Deletion of Mixed Lineage Kinase Domain Like Pseudokinase Aggravates Chronic Alcohol-Induced Liver Injury via Increasing Apoptosis.

BACKGROUND AND AIM: he mixed lineage kinase domain like pseudokinase (MLKL) is known to play a protective role in non-alcoholic fatty liver disease (NAFLD) via inhibition of necroptosis pathway. However, the role of MLKL in alcoholic liver disease (ALD) is not yet clear. METHOD: C57BL/6N wild-type (WT) and MLKL-knockout (KO) mice (8-10 weeks old) were randomly divided into eight groups. To establish ALD model of different durations, ethanol (EtOH) was fed to WT and MLKL KO for 10 days, 4 weeks, and 8 weeks. The control group was fed with Lieber-DeCarli control diet for 8 weeks. Mortality, degree of hepatic inflammation, and steatosis were compared among the groups. Bulk mRNA transcriptome analysis was performed. Abundance of transcript and gene expressions were calculated based on read count or Transcript by Million (TPM) value. RESULTS: Survival rate of MLKL KO mice compared to WT was similar until 4 weeks, but the survival of MLKL KO mice significantly decreased after 8 weeks in ALD model. There was no difference in degree of inflammation, steatosis, and NAS scores between EtOH-fed MLKL KO and EtOH-fed WT mice at 10 days. However, at 4 weeks and 8 weeks, the degree of hepatic steatosis, NAS, and inflammation were increased in MLKL KO mice. RNA transcriptome data showed that fatty acid synthesis, and lipogenesis, mitochondria, and apoptosis-related pathways were upregulated in EtOH-fed MLKL KO mice compared to EtOH-fed WT mice. Although hepatocyte apoptosis (BAX/BCL2 ratio, caspase-3, and TUNEL staining) increased after EtOH intake; however, apoptosis was more significantly increased in EtOH-fed MLKL KO mice compared to the WT group. At the same time, hepatic cFLIP was decreased in EtOH-fed MLKL KO mice compared to the WT group. CONCLUSION: MLKL deletion did not prevent chronic alcohol-induced liver damage independently of necroptosis and exacerbated hepatic steatosis by increasing hepatocyte apoptosis.

Novel Inhibitor of Mixed-Lineage Kinase Domain-Like Protein: The Antifibrotic Effects of a Necroptosis Antagonist.

The pseudokinase mixed-lineage kinase domain-like protein plays a crucial role in programmed cell death via necroptosis. We developed a novel mixed-lineage kinase domain-like inhibitor, P28, which demonstrated potent necroptosis inhibition and antifibrotic effects. P28 treatment directly inhibited mixed-lineage kinase domain-like phosphorylation and oligomerization after necroptosis induction, inhibited immune cell death after necroptosis, and reduced the expression of adhesion molecules. Additionally, P28 treatment reduced the level of activation of hepatic stellate cells and the expression of hepatic fibrosis markers induced by necroptosis stimulation. Unlike the necrosulfonamide treatment, the P28 treatment did not induce cytotoxicity. Finally, the cysteine covalent bonding of P28 was confirmed by liquid chromatography-tandem mass spectrometry.

Hepatic stellate cells activate and avoid death under necroptosis stimuli: Hepatic fibrosis during necroptosis.

BACKGROUND AND AIM: Necroptosis is an emerging cell death pathway that allows cells to undergo "cellular suicide" in a caspase-independent manner. We investigated the fate of hepatic stellate cells (HSCs) under necroptotic stimuli. METHODS AND RESULTS: The RNA level of mixed lineage kinase domain-like protein (MLKL) is higher in patients with non-alcoholic fatty liver disease than in healthy controls. Hepatic fibrosis was significantly lower in MLKL-KO bile duct ligation (KO-BDL) mice than in wild-type-BDL mice. Necroptotic stimuli caused the death of HT-29 and U937 cells. However, necroptotic stimuli activate HSCs instead of inducing cell death. MLKL inhibitors attenuated fibrogenic changes in HSCs during necroptosis. Unlike HT-29 and U937 cells, MLKL phosphorylation and oligomerization were not observed during necroptosis in HSCs. RNA sequencing showed that NF-κB signaling-related genes were upregulated in HSCs following necroptotic stimulation. Necroptotic stimuli in HSCs increased the nuclear expression of NF-κB, which decreased after MLKL inhibitor treatment. Induction of necroptosis in HSCs led to autophagosome activation and formation, which were attenuated by MLKL inhibitor treatment. CONCLUSION: HSCs avoid necroptosis due to the absence of MLKL phosphorylation and oligomerization and are activated through autophagosome and NF-κB pathways.

