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The aim of this study was to elucidate the mechanism underlying the effects of Kukoamine A (KuA) treatment on endotoxin-induced lung injury/inflammation. The study was performed in lipopolysaccharide (LPS)-exposed mouse models of lung injury and LPS-induced alveolar epithelial cell model. Relevant kits were used to detect levels of inflammation-related indicators, oxidative stress indicators, and mitochondrial function. Hematoxylin and eosin staining was to detect lung injury. Then, C-C motif chemokine receptor 5 (CCR5) overexpression plasmid was transfected into alveolar epithelial cells to investigate the mechanism of KuA in lung injury. The results showed that LPS induction increased the expression of inflammatory factors, oxidative stress markers, and mitochondrial dysfunction in both animal and cellular models. In the mouse model, KuA treatment improved lung tissue injury, decreased wet-to-dry ratio and MPO levels, reduced the expression of inflammatory factors, and ameliorated oxidative stress and mitochondrial dysfunction. The protective effect of KuA in the cell model remained whereas was markedly reversed after CCR5 overexpression. Taken together, KuA might improve LPS-induced lung injury by inhibiting CCR5. This might also provide a novel theory for KuA in the treatment of lung injury.

Periodontitis could lead to periodontal destruction such as the loss of alveolar bone. The issue that how to achieve the regeneration of alveolar bone and periodontal tissues under the inflammatory environment needs to be solved urgently. Bone morphogenetic protein 9 (BMP9) is one of the most potent osteoinductive BMPs and induces osteogenic differentiation of mesenchymal stem cells. The aim of this study is to explore the possible effect of BMP9 on the osteogenic differentiation of inflammatory periodontal ligament stem cells (PDLSCs).Human PDLSCs were cultured in osteoinductive medium with 1 μg/mL lipopolysaccharide Porphyromonas gingivitis (LPS-PG). Adenoviral vector expressing system was used to overexpress target genes. In vitro expression of osteogenic markers was assessed by quantitative reverse transcription polymerase chain reaction, Western blotting, alkaline phosphatase assay, and alizarin red staining. Subcutaneous implantation nude mice models were used to evaluate the effects of BMP9 on PDLSCs in vivo. Microcomputed tomography, hematoxylineosin staining, and trichrome staining were performed to assess ectopic bone formation.In the LPS-PG induced inflammatory environment, BMP9 promoted osteogenic differentiation of PDLSCs, but upregulated the expression of inflammatory markers (P 0.05); NEL-like protein 1 (NELL1) downregulated the expression of inflammation genes in PDLSCs induced by BMP9, while augmenting BMP9-induced osteogenesis of the cells both in vitro and in vivo. In the above process, the MAPK/p38/ERK signaling pathway was triggered by NELL1.The combination use of BMP9 and NELL1 might have the potential to promote the regeneration of alveolar bone in periodontitis.

This study aimed to identify and prepare peptides from selenium (Se)-enriched soybeans and determine whether dietary Se-enriched soybean peptides (Se-SPep) could inhibit lung injury in mice induced by fine particulate matter 2.5 (PM2.5). BALB/c mice were randomly divided into six groups. The mice in the prevention groups were pretreated with 378 mg kg

Semaphorins, a large group of highly conserved proteins, consist of eight subfamilies that are widely expressed in vertebrates, invertebrates, and viruses and exist in membrane-bound or secreted forms. First described as axon guidance cues during neurogenesis, semaphorins also perform physiological functions in other organ systems, such as bone homeostasis, immune response, and tumor progression. Semaphorin 7A (SEMA7A), also known as CDw108, is an immune semaphorin that modulates diverse immunoinflammatory processes, including immune cell interactions, inflammatory infiltration, and cytokine production. In addition, SEMA7A regulates the proliferation, migration, invasion, lymph formation, and angiogenesis of multiple types of tumor cells, and these effects are mediated by the interaction of SEMA7A with two specific receptors, PLXNC1 and integrins. Thus, SEMA7A is intimately related to the pathogenesis of multiple autoimmune and inflammation-related diseases and tumors. This review focuses on the role of SEMA7A in the pathogenesis of autoimmune disorders, inflammatory diseases, and tumors, as well as the underlying mechanisms. Furthermore, strategies targeting SEMA7A as a potential predictive, diagnostic, and therapeutic agent for these diseases are also addressed.

In vertebrates, the semaphorin family of proteins is composed of 21 members that are divided into five subfamilies, i.e. classes 3 to 7. Semaphorins play crucial roles in regulating multiple biological processes, such as neural remodeling, tissue regeneration, cancer progression, and, especially, in immunological regulation. Semaphorin 4D (SEMA4D), also known as CD100, is an important member of the semaphorin family and was first characterized as a lymphocyte-specific marker. SEMA4D has diverse effects on immunologic processes, including immune cell proliferation, differentiation, activation, and migration, through binding to its specific membrane receptors CD72, PLXNB1, and PLXNB2. Furthermore, SEMA4D and its underlying signaling have been increasingly linked with several immunological diseases. This review focuses on the significant immunoregulatory role of SEMA4D and the associated underlying mechanisms, as well as the potential application of SEMA4D as a diagnostic marker and therapeutic target for the treatment of immunological diseases.

Excessive light exposure is a principal environmental factor, which can cause damage to photoreceptors and retinal pigment epithelium (RPE) cells and may accelerate the progression of age-related macular degeneration (AMD). In this study, oxidative stress, endoplasmic reticulum (ER) stress and autophagy caused by light exposure were evaluated in vitro and in vivo. Light exposure caused severe photo-oxidative stress and ER stress in photoreceptors (661W cells) and RPE cells (ARPE-19 cells). Suppressing either oxidative stress or ER stress was protective against light damage in 661W and ARPE-19 cells and N-acetyl-L-cysteine treatment markedly inhibited the activation of ER stress caused by light exposure. Moreover, suppressing autophagy with 3-methyladenine significantly attenuated light-induced cell death. Additionally, inhibiting ER stress either by knocking down PERK signals or with GSK2606414 treatment remarkably suppressed prolonged autophagy and protected the cells against light injury. In vivo experiments verified neuroprotection via inhibiting ER stress-related autophagy in light-damaged retinas of mice. In conclusion, the above results suggest that light-induced photo-oxidative stress may trigger subsequent activation of ER stress and prolonged autophagy in photoreceptors and RPE cells. Suppressing ER stress may abrogate over-activated autophagy and protect the retina against light injury.

The zinc-finger protein ZBTB20 regulates development and metabolism in multiple systems, and is essential for postnatal survival in mice. However, its potential role in the cardiovascular system remains undefined. Here, we demonstrate that ZBTB20 is critically involved in the regulation of cardiac contractility and blood pressure in mice. At the age of 16 days, the relatively healthy Zbtb20-null mice exhibited hypotension without obvious change of heart rate or other evidence for heart failure. Moreover, Zbtb20 deletion led to a marked reduction in heart size, left ventricular wall thickness, and cell size of cardiomyocytes, which was largely proportional to the decreased body growth. Notably, echocardiographic and hemodynamic analyses showed that cardiac contractility was greatly impaired in the absence of ZBTB20. Mechanistically, ZBTB20 deficiency decreased cardiac ATP contents, and compromised the enzyme activity of mitochondrial complex I in heart as well as L-type calcium current density in cardiomyocytes. Furthermore, the developmental activation of some mitochondrial function-related genes was significantly attenuated in Zbtb20-null myocardium, which included Hspb8, Ckmt2, Cox7a1, Tfrc, and Ogdhl. Put together, these results suggest that ZBTB20 plays a crucial role in the regulation of heart development, energy metabolism, and contractility.

Cardiac fibroblast (CF) differentiation into myofibroblasts is a crucial cause of cardiac fibrosis, which increases in the extracellular matrix (ECM) stiffness. The increased stiffness further promotes CF differentiation and fibrosis. However, the molecular mechanism is still unclear. We used bioinformatics analysis to find new candidates that regulate the genes involved in stiffness-induced CF differentiation, and found that there were binding sites for the POU-domain transcription factor, POU2F1 (also known as Oct-1), in the promoters of 50 differentially expressed genes (DEGs) in CFs on the stiffer substrate. Immunofluorescent staining and Western blotting revealed that pathological stiffness upregulated POU2F1 expression and increased CF differentiation on polyacrylamide hydrogel substrates and in mouse myocardial infarction tissue. A chromatin immunoprecipitation assay showed that POU2F1 bound to the promoters of fibrosis repressors IL1R2, CD69, and TGIF2. The expression of these fibrosis repressors was inhibited on pathological substrate stiffness. Knockdown of POU2F1 upregulated these repressors and attenuated CF differentiation on pathological substrate stiffness (35 kPa). Whereas, overexpression of POU2F1 downregulated these repressors and enhanced CF differentiation. In conclusion, pathological stiffness upregulates the transcription factor POU2F1 to promote CF differentiation by inhibiting fibrosis repressors. Our work elucidated the crosstalk between CF differentiation and the ECM and provided a potential target for cardiac fibrosis treatment.

