The beneficial effect of GC was associated with a 20% decrease of intrahepatic NKT lymphocytes, significant lowering of serum IFN-gamma levels, and decreased STAT1 and STAT6 expression. In vitro administration of GC led to a 42% decrease of NKT cell proliferation in the presence of dendritic cells but not in their absence. Intraperitoneally administered radioactive GC was detected in the liver and bowel. Administration of GC led to amelioration of ConA hepatitis associated with an inhibitory effect on NKT lymphocytes. GC holds promise as a new immune-modulatory agent.     

Aqueous Extract of Gracilaria tenuistipitata Suppresses LPS-Induced NF-κB and MAPK Activation in RAW 264.7 and Rat Peritoneal Macrophages and Exerts Hepatoprotective Effects on Carbon Tetrachloride-Treated Rat

In addition to the previous investigations of bioactivity of aqueous extract of the edible Gracilaria tenuistipitata (AEGT) against H₂O₂-induced DNA damage and hepatitis C virus replication, the purpose of this study is to evaluate the potential therapeutic properties of AEGT against inflammation and hepatotoxicity using lipopolysaccharide (LPS)-stimulated mouse RAW 264.7 cells, primary rat peritoneal macrophages and carbon tetrachloride (CCl₄)-induced acute hepatitis model in rats. AEGT concentration-dependently inhibited the elevated RNA and protein levels of inducible nitric oxide synthase and cyclooxygenase-2, thereby reducing nitric oxide and prostaglandin E2 levels, respectively. Moreover, AEGT significantly suppressed the production of LPS-induced proinflammatory cytokines, including interleukin (IL)-1β, IL-6 and tumor necrosis factor-α. These inhibitory effects were associated with the suppression of nuclear factor-kappa B activation and mitogen-activated protein kinase phosphorylation by AEGT in LPS-stimulated cells. In addition, we highlighted the hepatoprotective and curative effects of AEGT in a rat model of CCl₄-intoxicated acute liver injury, which was evident from reduction in the elevated serum aspartate aminotransferase and alanine aminotransferase levels as well as amelioration of histological damage by pre-treatment or post-treatment of AEGT. In conclusion, the results demonstrate that AEGT may serve as a potential supplement in the prevention or amelioration of inflammatory diseases.     

腹腔镜胆囊切除术治疗胆囊结石合并急性胆囊炎的疗效及对免疫功能和生活质量的影响

摘要 目的：观察腹腔镜胆囊切除术治疗胆囊结石合并急性胆囊炎的疗效及对免疫功能和生活质量的影响。方法：本次研究为回顾性研究,分析2018年3月~2021年3月期间我院收治的98例胆囊结石合并急性胆囊炎患者的临床资料,根据手术方案的不同将患者分为A组（n=46,给予开腹手术）和B组（n=52,给予腹腔镜胆囊切除术）,记录两组患者围术期相关指标、肝功能、免疫功能、生活质量和并发症发生率。结果：B组术中出血量少于A组,切口大小短于A组,手术时间长于A组,住院时间、首次排气时间短于A组（P<0.05）。两组术后3 d总胆红素 （TBIL）、谷丙转氨酶（ALT）、谷草转氨酶（AST）均升高,但B组低于A组（P<0.05）。B组术后3 d CD3⁺、CD4⁺、NK细胞、CD4⁺/CD8⁺高于A组（P<0.05）,B组术后3 d CD8⁺低于A组（P<0.05）。B组术后3个月健康生活量表简表（SF-36）各维度评分高于A组（P<0.05）。B组术后并发症发生率虽低于A组,但组间对比差异无统计学意义（P>0.05）。结论：腹腔镜胆囊切除术治疗胆囊结石合并急性胆囊炎,虽然手术时间较开腹手术更长,但切口小、可促进患者术后恢复,对患者免疫功能、肝功能损害更轻,有利于提高患者生活质量。     

MyD88-dependent pathway accelerates the liver damage of Concanavalin A-induced hepatitis

