NEAT1 regulates VSMC differentiation and calcification in as long noncoding RNA NEAT1 enhances phenotypic and osteogenic switching of vascular smooth muscle cells in atherosclerosis via scaffolding EZH2.

Atherosclerosis (AS) is a significant contributor to cardio-cerebrovascular ischemia diseases, resulting in high mortality rates worldwide. During AS, vascular smooth muscle cells (VSMCs) play a crucial role in plaque formation by undergoing phenotypic and osteogenic switching. Long noncoding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) has previously been identified as a nuclear regulator that promotes tumorigenesis and metastasis, but its role in regulating VSMCs in AS remains unclear. Our study aimed to investigate the biological functions and specific mechanisms of NEAT1 in regulating VSMCs in AS. We found that NEAT1 was upregulated in the aortas of AS mouse models and dedifferentiated primary VSMCs. Silencing NEAT1 in vitro attenuated the proliferation, migration, and osteogenic differentiation of VSMCs, while NEAT1 overexpression had the opposite effect. Furthermore, NEAT1 promoted VSMC osteogenic differentiation and vascular calcification in both in vivo and in vitro vascular calcification models. We also discovered that NEAT1 directly activates enhancer of zeste homolog 2 (EZH2), an epigenetic enzyme that suppresses the expression of senescence- and antimigration-related genes, by translocating it into the nucleus. CUT&Tag assay revealed that NEAT1 guides EZH2 to the promoters of senescence-related genes (P16, P21, and TIMP3), methylating local histones to reduce their transcription. Our findings suggest that NEAT1 functions in AS by modulating the epigenetic function of EZH2, which enhances the proliferation, migration, and osteogenic differentiation of VSMCs. This study provides new insights into the molecular mechanisms underlying the pathogenesis of AS and highlights the potential of NEAT1 as a therapeutic target of AS.NEW & NOTEWORTHY Our study demonstrates that the upregulation of long noncoding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) promotes proliferation and migration during phenotypic switching of vascular smooth muscle cells in atherosclerosis. We also provide in vivo and in vitro evidence that NEAT1 accelerates vascular calcification. Our findings identified the direct interaction between enhancer of zeste homolog 2 (EZH2) and NEAT1 during atherosclerosis. NEAT1 is necessary for EZH2 to translocate from the cytoplasm to the nucleus, where EZH2 epigenetically inhibits the expression of genes related to senescence and antimigration.

O-GlcNAc of STING mediates antiviral innate immunity.

BACKGROUND: O-GlcNAcylation modification affects multiple physiological and pathophysiolocal functions of cells. Altered O-GlcNAcylation was reported to participate in antivirus response. Stimulator of interferon genes (STING) is an adaptor mediating DNA virus-induced innate immune response. Whether STING is able to be modified by O-GlcNAcylation and how O-GlcNAcylation affects STING-mediated anti-DNA virus response remain unknown. METHODS: Metabolomics analysis was used for detecting metabolic alterations in HSV-1 infection cells. Succinylated wheat germ agglutinin (sWGA), co-immunoprecipitation, and pull-down assay were employed for determining O-GlcNAcylation. Mutagenesis PCR was applied for the generation of STING mutants. WT and Sting1-/- C57BL/6 mice (KOCMP-72512-Sting1-B6NVA) were infected with HSV-1 and treated with O-GlcNAcylation inhibitor for validating the role of STING O-GlcNAcylation in antiviral response. RESULTS: STING was functionally activated by O-GlcNAcylation in host cells challenged with HSV-1. We demonstrated that this signaling event was initiated by virus infection-enhanced hexosamine biosynthesis pathway (HBP). HSV-1 (or viral DNA mimics) promotes glucose metabolism of host cells with a marked increase in HBP, which provides donor glucosamine for O-GlcNAcylation. STING was O-GlcNAcylated on threonine 229, which led to lysine 63-linked ubiquitination of STING and activation of antiviral immune responses. Mutation of STING T229 to alanine abrogated STING activation and reduced HSV-1 stimulated production of interferon (IFN). Application of 6-diazo-5-oxonorleucine (DON), an agent that blocks the production of UDP-GlcNAc and inhibits O-GlcNAcylation, markedly attenuated the removal of HSV-1 in wild type C57BL/6 mice, leading to an increased viral retention, elevated infiltration of inflammatory cells, and worsened tissue damages to those displayed in STING gene knockout mice. Together, our data suggest that STING is O-GlcNAcylated in HSV-1, which is crucial for an effective antiviral innate immune response. CONCLUSION: HSV-1 infection activates the generation of UDP-Glc-NAc by upregulating the HBP metabolism. Elevated UDP-Glc-NAc promotes the O-GlcNAcylation of STING, which mediates the anti-viral function of STING. Targeting O-GlcNAcylation of STING could be a useful strategy for antiviral innate immunity.

Self-Delivery Nanomedicines Reverse Thermal Resistance to Enhance Tumor Mild-Temperature Photothermal Therapy.