In this work, twenty-eight novel 17a-aza-D-homo-androster-17-one derivatives, which divided into two categories, were synthesized with commercial available starting material (dehydroepiandrosterone) via oximation reaction, Beckmann rearrangement, hydroxyl protection, N-alkylation and deprotection. All compounds were characterized by 1H NMR, 13C NMR and HRMS. The structure of 14 g was also identified by X-ray single crystal diffraction. The bioactivities, brine shrimp toxicity and cytotoxicity, of all derivatives were tested. The results indicated that compounds 11 h, 11i, 11 m, 11 s, 14 b and 14 g exhibited excellent toxicity against brine shrimp with LC50 values ranging from 5.34 to 16.89 μg/mL, and compounds 11 s and 14 g displayed significant cytotoxicity against HT29 cells and A549 cells with IC50 values of 9.70 μM and 8.85 μM, respectively. Structure-activity relationships are discussed based on the results obtained from our research, and some important determinants for further modification of steroids towards the development of novel drug candidates are identified.

To explore the central sensitization mechanism of pain in chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS).We randomly divided 40 adult male SPF SD rats, aged 3－4 weeks and weighing 250－350 g, into a normal control and a CP/CPPS model group. After modeling, we analyzed the state of infiltration of CD4+T cells into the L5－S2 spinal cord and detected the expression levels of GFAP and CR3 in the spinal cord tissue using flow cytometry, real-time fluorescent quantitative PCR (RT-qPCR) and immunofluorescence staining.Compared with the normal controls, the CP/CPPS model rats showed dramatically increased expression of CD4+T cells in the mononuclear cells of the L5－S2 spinal cord tissue (P0.01), mRNA expressions of interferon-γ (IFN-γ) and tumor necrosis factor α (TNF-α) secreted from the Th1 cells, interleukin (IL)-17 and retinoic acid-associated orphan receptor (ROR) γt secreted from the Th17 cells, cytokines IL-6 and IL-1β, and chemokines CCL2, CCL20 and CXCL10 (P0.01), and expressions of the molecular markers of Th1 and Th17 cells IFN-γ and IL-17 and those of astrocytes and microglias GFAP and CR3.CD4+T cells, specifically Th1 and Th17 cells, infiltrate L5－S2 spinal cord neurons in CP/CPPS model rats. The inflammatory factors secreted from these cells may damage the neuronal cells, affect nervous conduction, promote central sensitization and activate astrocytes and microglias, leading to the development and progression of pain.

BackgroundThe molecular mechanisms of acute otitis media (AOM) development, and the intercellular crosstalk within the multicellular ecosystem of AOM, are not clear.MethodsWe established a model of AOM in rats (with normal rats as controls) and undertook single-cell RNA sequencing (scRNA-seq) for the middle-ear mucosa (MEM). Cell clustering and trajectory analyses were undertaken using Seurat and Monocle 2 packages in R software. Pathway analyses were done by gene set enrichment analysis (GSEA). Cell–cell interactions were inferred by CellChat. Cell scores were calculated to identify cells with dual-feature.ResultsA total of 7023 cells from three samples of inflamed MEM and 5258 cells from three samples of healthy MEM underwent scRNA-seq, which identified 20 cell clusters belonging to eight major cell types. After exposure to lipopolysaccharide, the MEM underwent significant conversion of cell types characterized by rapid infiltration of macrophages and neutrophils. M2 macrophages seemed to play a key part in inflammatory intercellular crosstalk, which facilitated the maintenance and proliferation of macrophages, cell chemotaxis, and regulation of the proinflammatory activities of cytokines. Three rare cell clusters with phagocytosis-related dual-feature were also identified. They coexisted with professional phagocytes in the MEM, and displayed distinct immunoregulatory functions by maintaining a normal immune microenvironment or influencing inflammation progression.ConclusionsMacrophages might be the “master” initiators and regulators of the inflammatory response of the MEM to external stimuli. And their functions are fulfilled by a specific polarization status (M2) and sophisticated intercellular crosstalk via certain signaling pathways. Besides, the coexistence of professional phagocytes and non-professional phagocytes as well as their interplay in the MEM provides new clues for deciphering the underlying pathogenic mechanisms of AOM.

Rationale: Protein O-GlcNAcylation is dynamically regulated by 2 key enzymes, OGT (O-GlcNAc transferase) and OGA (O-GlcNAcase). Excessive protein O-GlcNAcylation contributes to dilated cardiomyopathy (DCM), but its regulatory mechanisms are not fully understood. The PRMT5 (protein arginine methyltransferase 5) is the major type II arginine methyltransferase, which plays critical physiological roles by symmetrically dimethylating various downstream targets including proteins involved in RNA splicing. However, its function in regulating protein O-GlcNAcylation and DCM is unexplored. Objective: To elucidate the physiological function of PRMT5 and the mechanism underlying its role in regulating cardiac O-GlcNAcylation and homeostasis. Methods and Results: Conditional gene knockout was used to study the in vivo function of PRMT5 in regulating cardiac homeostasis. An integrated analysis of transcriptomic and metabolomic profiles was performed to investigate the molecular mechanism. Adeno-associated virus 9-mediated gene delivery in the mouse was used to study the protein O-GlcNAcylation in Prmt5 deficiency-induced DCM. PRMT5 mRNA was decreased in human DCM hearts, and cardiomyocyte-specific Prmt5 deletion in mice resulted in DCM and heart failure. Transcriptomic and metabolomic profiling identified increased O-GlcNAcylation in the hearts of Prmt5 -knockout mice. Mechanistically, Prmt5 deletion suppressed OGA expression by inhibiting the transcription of Oga and triggering its aberrant splicing. Consistently, a positive correlation of PRMT5 and OGA was identified in human DCM hearts. Notably, gene therapy with adeno-associated virus 9 encoding the correctly spliced Oga normalized the cardiac protein O-GlcNAcylation levels and partially rescued the dilation and dysfunction of the hearts in Prmt5 -knockout mice. Conclusions: Our data demonstrate a novel function of PRMT5 in regulating protein O-GlcNAcylation to maintain cardiac homeostasis, suggesting that targeting the PRMT5-OGA axis could be a potential strategy for treating DCM.

Semaphorin 7A (SEMA7A), the only class VII semaphorin member, has been considered as a potent immunomodulatory regulator whose function in periapical lesions remains unclear. In our previous study, we found that SEMA7A was up-regulated in human periapical periodontitis and might be involved in the immune response and tissue destruction of periapical lesions. In this research, we aimed to further explore the specifical regulatory role of SEMA7A as well as its regulatory mechanisms in the inflammatory progression of periapical lesions.Human periodontal ligament cells (hPDLCs) were collected from intact, caries-free, and healthy third molars and stimulated with recombinant human/mouse SEMA7A (rSEMA7A). Real-time quantitative polymerase chain reaction (RT-qPCR), Western blot analysis, and enzyme-linked immunosorbent assay were used to detect the messenger RNA and protein levels of inflammatory cytokines and matrix metalloproteinases (MMPs) in hPDLCs. Twenty C57BL/6 mice were randomly divided into 4 groups: the healthy control group, pulp exposure group, pulp exposure and saline treatment group, and pulp exposure and rSEMA7A treatment group. Twenty microliters of sterile saline or 4 μg rSEMA7A were injected respectively into the buccal mucosa around the root apex at day 0, 7, and 14. Mandibular tissues were collected at day 21. Micro-computed tomographic and immunohistochemical staining were used to identify the bone destruction and inflammatory infiltration in periapical areas. Finally, an AKT inhibitor (LY294002) was used to pretreat hPDLCs before rSEMA7A stimulation to determine the role of AKT signaling activation in this process.After treatment with rSEMA7A, the messenger RNA and protein levels of interleukin (IL)-1β, IL-18, cyclooxygenase-2, MMP-1, and MMP-3 were remarkably up-regulated in hPDLCs. For in vivo experiments, compared with the other 3 groups, the treatment of rSEMA7A aggravated the osteolysis of alveolar bone and promoted the infiltration of immune cells into the apex area, along with increased expression levels of IL-1β, IL-18, MMP-1, and MMP-3. Furthermore, we found that the proinflammatory role of SEMA7A could be inhibited by the application of the AKT inhibitor (LY294002).SEMA7A likely aggravates the inflammatory reaction and bone destruction of existing periapical lesions. The proinflammatory role of SEMA7A in hPDLCs could partially be mediated through the AKT signaling transduction pathway.