We have explored the pathological role of the MyD88 signaling pathway via Toll-like receptors (TLRs) that mediate the recognition of pathogen-associated molecular patterns (PAMPs) in a murine model of autoimmune hepatitis induced by administering Concanavalin A (ConA). We first found that various TLRs and MyD88 molecules were expressed in liver of Con A-treated and untreated wild-type (WT) mice including liver macrophages. Flowcytometric analysis revealed that liver CD11b⁺CD11c⁻ and CD11b⁺CD11c⁺ antigen-presenting cells express TLR2, although NK and NKT cells did not. When WT and MyD88^−/− mice were intravenously administered with Con A, the severity of hepatitis was significantly lower in Con A-injected MyD88^−/− mice than in WT mice in terms of the histopathology, the levels of serum transaminase and pro-inflammatory cytokines (TNF-α, IFN-γ, and IL-6), and upregulation of CD80/CD86 and TNF-α on/in liver macrophages. The results provide evidence of a possible contribution of the TLRs-MyD88 signaling pathway in activating TLR-expressing liver macrophages in the autoimmune hepatitis model, and thus indicate that the strategy of blockade of pathological pathogens via the intestinal lumen may be feasible for the treatment of the disease.     

Ameliorated ConA-induced hepatitis in the absence of PKC-theta

Severe liver injury that occurs when immune cells mistakenly attack an individual's own liver cells leads to autoimmune hepatitis. In mice, acute hepatitis can be induced by concanavalin A (ConA) treatment, which causes rapid activation of CD1d-positive natural killer (NK) T cells. These activated NKT cells produce large amounts of cytokines, which induce strong inflammation that damages liver tissues. Here we show that PKC-θ(-/-) mice were resistant to ConA-induced hepatitis due to essential function of PKC-θ in NKT cell development and activation. A dosage of ConA (25 mg/kg) that was lethal to wild-type (WT) mice failed to induce death resulting from liver injury in PKC-θ(-/-) mice. Correspondingly, ConA-induced production of cytokines such as IFNγ, IL-6, and TNFα, which mediate the inflammation responsible for liver injury, were significantly lower in PKC-θ(-/-) mice. Peripheral NKT cells had developmental defects at early stages in the thymus in PKC-θ(-/-) mice, and as a result their frequency and number were greatly reduced. Furthermore, PKC-θ(-/-) bone marrow adoptively transferred to WT mice displayed similar defects in NKT cell development, suggesting an intrinsic requirement for PKC-θ in NKT cell development. In addition, upon stimulation with NKT cell-specific lipid ligand, peripheral PKC-θ(-/-) NKT cells produced lower levels of inflammatory cytokines than that of WT NKT cells, suggesting that activation of NKT cells also requires PKC-θ. Our results suggest PKC-θ is an essential molecule required for activation of NKT cell to induce hepatitis, and thus, is a potential drug target for prevention of autoimmune hepatitis.     

Death receptor-independent FADD signalling triggers hepatitis and hepatocellular carcinoma in mice with liver parenchymal cell-specific NEMO knockout