Peripheral blood mononuclear cell mitochondrial copy number and adenosine triphosphate inhibition test in NAFLD.

Background and aim: Non-alcoholic fatty liver disease (NAFLD) is associated with mitochondrial dysfunction. This study aims to develop biomarkers for assessing mitochondrial dysfunction in patients with NAFLD. Methods: Mitochondrion-associated transcriptome analysis was performed. Peripheral blood mononuclear cells obtained from patients with NAFLD (69) and healthy controls (19) were used to determine the mitochondrial DNA (mtDNA) copy number. A mitochondrial inhibition substrate test (ATP assay) was performed in HepG2 cells using the patient serum. Results: Hepatic mRNA transcriptome analysis showed that the gene expression related to mitochondrial functions (mitochondrial fusion, apoptotic signal, and mitochondrial envelope) increased in patients with steatohepatitis, but not in those with NAFL. Gene set enrichment analysis revealed that the upregulated expression of genes is related to the pathways of the tricarboxylic (TCA) cycle and deoxyribonucleic acid (DNA) replication in patients with steatohepatitis, but not in healthy controls. The mtDNA copy number in the peripheral blood mononuclear cells was 1.28-fold lower in patients with NAFLD than that in healthy controls (P <.0001). The mitochondrial inhibition substrate test showed that the cellular adenosine triphosphate (ATP) concentration was 1.2-fold times less in NAFLD patients than that in healthy controls (P <.0001). The mtDNA copy number and mitochondrial ATP inhibition substrate test demonstrated negative correlations with the degree of hepatic steatosis, whereas the ATP concentration showed a positive correlation with the mtDNA copy number. Conclusion: The mitochondrial copy number of peripheral blood mononuclear cells and mitochondrial ATP inhibition substrate can be used as biomarkers for assessing the mitochondrial dysfunction in patients with NAFLD.

Sacubitril/valsartan reduces endoplasmic reticulum stress in a rat model of doxorubicin-induced cardiotoxicity.

The induction of endoplasmic reticulum (ER) stress has been reported as a key contributor to the cardiotoxicity of doxorubicin. Previous in vitro and in vivo studies suggest that sacubitril/valsartan, a novel angiotensin receptor-neprilysin inhibitor, could be effective against doxorubicin-induced cardiotoxicity. However, the precise mechanisms are not fully understood. Therefore, we investigated whether the cardioprotective effects of sacubitril/valsartan are associated with ER stress modulation in a rat model of doxorubicin-induced cardiotoxicity. Male Sprague-Dawley rats were treated with intraperitoneal injections of doxorubicin (15 mg/kg; cumulative) or saline for 3 weeks. From the day before the first treatment, control animals were gavaged daily with water (n = 8), whereas doxorubicin-treated animals were gavaged daily with water (n = 8) or sacubitril/valsartan (60 mg/kg/day; n = 8) for 6 weeks. Echocardiography was performed 6 weeks after the initiation of doxorubicin. In addition, serum troponin I and N-terminal brain natriuretic peptide levels were determined, and the extent of apoptosis and protein levels related to ER stress in the cardiac tissue and doxorubicin-treated H9c2 cardiomyocytes were analyzed. Sacubitril/valsartan significantly reduced doxorubicin-induced cardiac dysfunction and apoptosis in the myocardium. In addition, sacubitril/valsartan significantly downregulated the expression levels of proteins related to apoptosis and ER stress, including BAX, caspase 3, GRP78, PERK, IRE-1α, ATF-6, eIF-2α, ATF-4, and CHOP, in the myocardium of a rat model of doxorubicin-induced cardiotoxicity in vivo and doxorubicin-treated H9c2 cardiomyocytes in vitro. Sacubitril/valsartan significantly alleviated doxorubicin-induced cardiotoxicity, which may be associated with the reduction of ER stress.

Postharvest Drying Techniques Regulate Secondary Metabolites and Anti-Neuroinflammatory Activities of Ganoderma lucidum.

