CLEC4E upregulation in gastric cancer: A potential therapeutic target correlating with tumor-associated macrophages.

Background: CLEC4E has been reported to promote lung cancer progression. Tumor-associated macrophages (TAMs) play an important role in tumorigenesis. Whether the expression of CLEC4E in TAMs is associated with gastric carcinogenesis remains unclear. Methods: The TIMER, UALCAN, UCSC Xena, and KM plotter databases are used to examine the expression of CLEC4E and its prognostic significance in gastric cancer (GC). Additionally, GO, KEGG, and GSEA analysis were conducted, and single-cell RNA-seq (scRNA-seq) datasets were utilized. The Coremine medical database was used to predict therapeutic drugs, and molecular docking was performed. Human GC samples were obtained, and co-culture models were constructed to evaluate the effects of CLEC4E in TAMs on tumor growth, migration, and invasion in vitro. Results: CLEC4E was significantly upregulated in GC, and high CLEC4E expression was associated with poor prognosis. Western blotting and immunostaining showed increased protein levels of CLEC4E in GC. GO, KEGG, and GSEA results indicated that CLEC4E is involved in immune response. Immune infiltration analysis demonstrated that CLEC4E expression positively correlated with multiple immune cell types. scRNA-seq analyses revealed that CLEC4E was predominantly expressed in myeloid cells specifically TAMs, in GC. In vitro experiments confirmed that MFC induced CLEC4E expression in TAMs to mediate tumor progression. Specifically targeting CLEC4E by si-CLEC4E or stigmasterol inhibited cancer cell migration and invasion. Conclusion: CLEC4E is a potential prognostic biomarker and new therapeutic target for GC that can be specifically targeted by stigmasterol.

Genetic and pharmacological targeting of XBP1 alleviates hepatic ischemia reperfusion injury by enhancing FoxO1-dependent mitophagy.

Hepatic ischemia reperfusion (I/R) injury is a common clinical complication. X-box binding protein 1 (XBP1), as a critical regulator of the endoplasmic reticulum stress, has been implicated in a variety of diseases. In this study, we aimed to investigate the effects and the underlying mechanism of XBP1 in the progression of hepatic I/R injury. Hepatocyte-specific XBP1 knockout mice, multiple viral delivery systems and specific pharmacological inhibitors were applied in vivo in a partial hepatic I/R injury mouse model and in vitro in a cell model of hypoxia-reoxygenation (H/R) injury. Mitophagy and autophagic flux were evaluated and fluorescence resonance energy transfer (FRET) as well as immunoprecipitation were performed. The results demonstrated that reperfusion for 6 h represented a critical timepoint in hepatic I/R injury and resulted in significant intracellular mitochondrial dysfunction; led to the breakdown of hepatocytes accompanied by the highest expression levels of XBP1. Hepatocyte-specific XBP1 knockout alleviated hepatic I/R injury via enhanced mitophagy, as demonstrated by the reduction in hepatocellular damage/necrosis and increased expression of mitophagy markers. Mechanistically, XBP1 interacted with FoxO1 directly and catalyzed the ubiquitination of FoxO1 for proteasomal degradation. Targeting XBP1 by genetic or pharmacological techniques potentiated the protein levels of FoxO1, further promoting the activity of the PINK1/Parkin signaling pathway, thus augmenting mitophagy and exerting hepatoprotective effects upon I/R injury. In conclusion, the inhibition of XBP1 potentiated FoxO1-mediated mitophagy in hepatic I/R injury. Specific genetic and pharmacological treatment targeting XBP1 in the perioperative 6 h prior to reperfusion exerted beneficial effects, thus providing a novel therapeutic approach.

Methyl eugenol protects the kidney from oxidative damage in mice by blocking the Nrf2 nuclear export signal through activation of the AMPK/GSK3ß axis.

Disrupted redox homeostasis contributes to renal ischemia-reperfusion (IR) injury. Abundant natural products can activate nuclear factor erythroid-2-related factor 2 (Nrf2), thereby providing therapeutic benefits. Methyl eugenol (ME), an analog of the phenolic compound eugenol, has the ability to induce Nrf2 activity. In this study, we investigated the protective effects of ME against renal oxidative damage in vivo and in vitro. An IR-induced acute kidney injury (AKI) model was established in mice. ME (20 mg·kg-1·d-1, i.p.) was administered to mice on 5 consecutive days before IR surgery. We showed that ME administration significantly attenuated renal destruction, improved the survival rate, reduced excessive oxidative stress and inhibited mitochondrial lesions in AKI mice. We further demonstrated that ME administration significantly enhanced Nrf2 activity and increased the expression of downstream antioxidative molecules. Similar results were observed in vitro in hypoxia/reoxygenation (HR)-exposed proximal tubule epithelial cells following pretreatment with ME (40 µmol·L-1). In both renal oxidative damage models, ME induced Nrf2 nuclear retention in tubular cells. Using specific inhibitors (CC and DIF-3) and molecular docking, we demonstrated that ME bound to the binding pocket of AMPK with high affinity and activated the AMPK/GSK3ß axis, which in turn blocked the Nrf2 nuclear export signal. In addition, ME alleviated the development of renal fibrosis induced by nonfatal IR, which is frequently encountered in the clinic. In conclusion, we demonstrate that ME modulates the AMPK/GSK3ß axis to regulate the cytoplasmic-nuclear translocation of Nrf2, resulting in Nrf2 nuclear retention and thereby enhancing antioxidant target gene transcription that protects the kidney from oxidative damage.

Prevention of alloimmune rejection using XBP1-deleted bone marrow-derived dendritic cells in heart transplantation.