Hepatocellular carcinoma (HCC) usually develops in the context of chronic hepatitis triggered by viruses or toxic substances causing hepatocyte death, inflammation and compensatory proliferation of liver cells. Death receptors of the TNFR superfamily regulate cell death and inflammation and are implicated in liver disease and cancer. Liver parenchymal cell-specific ablation of NEMO/IKKγ, a subunit of the IκB kinase (IKK) complex that is essential for the activation of canonical NF-κB signalling, sensitized hepatocytes to apoptosis and caused the spontaneous development of chronic hepatitis and HCC in mice. Here we show that hepatitis and HCC development in NEMO^LPC-KO mice is triggered by death receptor-independent FADD-mediated hepatocyte apoptosis. TNF deficiency in all cells or conditional LPC-specific ablation of TNFR1, Fas or TRAIL-R did not prevent hepatocyte apoptosis, hepatitis and HCC development in NEMO^LPC-KO mice. To address potential functional redundancies between death receptors we generated and analysed NEMO^LPC-KO mice with combined LPC-specific deficiency of TNFR1, Fas and TRAIL-R and found that also simultaneous lack of all three death receptors did not prevent hepatocyte apoptosis, chronic hepatitis and HCC development. However, LPC-specific combined deficiency in TNFR1, Fas and TRAIL-R protected the NEMO-deficient liver from LPS-induced liver failure, showing that different mechanisms trigger spontaneous and LPS-induced hepatocyte apoptosis in NEMO^LPC-KO mice. In addition, NK cell depletion did not prevent liver damage and hepatitis. Moreover, NEMO^LPC-KO mice crossed into a RAG-1-deficient genetic background-developed hepatitis and HCC. Collectively, these results show that the spontaneous development of hepatocyte apoptosis, chronic hepatitis and HCC in NEMO^LPC-KO mice occurs independently of death receptor signalling, NK cells and B and T lymphocytes, arguing against an immunological trigger as the critical stimulus driving hepatocarcinogenesis in this model.Liver cancer is one of the most common malignancies and the third leading cause of cancer-related deaths worldwide.^{1, 2} Liver cancer predominantly arises in the context of chronic inflammatory conditions, most notably in virus hepatitis (HBV and HCV).^{1, 2} Although infectious agents are the primary cause of liver cancer worldwide, the incidence in western countries is rising due to the increase in obesity and non-alcoholic steatohepatitis.³ The pathogenesis of hepatocellular carcinoma (HCC) is incompletely understood and it is plausible that the different underlying aetiologies determine a distinct context for liver carcinogenesis. However, the prevailing universal concept is that continuous liver parenchymal damage and hepatocyte cell death drive compensatory proliferation and within the context of a chronically inflamed liver tissue mutations and epigenetic changes accumulate eventually transforming hepatocytes into malignant cells. Therefore, understanding the tissue-intrinsic processes that determine cell death and chronic inflammation resulting in hepatocarcinogenesis is a critical need in order to design more effective therapeutic strategies.The nuclear factor κB (NF-κB) pathway is implicated in cancer development in particular in the context of chronic inflammation.^{4, 5} In relation to liver cancer, NF-κB signalling has been implicated in the pathogenesis of hepatitis, liver fibrosis, cirrhosis and HCC.^{6, 7} The IKK complex, composed of two catalytic subunits, IKK1/IKKα and IKK2/IKKβ, and a regulatory subunit termed NEMO/IKKγ, activates NF-κB by phosphorylating inhibitor of NF-κB (IκB) proteins targeting them for degradation by the proteasome and thus allowing the nuclear accumulation of NF-κB dimers.⁵ IKK2 is primarily responsible for targeting and degrading IκBα thus inducing canonical NF-κB activation, although the two kinases show some degree of functional redundancy in controlling canonical NF-κB signalling.^{5, 8} NEMO/IKKγ is indispensable for activation of canonical NF-κB signalling.^{9, 10, 11}NF-κB signalling was proposed to exhibit tumour promoter or tumour suppressor properties in different models of liver cancer. In the Mdr2^−/− mouse model of inflammation-driven liver carcinogenesis, NF-κB inhibition caused by transgenic IκBα super–repressor expression in hepatocytes inhibited HCC progression.¹² Moreover, hepatocyte-restricted ablation of IKK2 prevented hepatitis and liver tumorigenesis induced by overexpression of lymphotoxins α and β in hepatocytes.¹³ However, mice with hepatocyte-specific IKK2 ablation developed more tumours induced by a single injection of the chemical carcinogen diethylnitrosamine,¹⁴ revealing a tumour suppressor role of NF-κB in this context.Studies in mice lacking NEMO specifically in liver parenchymal cells (LPCs) further supported a tumour suppressor function of IKK/NF-κB signalling in liver cancer. NEMO^LPC-KO mice showed spontaneous hepatocyte apoptosis resulting in chronic steatohepatitis and the development of HCC by the age of 1 year.¹⁵ LPC-specific ablation of Fas-Associated with Death Domain (FADD or MORT1), an adapter protein essential for the recruitment of caspase-8 to the Death Inducing Signalling Complex and the induction of death receptor-mediated apoptosis,¹⁶ prevented both spontaneous and LPS-induced apoptosis of NEMO-deficient hepatocytes and the development of steatohepatitis.¹⁵ In addition, LPC-specific knockout of caspase-8 inhibited spontaneous hepatocyte apoptosis and HCC development in NEMO^LPC-KO mice, although it caused non-apoptotic hepatocyte death and cholestasis.¹⁷ Given the essential role of FADD and caspase-8 in mediating apoptosis downstream of death receptors,¹⁶ we hypothesized that death receptor-mediated apoptosis of NEMO-deficient hepatocytes drives the development of hepatitis and HCC in NEMO^LPC-KO mice. The three main death receptors of the TNF receptor superfamily that are capable of inducing caspase-8-mediated apoptosis are TNFR1, Fas/CD95 and TRAIL-R/DR5.¹⁶ To address the role of death receptor-induced apoptosis in triggering the spontaneous death of NEMO-deficient hepatocytes and the development of steatohepatitis and HCC, we generated and analysed NEMO^LPC-KO mice lacking TNFR1, Fas or TRAIL-R specifically in LPCs. Surprisingly, we found that LPC-specific knockout of each of the death receptors alone but also combined deficiency of TNFR1, Fas and TRAIL-R in LPCs did not prevent spontaneous hepatocyte apoptosis, hepatitis and HCC development in NEMO^LPC-KO mice. In addition, knockout of TNF in all cells also did not protect NEMO^LPC-KO mice from hepatocyte death, hepatitis and HCC. Collectively, these results demonstrate that TNFR1, Fas and TRAIL-R are not required for the development of chronic liver damage and HCC in NEMO^LPC-KO mice.     

