Acute exposure to polystyrene nanoparticles promotes liver injury by inducing mitochondrial ROS-dependent necroptosis and augmenting macrophage-hepatocyte crosstalk.

BACKGROUND: The global use of plastic materials has undergone rapid expansion, resulting in the substantial generation of degraded and synthetic microplastics and nanoplastics (MNPs), which have the potential to impose significant environmental burdens and cause harmful effects on living organisms. Despite this, the detrimental impacts of MNPs exposure towards host cells and tissues have not been thoroughly characterized. RESULTS: In the present study, we have elucidated a previously unidentified hepatotoxic effect of 20 nm synthetic polystyrene nanoparticles (PSNPs), rather than larger PS beads, by selectively inducing necroptosis in macrophages. Mechanistically, 20 nm PSNPs were rapidly internalized by macrophages and accumulated in the mitochondria, where they disrupted mitochondrial integrity, leading to heightened production of mitochondrial reactive oxygen species (mtROS). This elevated mtROS generation essentially triggered necroptosis in macrophages, resulting in enhanced crosstalk with hepatocytes, ultimately leading to hepatocyte damage. Additionally, it was demonstrated that PSNPs induced necroptosis and promoted acute liver injury in mice. This harmful effect was significantly mitigated by the administration of a necroptosis inhibitor or systemic depletion of macrophages prior to PSNPs injection. CONCLUSION: Collectively, our study suggests a profound toxicity of environmental PSNP exposure by triggering macrophage necroptosis, which in turn induces hepatotoxicity via intercellular crosstalk between macrophages and hepatocytes in the hepatic microenvironment.

Role of HSP27 in the multidrug sensitivity and resistance of colon cancer cells.

Multidrug resistance in cancer cells is a primary factor affecting therapeutic efficacy. Heat shock 27 kD protein 1 (HSP27) is associated with cell apoptosis and resistance to chemotherapy. However, the mechanisms underlying HSP27-associated pathways in colon cancer cells remain unclear. Therefore, the present study used short hairpin (sh) RNA to inhibit HSP27 expression in colon cancer cells in order to investigate the effects in vitro and in vivo. Flow cytometry was used to investigate cell apoptosis and a xenograft model was employed to examine the tumorigenesis. Protein expression was measured by Western blotting. The results revealed that suppression of HSP27 expression significantly increased cell apoptosis, inhibited tumor growth and enhanced sensitivity to the anti-cancer agents 5-fluorouracil (5-FU) and vincristine (VCR). shHSP27 significantly decreased the expression of notch receptor 1 and the phosphorylation level of Akt and mTOR, and enhanced the effect of 5-FU and VCR. In conclusion, HSP27 suppression enhanced the sensitivity of colon cancer cells to 5-FU and VCR, and increased colon cancer cell apoptosis with and without chemotherapy. Therefore, the development of novel therapeutic agents that inhibit the expression of HSP27 may offer a new treatment option for colon cancer.

Identification of FBXW7α-regulated genes in M1-polarized macrophages in colorectal cancer by RNA sequencing.

OBJECTIVES: To determine the FBXW7α-regulated genes in tumor-polarized macrophages in colorectal cancer. METHODS: This experimental study was performed between June 2017 and March 2019. FBXW7α siRNA transfected RAW264.7 cells, together with the control group, were co-cultured with the colon cancer cell line, Colon-26. M1  marker production from the macrophages was determined by ELISA and quantitative reverse transcription-polymerase chain reaction. Whole genomic differential expression between the FBXW7α siRNA group and the control group were determined by RNA-sequencing analysis. The target site of the microRNA-205 gene was predicted using Targetscan and was verified by the luciferase assay. By transfecting mimics or inhibitors of microRNA-205, we explored the role of FBXW7α/microRNA-205 axis in regulating the polarization of tumor-associated macrophages (TAM). RESULTS: FBXW7α knockdown in RAW264.7 enhanced the expression of cyclooxigenase (COX)-2 and inducible nitric oxide synthase (iNOS), mRNA expression and IL6, IL12, p40, and tumor necrosis factor-α (TNFα) production upon co-culture with Colon-26 cells in vitro. Further, compared with the control group, 648 genes in total were enhanced and 416 targets were downregulated in FBXW7α siRNA transfected cells, among which miR-205 was the most significantly upregulated. SMAD1 was identified as an miR-205 target. The FBXW7α/miR-205 axis might regulate TAM polarization by affecting SMAD1 expression. CONCLUSION: These results prove that the FBXW7α/miR-205 axis plays an important role in TAM polarization and could facilitate further exploration of its molecular mechanism.