During acute sympathetic stress, the overactivation of β-adrenergic receptors (β-ARs) causes cardiac fibrosis by triggering inflammation and cytokine expression. It is unknown whether exercise training inhibits acute β-AR overactivation-induced cytokine expression and cardiac injury. Here, we report that running exercise inhibited cardiac fibrosis and improved cardiac function in mice treated with isoproterenol (ISO), a β-AR agonist. A cytokine antibody array revealed that running exercise prevented most of the changes in cytokine expression induced by ISO. Specifically, ISO-induced upregulation of 18 cytokines was prevented by running exercise. A Kyoto encyclopedia of genes and genomes analysis of these cytokines revealed that Hedgehog and RAP1 signaling pathways were involved in the regulation of cytokine expression by exercise. The changes in the expression of some cytokines that were prevented by exercise were verified by an enzyme-linked immunosorbent assay and real-time PCR. In conclusion, running exercise prevented the cytokine expression changes after acute β-AR overactivation and therefore attenuated cardiac fibrosis. Acute sympathetic stress is an important risk factor for the patients with cardiovascular diseases, and the present study revealed that exercise training can prevent against the upregulation of cytokines and the subsequent cardiac injury induced by acute sympathetic stress, suggesting that exercise training may be beneficial for cardiovascular patients who are in risk of acute sympathetic stress. This finding provides a theoretical basis for the application of exercise training in patients who may suffer from acute sympathetic stress.

Retinal degeneration is a major cause of severe vision loss and irreversible blindness and is characterized by progressive damage to retinal photoreceptor cells. Resveratrol (RSV) serves as an activator of the histone deacetylase, Sirt1, and has been shown to exert anti-oxidative properties. In this study, we mimicked retinal degeneration by subjecting photoreceptors (661 W cells) to glucose deprivation (GD) or light exposure. Under these conditions, we investigated the mechanisms underlying GD- or light exposure-induced cell death and the protective effect of RSV. We found that GD and light exposure resulted in mitochondrial dysfunction, oxidative stress, and cell death. Treatment of injured cells with RSV decreased the production of reactive oxygen species (ROS), improved the ratio of reduced/oxidized glutathione (GSH/GSSG), mitochondrial membrane potential and morphology, and reduced apoptosis. We used the caspase inhibitor, z-VAD-fmk, and a lentiviral-mediated shRNA knockdown of PARP-1 to reveal that GD and light exposure-induced cell death have different underlying mechanisms; GD triggered a caspase-dependent cell death pathway, whereas light exposure triggered a PARP-dependent cell death pathway. The level of caspase-9 and caspase-3, upregulated following GD, were reduced by treatment with RSV. Similarly, the level of PARP-1 and AIF, upregulated following light exposure, were decreased by treatment with RSV. Additionally, treatment with RSV elevated the protein expression and enzymatic activity of Sirt1 and a Sirt1 inhibitor reduced the protective effect of RSV against insult-induced cellular injuries, indicating that RSV's protective effect may involve Sirt1 activation. Finally, we investigated the neuroprotection of RSV in vivo. Administration of RSV to mice under extreme light exposure led to a suppression of the light-induced thinning of the outer nuclear layer (ONL) detected by hematoxylin and eosin (H&E) staining and restored retinal function evaluated by electroretinography (ERG). Taken together, our findings provide evidence that treatment with RSV has neuroprotective effects on both GD and light exposure-induced cell death pathways in photoreceptor cells.

This study investigated the effect of sweet potato starch (SPS) and konjac glucomannan (KGM) on the textural, color, sensory, rheological properties, and microstructures of plant-based pork rinds. Plant-based gels were prepared using mixtures of soy protein isolate (SPI), soy oil, and NaHCO3 supplemented with different SPS and KGM concentrations. The texture profile analysis (TPA) results indicated that the hardness, cohesiveness, and chewiness of the samples improved significantly after appropriate SPS and KGM addition. The results obtained via a colorimeter showed no significant differences were found in lightness (L*) between the samples and natural pork rinds after adjusting the SPS and KGM concentrations. Furthermore, the rheological results showed that adding SPS and KGM increased both the storage modulus (G’) and loss modulus (G’’), indicating a firmer gel structure. The images obtained via scanning electron microscopy (SEM) showed that the SPS and KGM contributed to the formation of a more compact gel structure. A mathematical model allowed for a more objective sensory evaluation, with the 40% SPS samples and the 0.4% KGM samples being considered the most similar to natural pork rinds, which provided a comparable texture, appearance, and mouthfeel. This study proposed a possible schematic model for the gelling mechanism of plant-based pork rinds: the three-dimensional network structures of the samples may result from the interaction between SPS, SPI, and soybean oil, while the addition of KGM and NaHCO3 enabled a more stable gel structure.

Retinal detachment refers to the separation of the retinal neuroepithelium and pigment epithelium, usually involving the death of photoreceptor cells. Severe detachment may lead to permanent visual impairment if not treated properly and promptly. According to the underlying causes, retinal detachment falls into one of three categories: exudative retinal detachment, traction detachment, and rhegmatogenous retinal detachment. Like many other diseases, it is difficult to study the pathophysiology of retinal detachment directly in humans, because the human retinal tissues are precious, scarce and non-regenerative; thus, establishing experimental models that better mimic the disease is necessary. In this review, we summarize the existing models of the three categories of retinal detachment both in vivo and in vitro, along with an overview of their examination methods and the major strengths and weaknesses of each model.

Cardiac fibroblast (CF) differentiation plays a crucial role in cardiac fibrosis, which is a specific manifestation distinguishing pathological cardiac hypertrophy from physiological hypertrophy. The DNA-binding activity of paired box 6 (Pax6) has been shown to be oppositely regulated in physiological and pathological hypertrophy; however, it remains unclear whether Pax6 is involved in CF differentiation during cardiac fibrosis. We found that Pax6 is expressed in the heart of and CFs isolated from adult mice. Moreover, angiotensin II (Ang II) induced the downregulation of Pax6 mRNA and protein expression in fibrotic heart tissue and cardiac myofibroblasts. Pax6 knockdown in CFs promoted the expression of the myofibroblast marker α-smooth muscle actin (α-SMA) and the synthesis of the extracellular matrix (ECM) proteins collagen I and fibronectin. Furthermore, we validated the ability of Pax6 to bind to the promoter regions of Cxcl10 and Il1r2 and the intronic region of Tgfb1. Pax6 knockdown in CFs decreased CXC chemokine 10 (CXCL10) and interleukin-1 receptor 2 (IL-1R2) expression and increased transforming growth factor β1 (TGFβ1) expression, mimicking the effects of Ang II. In conclusion, Pax6 exerts an inhibitory effect on CF differentiation and ECM synthesis by transcriptionally activating the expression of the anti-fibrotic factors CXCL10 and IL-1R2 and repressing the expression of the pro-fibrotic factor TGFβ1. Therefore, maintaining Pax6 expression in CFs is essential for preventing CF differentiation, and provides a new therapeutic target for cardiac fibrosis.

Aims Rapid over-activation of β-adrenergic receptor (β-AR) upon stress leads to cardiac inflammation, a prevailing factor that underlies heart injury. However, mechanisms by which acute β-AR stimulation induce cardiac inflammation still remain unknown. Here, we set out to identify the crucial role of inflammasome/interleukin (IL)-18 in initiating and maintaining cardiac inflammatory cascades upon β-AR insult. Methods and results Male C57BL/6 mice were injected with a single dose of β-AR agonist, isoproterenol (ISO, 5 mg/kg body weight) or saline subcutaneously. Cytokine array profiling demonstrated that chemokines dominated the initial cytokines upregulation specifically within the heart upon β-AR insult, which promoted early macrophage infiltration. Further investigation revealed that the rapid inflammasome-dependent activation of IL-18, but not IL-1β, was the critical up-stream regulator for elevated chemokine expression in the myocardium upon ISO induced β1-AR-ROS signalling. Indeed, a positive correlation was observed between the serum levels of norepinephrine and IL-18 in patients with chest pain. Genetic deletion of IL-18 or the up-stream inflammasome component NLRP3 significantly attenuated ISO-induced chemokine expression and macrophage infiltration. In addition, IL-18 neutralizing antibodies selectively abated ISO-induced chemokines, proinflammatory cytokines and adhesion molecules but not growth factors. Moreover, blocking IL-18 early after ISO treatment effectively attenuated cardiac inflammation and fibrosis. Conclusion Inflammasome-dependent activation of IL-18 within the myocardium upon acute β-AR over-activation triggers cytokine cascades, macrophage infiltration and pathological cardiac remodelling. Blocking IL-18 at the early stage of β-AR insult can successfully prevent inflammatory responses and cardiac injuries.