Human umbilical cord mesenchymal stromal cells rescue mice from acetaminophen-induced acute liver failure

Background aimsAcute liver failure (ALF), a life-threatening disease characterized by the sudden loss of hepatic function, can occur after an accidental or intentional acetaminophen overdose.MethodsWith the use of an ALF mouse model, we examined both the preventive and therapeutic potential of intravenously administered human umbilical cord–derived mesenchymal stromal cells (hUCMSCs). Primary hUCMSCs were purified from freshly collected full-term umbilical cords and intravenously transplanted into BALB/c mice either before and after ALF induced by acetaminophen intoxication. We found that hUCMSCs significantly improved survival rates and relative liver weight of mice in both pre-ALF and post-ALF animals. Correspondingly, serum levels of markers that reflect hepatic injury (ie, aspartate aminotransferase, alanine aminotransferase and total bilirubin) were significantly attenuated in the group receiving hUCMSC therapy.ResultsMechanistically, we found that the protective potential of intravenously administered hUCMSCs was mediated by paracrine pathways that involved antioxidants (glutathione, superoxide dismutase), the reduction of inflammatory agents (tumor necrosis factor-α, interleukin-6) and elevated serum levels of hepatocyte growth factor.ConclusionsThrough these paracrine effects, intravenously administered hUCMSCs reduced hepatic necrosis/apoptosis and enhanced liver regeneration. Thus, our data demonstrate that intravenously administered hUCMSCs may be useful in the prevention or treatment of acetaminophen-induced ALF.     

扶正化瘀胶囊联合微生态制剂和恩替卡韦治疗乙型肝炎病毒感染失代偿期肝硬化的临床研究

目的:探讨扶正化瘀胶囊联合微生态制剂和恩替卡韦治疗乙型肝炎病毒(HBV)感染失代偿期肝硬化的临床效果。方法:选取2015年6月～2019年6月期间我院收治的HBV感染失代偿期肝硬化患者73例,根据随机数字表法分为对照组(n=36)和研究组(n=37),对照组患者予以微生态制剂和恩替卡韦治疗,研究组则在对照组的基础上联合扶正化瘀胶囊治疗,比较两组患者疗效、T淋巴细胞亚群、肝功能[天冬氨酸氨基转移酶(AST)、总胆红素(TBIL)]以及肝纤维化指标[透明质酸(HA)、层黏连蛋白(LN)],记录两组乙肝表面e抗原(HBeAg)、乙肝病毒基因(HBV-DNA)转阴情况,记录两组不良反应发生情况。结果:研究组治疗6个月后的临床总有效率为91.89%(34/37),高于对照组的72.22%(26/36)(P0.05)。两组治疗6个月后AST、TBIL、HA、LN、CD8+~均下降,且研究组低于对照组(P0.05)。两组治疗6个月后CD4+~、CD4+~/CD8+~升高,且研究组高于对照组(P0.05)。两组治疗6个月后HBeAg、HBV-DNA转阴率比较无统计学差异(P0.05)。两组不良反应发生率对比未见统计学差异(P0.05)。结论:扶正化瘀胶囊联合微生态制剂和恩替卡韦治疗HBV感染失代偿期肝硬化疗效显著,虽在HbeAg、HBV-DNA转阴率方面未见明显改善,但可有效改善肝功能,减轻肝纤维化,提高机体免疫功能,且不增加不良反应发生率,安全性较好。     

Therapeutic Potential of Stem Cells from Human Exfoliated Deciduous Teeth in Models of Acute Kidney Injury

