ABSTRACT
Acute liver injury (ALI) is a complex, life-threatening inflammatory liver disease, and persistent liver damage leads to rapid decline and even failure of liver function. However, the pathogenesis of ALI is still not fully understood, and no effective treatment has been discovered. Recent evidence shows that many circular RNAs (circRNAs) are associated with the occurrence of liver diseases. In this study we investigated the mechanisms of occurrence and development of ALI in lipopolysaccharide (LPS)-induced ALI mice. We found that expression of the circular RNA circDcbld2 was significantly elevated in the liver tissues of ALI mice and LPS-treated RAW264.7 cells. Knockdown of circDcbld2 markedly alleviates LPS-induced inflammatory responses in ALI mice and RAW264.7 cells. We designed and synthesized a series of hesperidin derivatives for circDcbld2, and found that hesperetin derivative 2a (HD-2a) at the concentrations of 2, 4, 8 µM effectively inhibited circDcbld2 expression in RAW264.7 cells. Administration of HD-2a (50, 100, 200 mg/kg. i.g., once 24 h in advance) effectively relieved LPS-induced liver dysfunction and inflammatory responses. RNA sequencing analysis revealed that the anti-inflammatory and hepatoprotective effects of HD-2a were mediated through downregulating circDcbld2 and suppressing the JAK2/STAT3 pathway. We conclude that HD-2a downregulates circDcbld2 to inhibit the JAK2/STAT3 pathway, thereby inhibiting the inflammatory responses in ALI. The results suggest that circDcbld2 may be a potential target for the prevention and treatment of ALI, and HD-2a may have potential as a drug for the treatment of ALI.
Subject(s)
Acute Lung Injury , Hesperidin , Animals , Mice , Lipopolysaccharides/pharmacology , Hesperidin/adverse effects , Down-Regulation , Acute Lung Injury/chemically induced , Acute Lung Injury/drug therapy , Acute Lung Injury/metabolism , Liver/metabolismABSTRACT
Circular RNA (circRNA) has been implicated in liver fibrosis and modulated by multiple elusive molecular mechanisms, while the effects of N6-methyladenosine (m6A) modification on circRNA are still elusive. Herein, we identify circIRF2 from our circRNA sequencing data, which decreased in liver fibrogenesis stage and restored in resolution stage, indicating that dysregulated circIRF2 may be closely associated with liver fibrosis. Gain/loss-of-function analysis was performed to evaluate the effects of circIRF2 on liver fibrosis at both the fibrogenesis and resolution in vivo. Ectopic expression of circIRF2 attenuated liver fibrogenesis and HSCs activation at the fibrogenesis stage, whereas downregulation of circIRF2 impaired mouse liver injury repair and inflammation resolution. Mechanistically, YTHDF2 recognized m6A-modified circIRF2 and diminished circIRF2 stability, partly accounting for the decreased circIRF2 in liver fibrosis. Microarray was applied to investigate miRNAs regulated by circIRF2, our data elucidate cytoplasmic circIRF2 may directly harbor miR-29b-1-5p and competitively relieve its inhibitory effect on FOXO3, inducing FOXO3 nuclear translocation and accumulation. Clinically, circIRF2 downregulation was prevalent in liver fibrosis patients compared with healthy individuals. In summary, our findings offer a novel insight into m6A modification-mediated regulation of circRNA and suggest that circIRF2 may be an exploitable prognostic marker and/or therapeutic target for liver fibrosis.