Tumoral thermal defense mechanisms considerably attenuate the therapeutic outcomes of mild-temperature photothermal therapy (PTT). Thus, developing a simple, efficient, and universal therapeutic strategy to sensitize mild-temperature PTT is desirable. Herein, we report self-delivery nanomedicines ACy NPs comprising a near-infrared (NIR) photothermal agent (Cypate), mitochondrial oxidative phosphorylation inhibitor (ATO), and distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), which have a high drug-loading efficiency that can reverse tumoral thermal resistance, thereby increasing mild-temperature PTT efficacy. ACy NPs achieved targeted tumor accumulation and performed NIR fluorescence imaging capability in vivo to guide tumor PTT for optimized therapeutic outcomes. The released ATO reduced intracellular ATP levels to downregulate multiple heat shock proteins (including HSP70 and HSP90) before PTT, which reversed the thermal resistance of tumor cells, contributing to the excellent results of mild-temperature PTT in vitro and in vivo. Therefore, this study provides a simple, biosafe, advanced, and universal heat shock protein-blocking strategy for tumor PTT.

Iron(II) Phthalocyanine-Catalyzed Homodimerization and Tandem Diamination of Diazo Compounds with Primary Amines: Access to Construct Substituted 2,3-Diaminosuccinonitriles in One-Pot.

We herein first report the homodimerization and tandem diamination of diazo compounds with primary amines catalyzed by the iron(II) phthalocyanine (PcFe(II)), which can construct one C-C bond and two C-N bonds within 20 min in one-pot. Compared to the traditional metal-catalyzed N-H insertion reaction between amines with diazo reagents, the developed reaction almost does not generate the N-H insertion product, but the homodimerization/tandem diamination product. The proposed mechanism studies indicate that primary amines play a crucial role in the homocoupling of diazo compounds via dimerization of iron(III)-acetonitrile radical generated from the reaction between diazoacetonitrile with PcFe(II) coordinated by bis(amines); the ß-hydride elimination is involved, and then, the attack of primary amines toward the carbon atoms on the formed C-C bond is followed. Moreover, this novel reaction can be used to effectively prepare substituted 2,3-diaminosuccinonitriles with high yields and even up to >99:1 d.r., encouragingly these products contain both 1,2-diamines and succinonitrile motifs, which are two classes of important organic compounds with significant applications in many yields. This reaction is also suitable for the gram-scale preparation of 2,3-bis(phenylamino)succinonitrile (2a) with a yield of 84%. Therefore, the developed reaction represents a new type of transformation.

Cholesterol mediates the effects of single and multiple environmental phenols in urine on obesity.

BACKGROUND: Overweight and obesity are among the leading chronic diseases worldwide. Environmental phenols have been renowned as endocrine disruptors that contribute to weight changes; however, the effects of exposure to mixed phenols on obesity are not well established. METHODS: Using data from adults in National Health and Nutrition Examination Survey, this study examined the individual and combined effects of four phenols on obesity. A combination of traditional logistic regression and two mixed models (weighted quantile sum (WQS) regression and Bayesian kernel-machine regression (BKMR)) were used together to assess the role of phenols in the development of obesity. The potential mediation of cholesterol on these effects was analyzed through a parallel mediation model. RESULTS: The results demonstrated that solitary phenols except triclosan were inversely associated with obesity (P-value < 0.05). The WQS index was also negatively correlated with general obesity (ß: 0.770, 95% CI: 0.644-0.919, P-value = 0.004) and abdominal obesity (ß: 0.781, 95% CI: 0.658-0.928, P-value = 0.004). Consistently, the BKMR model demonstrated the significant joint negative effects of phenols on obesity. The parallel mediation analysis revealed that high-density lipoprotein mediated the effects of all four single phenols on obesity, whereas low-density lipoprotein only mediated the association between benzophenol-3 and obesity. Moreover, Cholesterol acts as a mediator of the association between mixed phenols and obesity. Exposure to single and mixed phenols significantly and negatively correlated with obesity. Cholesterol mediated the association of single and mixed environmental phenols with obesity. CONCLUSIONS: Assessing the potential public health risks of mixed phenols helps to incorporate this information into practical health advice and guidance.

Effects of different proportions of fruit tree branches on nicotine content and microbial diversity during composting of tobacco waste.

Adding fruit tree branches to the compost pile in appropriate proportions is one of the methods used to address the challenge of tobacco waste recycling. However, the effects of different proportions of fruit tree branches on nicotine concentration and microbial diversity during tobacco waste composting have not been reported. In this study, a composting system with tobacco waste, cow dung, and fruit tree branches was established in a laboratory fermenter to assess the impact of adding 10%, 20%, and 30% fruit tree branches on quantity changes. In addition, the relationships between nicotine degradation, compost properties, enzyme activities, and microbial diversities were determined using biochemical assay methods and high-throughput sequencing. The results showed that adding appropriate proportions of fruit branch segments affected changes in physical and chemical properties during composting and promoted tobacco waste compost maturity. Aerobic composting effectively degraded nicotine in tobacco waste. Increased proportions of fruit branch segments led to elevations in nicotine degradation rates and enzyme activities related to lignocellulose degradation. The addition of fruit branches influenced the relative abundance and species of dominant bacteria and fungi at the phylum and genus levels. However, it did not significantly affect the relative abundance of the main bacterial genera involved in nicotine degradation. Nevertheless, it reduced the sensitivity of enzyme activity to nicotine content within heaps, increasing reliance on total nitrogen changes. The results of this study provide a theoretical basis for the utilization of tobacco waste in composting systems and indicate that fruit tree branches can enhance nicotine degradation efficiency during tobacco waste composting.