BACKGROUND: Genetically modified dendritic cells (DCs) modulate the alloimmunity of T lymphocytes by regulating antigen presentation. METHODS: We generated mice with specific deletion of the X-box-binding protein 1 (XBP1) allele in bone marrow cells and cultured bone marrow-derived DCs (Xbp1-/- BMDCs) from these animals. We then tested the phenotype of Xbp1-/- BMDCs, evaluated their capability to activate allogeneic T cells and investigated their mechanistic actions. We developed a mouse model of allogeneic heart transplantation in which recipients received PBS, Xbp1-/- BMDCs, a suboptimal dose of cyclosporine A (CsA), or Xbp1-/- BMDCs combined with a suboptimal dose of CsA to evaluate the effects of Xbp1-/- BMDC transfusion on alloimmunity and on the survival of heart allografts. RESULTS: The deletion of XBP1 in BMDCs exploited the IRE1-dependent decay of TAPBP mRNA to reduce the expression of MHC-I on the cell surface, altered the capability of BMDCs to activate CD8+ T cells, and ultimately suppressed CD8+ T-cell-mediated allogeneic rejection. The adoptive transfer of Xbp1-/- BMDCs inhibited CD8+ T-cell-mediated rejection. In addition, XBP1-deficient BMDCs were weak stimulators of allogeneic CD4+ T cells despite expressing high levels of MHC-II and costimulatory molecules on their cell surface. Moreover, the adoptive transfer of Xbp1-/- BMDCs inhibited the production of circulating donor-specific IgG. The combination of Xbp1-/- BMDCs and CsA treatment significantly prolonged the survival of allografts compared to CsA alone. CONCLUSIONS: The deletion of XBP1 induces immunosuppressive BMDCs, and treatment with these immunosuppressive BMDCs prevents alloimmune rejection and improves the outcomes of heart transplantation. This finding provides a promising therapeutic target in combating transplant rejection and expands knowledge of inducing therapeutic DCs.

Methyl eugenol attenuates liver ischemia reperfusion injury via activating PI3K/Akt signaling.

BACKGROUND: Liver ischemia reperfusion injury (LIRI) often occurs during liver transplantation, resection, and various circulatory shock procedures, leading to severe metabolic disorders, inflammatory immune responses, oxidative stress injury, and cell apoptosis. Methyl eugenol (ME) is structurally similar to eugenol and has anti-inflammatory and apoptotic pharmacological effects. However, whether ME protects the liver from LIRI damage requires further investigation. METHODS: We established a partially warm LIRI model by subjecting C57BL/6J mice to 60 min of ischemia, followed by reperfusion for 6 h. We also established a hypoxia-reoxygenation injury (H/R) cell model by subjecting AML12 (a mouse liver cell line) cells to 24 h hypoxia, followed by 18 h normoxia. The extent of liver injury was assessed by serum transaminase concentrations, hematoxylin and eosin staining, quantitative real-time PCR, myeloperoxidase activity, and TUNEL analysis. Apoptosis was detected using flow cytometry. The protein levels of p-PI3K, PI3K, p-Akt, Akt, p-Bad, Bad, Bcl-2, Bax, and cleaved caspase-3 were detected by western blotting. LY294002, an inhibitor of PI3K/Akt signaling, was used to elucidate the relationship between ME and PI3K/Akt signaling. RESULTS: ME successfully alleviated LIRI-induced liver injury, inflammatory response, and apoptosis induced, as well as liver cell injury induced by hypoxia reoxygenation. ME is known to activate the PI3K/Akt signaling pathway in hepatocyte injury in vivo and in vitro, and when this signaling pathway is inhibited, the protective effect of ME is abrogated. CONCLUSIONS: The use of ME is a potential therapeutic approach for regulating LIRI by activating PI3K/Akt signaling.

[Effect of methyl eugenol on hypoxia/reoxygenation injury of human renal tubular epithelial cells and its mechanism].

This study aimed to investigate the effect of methyl eugenol(ME) on hypoxia/reoxygenation(H/R)-induced injury of human renal tubular epithelial HK-2 cells and its mechanism. The viability of HK-2 cells cultured with different concentrations of ME and exposed to H/R was detected by cell counting kit-8(CCK-8) assay. The effect of ME on the morphology of HK-2 cells was observed under an inverted microscope. The content of intracellular reactive oxygen species in different groups was detected after 2',7'-dichlorodihydrofluorescein diacetate(DCFH-DA) fluorescence staining. Cell apoptosis was determined by flow cytometry. Changes in mitochondrial membrane potential were monitored by JC-1 dye. The concentrations of nuclear factor erythroid 2 related factor 2(Nrf2), heme oxygenase-1(HO-1), and nicotinamide adenine dinucleotide phosphatase oxidase 4(Nox4) were measured by Western blot, followed by the assay of Nrf2 concentration changes in cytoplasm and nucleus by confocal fluorescence staining. The results showed that when the concentration of ME was 0-40 µmol·L~(-1), the activity of HK-2 cells was not affected. Compared with the model group, ME enhanced the activity of HK-2 cells and the cell morphology was normal. As revealed by further experiments, ME inhibited the release of reactive oxygen species and the decline in mitochondrial membrane potential of HK-2 cells after H/R injury, promoted Nrf2/HO-1 expression and Nrf2 translocation to the nucleus, and down-regulated the expression of Nox4, thereby significantly reducing apoptosis. This protective effect of ME could be reversed by the specific Nrf2 inhibitor ML385. These findings have preliminarily proved that ME effectively protected HK-2 cells against H/R injury, which might be related to its promotion of Nrf2/HO-1 signaling pathway and inhibition of Nox4. Such exploration on the possible mechanism of ME in the treatment of renal ischemia-reperfusion injury(IRI) and protection of organ function from the perspective of antioxidant stress has provided reference for related research on the treatment of acute kidney injury with traditional Chinese medicine.