Kindlin-2 plays an important role in the regulation of cardiac structure and function. Depletion of Kindlin-2 contributes to cardiac hypertrophy and progressive heart failure, however, the precise mechanisms involved in this process remain unclear. GATA4 is a critical transcription factor in regulating cardiogenesis. We found that Kindlin-2 suppresses the expression of GATA4 through binding to its promoter and prevents cardiomyocytes from hypertrophy induced by isoproterenol (ISO) treatment. Mechanistically, Kindlin-2 interacts with histone methyltransferase SUV39H1 and recruits it to GATA4 promoter leading to the occupancy of histone H3K9 di- and tri-methylation. Furthermore, to confirm the function of Kindlin-2 in vivo, we generated mice with targeted deletion of cardiac Kindlin-2. We found that 6-month-old Kindlin-2 cKO mice have developed hypertrophic cardiomyopathy and that this pathological process can be accelerated by ISO-treatment. GATA4 expression was markedly activated in cardiac tissues of Kindlin-2 cKO mice compared to wild-type animals. Collectively, our data revealed that Kindlin-2 suppresses GATA4 expression by triggering histone H3K9 methylation in part and protects heart from pathological hypertrophy.

To analyze the protective effect of PARP inhibitors on light-damaged retina and explore its possible mechanism from the perspective of ciliopathy. A systematic review of the literature was performed to investigate the protection of PARP inhibition on light-damaged cilia. PubMed database was retrieved to find the relevant studies and 119 literatures were involved in the review. In retina, the outer segment of photoreceptor is regarded as a special type of primary cilium, so various retinal diseases actually belong to a type of ciliopathy. The retina is the only central nervous tissue exposed to light, but poly (ADP-ribose) polymerase (PARP), as a nuclear enzyme repairing DNA breaks, is overactivated during the light-induced DNA damage, and is involved in the cell death cascade. Studies show that both ATR and phosphorylated Akt colocalize with cilium and play an important role in regulating ciliary function. PARP may function at ATR or PI3K/Akt signal to exert protective effect on cilia. PARP inhibitors may protect the cilia/OS of photoreceptor during light-induced damage, which the possible mechanism may be involved in the activation of ATR and PI3K/Akt signal.

To investigate the protective effects of shen-mai injection on intestinal barrier function in the early stage of 30% 3° scald, and to provide experimental basis for the prevention and control of enterogenic infection.A total of 60 Wistar rats were randomly divided into 6 groups: normal control group (without treatment), model control group (with 30% total body surface area (TBSA) fully thickness burn on the back), hexadecadrol (5 mg/kg) group, Shenmai injection (5, 10, 15 mg/kg) groups, with 10 rats in each group. After burned by scald apparatus, rats in each group were treated with drugs immediately by intraperitoneal injection once a day. At 72 hours after burned, the levels of plasma endotoxin, diamine oxidase (DAO), tumor necrosis factor-alpha (TNF-α), interleukins-6(IL-6) in all rats were detected and the mesenteric lymph nodes, liver and spleen were homogenized to culture for bacteria. The change of secretory immunoglobulin A (sIgA) in intestinal mucosa was measured.Compared with normal control group, bacterial translocation quantity in mesenteric lymph nodes(MLN), liver, and spleen, and the plasma levels of DAO, endotoxin, TNF-α, IL-6 and the level of sIgA in intestinal mucosa in model control group were increased significantly (P＜0.01); compared with model control group, bacterial translocation quantity in MLN, liver, and spleen, and the plasma levels of DAO, endotoxin, TNF-α, IL-6 and the level of sIgA in intestinal mucosa in hexadecadrol (5 mg/kg) group and shen-mai injection (5, 10, 15 mg/kg) groups were decreased significantly (P＜0.05 or P＜0.01).Shen-mai injection can alleviate intestinal mucosa injury caused by severe scald, and the effects are similar with those of dexamethasone, and the effect is better in the high-dose group.

Background Little information about the prevalence of gastrointestinal parasites in yaks (Bos grunniens) in northwest China is available. Therefore, the objective of the study was to quantify faecal egg counts of gastrointestinal parasites (helminths and coccidia) in free-range yaks from Gannan Tibetan Autonomous Prefecture, Gansu Province, Northwest China. Results Parasites were detected in 290 of 733 (39.56%) faecal samples. The results showed that Strongylidae, Trichuris spp. and Eimeria spp. were detected all year round, Strongyloides papillosus was detected in autumn and summer, and Nematodirus spp. was detected in both autumn and spring. In contrast, Fasciola spp. was only detected in spring. The prevalence rates of parasitic infections in different seasons were significantly different. Conclusions To our knowledge, this is the first investigation of gastrointestinal parasites in yaks (Bos grunniens) in Gansu, China. The results demonstrated a high prevalence of gastrointestinal parasitic infections, specifically GN infections, in yaks in GTAP and these infections can cause economic losses to the local cattle industry.

Acute sympathetic stress causes excessive secretion of catecholamines and induces cardiac injuries, which are mainly mediated by β-adrenergic receptors (β-ARs). However, α1-adrenergic receptors (α1-ARs) are also expressed in the heart and are activated upon acute sympathetic stress. In the present study, we investigated whether α1-AR activation induced cardiac inflammation and the underlying mechanisms. Male C57BL/6 mice were injected with a single dose of α1-AR agonist phenylephrine (PE, 5 or 10 mg/kg, s.c.) with or without pretreatment with α-AR antagonist prazosin (5 mg/kg, s.c.). PE injection caused cardiac dysfunction and cardiac inflammation, evidenced by the increased expression of inflammatory cytokine IL-6 and chemokines MCP-1 and MCP-5, as well as macrophage infiltration in myocardium. These effects were blocked by prazosin pretreatment. Furthermore, PE injection significantly increased the expression of NOD-like receptor protein 3 (NLRP3) and the cleavage of caspase-1 (p20) and interleukin-18 in the heart; similar results were observed in both Langendorff-perfused hearts and cultured cardiomyocytes following the treatment with PE (10 μM). Moreover, PE-induced NLRP3 inflammasome activation and cardiac inflammation was blocked in Nlrp3-/- mice compared with wild-type mice. In conclusion, α1-AR overactivation induces cardiac inflammation by activating NLRP3 inflammasomes.

Senescent hearts exhibit defective responses to β-adrenergic receptor (β-AR) over-activation upon stress, leading to more severe pathological cardiac remodelling. However, the underlying mechanisms remain unclear. Here, we investigated the role of adenosine monophosphate-activated protein kinase (AMPK) in protecting against ageing-associated cardiac remodelling in mice upon β-AR over-activation. 10-week-old (young) and 18-month-old (old) mice were subcutaneously injected with the β-AR agonist isoproterenol (ISO; 5 mg/kg). More extensive cardiac fibrosis was found in old mice upon ISO exposure than in young mice. Meanwhile, ISO treatment decreased AMPK activity and increased β-arrestin 1, but not β-arrestin 2, expression, and the effects of ISO on AMPK and β-arrestin 1 were greater in old mice than in young mice. Similarly, young AMPKα2-knockout (KO) mice showed more extensive cardiac fibrosis upon ISO exposure than that was observed in age-matched wild-type (WT) littermates. The extent of cardiac fibrosis in WT old mice was similar to that in young KO mice. Additionally, AMPK activities were decreased and β-arrestin 1 expression increased in KO mice. In contrast, the AMPK activator metformin decreased β-arrestin 1 expression and attenuated cardiac fibrosis in both young and old mice upon ISO exposure. In conclusion, more severe cardiac fibrosis is induced by ISO in old mice than in young mice. A decrease in AMPK activity, which further increases β-arrestin 1 expression, is the central mechanism underlying the ageing-related cardiac fibrosis induced by ISO. The AMPK activator metformin is a promising therapeutic agent for treating ageing-related cardiac remodelling upon β-AR over-activation.