Qi-Long-Tian capsule alleviates pulmonary fibrosis development by modulating inflammatory response and gut microbiota.

Pulmonary fibrosis (PF) is a chronic, progressive, and fibrotic interstitial lung disease with a high mortality rate. Qi-Long-Tian (QLT) capsule is an herbal formula with great potential for antifibrotic effects, consisting of San Qi (Notoginseng Radix et Rhizoma), Di Long [Pheretima aspergillum (E. Perrier)], and Hong Jingtian (Rhodiolae Crenulatae Radix et Rhizoma), and has been used in clinical practice for many years. To explore the relationship between the effects of Qi-Long-Tian capsule and gut microbiota of PF mice, pulmonary fibrosis model were established by tracheal drip injection of bleomycin. Thirty-six mice were randomly divided into 6 groups: control group (control), model group (model), QLT capsule low dose group (QL), QLT capsule medium dose group (QM), QLT capsule high dose group (QH), and pirfenidone group (PFD). After 21 days of treatment, after pulmonary function tests, the lung tissues, serums, and enterobacterial samples were collected for further analysis. HE staining and Masson's staining were used to detect changes as the main indicators of PF in each group, and the expression of hydroxyproline (HYP) related to collagen metabolism was detected by and alkaline hydrolysis method. qRT-PCR and ELISA were used to detect the mRNA and protein expressions of pro-inflammatory factors include interleukin 1ß (IL-1ß), interleukin 6 (IL-6), transforming growth factor ß1 (TGF-ß1), tumor necrosis factor α (TNF-α) in lung tissues and serums, and the inflammation-mediating factors include tight junction protein (ZO-1, Claudin, Occludin). ELISA was used to detect the protein expressions of secretory immunoglobulin A (sIgA), short-chain fatty acids (SCFAs), and lipopolysaccharide (LPS) in colonic tissues. 16sRNA gene sequencing was used to detect changes in the abundance and diversity of intestinal flora in the control, model, and QM groups, to search for differential genera, and analyze the correlation with inflammatory factors. QLT capsule effectively improved the status of pulmonary fibrosis and reduced HYP. In addition, QLT capsule significantly reduced the abnormal levels of pro-inflammatory factors, including IL-1ß, IL-6, TNF-α, and TGF-ß in lung tissue and serum, while improving the levels of pro-inflammatory related factors ZO-1, Claudin, Occludin, sIgA, SCFAs, and reducing LPS in the colon. The comparison between the alpha diversity and beta diversity in enterobacteria suggested that the composition of the gut flora in the control, model, and QLT capsule groups were different. QLT capsule significantly increased the relative abundance of Bacteroidia (which might limit the onset of inflammation) and decreased the relative abundance of Clostridia (which might promote inflammation). In addition, these two enterobacteria were closely associated with pro-inflammatory-related indicators and pro-inflammatory factors in PF. All these results suggest that QLT capsule intervenes in pulmonary fibrosis by regulating the differential genera of intestinal flora, increasing immunoglobulin secretion, repairing the intestinal mucosal barrier, reducing LPS entry into the blood, and decreasing inflammatory factor secretion in the serum, which in turn alleviates pulmonary inflammation. This study clarifies the therapeutic mechanism of QLT capsule in PF and provides a theoretical basis for it. It provides a theoretical basis for its further clinical application.

MMP-3-mediated cleavage of OPN is involved in copper oxide nanoparticle-induced activation of fibroblasts.

