Complement plays a role in both hepatic ischemia reperfusion (IR) injury (IRI) and liver regeneration, but it is not clear how complement is activated in either process. We investigated the role of ...self‐reactive immunoglobulin M (IgM) antibodies in activating complement after hepatic IR and liver resection. Natural IgM antibodies that recognize danger‐associated molecular patterns (neoepitopes) activate complement following both hepatic IR and liver resection. Antibody‐deficient Rag1–/– mice were protected from hepatic IRI, but had increased hepatic injury and an impaired regenerative response after 70% partial hepatectomy (PHx). We identified two IgM monoclonal antibodies (mAbs) that specifically reversed the effect of Rag1 deficiency in both models; B4 (recognizes Annexin IV) and C2 (recognizes subset of phospholipids). Focusing on the B4‐specific response, we demonstrated sinusoidal colocalization of IgM and C3d in Rag1–/– mice that were reconstituted with B4 mAb, and furthermore that the Annexin IV neoepitope is specifically and similarly expressed after both hepatic IR and PHx in wild‐type (WT) mice. A single‐chain antibody construct (scFv) derived from B4 mAb blocked IgM binding and reduced injury post‐IR in WT mice, although, interestingly, B4scFv did not alter regeneration post‐PHx, indicating that anti‐Annexin IV antibodies are sufficient, but not necessary, for the regenerative response in the context of an entire natural antibody repertoire. We also demonstrated expression of the B4 neoepitope in postischemic human liver samples obtained posttransplantation and a corollary depletion in IgM recognizing the B4 and C2 neoepitopes in patient sera following liver transplantation. Conclusion: These data indicate an important role for IgM in hepatic IRI and regeneration, with a similar cross‐species injury‐specific recognition system that has implications for the design of neoepitope targeted therapeutics. (Hepatology 2018;67:721‐735).
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•Cell cycle arrest may be the main inhibition effect of curcumol on hepatocellular carcinoma cells.•CDCA2, CDCA3, CDCA8, CDC25C, SKP2 and PLK1 involved in the cell cycle pathway were ...essential for the proliferation inhibition of curcumol on HCC.•PLK1 was identified as a potential target of curcumol in HCC cells.
Curcumol is a natural drug isolated from Chinese medicinal plant Rhizoma Curcumae. Though many studies have revealed its anti-tumor mechanism, there are still few studies reveal its comprehensive anti-cancer effect used multi-omics method. In this study, we aim to comprehensively analyze its anti-cancer mechanism and find its potential target in human hepatocellular carcinoma cells (HCC). In total, 1330 differential genes and 1381 differential proteins were significantly altered by curcumol in HCC. Transcriptomics and proteomics combined analysis revealed that 47 downregulated of the 97 common molecules regulated by curcumol were mainly enriched in the cell cycle pathway, included Cell Division Cycle Associated 2 (CDCA2), CDCA3, CDCA8, CDC25C, S-phase kinase-associated protein 2 (SKP2) and Polo-like kinase 1(PLK). Furthermore, silenced PLK1 inhibited the cell growth and reversed curcumol’s effect on HCC. This paper provided a comprehensive understanding of the effect of curcumol and identified PLK1 was its potential target in HCC cells.
It has been well established that patients with diabetes or metabolic syndrome (MetS) have increased prevalence and severity of periodontitis, an oral infection initiated by bacteria and ...characterized by tissue inflammation and destruction. To understand the underlying mechanisms, we have shown that saturated fatty acid (SFA), which is increased in patients with type 2 diabetes or MetS, and LPS, an important pathogenic factor for periodontitis, synergistically stimulate expression of proinflammatory cytokines in macrophages by increasing ceramide production. However, the mechanisms by which increased ceramide enhances proinflammatory cytokine expression have not been well understood. Since sphingosine 1 phosphate (S1P) is a metabolite of ceramide and a bioactive lipid, we tested our hypothesis that stimulation of ceramide production by LPS and SFA facilitates S1P production, which contributes to proinflammatory cytokine expression. Results showed that LPS and palmitate, a major SFA, synergistically increased not only ceramide, but also S1P, and stimulated sphingosine kinase (SK) expression and membrane translocation in RAW264.7 macrophages. Results also showed that SK inhibition attenuated the stimulatory effect of LPS and palmitate on IL‐6 secretion. Moreover, results showed that S1P enhanced the stimulatory effect of LPS and palmitate on IL‐6 secretion. Finally, results showed that targeting S1P receptors using either S1P receptor antagonists or small interfering RNA attenuated IL‐6 upregulation by LPS and palmitate. Taken together, this study demonstrated that LPS and palmitate synergistically stimulated S1P production and S1P in turn contributed to the upregulation of proinflammatory cytokine expression in macrophages by LPS and palmitate.