Triple TOF MS/MS was used to identify adducts between rosmarinic acid (RosA)-derived quinones and meat proteins in a gel model under oxidative stress. Seventy-five RosA-modified peptides responded to 67 proteins with adduction of RosA. RosA conjugated with different amino acids in proteins, and His, Arg, and Lys adducts with RosA were identified for the first time in meat. A total of 8 peptides containing Cys, 14 peptides containing His, 48 peptides containing Arg, 64 peptides containing Lys, and 5 peptides containing N-termini that which participated in adduction reaction with RosA were identified, respectively. Seventy-seven adduction sites were subdivided into all adducted proteins including 2 N-terminal adduction sites, 3 Cys adduction sites, 4 His adduction sites, 29 Arg adduction sites, and 39 Lys adduction sites. Site occupancy analyses showed that approximately 80.597% of the proteins carried a single RosA-modified site, 14.925% retained two sites, 1.492% contained three sites, and the rest 2.985% had four or more sites. Large-scale triple TOF MS/MS mapping of RosA-adducted sites reveals the adduction regulations of quinone and different amino acids as well as the adduction ratios, which clarify phenol-protein adductions and pave the way for industrial meat processing and preservation.

Aims The H19 lncRNA, a highly abundant and conserved imprinted gene, has been implicated in many essential biological processes and diseases. However, the function of H19 in the heart remains unknown. In this study, we investigated the function and underlying mechanism of H19 in regulating cardiomyocyte hypertrophy. Methods and results We first detected the expression of H19 and its encoded miR-675 in both normal and diseased hearts and verified their up-regulations in pathological cardiac hypertrophy and heart failure. Adenovirus-mediated expression and a siRNA-mediated silence of H19 showed that H19 overexpression reduced cell size both at baseline and in response to phenylephrine, whereas knock-down of H19 induced cardiomyocyte hypertrophy. Overexpression or knock-down of miR-675 in cardiomyocytes demonstrated that miR-675 also inhibited cardiomyocyte hypertrophy. Moreover, inhibition of miR-675 reversed the reduction of cardiomyocyte size in H19-overexpressing cardiomyocytes, while infection with an adenovirus carrying H19 fragment without pre-miR-675 (H19-Tru) or with mutant sequences of pre-miR-675 (H19-Mut) failed to reduce cardiomyocyte size, indicating that miR-675 mediated the inhibitory effect of H19 on cardiomyocyte hypertrophy. We also identified that CaMKIIδ was a direct target of miR-675 and partially mediated the effect of H19 on cardiomyocyte hypertrophy. Furthermore, in vivo silencing of miR-675 using a specific antagomir in a pressure overload-induced mouse model of heart failure increased cardiac CaMKIIδ expression and exacerbated cardiac hypertrophy. Conclusion These findings reveal a novel function of H19-miR-675 axis targeting CaMKIIδ as a negative regulator of cardiac hypertrophy, suggesting its potential therapeutic role in cardiac diseases.

The mammalian target of rapamycin (mTOR) pathway is a crucial cellular signaling hub, which integrates internal and external cues to modulate the cell cycle, protein synthesis and metabolism. The present study hypothesized that inhibiting mTOR signaling may induce cells to enter lower and more stable bioenergetic states, in which neurons have greater resistance to various insults. Neurotrophin withdrawal from photoreceptor cells (661W cells) was mimicked using serum deprivation, and the neuroprotective mechanisms were studied following suppression of the mTOR pathway. Treatment with an mTOR specific inhibitor, rapamycin, reduced intracellular levels of reactive oxygen species, suppressed oxidative stress, and attenuated mitochondrial dysfunction. In addition, inhibiting mTOR signaling induced a G2/M cell cycle arrest, thus providing an opportunity to repair damaged DNA and block the cell death cascade. These results suggested that inhibition of mTOR had a neuroprotective effect on serum‑deprived 661W cells. In conclusion, the mTOR pathway is a critical molecular signal for cell cycle regulation and energy metabolism, and inhibiting the mTOR pathway may attenuate neurotrophin withdrawal‑induced damage. These observations may provide evidence for the treatment of retinal degenerative disease, since inducing neurons into a lower and more stable bioenergetic state by blocking mTOR signaling may slow the progression of neurodegenerative diseases.

Aims Overexpression of either member of the miR-199 family, miR-199a-5p or miR-199b-5p (hereinafter referred to as miR-199a or miR-199b), promotes pathological cardiac hypertrophy, but little is known about the role of endogenous miR-199 in cardiac development and disease. Our study aimed to determine the physiological function of the endogenous miR-199 family in cardiac homeostasis maintenance. Methods and results We generated a sponge transgenic mouse model with a specific disruption of miR-199 in the heart. To our surprise, we found that knockdown of endogenous miR-199 caused physiological cardiac hypertrophy characterized by an increased heart weight and cardiomyocyte size, but with normal cardiac morphology and function. Furthermore, we also identified PGC1α as the target gene of the miR-199 family, and PGC1α was also increased in sponge transgenic mice. Conclusions Inhibition of endogenous miR-199 led to physiological cardiac hypertrophy probably due to the upregulation of PGC1α, uncovering a surprising role for endogenous miR-199 in the maintenance of cardiac homeostasis.

Basal autophagy is tightly regulated by transcriptional and epigenetic factors to maintain cellular homeostasis. Dysregulation of cardiac autophagy is associated with heart diseases, including cardiac hypertrophy, but the mechanism governing cardiac autophagy is rarely identified. To analyze the in vivo function of miR-199a in cardiac autophagy and cardiac hypertrophy, we generated cardiac-specific miR-199a transgenic mice and showed that overexpression of miR-199a was sufficient to inhibit cardiomyocyte autophagy and induce cardiac hypertrophy in vivo. miR-199a impaired cardiomyocyte autophagy in a cell-autonomous manner by targeting glycogen synthase kinase 3β (GSK3β)/mammalian target of rapamycin (mTOR) complex signaling. Overexpression of autophagy related gene 5 (Atg5) attenuated the hypertrophic effects of miR-199a overexpression on cardiomyocytes, and activation of autophagy using rapamycin was sufficient to restore cardiac autophagy and decrease cardiac hypertrophy in miR-199a transgenic mice. These results reveal a novel role of miR-199a as a key regulator of cardiac autophagy, suggesting that targeting miRNAs controlling autophagy as a potential therapeutic strategy for cardiac disease.

Kindlin-2, as an integrin-interacting protein, was known to be required for the maintenance of cardiac structure and function in zebrafish. However, the mechanism remains unclear. We found that Kindlin-2 interacts and colocalizes with α-actinin-2 at the Z-disc of mouse cardiac muscles and there Kindlin-2 also interacts with β1 integrin. Knockdown of Kindlin-2 influences the association of β1 integrin with α-actinin-2 and disrupts the structure of the Z-disc and leads to cardiac dysfunction. Our data indicated that Kindlin-2 is a novel α-actinin-2-interacting protein and plays an important role in the regulation of cardiac structure and function.

Atrazine (2-chloro-4-ethytlamino-6-isopropylamine-1,3,5-triazine, ATR) is widely used as a broad-spectrum herbicide. Animal studies have demonstrated that ATR exposure can cause cell death in dopaminergic neurons. The molecular mechanisms underlying ATR-induced neuronal cell death, however, are unknown. In this study, we investigated the autophagy and apoptosis induced by ATR in dopaminergic neurons in vivo. Wistar rats were administered with ATR at doses of 10, 50 and 100 mg/kg body weight by oral gavage for three months. In terms of histopathology, the expression of autophagy- and apoptosis-related genes as well as proteins related to the Beclin-1/B-cell lymphoma 2 (Bcl-2) autophagy and apoptosis pathways were examined in the rat nigrostriatal dopaminergic system. We observed degenerative micromorphology indicative of neuronal apoptosis and mitochondrial autophagy by electron microscopy in ATR-exposed rat striatum. The rat ventral mesencephalon in the ATR-exposed groups also showed increased expression of Beclin-1, LC3-II, Bax and Caspase-9, and decreased expression of tyrosine hydroxylase (TH), Bcl-xl and Bcl-2. These findings indicate that ATR may induce autophagy- and apoptosis-related changes in doparminergic neurons. Furthermore, this induction may be regulated by the Beclin-1 and Bcl-2 autophagy and apoptosis pathways, and this may help to better understand the mechanism underlying the neurotoxicity of ATR.

mukaida@staff.kanazawa-u.ac.jp, Patients with pancreatic ductal adenocarcinoma (PDAC) are frequently complicated with metastatic disease or locally advanced tumors, and consequently need chemotherapy. Gemcitabine is commonly used for PDAC treatment, but with limited efficacy. The capacity of gemcitabine to generate reactive oxygen species (ROS) in human pancreatic cancer cells, prompted us to examine its effects on the expression of pro-inflammatory cytokines and chemokines. We observed that gemcitabine enhanced selectively the expression of CXCL8 in human pancreatic cancer cells through ROS generation and NF-κB activation. In vitro blocking of CXCL8 failed to modulate gemcitabine-mediated inhibition of cell proliferation in human pancreatic cancer cells. Gemcitabine also enhanced CXCL8 expression in pancreatic cancer cells in xenografted tumor tissues. Moreover, anti-CXCL8 antibody treatment in vivo attenuated tumor formation as well as intra-tumoral vascularity in nude mice, which were transplanted with Miapaca-2 cells and treated with gemcitabine. Thus, gemcitabine-induced CXCL8 may counteract the drug through inducing neovascularization.