BACKGROUND: Copper oxide nanoparticles (Nano-CuO) are one of the most produced and used nanomaterials. Previous studies have shown that exposure to Nano-CuO caused acute lung injury, inflammation, and fibrosis. However, the mechanisms underlying Nano-CuO-induced lung fibrosis are still unclear. Here, we hypothesized that exposure of human lung epithelial cells and macrophages to Nano-CuO would upregulate MMP-3, which cleaved osteopontin (OPN), resulting in fibroblast activation and lung fibrosis. METHODS: A triple co-culture model was established to explore the mechanisms underlying Nano-CuO-induced fibroblast activation. Cytotoxicity of Nano-CuO on BEAS-2B, U937* macrophages, and MRC-5 fibroblasts were determined by alamarBlue and MTS assays. The expression or activity of MMP-3, OPN, and fibrosis-associated proteins was determined by Western blot or zymography assay. Migration of MRC-5 fibroblasts was evaluated by wound healing assay. MMP-3 siRNA and an RGD-containing peptide, GRGDSP, were used to explore the role of MMP-3 and cleaved OPN in fibroblast activation. RESULTS: Exposure to non-cytotoxic doses of Nano-CuO (0.5 and 1 µg/mL) caused increased expression and activity of MMP-3 in the conditioned media of BEAS-2B and U937* cells, but not MRC-5 fibroblasts. Nano-CuO exposure also caused increased production of cleaved OPN fragments, which was abolished by MMP-3 siRNA transfection. Conditioned media from Nano-CuO-exposed BEAS-2B, U937*, or the co-culture of BEAS-2B and U937* caused activation of unexposed MRC-5 fibroblasts. However, direct exposure of MRC-5 fibroblasts to Nano-CuO did not induce their activation. In a triple co-culture system, exposure of BEAS-2B and U937* cells to Nano-CuO caused activation of unexposed MRC-5 fibroblasts, while transfection of MMP-3 siRNA in BEAS-2B and U937* cells significantly inhibited the activation and migration of MRC-5 fibroblasts. In addition, pretreatment with GRGDSP peptide inhibited Nano-CuO-induced activation and migration of MRC-5 fibroblasts in the triple co-culture system. CONCLUSIONS: Our results demonstrated that Nano-CuO exposure caused increased production of MMP-3 from lung epithelial BEAS-2B cells and U937* macrophages, which cleaved OPN, resulting in the activation of lung fibroblasts MRC-5. These results suggest that MMP-3-cleaved OPN may play a key role in Nano-CuO-induced activation of lung fibroblasts. More investigations are needed to confirm whether these effects are due to the nanoparticles themselves and/or Cu ions.

High glucose enhances the activation of NLRP3 inflammasome by ambient fine particulate matter in alveolar macrophages.

BACKGROUND: Epidemiological studies have demonstrated that individuals with preexisting conditions, including diabetes mellitus (DM), are more susceptible to air pollution. However, the underlying mechanisms remain unclear. In this study, we proposed that a high glucose setting enhances ambient fine particulate matter (PM2.5)-induced macrophage activation and secretion of the proinflammatory cytokine, IL-1ß, through activation of the NLRP3 inflammasome, altering the balance between matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs). RESULTS: Exposure of mouse alveolar macrophages to non-cytotoxic doses of PM2.5 led to upregulation of IL-1ß, activation of the NLRP3 inflammasome, increased nuclear translocation of the transcription factor NF-κB, increased generation of reactive oxygen species (ROS), and increased expression and enzymatic activity of MMP-9; these effects were enhanced when cells were pretreated with high glucose. However, pretreatment in a high glucose setting alone did not induce significant changes. ROS generation following PM2.5 exposure was abolished when cells were pretreated with ROS scavengers such as Trolox and superoxide dismutase (SOD), or with an NADPH oxidase inhibitor, DPI. Pretreatment of cells with DPI attenuated the effects of a high glucose setting on PM2.5-induced upregulation of IL-1ß, activation of the NLRP3 inflammasome, and nuclear translocation of NF-κB. In addition, enhancement of PM2.5-induced expression and enzymatic activity of MMP-9 following high glucose pretreatment was not observed in primary alveolar macrophages obtained from NLRP3 or IL-1R1 knockout (KO) mice, where pro-IL-1ß cannot be cleaved to IL-1ß or cells are insensitive to IL-1ß, respectively. CONCLUSIONS: This study demonstrated that exposure of mouse alveolar macrophages to PM2.5 in a high glucose setting enhanced PM2.5-induced production of IL-1ß through activation of the NLRP3 inflammasome and nuclear translocation of NF-κB due to PM2.5-induced oxidative stress, leading to MMP-9 upregulation. The key role of NADPH oxidase in PM2.5-induced ROS generation and activation of the IL-1ß secretion pathway and the importance of IL-1ß secretion and signaling in PM2.5-induced increases in MMP-9 enzymatic activity were also demonstrated. This study provides a further understanding of the potential mechanisms underlying the susceptibility of individuals with DM to air pollution and suggests potential therapeutic targets.

Cadherin-11-Interleukin-6 Signaling between Cardiac Fibroblast and Cardiomyocyte Promotes Ventricular Remodeling in a Mouse Pressure Overload-Induced Heart Failure Model.

Heart failure is a serious and life-threatening disease worldwide. Cadherin-11 (Cad-11) is highly expressed in the heart and closely associated with inflammation. There is currently limited understanding on how Cad-11 contributes to cardiac remodeling and its underline molecular mechanism. We found an increased expression of Cad-11 in biopsy heart samples from heart failure patients, suggesting a link between Cad-11 and heart failure. To determine the role of Cad-11 in cardiac remodeling, Cad-11-deficient mice were used in a well-established mouse transverse aortic constriction (TAC) model. Loss of Cad11 greatly improved pressure overload-induced LV structural and electrical remodeling. IL (interleukin)-6 production was increased following TAC in WT mice and this increase was inhibited in cadherin-11-/- mice. We further tested the effect of IL-6 on myocyte hypertrophy and fibrosis in a primary culture system. The addition of hCad-11-Fc to cultured cardiac fibroblasts increased IL-6 production and fibroblast cell activation, whereas neutralizing IL-6 with an IL-6 antibody resulted in alleviating the fibroblast activation induced by hCad-11-Fc. On the other hand, cardiomyocytes were promoted to cardiomyocyte hypertrophy when cultured in condition media collected from cardiac fibroblasts stimulated by hCad-11-Fc.Similarly, neutralizing IL-6 prevented cardiomyocyte hypertrophy. Finally, we found that MAPKs and CaMKII-STAT3 pathways were activated in both hCad-11-Fc stimulated fibroblasts and cardiomyocytes treated with hCad-11-Fc stimulated fibroblast condition medium. IL-6 neutralization inhibited such MAPK and CaMKII-STAT3 signaling activation. These data demonstrate that Cad-11 functions in pressure overload-induced ventricular remodeling through inducing IL-6 secretion from cardiac fibroblasts to modulate the pathophysiology of neighboring cardiomyocytes.

