Mecp2 promotes the anti-inflammatory effect of alpinetin via epigenetic modification crosstalk.

In recent years, inflammatory disorders have emerged as a significant concern for human health. Through ongoing research on anti-inflammatory agents, alpinetin has shown promising anti-inflammatory properties, including involvement in epigenetic modification pathways. As a crucial regulator of epigenetic modifications, Mecp2 may play a role in modulating the epigenetic effects of alpinetin, potentially impacting its anti-inflammatory properties. To test this hypothesis, two key components, p65 (a member of NF-KB family) and p300 (a type of co-activator), were screened by the expression profiling microarray, which exhibited a strong correlation with the intensity of LPS stimulation in mouse macrophages. Meanwhile, alpinetin demonstrates the anti-inflammatory properties through its ability to disrupt the synthesis of p65 and its interaction with promoters of inflammatory genes, yet it did not exhibit similar effects on p300. Additionally, Mecp2 can inhibit the binding of p300 by attaching to the methylated inflammatory gene promoter induced by alpinetin, leading to obstacles in promoter acetylation and subsequently impacting the binding of p65, ultimately enhancing the anti-inflammatory capabilities of alpinetin. Similarly, in a sepsis mouse model, it was observed that homozygotes overexpressing Mecp2 showed a greater reduction in organ damage and improved survival rates compared to heterozygotes when administered by alpinetin. However, blocking the expression of DNA methyltransferase 3A (DNMT3A) resulted in the loss of Mecp2's anti-inflammatory assistance. In conclusion, Mecp2 may augment the anti-inflammatory effects of alpinetin through epigenetic 'crosstalk', highlighting the potential efficacy of a combined therapeutic strategy involving Mecp2 and alpinetin for anti-inflammatory intervention.

Sweroside attenuates podocyte injury and proteinuria in part by activating Akt/BAD signaling in mice.

In this study, we investigated the effects of sweroside on podocyte injury in diabetic nephropathy (DN) mice and elucidated its molecular mechanisms. We conducted in vivo experiments using a C57BL/6 mice model of DN to explore the effects of sweroside on proteinuria and podocyte injury in DN mice. In in vitro experiments, conditionally immortalized mouse podocytes were treated with high glucose and sweroside, and the protective effects of sweroside on podocyte injury were analyzed. In vitro, Akt/BAD pathways were detected using gene siRNA silencing assays and found to be involved in the protective roles of sweroside in high glucose-mediated podocyte injury. In vivo, sweroside significantly decreased albuminuria in DN mice (p < 0.01). periodic acid-Schiff staining showed that sweroside alleviated the glomerular volume and mesangium expansion in DN mice. Consistently, western blot and reverse transcription-polymerase chain reaction analyses showed that the profibrotic molecule expression in the glomeruli declined in sweroside-treated DN mice. Immunofluorescent results showed that sweroside preserved nephrin and podocin expression, and transmission electron microscopy showed that sweroside attenuated podocyte injury. In DN mice, sweroside decreased podocyte apoptosis, and increased nephrin, podocin expression and decreased desmin and HIF1α expression. These results confirmed that sweroside ameliorated albuminuria, glomerulomegaly, and glomerulosclerosis in these mice. Experiments in vitro revealed that sweroside improved HG-induced podocyte injury and apoptosis. Sweroside stimulated activation of the Akt/BAD pathway and upregulated Bcl-2-associated death promoter (BAD) and p-Akt. Overall, sweroside protected podocytes from injury and prevented the progression of DN, providing a novel strategy for the treatment of DN.

Sweroside alleviates hepatic steatosis in part by activating AMPK/mTOR-mediated autophagy in mice.

In this study, we investigated the effect of sweroside (SOS) on hepatic steatosis in mice and elucidated its molecular mechanisms. We conducted in vivo experiments using a C57BL/6 mice model of nonalcohol fatty liver disease (NAFLD) to explore the effect of SOS on hepatic steatosis in NAFLD mice. In in vitro experiments, primary mouse hepatocytes were treated with palmitic acid and SOS, and the protective effects of SOS on inflammation, lipogenesis, and fat deposition were analyzed. Autophagy-related protein levels and their related signaling pathways were evaluated in both in vivo and in vitro experiments. The results demonstrated that SOS decreased the high-fat-induced intrahepatic lipid content both in vivo and in vitro. The autophagy level in the liver was decreased in NAFLD mice but was reactivated following SOS intervention. SOS intervention was found to partially activate autophagy via the adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway. Consequently, when the AMPK/mTOR pathway was suppressed or autophagy was inhibited, the beneficial effects of SOS intervention on hepatic steatosis were diminished. These results indicate that SOS intervention attenuates hepatic steatosis by promoting autophagy in the liver of NAFLD mice, in part by activating the AMPK/mTOR signaling pathway.