Background DBZ (Danshensu Bingpian Zhi), a synthetic derivative of a natural compound found in traditional Chinese medicine, has been reported to suppress lipopolysaccharide‐induced macrophage activation and lipid accumulation in vitro. The aim of this study was to assess whether DBZ could attenuate atherosclerosis at early and advanced stages. Methods and Results The effects of DBZ on the development of atherosclerosis were studied using apolipoprotein E–deficient (apoE −/− ) mice. For early treatment, 5‐week‐old apoE −/− mice were fed a Western diet and treated daily by oral gavage with or without DBZ or atorvastatin for 10 weeks. For advanced treatment, 5‐week‐old apoE −/− mice were fed a Western diet for 10 weeks to induce atherosclerosis, and then they were randomly divided into 4 groups and subjected to the treatment of vehicle, 20 mg/kg per day DBZ , 40 mg/kg per day DBZ , or 10 mg/kg per day atorvastatin for the subsequent 10 weeks. We showed that early treatment of apoE −/− mice with DBZ markedly reduced atherosclerotic lesion formation by inhibiting inflammation and decreasing macrophage infiltration into the vessel wall. Treatment with DBZ also attenuated the progression of preestablished diet‐induced atherosclerotic plaques in apoE −/− mice. In addition, we showed that DBZ may affect LXR (liver X receptor) function and that treatment of macrophages with DBZ suppressed lipopolysaccharide‐stimulated cell migration and oxidized low‐density lipoprotein–induced foam cell formation. Conclusions DBZ potentially has antiatherosclerotic effects that involve the inhibition of inflammation, macrophage migration, leukocyte adhesion, and foam cell formation. These results suggest that DBZ may be used as a therapeutic agent for the prevention and treatment of atherosclerosis.

1. Chronic injection with the β-adrenoceptor (β-AR) agonist isoproterenol (ISO) has been commonly used as an animal model of β-AR-induced cardiac remodelling and heart failure. This ISO-treated model usually exhibits significantly decreased conscious heart rate (HR). However, the HR in treatment groups is usually adjusted to the same levels by anaesthesia to assess cardiac geometry and function. In the present study, we report a method of echocardiographic assessment that represents the true cardiac geometry and function under conditions of ISO withdrawal. 2. Briefly, C57BL/6 mice were treated with 5 mg/kg per day ISO for 12 weeks. Cardiac geometry and function were assessed by high-resolution echocardiography in vehicle (saline) - and ISO-treated mice that were either conscious or anaesthetized using different concentrations of isoflurane. 3. The cardiac β-AR response was decreased in ISO-treated mice, as evidenced by markedly decreased conscious HR. Vehicle- and ISO-treated mice did not differ in terms of cardiac geometry or function when HR was adjusted to the same level (400 b.p.m.) in both treatment groups, but cardiac geometry and function did differ when a low (1%) rather than high (1.5% or 2%) isoflurane concentration was used to adjust HR. Furthermore, 3 day ISO withdrawal eliminated the difference in conscious HR between the two groups. In addition, the groups differed in cardiac geometry and function regardless of the isoflurane concentration used. 4. In conclusion, using isoflurane to decrease the HR of treated groups to the same level may mask left ventricular dysfunction in ISO-treated mice. Withdrawal of ISO eliminated the difference in basal HR between the ISO-treated and control groups on echocardiography, allowing a more accurate assessment of cardiac pathological and functional changes.

The aim of this study was to examine the therapeutic effect of oxymatrine, a monomer isolated from the medicinal plant Sophora flavescens Ait, on the hepatic lipid metabolism in non-alcoholic fatty liver (NAFLD) rats and to explore the potential mechanism. Rats were fed with high fructose diet for 8 weeks to establish the NAFLD model, then were given oxymatrine treatment (40, 80, and 160 mg/kg, respectively) for another 8 weeks. Body weight gain, liver index, serum and liver lipids, and histopathological evaluation were measured. Enzymatic activity and gene expression of the key enzymes involved in the lipogenesis and fatty acid oxidation were assayed. The results showed that oxymatrine treatment reduced body weight gain, liver weight, liver index, dyslipidemia, and liver triglyceride level in a dose dependant manner. Importantly, the histopathological examination of liver confirmed that oxymatrine could decrease the liver lipid accumulation. The treatment also decreased the fatty acid synthase (FAS) enzymatic activity and increased the carnitine palmitoyltransferase 1A (CPT1A) enzymatic activity. Besides, oxymatrine treatment decreased the mRNA expression of sterol regulatory element binding transcription factor 1(Srebf1), fatty acid synthase (Fasn), and acetyl CoA carboxylase (Acc), and increased the mRNA expression of peroxisome proliferator activated receptor alpha (Pparα), carnitine palmitoyltransferase 1A (Cpt1a), and acyl CoA oxidase (Acox1) in high fructose diet induced NAFLD rats. These results suggested that the therapeutic effect of oxymatrine on the hepatic steatosis in high fructose diet induced fatty liver rats is partly due to down-regulating Srebf1 and up-regulating Pparα mediated metabolic pathways simultaneously.

Kindlin-2, a member of the Kindlin family focal adhesion proteins, plays an important role in cardiac development. It is known that defects in the Z-disc proteins lead to hypertrophic cardiomyopathy (HCM) or dilated cardiomyopathy (DCM). Our previous investigation showed that Kindlin-2 is mainly localized at the Z-disc and depletion of Kindlin-2 disrupts the structure of the Z-Disc. Here, we reported that depletion of Kindlin-2 leads to the disordered myocardial fibers, fractured and vacuolar degeneration in myocardial fibers. Interestingly, depletion of Kindlin-2 in mice induced cardiac myocyte hypertrophy and increased the heart weight. Furthermore, decreased expression of Kindlin-2 led to cardiac dysfunction and also markedly impairs systolic function. Our data indicated that Kindlin-2 not only maintains the cardiac structure but also is required for cardiac function.

To evaluate the effect of sodium tanshinone IIA sulfonate (STS) in preventing postoperative peritoneal adhesions in rats and explore the mechanisms.Sixty SD rats were randomized into 4 equal groups, including a blank control group, adhesion model group, and high-, moderate-, and low-dose STS-treated groups, and were subjected to injuries of the parietal peritoneum and cecum to induce peritoneal adhesions, followed by intraperitoneal administration of saline and STS at the doses of 20, 10, and 5 mg/kg for 7 consecutive days, respectively. Another 15 untreated rats served as the blank control group. The adhesion scores in each group were recorded after the treatments; the activity of tissue-type plasminogen activator (tPA) in peritoneal lavage fluid was measured, tPA/PAI-1 protein ratio in the peritoneal tissue was determined by ELISA, and the expressions of TGF-β1 and collagen I were detected by immunohistochemistry. The anastomotic healing model was used to assess the impact of STS on wound healing.Intraperitoneal administration of STS effectively prevented peritoneal adhesion without affecting anastomotic healing in the rats. Compared with the adhesion model group, the STS-treated groups showed increased peritoneal lavage fluid tPA activity and tPA/PAI-1 ratio in the ischemic tissues with lowered TGF-β1 and collagen I expressions in the ischemic tissues.Intraperitoneal administration of STS can prevent peritoneal adhesion and enhance local fibrinolysis in rats, and these effects may be mediated by TGF-β signaling pathway.

Recent studies have begun to reveal critical roles of microRNAs (miRNAs) in the pathogenesis of cardiac hypertrophy and dysfunction. In this study, we tested whether a transforming growth factor-β (TGF-β)-regulated miRNA played a pivotal role in the development of cardiac hypertrophy and heart failure (HF). We observed that miR-27b was upregulated in hearts of cardiomyocyte-specific Smad4 knockout mice, which developed cardiac hypertrophy. In vitro experiments showed that the miR-27b expression could be inhibited by TGF-β1 and that its overexpression promoted hypertrophic cell growth, while the miR-27b suppression led to inhibition of the hypertrophic cell growth caused by phenylephrine (PE) treatment. Furthermore, the analysis of transgenic mice with cardiomyocyte-specific overexpression of miR-27b revealed that miR-27b overexpression was sufficient to induce cardiac hypertrophy and dysfunction. We validated the peroxisome proliferator-activated receptor-γ (PPAR-γ) as a direct target of miR-27b in cardiomyocyte. Consistently, the miR-27b transgenic mice displayed significantly lower levels of PPAR-γ than the control mice. Furthermore, in vivo silencing of miR-27b using a specific antagomir in a pressure-overload-induced mouse model of HF increased cardiac PPAR-γ expression, attenuated cardiac hypertrophy and dysfunction. The results of our study demonstrate that TGF-β1-regulated miR-27b is involved in the regulation of cardiac hypertrophy, and validate miR-27b as an efficient therapeutic target for cardiac diseases.