Transcriptome-wide association study identifies PSMB9 as a susceptibility gene for coal workers' pneumoconiosis.

Coal workers' pneumoconiosis (CWP) is a type of typical occupational lung disease caused by prolonged inhalation of coal mine dust. The individuals' different genetic background may underlie their different susceptibility to develop pneumoconiosis, even under the same exposure level. This study aimed to identify susceptibility genes associated with CWP. Based on our previous genome-wide association study (GWAS, 202 CWP cases vs. 198 controls) and gene expression data obtained by analyzing human lungs and whole blood from the Genotype-Tissue Expression (GTEx) Portal, a transcriptome-wide association study (TWAS) was applied to identify CWP risk-related genes. Luciferase report gene assay, qRT-PCR, Western blot, immunofluorescence assay, and TUNEL assay were conducted to explore the potential role of the candidate gene in CWP. Proteasome 20S subunit beta 9 (PSMB9) was identified as a strong risk-related gene of CWP in both lungs and whole blood (Lungs: PTWAS  = 4.22 × 10-4 ; Whole blood: PTWAS  = 2.11 × 10-4 ). Single nucleotide polymorphisms (SNPs) rs2071480 and rs1351383, which locate in the promoter region and the first intron of the PSMB9 gene, were in high linkage disequilibrium (LD, r2  = 0.98) with the best GWAS SNP rs4713600 (G>T, OR = 0.55, 95% CI: 0.42-0.74, P = 6.86 × 10-5 ). Both rs2071480 and rs1351383 significantly enhanced the transcriptional activity of PSMB9. Functional experiments revealed that silica exposure remarkably reduced the PSMB9 expression and caused cell apoptosis, while overexpression of PSMB9 markedly abolished silica-induced cell apoptosis. We here identified PSMB9 as a novel susceptibility gene for CWP and provided important insights into the further exploration of the CWP pathogenesis.

Cadherin-11 deficiency mitigates high-fat diet-induced inflammatory atrial remodeling and vulnerability to atrial fibrillation.

Atrial fibrillation (AF) is the most common cardiac arrhythmia nowadays. The occurrence of AF is closely associated with obesity. Cadherin-11 (Cad-11), as a member of the cadherin family, can make a contribution to diet-induced obesity and it will be informative to know whether Cad-11 exerts its effects on atrial remodeling and AF vulnerability in a diet-induced obesity model. In this study, we demonstrated that the expression of Cad-11 was significantly upregulated in the left atrium of AF patients with obesity and mice following 16 weeks of high-fat diet (HFD) feeding. Further confirmed that Cad-11 could regulate the activity of atrial fibroblasts by participating in inducing proinflammatory cytokines production. At animal levels, we found that although there was a lack of statistical difference in body weight, Cad-11-/- mice could markedly improve impaired glucose tolerance and hyperlipidemia. Adverse atrial structural remodeling, including atrial enlargement, inflammation, and fibrosis provoked by HFD feeding were mitigated in Cad-11-/- mice. Mechanistically, Cad-11 activated mitogen-activated protein kinases and nuclear factor-κB for interleukin-6 production in atrial fibroblasts that may contribute to the atrial fibrosis process in obesity-related AF, suggesting Cad-11 might be a new therapeutic target for obesity-related AF.

Lupus gut microbiota transplants cause autoimmunity and inflammation.

BACKGROUND: The etiology of systemic lupus erythematosus (SLE) is multifactorial. Recently, growing evidence suggests that the microbiota plays a role in SLE, yet whether gut microbiota participates in the development of SLE remains largely unknown. To investigate this issue, we carried out 16 s rDNA sequencing analyses in a cohort of 18 female un-treated active SLE patients and 7 female healthy controls, and performed fecal microbiota transplantation from patients and healthy controls to germ-free (GF) mice. RESULTS: Compared to the healthy controls, we found no significant different microbial diversity but some significantly different species in SLE patients including Turicibacter genus and other 5 species. Fecal transfer from SLE patients to GF mice caused GF mice to develop a series of lupus-like phenotypic features, including increased serum autoimmune antibodies, imbalanced cytokines, altered distribution of immune cells in mucosal and peripheral immune response, and upregulated expression of genes related to SLE in recipient mice that received SLE fecal microbiota transplantation (FMT). Moreover, the metabolism of histidine was significantly altered in GF mice treated with SLE patient feces, as compared to those which received healthy fecal transplants. CONCLUSIONS: Overall, our results describe a causal role of aberrant gut microbiota in contributing to the pathogenesis of SLE. The interplay of gut microbial and histidine metabolism may be one of the mechanisms intertwined with autoimmune activation in SLE.