Subject(s)
MicroRNAs , RNA, Circular , Mice , Animals , Humans , RNA, Circular/genetics , RNA, Circular/metabolism , Hepatic Stellate Cells/metabolism , Liver Cirrhosis/pathology , MicroRNAs/genetics , MicroRNAs/metabolism , Transcription Factors/metabolism , Forkhead Box Protein O3/genetics , RNA-Binding Proteins/metabolismABSTRACT
Hepatic fibrosis is an essential pathology of multiple chronicliverdiseases. The aim of this study was to investigate the role of miR-301a-3p in hepatic fibrosis. We found that miR-301a-3p was upregulated in hepatic fibrosis patients and in culture-activated human hepatic stellate cells (HSCs). Interestingly, miR-301a-3p expression was increased in hepatic fibrosis progression mice while decreased in hepatic fibrosis recovery mice, indicating that miR-301a-3p may participate in the hepatic fibrosis pathology. Functionally, the effects of miR-301a-3p both on hepatic fibrosis progression and regression were assessed in vivo. Inhibiting miR-301a-3p amelioratedmouse liver fibrogenesis and collagen deposition and suppressed HSC activation and fibrogenic factor expression. Whereas, in hepatic fibrosis regression, upregulating miR-301a-3p impaired mouse hepatic fibrosis recovery by inducing HSC activation and triggering inflammation. Consistently, gain-of-function and loss-of-function analysis of miR-301a-3p were performed to evaluate its effects on human HSCs LX-2 cell. We found that suppressing miR-301a-3p inhibited LX-2 cell activation and proliferation, and induced LX-2 cell apoptosis, accompaniedby decreased fibrotic mediators expression. Collectively, these findings suggest miR-301a-3p drives liver fibrogenesis and HSC activation in hepatic fibrosis. Mechanistically, we demonstrated miR-301a-3p binds directly to phosphatase and tensin homolog (PTEN) by luciferase reporter analysis, pull-down, and RIP assay. Indicating that miR-301a-3p plays a critical rolein promotingliverfibrogenesis viamodulating the PTEN/platelet derived growth factor ß (PDGFR-ß) pathway. In conclusion, our findings demonstrate that miR-301a-3p expression is closely correlated with hepatic fibrosis pathology, and that enhancing miR-301a-3p maintains the HSC profibrogenic phenotype, triggers inflammatoryresponses, promotes fibrogenic factor production, and further exacerbates liver fibrogenesis. These findings suggest that miR-301a-3p may serve as a promising diagnostic and prognosis biomarker for hepatic fibrosis treatment.
Subject(s)
Hepatic Stellate Cells , MicroRNAs/metabolism , Animals , Cell Proliferation , Hepatic Stellate Cells/pathology , Humans , Liver Cirrhosis/metabolism , Mice , MicroRNAs/genetics , PTEN Phosphohydrolase/genetics , PTEN Phosphohydrolase/metabolism , Proto-Oncogene Proteins c-sis/pharmacology , Signal TransductionABSTRACT
Alternative oxidase (AOX) has a well-established involvement in plant growth and stress tolerance in many studies. In this study, we isolated and characterized the AOX2 from Cerasus humilis. The ChAOX2 Open Reading Frame (ORF) contains 1029 nucleotides and encodes 342 amino acid residues. The inferred amino acid sequence of ChAOX2 shared the highest sequence similarity with a homolog from Prunus yedoensis. The ChAOX2 transcripts were relatively abundant in the old leaves and significantly up-regulated by salt stress. Subcellular localization analysis showed that ChAOX2 was located in the mitochondria. We transformed ChAOX2 into wild-type Arabidopsis thaliana and found that compared with wild-type and aox mutant lines, heterotopic expression of ChAOX2 increased proline content, and peroxidase and superoxide dismutase activities, while decreasing relative conductivity and the reactive oxygen species level. Further, the ratio of alternate respiration to the total respiration in plants that overexpressed ChAOX2 was significantly higher than that in wild-type and mutant plants under salt stress. These results indicate that ChAOX2 plays a key role in salt tolerance.
ABSTRACT
To investigate the protective mechanism of violaxanthin de-epoxidase (VDE) zeaxanthin in Cerasus humilis under drought and salt-stress conditions, we cloned the entire cDNA sequence of ChVDE from C. humilis and generated ChVDE-overexpression (OE) and ChVDE-complementation (CE) Arabidopsis plants. The open reading frame of ChVDE contained 1,446 bp nucleotides and encoded 481 amino acids. The ChVDE showed the highest similarity with those of Camellia sinensis and Citrus sinensis. Subcellular localization analysis showed that ChVDE was located in the chloroplasts. OE plants showed stronger root growth and higher levels of total chlorophyll as compared to WT and VDE mutant (npq1-2) plants. Moreover, the relative de-epoxidation state of the xanthophyll cycle pigments (A + Z)/(V + A+Z) was higher in OE plants than in the controls. OE plants had enhanced photosynthetic rates, respiration rates, and transpiration rates compared with the WT or npq1-2 plants after drought or salt treatment. Collectively, our results demonstrate that ChVDE plays a positive role in both drought and salt tolerance.