LPS and palmitate synergistically increase S1P and, in turn, S1P contributes to upregulation of proinflammatory genes by LPS and palmitate.
TRAF-binding domain (Trabid), one of deubiquitination enzymes, was recently reported to activate Wnt/ β-catenin signaling pathway. However, the role of Trabid in tumors including hepatocellular ...carcinoma (HCC) and the underlying mechanisms controlling its activity remain poorly understood. Here, we report that Trabid is significantly downregulated in HCC tumor samples and cell lines compared with normal controls and that its expression level is negatively correlated with HCC pathological grading, recurrence, and metastasis. The reintroduction of Trabid expression in tumor cells significantly decreases HCC progression as well as pulmonary metastasis. The effect of Trabid on HCC development occurs at least partially through regulation of Twist1 activity. Mechanistically, Trabid forms a complex with Twist1 and specifically cleaves RNF8-induced K63-linked poly-ubiquitin chains from Twist1, which enhances the association of Twist1 with β-TrCP1 and allows for subsequent K48-linked ubiquitination of Twist1. Knockdown of Trabid increases K63-linked ubiquitination, but abrogates K48-linked ubiquitination and degradation of Twist1, thus enhancing HCC growth and metastasis. Interestingly, Twist1 negatively regulates the promoter activity of Trabid, indicating that a double-negative feedback loop exists. Our findings also identify an essential role for activation of Trabid by AKT-mediated phosphorylation at Ser78/Thr117 in negatively regulating Twist1 signaling, which further provides insights into the mechanisms by which Trabid regulates Twist1 ubiquitination. Our results reveal that Trabid is a previously unrecognized inhibitor of HCC progression and metastasis, which sheds light on new strategies for HCC treatment.
Obesity increases the risk of developing diabetes mellitus. Clinical studies suggest that risk factors like palmitic acid (PA) and lipopolysaccharide (LPS) exist simultaneously in diabetes with ...obesity. Combination of PA and LPS even at low concentration can induce strong inflammatory reaction. Monocyte chemoattractant protein-1 (MCP-1) is an important inflammatory chemokine related to insulin resistance and type II diabetes. Our previous study using PCR array revealed that LPS and PA synergistically induce MCP-1 mRNA expression in macrophage cells RAW264.7, while the protein expression of MCP-1 in this case was not investigated. Moreover, the underling mechanism in the synergistic effect of MCP-1 expression or production induced by treatment of LPS and PA combination remains unclear.
Protein secretion of MCP-1 was measured by the enzyme-linked immunosorbent assay (ELISA) and mRNA levels of MCP-1 and Toll-like receptor 4 (TLR4) were measured by real-time PCR. Statistical analysis was conducted using SPSS software.
LPS could increase MCP-1 transcription as well as secretion in RAW264.7, and PA amplified this effect obviously. Meanwhile, combination of LPS with PA increased TLR4 mRNA expression while LPS alone or PA alone could not, TLR4 knockdown inhibited MCP-1 transcription/secretion induced by LPS plus PA. Moreover, not NF-κB inhibitor but inhibitors of mitogen-activated protein kinase (MAPK) signaling pathways, including c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 MAPK were found to block MCP-1 generation stimulated by LPS plus PA.