Rationale: Aortic aneurysm is a life-threatening cardiovascular disorder caused by the predisposition for dissection and rupture. Genetic studies have proved the involvement of the transforming growth factor-β (TGF-β) pathway in aortic aneurysm. Smad4 is the central mediator of the canonical TGF-β signaling pathway. However, the exact role of Smad4 in smooth muscle cells (SMCs) leading to the pathogenesis of aortic aneurysms is largely unknown. Objective: To determine the role of smooth muscle Smad4 in the pathogenesis of aortic aneurysms. Methods and Results: Conditional gene knockout strategy combined with histology and expression analysis showed that Smad4 or TGF-β receptor type II deficiency in SMCs led to the occurrence of aortic aneurysms along with an upregulation of cathepsin S and matrix metallopeptidase-12, which are proteases essential for elastin degradation. We further demonstrated a previously unknown downregulation of matrix metallopeptidase-12 by TGF-β in the aortic SMCs, which is largely abrogated in the absence of Smad4. Chemotactic assay and pharmacologic treatment demonstrated that Smad4-deficient SMCs directly triggered aortic wall inflammation via the excessive production of chemokines to recruit macrophages. Monocyte/macrophage depletion or blocking selective chemokine axis largely abrogated the progression of aortic aneurysm caused by Smad4 deficiency in SMCs. Conclusions: The findings reveal that Smad4-dependent TGF-β signaling in SMCs protects against aortic aneurysm formation and dissection. The data also suggest important implications for novel therapeutic strategies to limit the progression of the aneurysm resulting from TGF-β signaling loss-of-function mutations.

Giardia duodenalis is one of the most common enteric parasites of humans and animals, including companion animals, livestock and wildlife. To date, the information about the prevalence and molecular characterization of G. duodenalis infection in white yaks was limited. In the present study, a total of 208 white yak fecal samples were collected from Tianzhu Tibetan Autonomous County (TTAC) between September 2013 and March 2014. Of the 208 white yak fecal samples, four samples (1.92%, all collected in March 2014) tested G. duodenalis-positive by PCR amplification of triosephosphate isomerase (tpi) gene. Sequence analysis confirmed the presence of G. duodenalis assemblage E. The present study revealed the presence and genotype of G. duodenalis in white yaks for the first time, and extended the host range of G. duodenalis. These results provided useful information for further genotyping or subtyping studies of G. duodenalis in white yaks.

To study the effect Qige powder on esophageal cancer angiogenesis.Inhibitive effect of Qige powder ethanol extract on proliferation of Esophageal cancer cell line Eca-9706 was detected by MTT assay. The Eca-9706 cells conditioned medium in Qige powder IC50 concentration were collected. Angiogenesis, as well as proliferation, migration and tube formation of human umbilical vein endothelial cells were observed by Chick embryo chorioallantoic membrane model(CAM), MTT assay, the migration and tube formation assay. VEGF and IL-6 contents in culture medium supernatant of Eca-9706 cells and human umbilical vein endothelial cells were detected by ELISA.Qige powder ethanol extract could inhibit the proliferation of Eca-9706 cells, with a certain dose-effect relationship, IC50 value was 96 µg/mL. Eca-9706 cells conditioned medium could significantly increase the CAM generating total vessel area, human umbilical vein endothelial cells proliferation,migration and tube formation, while the Eca-9706 cell conditioned medium of Qige powder ethanol extract could reduce CAM angiogenesis, human umbilical vein endothelial cells proliferation and tube formation, but increase endothelial cells migration. Qige powder ethanol extract could reduce endothelial cells secreting VEGF and IL-6 while increase Eca-9706 cells secreting.Qige powder ethanol extract can inhibit angiogenesis, endothelial cell proliferation, and tube formation induced by Eca-9706 cells, while reduce VEGF and IL-6 secretion of endothelial cells.

Regular exercise can protect the heart against external stimuli, but the mechanism is not well understood. We determined the role of adenosine monophosphate-activated protein kinase (AMPK) in regulating swimming exercise-mediated cardiac protection against β-adrenergic receptor overstimulation with isoproterenol (ISO) in mice. Ten-week-old AMPKα2+/+ and AMPKα2-knockout (AMPKα2-/-) littermates were subjected to 4 weeks of swimming training (50 min daily, 6 days a week) or housed under sedentary conditions. The mice received daily subcutaneous injection of ISO (5 mg/kg/d), a nonselective β-adrenergic receptor agonist, during the last 2 weeks of swimming training. Swimming training alleviated ISO-induced cardiac fibrosis in AMPKα2+/+ mice but not AMPKα2-/- mice. Swimming training activated cardiac AMPK in AMPKα2+/+ mice. Furthermore, swimming training attenuated ISO-induced production of reactive oxygen species (ROS) and expression of NADPH oxidase and promoted the expression of antioxidant enzymes in AMPKα2+/+ mice but not AMPKα2-/- mice. In conclusion, swimming training attenuates ISO-induced cardiac fibrosis by inhibiting the NADPH oxidase–ROS pathway mediated by AMPK activation. Our findings provide a new mechanism for the cardioprotective effects of exercise.

The mammalian target of rapamycin (mTOR) signaling pathway in pulmonary fibrosis was investigated in cell and animal models. mTOR overactivation in alveolar epithelial cells (AECs) was achieved in the conditional and inducible Tsc1 knock-down mice SPC-rtTA/TetO-Cre/Tsc1(fx/+) (STT). Doxycycline caused Tsc1 knock-down and consequently mTOR activation in AECs for the STT mice. Mice treated with bleomycin exhibited increased mortality and pulmonary fibrosis compared with control mice. In wild-type C57BL/6J mice, pretreatment with rapamycin attenuated the bleomycin-mediated mortality and fibrosis. Rapamycin-mediated mouse survival benefit was inhibited by chloroquine, an autophagy inhibitor. Autophagosomes were decreased in the lungs after bleomycin exposure. Rapamycin induced the production of autophagosomes and diminished p62. We concluded that mTOR overactivation in AECs and compromised autophagy in the lungs are involved in the pathogenesis of pulmonary fibrosis. The suppression of mTOR and enhancement of autophagy may be used for treatment of pulmonary fibrosis.

The Na-K-2Cl cotransporter 2 (NKCC2) was thought to be kidney specific. Here we show expression in the brain hypothalamo-neurohypophyseal system (HNS), wherein upregulation follows osmotic stress. The HNS controls osmotic stability through the synthesis and release of the neuropeptide hormone, arginine vasopressin (AVP). AVP travels through the bloodstream to the kidney, where it promotes water conservation. Knockdown of HNS NKCC2 elicited profound effects on fluid balance following ingestion of a high-salt solution-rats produced significantly more urine, concomitant with increases in fluid intake and plasma osmolality. Since NKCC2 is the molecular target of the loop diuretics bumetanide and furosemide, we asked about their effects on HNS function following disturbed water balance. Dehydration-evoked GABA-mediated excitation of AVP neurons was reversed by bumetanide, and furosemide blocked AVP release, both in vivo and in hypothalamic explants. Thus, NKCC2-dependent brain mechanisms that regulate osmotic stability are disrupted by loop diuretics in rats.

Besides NO, neuronal NO synthase (nNOS) also produces superoxide (O(2)(-.) at low levels of L-arginine. Recently, heat shock protein 90 (hsp90) was shown to facilitate NO synthesis from eNOS and nNOS. However, the effect of hsp90 on the O(2)(-.) generation from NOS has not been determined yet. The interrelationship between its effects on O(2)(-.) and NO generation from NOS is also unclear. Therefore, we performed electron paramagnetic resonance measurements of O(2)(-.) generation from nNOS to study the effect of hsp90. Purified rat nNOS generated strong O(2)(-.) signals in the absence of L-arginine. In contrast to its effect on NO synthesis, hsp90 dose-dependently inhibited O(2)(-.) generation from nNOS with an IC(50) of 658 nM. This inhibition was not due to O(2)(-.) scavenging because hsp90 did not affect the O(2)(-.) generated by xanthine oxidase. At lower levels of L-arginine where marked O(2)(-.) generation occurred, hsp90 caused a more dramatic enhancement of NO synthesis from nNOS as compared to that under normal L-arginine. Significant O(2)(-.) production was detected from nNOS even at intracellular levels of L-arginine. Adding hsp90 prevented this O(2)(-.) production, leading to enhanced nNOS activity. Thus, these results demonstrated that hsp90 directly inhibited O(2)(-.) generation from nNOS. Inhibition of O(2)(-.) generation may be an important mechanism by which hsp90 enhances NO synthesis from NOS.