LncRNA CASC11 upregulation promotes HDAC4 to alleviate oxidized low-density lipoprotein-induced injury of cardiac microvascular endothelial cells.

Long noncoding RNAs (LncRNAs) are essential to regulate the pathogenesis of coronary artery disease (CAD). This study was conducted to analyze the functionality of long noncoding RNA cancer susceptibility candidate 11 (lncRNA CASC11) in oxidized low-density lipoprotein (ox-LDL)-induced injury of cardiac microvascular endothelial cells (CMECs). CMECs were treated with ox-LDL to induce the CAD cell model. The cellular expression levels of CASC11 and histone deacetylase 4 (HDAC4) were determined by real-time quantitative polymerase chain reaction or Western blot assay. Cell absorbance, apoptosis, angiogenesis, and inflammation were evaluated by cell counting kit-8, flow cytometry, tube formation, and enzyme-linked immunosorbent assays. The subcellular localization of CASC11 was examined by the nuclear/cytoplasmic fractionation assay. The binding of human antigen R (HuR) to CASC11 and HDAC4 was analyzed by RNA immunoprecipitation. HDAC4 stability was determined after actinomycin D treatment. CASC11 was found to be decreased in the CAD cell model. CASC11 upregulation increased cell viability and angiogenesis and reduced apoptosis and inflammation. CASC11 bound to HuR and improved HDAC4 expression. HDAC4 downregulation counteracted the protective role of CASC11 overexpression in CMECs. In summary, CASC11 alleviated ox-LDL-induced injury of CMECs by binding to HuR and stabilizing HDAC4.

Protective Effect of Galangin Methylation Modification Based on Cell Imaging on Inflammatory Lung Injury and Its Molecular Mechanism.

Background: Recently, inflammation has become a major threat to human health. Studies have confirmed that some Chinese traditional medicine ingredients may effectively interfere with the expression of inflammatory mediators through epigenetic modification, showing a great potential of the application. Objective: To investigate the role of the PPAR/DNMT3A pathway in the reversal of galangin-mediated inflammatory lung injury, promote the development of new anti-inflammatory drugs, reduce the side effects of chemical synthetic drugs on the body, and prove the effectiveness and safety of galangin in inhibiting inflammatory response and injury. Methods: 120 rats were randomly divided into 6 groups: (Group 1) LPS group; (Group 2) LPS + galangin group; (Group 3) LPS + galangin + GW9662 group; (Group 4) LPS + galangin + DNMT3A siRNA group; (Group 5) LPS + galangin + siRNA negative group; (Group 6) control group. The model of inflammatory lung injury was established by intrathecal instillation of LPS in the first five groups and NS in the control group. SD survival rate was recorded every 24 hours after modeling, lasting for 168 hours. The lung tissues were taken 168 hours after the establishment of the model. The pathological morphology of lung tissue was observed after the staining under the light microscope, and the lung dry/wet weight ratio was calculated after drying. After NS was perfused into lung tissue, the lavage fluid was collected and the levels of IL-6 and TNF-a were measured by ELISA. The contents of PPAR, DNMT3A, phosphorylated p65, and ERK in monocytes were detected by the WB method, and the binding contents of p65 and AP-1 in the promoter regions of IL-6 and TNF-a genes were detected by the Chip-qPCR method. Results: Intraperitoneal injection of galangin could inhibit the synthesis of alveolar inflammatory factors (TFs) in the SD model of lung injury induced by LPS, reduce the degree of pathological injury of lung tissue, and improve the survival rate of the SD model. GW9662 can completely reverse the protective effect, while DNMT3A interference can only partially block its protective effect. In addition, galangin could significantly inhibit the LPS-induced expression of p65 and AP-1 in alveolar monocytes and their binding content in the promoter region of inflammatory genes by activating PPAR/DNMT3A pathway. GW9662 could completely reverse the inhibitory effect of galangin. DNMT3A interference could restore the binding content of transcription factors at the promoter of the inflammatory gene but had no significant effect on its synthesis. Conclusion: Galangin can interfere with the binding of transcription factors to inflammatory gene promoters through the methylation modification induced by PPAR/DNMT3A pathway, so as to inhibit the synthesis of inflammatory molecules and reverse inflammatory lung injury.