EZH2 as an Epigenetic Regulator of Cardiovascular Development and Diseases.

ABSTRACT: Enhancer of zeste homolog 2(EZH2) is an enzymatic subunit of polycomb repressive complex 2 (PRC2) and is responsible for catalyzing mono-, di-, and trimethylation of histone H3 at lysine-27(H3K27me1/2/3). Many noncoding RNAs or signaling pathways are involved in EZH2 functional alterations. This new epigenetic regulation of target genes is able to silence downstream gene expression and modify physiological and pathological processes in heart development, cardiomyocyte regeneration, and cardiovascular diseases, such as hypertrophy, ischemic heart diseases, atherosclerosis, and cardiac fibrosis. Targeting the function of EZH2 could be a potential therapeutic approach for cardiovascular diseases.

Effects of metal nanoparticles on tight junction-associated proteins via HIF-1α/miR-29b/MMPs pathway in human epidermal keratinocytes.

BACKGROUND: The increasing use of metal nanoparticles in industry and biomedicine raises the risk for unintentional exposure. The ability of metal nanoparticles to penetrate the skin ranges from stopping at the stratum corneum to passing below the dermis and entering the systemic circulation. Despite the potential health risks associated with skin exposure to metal nanoparticles, the mechanisms underlying the toxicity of metal nanoparticles on skin keratinocytes remain unclear. In this study, we proposed that exposure of human epidermal keratinocytes (HaCaT) to metal nanoparticles, such as nickel nanoparticles, dysregulates tight-junction associated proteins by interacting with the HIF-1α/miR-29b/MMPs axis. METHODS: We performed dose-response and time-response studies in HaCaT cells to observe the effects of Nano-Ni or Nano-TiO2 on the expression and activity of MMP-2 and MMP-9, and on the expression of tight junction-associated proteins, TIMP-1, TIMP-2, miR-29b, and HIF-1α. In the dose-response studies, cells were exposed to 0, 10, or 20 µg/mL of Nano-Ni or Nano-TiO2 for 24 h. In the time-response studies, cells were exposed to 20 µg/mL of Nano-Ni for 12, 24, 48, or 72 h. After treatment, cells were collected to either assess the expression of mRNAs and miR-29b by real-time PCR or to determine the expression of tight junction-associated proteins and HIF-1α nuclear accumulation by Western blot and/or immunofluorescent staining; the conditioned media were collected to evaluate the MMP-2 and MMP-9 activities by gelatin zymography assay. To further investigate the mechanisms underlying Nano-Ni-induced dysregulation of tight junction-associated proteins, we employed a HIF-1α inhibitor, CAY10585, to perturb HIF-1α accumulation in one experiment, and transfected a miR-29b-3p mimic into the HaCaT cells before Nano-Ni exposure in another experiment. Cells and conditioned media were collected, and the expression and activities of MMPs and the expression of tight junction-associated proteins were determined as described above. RESULTS: Exposure of HaCaT cells to Nano-Ni resulted in a dose-dependent increase in the expression of MMP-2, MMP-9, TIMP-1, and TIMP-2 and the activities of MMP-2 and MMP-9. However, exposure of cells to Nano-TiO2 did not cause these effects. Nano-Ni caused a dose-dependent decrease in the expression of miR-29b and tight junction-associated proteins, such as ZO-1, occludin, and claudin-1, while Nano-TiO2 did not. Nano-Ni also caused a dose-dependent increase in HIF-1α nuclear accumulation. The time-response studies showed that Nano-Ni caused significantly increased expressions of MMP-2 at 24 h, MMP-9 at 12, 24, and 48 h, TIMP-1 from 24 to 72 h, and TIMP-2 from 12 to 72 h post-exposure. The expression of miR-29b and tight junction-associated proteins such as ZO-1, occludin, and claudin-1 decreased as early as 12 h post-exposure, and their levels declined gradually over time. Pretreatment of cells with a HIF-1α inhibitor, CAY10585, abolished Nano-Ni-induced miR-29b down-regulation and MMP-2/9 up-regulation. Introduction of a miR-29b-3p mimic into HaCaT cells by transfection before Nano-Ni exposure ameliorated Nano-Ni-induced increased expression and activity of MMP-2 and MMP-9 and restored Nano-Ni-induced down-regulation of tight junction-associated proteins. CONCLUSION: Our study herein demonstrated that exposure of human epidermal keratinocytes to Nano-Ni caused increased HIF-1α nuclear accumulation and increased transcription and activity of MMP-2 and MMP-9 and down-regulation of miR-29b and tight junction-associated proteins. Nano-Ni-induced miR-29b down-regulation was through Nano-Ni-induced HIF-1α nuclear accumulation. Restoration of miR-29b level by miR-29b-3p mimic transfection abolished Nano-Ni-induced MMP-2 and MMP-9 activation and down-regulation of tight junction-associated proteins. In summary, our results demonstrated that Nano-Ni-induced dysregulation of tight junction-associated proteins in skin keratinocytes was via HIF-1α/miR-29b/MMPs pathway.