LPS and PA synergistically induced MCP-1 secretion in RAW264.7 macrophage cells, in which MCP-1 transcription mediated by MAPK/TLR4 signaling pathways was involved. Combined treatment of PA and LPS in RAW264.7 cells mimics the situation of diabetes with obesity that has higher level of PA and LPS, MAPK/TLR4/ MCP-1 might be potential therapeutic targets for diabetes with obesity.
It is well known that saturated fatty acids (SFAs) and unsaturated fatty acid, in particular omega-3 polyunsaturated fatty acids (n-3 PUFAs), have different effects on inflammatory signaling: SFAs ...are pro-inflammatory but n-3 PUFAs have strong anti-inflammatory properties. We have reported that palmitic acid (PA), a saturated fatty acid, robustly amplifies lipopolysaccharide (LPS) signaling to upregulate proinflammatory gene expression in macrophages. We also reported that the increased production of ceramide (CER) via sphingomyelin (SM) hydrolysis and CER de novo synthesis plays a key role in the synergistic effect of LPS and PA on proinflammatory gene expression. However, it remains unclear if n-3 PUFAs are capable of antagonizing the synergistic effect of LPS and PA on gene expression and CER production. In this study, we employed the above macrophage culture system and lipidomical analysis to assess the effect of n-3 PUFAs on proinflammatory gene expression and CER production stimulated by LPS and PA. Results showed that DHA strongly inhibited the synergistic effect of LPS and PA on proinflammatory gene expression by targeting nuclear factor kappa B (NFκB)-dependent gene transcription. Results also showed that DHA inhibited the cooperative effect of LPS and PA on CER production by targeting CER de novo synthesis, but not SM hydrolysis. Furthermore, results showed that myriocin, a specific inhibitor of serine palmitoyltransferase, strongly inhibited both LPS-PA-stimulated CER synthesis and proinflammatory gene expression, indicating that CER synthesis is associated with proinflammatory gene expression and that inhibition of CER synthesis contributes to DHA-inhibited proinflammatory gene expression. Taken together, this study demonstrates that DHA antagonizes the boosting effect of PA on LPS signaling on proinflammatory gene expression by targeting both NFκB-dependent transcription and CER de novo synthesis in macrophages.
Periodontal disease is more prevalent and severe in patients with diabetes than in nondiabetic patients. In addition to diabetes, a large number of studies have demonstrated an association between ...obesity and chronic periodontal disease. However, the underlying mechanisms have not been well understood. Since plasma free fatty acids (FAs) are elevated in obese patients and saturated FAs such as palmitic acid (PA) have been shown to increase host inflammatory response, we sought to find out how PA interacts with lipopolysaccharide (LPS), an important pathological factor involved in periodontal disease, to enhance inflammation. We found that whereas low concentration of LPS (1 ng/ml) stimulated interleukin (IL)-6 expression in RAW 264.7 macrophages, PA further augmented it fourfold. Besides IL-6, PA amplified the stimulatory effect of LPS on a large amount of Toll-like receptor (TLR)4-mediated expression of proinflammatory signaling molecules such as IL-1 receptor-associated kinase-like 2 and proinflammatory molecules, including monocyte chemotactic protein-1 and colony-stimulating factor. We also observed that PA augmented TLR4 but not TLR2 signal, and the augmentation was mediated by nuclear factor-κB (NF-κB) pathways. To further elucidate the regulatory mechanism whereby PA amplifies LPS signal, our studies showed that PA and LPS synergistically increased hydrolysis of sphingomyelin by stimulating acid sphingomyelinase (ASMase) activity, which contributed to a marked increase in ceramide production and IL-6 upregulation. Taken together, this study has demonstrated that PA markedly augments TLR4-mediated proinflammatory signaling triggered by low concentration of LPS in macrophages, and ASMase plays a key role in the augmentation.
Liver cancer is one of the most common tumors with the seventh-highest incidence and the third-highest mortality. Many studies have shown that small extracellular vesicles (sEVs) play an important ...role in liver cancer. Here, we report comprehensive signatures for sEV proteins from plasma obtained from patients with hepatocellular carcinoma (HCC), which might be valuable for the evaluation and diagnosis of HCC.