[Methyl CpG-binding protein 2 (MeCP2) inhibits the activity of methylated IL-6 promoter of HEK293 cells and its molecular mechanism].

Objective To investigate the underlying molecular mechanism of methyl-CpG-binding protein 2 (MeCP2) inhibiting interleukin 6 (IL-6) transcriptional activity by observing the sequence of methylated IL-6 promoter, overexpression of MeCP2, and transcription factor P300 in HEK293 cells. Methods The binding site of P300 in the IL-6 promoter region was confirmed by electrophoretic mobility shift assay (EMSA); the IL-6 promoter sequence was ligated into luciferase reporter plasmid and transfected into HEK293 cells. The methylation of the promoter was mediated by clustered regularly interspaced short palindromic repeats-deactivated Cas9 (CRISPR-dCas9)-mediated DNA methyltransferase 3A (DNMT3A) transfection, and then MeCP2 and P300 overexpression plasmids were transfected. The bisulfate sequencing PCR(BSP)was used to analyze the cytosine methylation in the IL-6 promoter region of each group. The contents of intracellular MeCP2 and P300 were detected by the Western blot. A chemiluminescence detector was used to determine the luciferase activity of HEK293 cells. The binding level of P300 and MeCP2 in the IL-6 promoter region was analyzed by chromatin immunoprecipitation followed by sequencing(ChIP-seq). Results EMSA confirmed the presence of P300 binding sites in the IL-6 promoter of mice. CRISPR-dCas9-DNMT3A transfection into HEK293 cells successfully methylated the IL-6 promoter. MeCP2 and P300 overexpression plasmid steadfastly synthesized the target protein and was not affected by other transfection. Compared with the unmodified promoter, methylation could reduce the transcriptional activity of the promoter. When P300 was overexpressed, MeCP2 could further inhibit the transcriptional activity of the promoter, when compared with methylation alone. Also, overexpression of P300 could not promote the transcriptional activity of IL-6 promoter after the methylation modified promoter combined with MeCP2, while the overexpression of P300 enhanced the transcriptional activity when the promoter was not methylated or MeCP2 was not overexpressed. ChIP-seq analysis revealed that the methylated IL-6 promoter showed no difference in binding to P300; however, when combined with MeCP2, the binding capacity would be repressed. Conclusion The combination of MeCP2 with methylated IL-6 promoter can inhibit the binding of the transcription factor to the promoter, thereby impeding the transcriptional activity of the promoter.

Uncovering a Key Role of ETS1 on Vascular Abnormality in Glioblastoma.

Glioblastoma (GBM) is the most aggressive type of brain tumor. Microvascular proliferation and abnormal vasculature are the hallmarks of the GBM, aggravating disease progression and increasing patient morbidity. Here, we uncovered a key role of ETS1 on vascular abnormality in glioblastoma. ETS1 was upregulated in endothelial cells from human tumors compared to endothelial cells from paired control brain tissue. Knockdown of Ets1 in mouse brain endothelial cells inhibited cell migration and proliferation, and suppressed expression of genes associated with vascular abnormality in GBM. ETS1 upregulation in tumor ECs was dependent on TGFß signaling, and targeting TGFß signaling by inhibitor decreased tumor angiogenesis and vascular abnormality in CT-2A glioma model. Our results identified ETS1 as a key factor regulating tumor angiogenesis, and suggested that TGFß inhibition may suppress the vascular abnormality driven by ETS1.

Immune-Related Gene Expression Analysis Revealed Three lncRNAs as Prognostic Factors for Colon Cancer.