Chloroquine attenuates lipopolysaccharide-induced inflammatory responses through upregulation of USP25.

Lipopolysaccharide (LPS) is a key pathogenic factor in sepsis, and its recognition by toll-like receptor 4 (TLR4) can activate two district signaling pathways, leading to activation of transcription factors including NF-κB and interferon regulatory factor 3 (IRF3). Chloroquine (CQ) has been shown to affect LPS-TLR4 colocalization and inhibit both MyD88-dependent and TRAM/TRIF-dependent pathways, though the mechanism involved is still poorly understood. Here, we found that the ubiquitin-proteasome system might be involved in this process. CQ increased USP25, a deubiquitinating enzyme, as well as mRNA and protein expression in a dose-dependent manner, which might to some degree be involved in CQ attenuation of LPS-induced macrophage activation. Overexpression of USP25 decreased LPS-induced inflammatory cytokines like TNF-α, IL-6, and IFN-ß, while specific siRNA-mediated USP25 silencing increased TNF-α, IL-6, and IFN-ß production and secretion. In addition, USP25 deletion strengthened mitogen-activated protein kinase (MAPKs) phosphorylation and IκB degradation. Moreover, USP25 interference increased NF-κB and IRF3 nuclear translocation. Taken together, our data demonstrated a new possible regulator of LPS-induced macrophage activation mediated by CQ, through upregulation of USP25.

Chloroquine attenuates LPS-mediated macrophage activation through miR-669n-regulated SENP6 protein translation.

Chloroquine (CQ) has been shown to inhibit Toll-like receptor 4 (TLR4)-mediated monocyte and macrophage activation induced by lipopolysaccharide (LPS). However, the underlying mechanisms have not been completely elucidated. Recently, SUMO-specific protease 6 (SENP6) has been reported to suppress LPS-induced activation of macrophages through deSUMOlation of NF-κB essential modifier (NEMO). Here, we studied whether this molecular pathway may also be involved in CQ/LPS model. We found that CQ dose-dependently increased SENP6 protein, but not mRNA, in mouse macrophages, RAW264.7 cells. Overexpression of SENP6 in RAW264.7 cells significantly decreased the LPS-induced release of pro-inflammatory proteins, TNF-α, IL-6 and IFN-γ, while depletion of SENP6 in RAW264.7 cells significantly increased these proteins. Moreover, in LPS-treated RAW264.7 cells, CQ dose-dependently decreased the levels of microRNA-669n (miR-669n), which bound to 3'-UTR of SENP6 mRNA to inhibit its translation. Overexpression of miR-669n decreased SENP6, resulting in increased production of TNF-α, IL-6 and IFN-γ in RAW264.7 cells, while depletion of miR-669n increased SENP6, resulting in decreased production of TNF-α, IL-6 and IFN-γ in RAW264.7 cells. In vivo, delivery of miR-669n plasmids augmented the effects of LPS, while delivery of antisense of miR-669n (as-miR-669n) plasmids abolished the effects of LPS. Taken together, our data demonstrate a previously unappreciated molecular control of LPS-induced macrophage activation by CQ, through miR-669n-regulated SENP6 protein translation.

Genome-wide analysis of long noncoding RNA profile in human gastric epithelial cell response to Helicobacter pylori.