Ganoderma lucidum extract is a potent traditional remedy for curing various ailments. Drying is the most important postharvest step during the processing of Ganoderma lucidum. The drying process mainly involves heat (36 h at 60 °C) and freeze-drying (36 h at -80 °C). We investigated the effects of different postharvest drying protocols on the metabolites profiling of Ganoderma lucidum using GC-MS, followed by an investigation of the anti-neuroinflammatory potential in LPS-treated BV2 microglial cells. A total of 109 primary metabolites were detected from heat and freeze-dried samples. Primary metabolite profiling showed higher levels of amino acids (17.4%) and monosaccharides (8.8%) in the heat-dried extracts, whereas high levels of organic acids (64.1%) were present in the freeze-dried samples. The enzymatic activity, such as ATP-citrate synthase, pyruvate kinase, glyceraldehyde-3-phosphatase dehydrogenase, glutamine synthase, fructose-bisphosphate aldolase, and D-3-phosphoglycerate dehydrogenase, related to the reverse tricarboxylic acid cycle were significantly high in the heat-dried samples. We also observed a decreased phosphorylation level of the MAP kinase (Erk1/2, p38, and JNK) and NF-κB subunit p65 in the heat-dried samples of the BV2 microglia cells. The current study suggests that heat drying improves the production of ganoderic acids by the upregulation of TCA-related pathways, which, in turn, gives a significant reduction in the inflammatory response of LPS-induced BV2 cells. This may be attributed to the inhibition of NF-κB and MAP kinase signaling pathways in cells treated with heat-dried extracts.

The Role of Tumor Necrosis Factor Alpha (TNF-α) in Autoimmune Disease and Current TNF-α Inhibitors in Therapeutics.

Tumor necrosis factor alpha (TNF-α) was initially recognized as a factor that causes the necrosis of tumors, but it has been recently identified to have additional important functions as a pathological component of autoimmune diseases. TNF-α binds to two different receptors, which initiate signal transduction pathways. These pathways lead to various cellular responses, including cell survival, differentiation, and proliferation. However, the inappropriate or excessive activation of TNF-α signaling is associated with chronic inflammation and can eventually lead to the development of pathological complications such as autoimmune diseases. Understanding of the TNF-α signaling mechanism has been expanded and applied for the treatment of immune diseases, which has resulted in the development of effective therapeutic tools, including TNF-α inhibitors. Currently, clinically approved TNF-α inhibitors have shown noticeable potency in a variety of autoimmune diseases, and novel TNF-α signaling inhibitors are being clinically evaluated. In this review, we briefly introduce the impact of TNF-α signaling on autoimmune diseases and its inhibitors, which are used as therapeutic agents against autoimmune diseases.

Role of Autophagy in Granulocyte-Colony Stimulating Factor Induced Anti-Apoptotic Effects in Diabetic Cardiomyopathy.

BACKGROUND: We previously, reported that granulocyte-colony stimulating factor (G-CSF) reduces cardiomyocyte apoptosis in diabetic cardiomyopathy. However, the underlying mechanisms are not yet fully understood. Therefore, we investigated whether the mechanisms underlying of the anti-apoptotic effects of G-CSF were associated with autophagy using a rat model of diabetic cardiomyopathy. METHODS: Diabetic cardiomyopathy was induced in rats through a high-fat diet combined with low-dose streptozotocin and the rats were then treated with G-CSF for 5 days. Rat H9c2 cardiac cells were cultured under high glucose conditions as an in vitro model of diabetic cardiomyopathy. The extent of apoptosis and protein levels related to autophagy (Beclin-1, microtubule-binding protein light chain 3 [LC3]-II/LC3-I ratio, and P62) were determined for both models. Autophagy determination was performed using an Autophagy Detection kit. RESULTS: G-CSF significantly reduced cardiomyocyte apoptosis in the diabetic myocardium in vivo and led to an increase in Beclin-1 level and the LC3-II/LC3-I ratio, and decreased P62 level. Similarly, G-CSF suppressed apoptosis, increased Beclin-1 level and LC3-II/LC3-I ratio, and decreased P62 level in high glucose-induced H9c2 cardiac cells in vitro. These effects of G-CSF were abrogated by 3-methyladenine, an autophagy inhibitor. In addition, G-CSF significantly increased autophagic flux in vitro. CONCLUSION: Our results suggest that the anti-apoptotic effect of G-CSF might be significantly associated with the up-regulation of autophagy in diabetic cardiomyopathy.

Fucoxanthin from microalgae Phaeodactylum tricornutum inhibits pro-inflammatory cytokines by regulating both NF-κB and NLRP3 inflammasome activation.