Nickel nanoparticle-induced cell transformation: involvement of DNA damage and DNA repair defect through HIF-1α/miR-210/Rad52 pathway.

BACKGROUND: Nickel nanoparticles (Nano-Ni) are increasingly used in industry and biomedicine with the development of nanotechnology. However, the genotoxic and carcinogenic effects of Nano-Ni and the underlying mechanisms are still unclear. METHODS: At first, dose-response (0, 10, 20, and 30 µg/mL) and time-response (0, 3, 6, 12, and 24 h) studies were performed in immortalized normal human bronchial epithelial cells BEAS-2B to observe the effects of Nano-Ni on DNA damage response (DDR)-associated proteins and the HIF-1α/miR-210/Rad52 pathway by real-time PCR or Western blot. Then, a Hsp90 inhibitor (1 µM of 17-AAG, an indirect HIF-1α inhibitor), HIF-1α knock-out (KO) cells, and a miR-210 inhibitor (20 nM) were used to determine whether Nano-Ni-induced Rad52 down-regulation was through HIF-1α nuclear accumulation and miR-210 up-regulation. In the long-term experiments, cells were treated with 0.25 and 0.5 µg/mL of Nano-Ni for 21 cycles (~ 150 days), and the level of anchorage-independent growth was determined by plating the cells in soft agar. Transduction of lentiviral particles containing human Rad52 ORF into BEAS-2B cells was used to observe the role of Rad52 in Nano-Ni-induced cell transformation. Nano-Ni-induced DNA damage and dysregulation of HIF-1α/miR-210/Rad52 pathway were also investigated in vivo by intratracheal instillation of 50 µg per mouse of Nano-Ni. gpt delta transgenic mice were used to analyze mutant frequency and mutation spectrum in mouse lungs after Nano-Ni exposure. RESULTS: Nano-Ni exposure caused DNA damage at both in vitro and in vivo settings, which was reflected by increased phosphorylation of DDR-associated proteins such as ATM at Ser1981, p53 at Ser15, and H2AX. Nano-Ni exposure also induced HIF-1α nuclear accumulation, miR-210 up-regulation, and down-regulation of homologous recombination repair (HRR) gene Rad52. Inhibition of or knocking-out HIF-1α or miR-210 ameliorated Nano-Ni-induced Rad52 down-regulation. Long-term low-dose Nano-Ni exposure led to cell malignant transformation, and augmentation of Rad52 expression significantly reduced Nano-Ni-induced cell transformation. In addition, increased immunostaining of cell proliferation markers, Ki-67 and PCNA, was observed in bronchiolar epithelial cells and hyperplastic pneumocytes in mouse lungs at day 7 and day 42 after Nano-Ni exposure. Finally, using gpt delta transgenic mice revealed that Nano-Ni exposure did not cause increased gpt mutant frequency and certain DNA mutations, such as base substitution and small base insertions/deletions, are not the main types of Nano-Ni-induced DNA damage. CONCLUSIONS: This study unraveled the mechanisms underlying Nano-Ni-induced cell malignant transformation; the combined effects of Nano-Ni-induced DNA damage and DNA repair defects through HIF-1α/miR-210/Rad52 pathway likely contribute to Nano-Ni-induced genomic instability and ultimately cell transformation. Our findings will provide information to further elucidate the molecular mechanisms of Nano-Ni-induced genotoxicity and carcinogenicity.

miR-770-5p inhibits the activation of pulmonary fibroblasts and silica-induced pulmonary fibrosis through targeting TGFBR1.

Silicosis is a devastating interstitial lung disease arising from long-term exposure to inhalable silica. Regrettably, no therapy currently can effectively reverse the silica-induced fibrotic lesion. Emerging evidence has indicated that the dysregulation of microRNAs is involved in silica-induced pulmonary fibrosis. The aim of this study is to explore the expression pattern and underlying mechanisms of miR-770-5p in silica-induced pulmonary fibrosis. Consistent with our previous miRNA microarray analysis, the results of qRT-PCR showed that miR-770-5p expression was downregulated in silica-induced pulmonary fibrosis in humans and animal models. Administration of miR-770-5p agomir significantly reduced the fibrotic lesions in the lungs of mice exposed to silica dust. MiR-770-5p also exhibited a dramatic reduction in TGF-ß1-activated human pulmonary fibroblasts (MRC-5). Transfection of miR-770-5p mimics significantly decreased the viability, migration ability, and S/G0 phase distribution, as well as the expression of fibronectin, collagen I, and α-SMA in TGF-ß1-treated MRC-5 cells. Transforming growth factor-ß receptor 1 (TGFBR1) was confirmed as a direct target of regulation by miR-770-5p. The expression of TGFBR1 was significantly increased in pulmonary fibrosis. Knockdown of TGFBR1 blocked the transduction of the TGF-ß1 signaling pathway and attenuated the activation of MRC-5 cells, while overexpression of TGFBR1 effectively restored the activation of MRC-5 cells inhibited by miR-770-5p. Together, our results demonstrated that miR-770-5p exerted an anti-fibrotic effect in silica-induced pulmonary fibrosis by targeting TGFBR1. Targeting miR-770-5p might provide a new therapeutic strategy to prevent the abnormal activation of pulmonary fibroblasts in silicosis.