To investigate the expression of nesfatin-1/NUCB2 and ghrelin in the gastric mucosa of rats with intrauterine growth retardation (IUGR) and its significance.The IUGR animal model was established by feeding rats low-protein diets during their pregnancy. Newborn rats were divided into catch-up growth, non-catch-up growth and control groups. Protein and mRNA levels of nesfatin-1/NUCB2 and ghrelin in the gastric mucosa of rats were determined by RT-PCR and Western blot, respectively.Nesfatin-1/NUCB2 mRNA and protein were expressed in the gastric mucosa of rats immediately after birth, and their expression increased in an age-dependent manner in all three groups. Furthermore, the level of nesfatin-1/NUCB2 in the catch-up growth group was higher than that in the control group before weaning, whereas there was no significant difference in nesfatin-1/NUCB2 expression between the two groups after weaning. The level of nesfatin-1/NUCB2 in the non-catch-up growth group was lower than that in the catch-up growth group during the whole observation period. The level of ghrelin in the catch-up growth group was higher than that in the control group starting from day 12 after birth, whereas there was no significant difference in ghrelin expression between the two groups after weaning. The level of ghrelin in the non-catch-up growth group was lower compared with those in the catch-up growth and control groups from days 12 to 28 after birth.Nesfatin-1 and ghrelin are co-expressed in the gastric mucosa of rats with IUGR after birth and interact with each other to produce long-term nutritional regulation.

To observe effect of Invigorating Lung and Kidney Prescription (ILKP) on secretion of cytokines induced by Lipopolysaccharide (LPS) and cigarette smoke extract (CSE) in vitro and discuss prescription regularity of ILKP and reveal molecular mechanism of ILKP treatment on COPD.Alveolar epithelial cells A549, airway epithelial cells H292 and monocyte macrophage THP-1 were stimulated with LPS and CSE, in which samples were divided into normal group (20% normal rat serum), model group (20% normal rat serum and 5% CSE with 75 pg/mL LPS), ILKP group and its Components [20% Replenishing qi group (1), Replenishing Lung and Kidney group (2), Activating blood group (3), Reduce phlegm group (4), Replenishing qi with Replenishing Lung and Kidney group (5), Replenishing qi with Activating blood group (6), Replenishing qi with Reduce phlegm group (7), Replenishing qi with Replenishing Lung and Kidney and Activating blood group (8), Replenishing qi with Replenishing Lung and Kidney and Reduce phlegm group (9), Replenishing qi with Activating blood and Reduce phlegm group (10), ILKP serum group (11)]. The cytokines were detected by ELISA and the transcription factors of cytokines were analyzed with Network tool.Compared with the normal group,the secretion of MMP-9, GM-CSF and TGF-beta by THP-1 cells,TIMP by A549 cells,TNF-alpha, MMP-9, GM-CSF, CD36 and TIMP-1 by H292 cells all were increased, while secretion of TNF-alpha, PPAP2B, IL-3 and SOD decreased by THP-1 cells, MMP-9, TNF-alpha, TGF-beta and IL-3 by A549 cells, IL-8 by H292 cells all were decreased. Compared with model group, for THP-1 cells, the secretion of GM-CSF in 3, 6, 10 and 11 prescription groups,TGF-beta in 10 and 11 prescription groups all were decreased. MMP-9 decreased in 9 and 5 prescription groups while it increased in 1, 2, 3, 4, 7, 8, 10 and 11 prescription groups. TNF-alpha was increased in 1, 2, 3, 4, 6, 8, and 9 prescription groups, as it decreased in 11 prescription group. Except 2 prescription group, IL-3 was increased in all other drugs groups. SOD in 2, 4, 5, 8, 9, 11 prescription groups, and PPAP2B in 1, 2, 3, 4, 5, 6, 7 prescription groups were increased while PPAP2B was decreased in 11 prescription group. For A549 cells, the secretion of TIMP-1 was increased in 1, 2, 3, 4, and 5 prescription groups, while it was decreased in 11 prescription groups; Except 1 and 4 prescription group, MMP-9 was increased in other groups; Except TGF-beta was increased in 11 prescription groups, it was increased in other groups; IL-3 was increased in 1, 2, 4, 5, 11 and 3 prescription groups. For H292 cells, the secretion of GM-CSF in 5,6,7 and 2 prescription groups,TNF-alpha in 1, 2, 4, 6, 7, 8, 9 and 10 prescription groups, MMP-9 in 4, 8, 7 and 9 prescription groups all were decreased.The effect of ILKP and its components on secretion of cytokines induced by LPS and CSE and its target cells are different, in which Activating blood prescription focuses on inflammation cytokines, Replenishing Lung and Kidney prescription on Lipid metabolism and redox regulation. The effect of ILKPA is the most widely, followed by Activating blood Replenishing Lung and Kidney, and Reduce phlegm prescription on Protease regulation.

This is a study on the role of tuberous sclerosis complex1 (TSC1) mutation and mTOR activation in endothelial cells during angiogenic and embryonic development. Past studies had shown that Tsc1/Tsc2 mutant genes lead to overactivation of mTOR in the regulating pathways in developing fetus. We used conditional Cre-loxp gene knockout approach to delete Tsc1 in mice's endothelial cells in our experimental models. Similarly, activation of mTOR signaling in endothelial cells of these embryos (Tie2-Cre/Tsc1(-/-)) was found. Majority of Tie2-Cre/Tsc1(-/-) embryos died at embryonic day 14.5 in utero. Cardiovascular defects, subcutaneous edema and hemorrhage were present among them. Whole-mount immunostaining in these embryos revealed a disorganized vascular network, defective sprouting of vessels in yolk sac and thickening of the labyrinth layer in the placenta. A thinner ventricular wall with disorganized trabeculae was present in the hearts of Tie2-Cre/Tsc1(-/-) embryos. Endothelial cells in Tsc1-deficient mice showed defective mitochondrial and endoplasmic reticular morphology, but no significant change was observed in cell junctions. The mutant embryos displayed significantly reduced cell proliferation, increased apoptosis and disturbed expression of angiogenic factors. A cohort of mice was treated prenatally with mTOR inhibitor rapamycin. The offspring of these mutant mice survived up to 22 days after birth. It was concluded that physiological TSC1-mTOR signaling in endothelial cells is crucial for vascular development and embryogenesis. We postulated that disruption of normal angiogenic pathways through hyperactive mTOR signaling maybe the mechanism that lead to deranged vascular pathogenesis in the tuberous sclerosis complex.

AimsIt has been reported that cardiac ankyrin repeat protein is associated with heart development and diseases. This study is aimed to investigate the role of CARP in heart hypertrophy in vivo.Methods and resultsWe generated a cardiac-specific CARP-overexpressing transgenic mouse. Although such animals did not display any overt physiological abnormality, they developed less cardiac hypertrophy in response to pressure overload than did wildtype mice, as indicated by heart weight/body weight ratios, echocardiographic and histological analyses, and expression of hypertrophic markers. These mice also exhibited less cardiac hypertrophy after infusion of isoproterenol. To gain a molecular insight into how CARP attenuated heart hypertrophy, we examined expression of the mitogen-activated protein kinase cascade and found that the concentrations of phosphorylated ERK1/2 and MEK were markedly reduced in the hearts of transgenic mice subjected to pressure overload. In addition, the expressions of TGF-β and phosphorylated Smad3 were significantly downregulated in the hearts of CARP Tg mice in response to pressure overload. Furthermore, addition of human TGF-β1 could reverse the inhibitory effect of CARP on the hypertrophic response induced by phenylephrine in cardiomyocytes. It was also evidenced that the inhibitory effect of CARP on cardiac hypertrophy was not attributed to apoptosis.ConclusionCARP attenuates cardiac hypertrophy, in which the ERK and TGF-β pathways may be involved. Our findings highlight the significance of CARP as an anti-hypertrophic factor in therapy of cardiac hypertrophy.