Pro-inflammatory cytokines such as IL-1ß, IL-6, and TNF-α are mediated by the activation of various kinds of signaling pathways in the innate immune system. Particularly, NF-κB and NLRP3 inflammasome signaling are involved in the production and secretion of these cytokines. Each signaling is participated in the two steps necessary for IL-1ß, a representative pro-inflammatory cytokine, to be processed into a form secreted by cells. In the priming step stimulated by LPS, pro-IL-1ß is synthesized through NF-κB activation. Pro-IL-1ß cleavages into mature IL-1ß by formed NLRP3 inflammasome in the activation step induced by ATP. The mature form of IL-1ß is subsequently secreted out of the cell, causing inflammation. Moreover, IL-6 and TNF-α are known to increase in NLRP3 inflammasome-mediated conditions. Here, we found that fucoxanthin, one of the major components of Phaeodactylum tricornutum, has an inhibitory effect on NF-κB and NLRP3 inflammasome activation induced by the combination of LPS and ATP in bone marrow-derived immune cells as well as astrocytes. Fucoxanthin, which is abundant in the EtOH fraction of Phaeodactylum tricornutum extracts, has shown to have less cell toxicity and found to decrease the production of major pro-inflammatory cytokines such as IL-1ß, IL-6, and TNF-α. Fucoxanthin has also shown to suppress the expression of cleaved caspase-1 and the oligomerization of ASC, which are the main components of the NLRP3 inflammasome. Furthermore, phosphorylated IκBα and pro-IL-1ß expression decreased in the presence of fucoxanthin, suggesting that fucoxanthin can negatively regulate the priming step of inflammasome signaling. Thus, our results provide reliable evidence that fucoxanthin may serve as a key candidate in the development of potential therapeutic agents for inflammatory diseases as well as neurodegenerative diseases caused by NF-κB and NLRP3 inflammasome activation.

Modified method for effective primary vascular smooth muscle progenitor cell culture from peripheral blood.

In previous studies, vascular smooth muscle progenitor cells (vSMPCs) isolated from peripheral blood mononuclear cells (PBMCs) were cultured using medium containing platelet-derived growth factor-BB (PDGF-BB) for 4 weeks. However, this method requires long culture periods of up to 4 weeks and yields low cell counts. Therefore, we proposed the modified method to improve the cell yield and purity and to reduce the cell culture period. PBMCs were isolated from human peripheral blood and cultured by the conventional method using medium containing PDGF-BB alone or the modified method using medium containing PDGF-BB, basic fibroblast growth factor (bFGF), and insulin-transferrin-selenium ITS for 4 weeks. The purity of vSMPCs was analyzed for the expression of a- smooth muscle actin (SMA) by flow cytometry and significantly higher in the modified method than conventional methods at the 1st and 2nd weeks. Also, mRNA expression of a-SMA by real-time PCR was significantly higher in the modified method than conventional method at the 2 weeks. The yield of vSMPCs by trypan blue exclusion assay was significantly higher in the modified method than conventional method at the 1st, 2nd and 3rd weeks. The primary culture using the modified method with PDGF-BB, bFGF, and ITS not only improved cell purity and yield, but also shortened the culture period, compared to the conventional culture method for vSMPCs. The modified method will be a time-saving and useful tool in various studies related to vascular pathology.

Granulocyte Colony Stimulating Factor Ameliorates Hepatic Steatosis Associated with Improvement of Autophagy in Diabetic Rats.

Background: We previously reported that the granulocyte colony stimulating factor (G-CSF) ameliorated hepatic steatosis with the enhancement of ß-oxidation-related gene expression. However, the mechanisms underlying this process remain unclear. This study aimed to determine whether the improvement of hepatic steatosis by G-CSF was associated with autophagy in a rat model of diabetes. Methods: Eight rats were fed a standard diet, and 16 rats were fed high-fat diet (HFD) for 5 weeks. All HFD-fed rats were then injected with streptozotocin (STZ). One week later, HFD rats injected with STZ were randomly treated with either G-CSF (200 µg/kg/day; diabetes mellitus (DM)/G-CSF) or saline (DM/saline) for 5 consecutive days. Four weeks later, serum biochemical and histology analyses were conducted. The expression of autophagy-associated proteins was determined by Western blotting. The mRNA expression of ß-oxidation-related genes was determined by quantitative real-time polymerase chain reaction. HepG2 cells were cultured under high glucose (HG) conditions with G-CSF treatment, followed by Oil Red O staining for quantification of lipids. Results: Histological analysis showed lower lipid accumulation in the DM/G-CSF group than in the DM/saline-treated rats. Protein levels of LC3 and beclin-1 were higher, and those of p62 were lower in the DM/G-CSF rats than in the DM/saline-treated rats. The mRNA expression of ß-oxidation-related genes was higher in DM/G-CSF rats than in the DM/saline-treated rats. Quantification of lipid levels in HepG2 cells cultured with HG and G-CSF treatment revealed no significant differences. Conclusions: Our data suggested that G-CSF potentially improves hepatic steatosis and autophagy in the liver of diabetic rats.