LncRNA-PVT1 activates lung fibroblasts via miR-497-5p and is facilitated by FOXM1.

It is little known about the lncRNA-PVT1 effect on occupational pulmonary fibrosis, although researches show it plays an essential role in cancer. Studies reveal that lung fibroblast activation is one of the key events in silica-induced fibrosis. Here, we found that lncRNA-PVT1 promoted the proliferation, activation, and migration of lung fibroblasts. The isolation of cytoplasmic and nuclear RNA assay and fluorescence in situ hybridization experiment showed that lncRNA-PVT1 was abundantly expressed in the cytoplasm. Luciferase reporter gene assay and RNA pull-down experiment indicated that the cytoplasmic-localized lncRNA-PVT1 could competitively bind miR-497-5p. MiR-497-5p was further observed to attenuate silica-induced pulmonary fibrosis by targeting Smad3 and Bcl2. Moreover, the transcription factor FOXM1 acted as a profibrotic factor by elevating lncRNA-PVT1 transcription in lung fibroblasts. Inhibition of FOXM1 expression with thiostrepton alleviated silica-induced pulmonary fibrosis in vivo. Collectively, we revealed that FOXM1-facilitated lncRNA-PVT1 activates lung fibroblasts via miR-497-5p during silica-induced pulmonary fibrosis, which may provide potential therapeutic targets for pulmonary fibrosis.

Combined Therapy of Catheter Ablation and Left Atrial Appendage Closure for Patients with Atrial Fibrillation: A Case-Control Study.

AIM: The feasibility and safety of performing the combined procedure of catheter ablation (CA) and left atrial appendage closure (LAAC) for atrial fibrillation (AF) have been reported by observational studies without controls. The aim of this study was to compare the procedural and long-term outcomes of combined procedures with isolated CA or LAAC. METHODS AND RESULTS: This study included patients who underwent combined CA and LAAC (combined group), CA alone (CA-only group), or LAAC alone (LAAC-only group). Propensity score matching was used to select controls from the CA-only and LAAC-only groups. Each group contained 76 subjects. The procedures were successfully performed in all the patients. Procedure-related complications of the combined group included one pericardial effusion and two groin haematomas, which did not differ significantly with those of the CA-only group (3.9% vs. 2.6%, P=0.650) or the LAAC-only group (3.9% vs. 2.6%, P=0.650), respectively. The AF-free rate of the combined group was comparable with that of the CA-only group after a mean of 2 years follow-up (67.1% vs. 69.7%, P=0.727). Compared with the LAAC-only group, the combined group achieved similar complete occlusion rate at implant (94.7% vs. 93.4%) and at 45 days (82.9% vs. 85.5%). At the end of follow-up, ischemic stroke and bleeding events of the combined group were low (3.9%) and were comparable with those of the CA-only group (5.3%) and the LAAC-only group (2.6%). CONCLUSIONS: The combination of AF-CA and LAAC is safe and efficacious compared with single procedures alone.

Effects of Hetiao Jianpi Decoction on Intestinal Injury and Repair in Rats with Antibiotic-Associated Diarrhea.

BACKGROUND Through observing the changes of indexes of the intestinal mucosal barrier and intestinal flora in rats, we explored the mechanism by which Hetiao Jianpi Decoction (HTJPD) treats antibiotic-associated diarrhea (AAD) by repairing intestinal mucosal injury and regulating intestinal flora. MATERIAL AND METHODS Samples of colon tissues were collected for HE staining. Enzyme-linked immunosorbent assay (ELISA) was used to assess levels of diamine oxidase (DAO) and D-lactic acid in rat plasma and the expression of secretory immunoglobulin A (SIgA) in colon tissue. We assessed the abundance of intestinal contents by high-throughput sequencing of the 16S rRNA gene. RESULTS Compared with the Model group, the muscle layer and intestinal mucosal edema were improved, and the continuity was restored; the levels of DAO and D-lactic acid in plasma decreased, and the SIgA level were increased in the HTJPD group. The structure of the intestinal flora changed, as indicated by increased levels of certain beneficial bacteria (Verrucomicrobia, Actinobacteria, CF231, and Akkermansia), decreased levels of pathogenic bacteria (Spirochaetes and Treponema), and increased species diversity. CONCLUSIONS By improving the permeability and immune function of the intestinal mucosa, Hetiao Jianpi decoction prevented the occurrence of AAD by repairing the intestinal mucosal damage and regulating the structure and diversity of intestinal flora.