BackgroundAcute kidney injury (AKI) is a critical condition associated with high mortality. However, the available treatments for AKI are limited. Stem cells from human exfoliated deciduous teeth (SHED) have recently gained attention as a novel source of stem cells. The purpose of this study was to clarify whether SHED have a therapeutic effect on AKI induced by ischemia-reperfusion injury.MethodsThe left renal artery and vein of the mice were clamped for 20 min to induce ischemia. SHED, bone marrow derived mesenchymal stem cells (BMMSC) or phosphate-buffered saline (control) were administered into the subrenal capsule. To confirm the potency of SHED in vitro, H₂O₂ stimulation assays and scratch assays were performed.ResultsThe serum creatinine and blood urea nitrogen levels of the SHED group were significantly lower than those of the control group, while BMMSC showed no therapeutic effect. Infiltration of macrophages and neutrophils in the kidney was significantly attenuated in mice treated with SHED. Cytokine levels (MIP-2, IL-1β, and MCP-1) in mice kidneys were significantly reduced in the SHED group. In in vitro experiments, SHED significantly decreased MCP-1 secretion in tubular epithelial cells (TEC) stimulated with H₂O₂. In addition, SHED promoted wound healing in the scratch assays, which was blunted by anti-HGF antibodies.DiscussionSHED attenuated the levels of inflammatory cytokines and improved kidney function in AKI induced by IRI. SHED secreted factors reduced MCP-1 and increased HGF expression, which promoted wound healing. These results suggest that SHED might provide a novel stem cell resource, which can be applied for the treatment of ischemic kidney injury.     

数字减影血管造影引导下肝动脉化疗栓塞对肝癌患者肝功能、细胞免疫功能及肿瘤标志物的影响

摘要 目的：探讨数字减影血管造影（DSA）引导下肝动脉化疗栓塞（TACE）对肝癌患者肝功能、细胞免疫功能及肿瘤标志物的影响。方法：随机选取2021年1月～2022年1月来我院就诊的行DSA引导下TACE化疗的肝癌患者50例为研究组,另随机选取同期行常规治疗的肝癌患者50例为对照组,对比两组临床总有效率、肝功能、细胞免疫功能及肿瘤标志物和不良反应情况。结果：研究组的临床总有效率为56.00%（28/50）高于对照组的34.00%（17/50）,差异有统计学意义（P<0.05）。两组治疗结束后谷草转氨酶（AST）和谷丙转氨酶（ALT）升高,但研究组低于对照组（P<0.05）。两组治疗结束后CD8⁺升高,但研究组低于对照组（P<0.05）;CD4⁺、CD4⁺/CD8⁺、CD3⁺均下降,但研究组高于对照组（P<0.05）。两组治疗结束后甲胎蛋白（AFP）、糖链抗原（CA）242、CA724、磷脂酰肌醇蛋白聚糖-3（GPC3）下降,且研究组低于对照组（P<0.05）。对照组（30.00%）、研究组（24.00%）的不良反应发生率组间对比无差异（P>0.05）。结论：DSA引导下TACE化疗治疗肝癌患者,疗效可靠,可有效阻止疾病进展,同时减轻化疗所致的肝损伤和细胞免疫功能损伤。     

The Role of Regulatory CD4 T Cells in Maintaining Tolerance in a Mouse Model of Autoimmune Hepatitis

Background

The role of regulatory CD4 T cells (Treg) in immune-mediated liver disease is still under debate. It remains disputed whether Treg suppress T cell-mediated hepatitis in vivo and whether hepatic regulatory T cells are functional in patients with autoimmune hepatitis.

Methods

We used TF-OVA mice, which express ovalbumin in hepatocytes, to investigate the impact of Treg in a model of autoimmune hepatitis. Treg isolated from inflamed livers of TF-OVA mice were tested for their functionality in vitro. By employing double transgenic TF-OVAxDEREG (DEpletion of REGulatory T cells) mice we analyzed whether Treg-depletion aggravates autoimmune inflammation in the liver in vivo.

Results

CD25⁺Foxp3⁺ CD4 T cells accumulated in the liver in the course of CD8 T cell-mediated hepatitis. Treg isolated from inflamed livers were functional to suppress CD8 T-cell proliferation in vitro. Depletion of Treg in TF-OVAxDEREG mice dramatically amplified T cell-mediated hepatitis. Repeated administration of antigen-specific CD8 T cells led to a second wave of inflammation only after depletion of Treg.

Conclusion

Our data add to the evidence for an important role of Treg in autoimmune hepatitis and show that Treg reduce the severity of T-cell mediated hepatitis in vivo. They constitute a key immune cell population that actively maintains a tolerogenic milieu in the liver and protects the liver against repeated inflammatory challenges.