Long noncoding RNAs (lncRNAs) are an important class of pervasive genes, and their misregulation has been shown in various types of diseases. However, the relationship between lncRNAs and the immune response to pathogen infection has been rarely reported. Helicobacter pylori is a major human pathogenic bacterium that causes gastritis, peptic ulcer disease, mucosa-associated lymphoid tissue lymphoma, and gastric cancer. The regulatory mechanism of the H. pylori-induced immune response is not yet clear. In the present study, we identified nonoverlapping signatures of a small number of lncRNAs that were aberrantly expressed in H. pylori-infected gastric epithelial cells using microarray analysis followed by bioassays. From microarray data, we found that 23 lncRNAs were upregulated and 21 were downregulated. Five lncRNAs, XLOC_004562, XLOC_005912, XLOC_000620, XLOC_004122, and XLOC_014388, were further evaluated using quantitative reverse transcription-PCR, and the results matched well with microarray data. In addition, XLOC_004122 and XLOC_014388 were decreased in gastric mucosal tissues of H. pylori-positive patients. Differentially expressed lncRNAs may play a partial or key role in the immune response to H. pylori, and this may provide potential targets for the future treatment of H. pylori-related diseases.

Kukoamine B, a novel dual inhibitor of LPS and CpG DNA, is a potential candidate for sepsis treatment.

BACKGROUND AND PURPOSE: Lipopolysaccharides (LPS) and oligodeoxynucleotides containing CpG motifs (CpG DNA) are important pathogenic molecules for the induction of sepsis, and thus are drug targets for sepsis treatment. The present drugs for treating sepsis act only against either LPS or CpG DNA. Hence, they are not particularly efficient at combating sepsis as the latter two molecules usually cooperate during sepsis. In this study, a natural alkaloid compound kukoamine B (KB) is presented as a potent dual inhibitor for both LPS and CpG DNA. EXPERIMENTAL APPROACH: The affinities of KB for LPS and CpG DNA were assessed using biosensor technology. Direct interaction of KB with LPS and CpG DNA were evaluated using neutralization assays. Selective inhibitory activities of KB on pro-inflammatory signal transduction and cytokine expression induced by LPS and CpG DNA were analysed by cellular assays. Protective effects of KB in a sepsis model in mice were elucidated by determining survival and circulatory LPS and tumour necrosis factor-alpha (TNF-α) concentrations. KEY RESULTS: KB had high affinities for LPS and CpG DNA. It neutralized LPS and CpG DNA and prevented them from interacting with mouse macrophages. KB selectively inhibited LPS- and CpG DNA-induced signal transduction and expression of pro-inflammatory mediators without interfering with signal pathways or cell viability in macrophages. KB protected mice challenged with heat-killed Escherichia coli, and reduced the circulatory levels of LPS and TNF-α. CONCLUSIONS AND IMPLICATIONS: This is the first report of a novel dual inhibitor of LPS and CpG DNA. KB is worthy of further investigation as a potential candidate to treat sepsis.

Dual targets guided screening and isolation of Kukoamine B as a novel natural anti-sepsis agent from traditional Chinese herb Cortex lycii.

Treating sepsis remains challenging at present. Bacterial lipopolysaccharide (LPS) and bacterial DNA/CpG DNA are important pathogenic molecules and drug targets for sepsis. It is thus a promising strategy to treat sepsis by discovering agents that neutralize LPS and CpG DNA simultaneously. In this study, we present evidences of the biosensor based screening and isolation of active anti-sepsis fractions and monomers from traditional Chinese herbs using dual targets (LPS and CpG DNA) guided drug discovery strategy. Firstly, LPS or CpG DNA was immobilized on surfaces of cuvettes in the biosensor to establish a screening platform. Then, Cortex lycii with both highest affinities was selected out from one hundred and fourteen traditional Chinese herbs. In subsequent experiments, chromatography was utilized and coupled with the biosensor to purify fractions with a higher affinity for LPS and CpG DNA. In line with affinity assay, these fractions were shown to neutralize LPS and CpG DNA and inhibit their activity in vitro and in vivo. Lastly, the contributing monomer Kukoamine B (KB) was purified. KB neutralized LPS and CpG DNA in vitro. It inhibited TLR4, TLR9 and MyD88 mRNA expressions up-regulated by LPS and CpG DNA, and also attenuated the LPS and CpG DNA elicited nuclear translocation of NF-κB p65 protein in RAW264.7 cells. It also protected mice from lethal challenge of heat-killed E. coli, a mixture of LPS and CpG DNA. In conclusion, we presented a dual target guided discovery of a novel anti-sepsis agent KB from traditional Chinese herbs via combination of biosensor technology and chromatography methods.