We extracted sEVs from the plasma of controls and patients with HCC. Differentially expressed proteins in the sEVs were analyzed using label-free quantification and bioinformatic analyses. Western blotting (WB) was used to validate the abovementioned sEV proteins.
Proteomic analysis was performed for plasma sEVs from 21 patients with HCC and 15 controls. Among the 335 identified proteins in our study, 27 were significantly dysregulated, including 13 upregulated proteins that were involved predominantly in the complement cascade (complement C1Q subcomponent subunit B (C1QB), complement C1Q subcomponent subunit C (C1QC), C4B-binding protein alpha chain (C4BPA), and C4B-binding protein beta chain (C4BPB)) and the coagulation cascade (F13B, fibrinogen alpha chain (FGA), fibrinogen beta chain (FGB), and fibrinogen gamma chain (FGG)). We verified increased levels of the C1QB, C1QC, C4BPA, and C4BPB proteins in the plasma sEVs from patients with HCC in both the discovery cohort and validation cohort.
The complement cascade in sEVs was significantly involved in HCC progression. C1QB, C1QC, C4BPA, and C4BPB were highly abundant in the plasma sEVs from patients with HCC and might represent molecular signatures.
Neutral ceramidase (NCDase) is a class of ceramidases, a key enzyme in ceramide degradation. Recently, it was observed that NCDase activity was suppressed by saturated fatty acids to increase ...ceramide content in rat muscle. However, little is known about its changes in activity and roles in palmitate (Palm)-induced lipotoxicity in pancreatic β cells. Here, we demonstrated that Palm treatment significantly down-regulated NCDase activity, mRNA and protein levels in rat INS-1 cells. In addition, Palm caused a significant accumulation of ceramide, while SPH level remained unchanged, suggesting that inhibition of NCDase activity led to no change of SPH level after treatment with Palm for 24 h. Furthermore, NCDase overexpression significantly reduced Palm-induced apoptosis in INS-1 cells. Conversely, NCDase siRNA knockdown markedly exacerbated Palm-induced apoptosis. In conclusion, Palm treatment suppressed the activity of NCDase and down-regulated its mRNA and protein expression. Furthermore, NCDase inhibition was involved in Palm-induced apoptosis by blocking ceramide degradation in INS-1 cells.
Increasing evidence suggests a vital role of the pre-metastatic niche in the formation of distant metastasis of many cancers. However, how the pre-metastatic niche is formed and promotes pulmonary ...metastasis of hepatocellular carcinoma (HCC) remains unknown.
Orthotopic liver tumor models and RNA-Seq were used to identify dysregulated genes in the pre-metastatic lung. Il1b knockout (Il1b
) mice and lentivirus-mediated gene knockdown/overexpression were utilized to demonstrate the role of interleukin 1 beta (IL-1β)/serum amyloid A3 (SAA3) in the pre-metastatic niche formation and pulmonary metastasis. The potential molecular mechanisms were investigated by RNA-Seq, real-time quantitative PCR (qPCR), western blotting, immunohistochemistry (IHC), flow cytometry, luciferase reporter assay, double immunofluorescent staining and H&E staining.
Accumulation of myeloid cells and upregulation of IL-1β were observed in the pre-metastatic lung of orthotopic liver tumor models. Myeloid cells accumulation and pulmonary metastasis were suppressed in Il1b
mice and Il1r1-silencing mice. Mechanistically, SAA3 and matrix metallopeptidase 9 (MMP9) were identified as potential downstream targets of IL-1β. Overexpression of SAA3 in the lungs of Il1b
mice restored myeloid cells accumulation and pulmonary metastasis of the orthotopic HCC xenografts. Moreover, alveolar macrophages-derived IL-1β dramatically enhanced SAA3 expression in alveolar epithelial cells in an NF-κB dependent manner and increased MMP9 levels in an autocrine manner. Furthermore, SAA3 recruited myeloid cells to the lung without affecting the expression of MMP9 in myeloid cells.
Our study suggests a key role of pulmonary IL-1β and SAA3 in creating a permissive lung pre-metastatic niche by enhancing MMP9 expression and recruiting myeloid cells, respectively, thus promoting pulmonary metastasis of HCC.
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