Colorectal cancer (CRC) is one of the most common cancers. Almost 80% of CRC cases are colon adenocarcinomas (COADs). Several studies have indicated the role of immunotherapy in the treatment of various cancers. Our study aimed to identify immune-related long non-coding RNAs (lncRNAs) and to use them to construct a risk assessment model for evaluating COAD prognosis. Using differential expression, correlation, and Cox regression analyses, we identified three immune-related differentially expressed lncRNAs (IR-DELs) and used them to construct a risk assessment model. The area under the curve (AUC) for each receiver operating characteristic (ROC) curve at 3-, 5-, and 10-years were greater than 0.6. In addition, the risk assessment model was correlated with several immune cells and factors. The three IR-DELs (AC124067.4, LINC02604, and MIR4435-2HG) identified in this study can be used to predict outcomes for patients with COAD and might help in identifying those who can benefit from anti-tumor immunotherapy.

MicroRNA related prognosis biomarkers from high throughput sequencing data of kidney renal clear cell carcinoma.

BACKGROUND: Kidney renal clear cell carcinoma (KIRC) is the most common type of kidney cell carcinoma which has the worst overall survival rate. Almost 30% of patients with localized cancers eventually develop to metastases despite of early surgical treatment carried out. MicroRNAs (miRNAs) play a critical role in human cancer initiation, progression, and prognosis. The aim of our study was to identify potential prognosis biomarkers to predict overall survival of KIRC. METHODS: All data were downloaded from an open access database The Cancer Genome Atlas. DESeq2 package in R was used to screening the differential expression miRNAs (DEMs) and genes (DEGs). RegParallel and Survival packages in R was used to analysis their relationships with the KIRC patients. David version 6.8 and STRING version 11 were used to take the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. RESULTS: We found 2 DEGs (TIMP3 and HMGCS1) and 3 DEMs (hsa-miR-21-5p, hsa-miR-223-3p, and hsa-miR-365a-3p) could be prognosis biomarkers for the prediction of KIRC patients. The constructed prognostic model based on those 2 DEGs could effectively predict the survival status of KIRC. And the constructed prognostic model based on those 3 DEMs could effectively predict the survival status of KIRC in 3-year and 5-year. CONCLUSION: The current study provided novel insights into the miRNA related mRNA network in KIRC and those 2 DEGs biomarkers and 3 DEMs biomarkers may be independent prognostic signatures in predicting the survival of KIRC patients.

Inhibitory effect of alpinetin on IL-6 expression by promoting cytosine methylation in CpG islands in the IL-6 promoter region.

BACKGROUND: Alpinetin is a flavonoid which exerts antibacterial and anti-inflammatory functions. In order to prove that the induced methylation is an important mechanism for alpinetin in regulating the expression of inflammatory factor Interleukin-6 (IL-6), we detected the dinucleotide methylation status of CpG islands in the IL-6 promoter region and IL-6 level after treatment of RAW246.7 murine macrophages with alpinetin. METHODS: After RAW246.7 murine macrophages were treated with alpinetin, alpinetin + GW9662 (the peroxisome proliferator-activated receptor (PPAR) antagonist), and alpinetin + DNA methyltransferase 3 alpha (DNMT3A) siRNA for 96 hr, CpG islands were analyzed using time-of-flight mass spectrophotometry (TOF-MS) and bisulfite sequencing polymerase chain reaction (BSP). Dinucleotide methylation status of the CpG islands in the IL-6 promoter region was analyzed by methylation-specific Polymerase Chain Reaction (PCR). IL-6 level was detected using the enzyme-linked immunosorbent assay (ELISA) method. Pearson's correlation analysis was conducted to test for potential correlation between the methylation status of CpG islands in the IL-6 promoter region and IL-6 level in RAW 246.7 cells. RESULTS: Alpinetin promoted dinucleotide methylation status of two CpG islands in the IL-6 promoter region stretching 500-2500 bp upstream of the transcriptional start site (TSS) (p < .05). This promoting effect was more significant for the CpG island stretching 500-1500 bp long. The methylation ratio of dinucleotide at this position was significantly inversely correlated with the level of IL-6 (p < .05). PPAR antagonist GW9662 and interference of DNMT3A could reverse both the alpinetin-induced methylation and inhibitory effects on IL-6 expression. CONCLUSION: Alpinetin could induce dinucleotide methylation status of CpG islands in the IL-6 promoter region by activating methyltransferase, thus inhibiting IL-6 expression in murine macrophages.