A novel small-molecule PCSK9 inhibitor E28362 ameliorates hyperlipidemia and atherosclerosis.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the epidermal growth factor precursor homologous domain A (EGF-A) of low-density lipoprotein receptor (LDLR) in the liver and triggers the degradation of LDLR via the lysosomal pathway, consequently leading to an elevation in plasma LDL-C levels. Inhibiting PCSK9 prolongs the lifespan of LDLR and maintains cholesterol homeostasis in the body. Thus, PCSK9 is an innovative pharmacological target for treating hypercholesterolemia and atherosclerosis. In this study, we discovered that E28362 was a novel small-molecule PCSK9 inhibitor by conducting a virtual screening of a library containing 40,000 compounds. E28362 (5, 10, 20 µM) dose-dependently increased the protein levels of LDLR in both total protein and the membrane fraction in both HepG2 and AML12 cells, and enhanced the uptake of DiI-LDL in AML12 cells. MTT assay showed that E28362 up to 80 µM had no obvious toxicity in HepG2, AML12, and HEK293a cells. The effects of E28362 on hyperlipidemia and atherosclerosis were evaluated in three different animal models. In high-fat diet-fed golden hamsters, administration of E28362 (6.7, 20, 60 mg·kg-1·d-1, i.g.) for 4 weeks significantly reduced plasma total cholesterol (TC), triglyceride (TG), low-density lipoprotein-cholesterol (LDL-C) and PCSK9 levels, and reduced liver TC and TG contents. In Western diet-fed ApoE-/- mice (20, 60 mg·kg-1·d-1, i.g.) and human PCSK9 D374Y overexpression mice (60 mg·kg-1·d-1, i.g.), administration of E28362 for 12 weeks significantly decreased plasma LDL-C levels and the area of atherosclerotic lesions in en face aortas and aortic roots. Moreover, E28362 significantly increased the protein expression level of LDLR in the liver. We revealed that E28362 selectively bound to PCSK9 in HepG2 and AML12 cells, blocked the interaction between LDLR and PCSK9, and induced the degradation of PCSK9 through the ubiquitin-proteasome pathway, which finally resulted in increased LDLR protein levels. In conclusion, E28362 can block the interaction between PCSK9 and LDLR, induce the degradation of PCSK9, increase LDLR protein levels, and alleviate hyperlipidemia and atherosclerosis in three distinct animal models, suggesting that E28362 is a promising lead compound for the treatment of hyperlipidemia and atherosclerosis.

Trichostatin C Synergistically Interacts with DNMT Inhibitor to Induce Antineoplastic Effect via Inhibition of Axl in Bladder and Lung Cancer Cells.

Aberrant epigenetic modifications are fundamental contributors to the pathogenesis of various cancers. Consequently, targeting these aberrations with small molecules, such as histone deacetylase (HDAC) inhibitors and DNA methyltransferase (DNMT) inhibitors, presents a viable strategy for cancer therapy. The objective of this study is to assess the anti-cancer efficacy of trichostatin C (TSC), an analogue of trichostatin A sourced from the fermentation of Streptomyces sp. CPCC 203909. Our investigations reveal that TSC demonstrates potent activity against both human lung cancer and urothelial bladder cancer cell lines, with IC50 values in the low micromolar range. Moreover, TSC induces apoptosis mediated by caspase 3/7 and arrests the cell cycle at the G2/M phase. When combined with the DNMT inhibitor decitabine, TSC exhibits a synergistic anti-cancer effect. Additionally, protein analysis elucidates a significant reduction in the expression of the tyrosine kinase receptor Axl. Notably, elevated concentrations of TSC correlate with the up-regulation of the transcription factor forkhead box class O1 (FoxO1) and increased levels of the proapoptotic proteins Bim and p21. In conclusion, our findings suggest TSC as a promising anti-cancer agent with HDAC inhibitory activity. Furthermore, our results highlight the potential utility of TSC in combination with DNMT inhibitors for cancer treatment.

The novel small molecule E0924G dually regulates bone formation and bone resorption through activating the PPARδ signaling pathway to prevent bone loss in ovariectomized rats and senile mice.

Osteoporosis is particularly prevalent among postmenopausal women and the elderly. In the present study, we investigated the effect of the novel small molecule E0924G (N-(4-methoxy-pyridine-2-yl)-5-methylfuran-2-formamide) on osteoporosis. E0924G significantly increased the protein expression levels of osteoprotegerin (OPG) and runt-related transcription factor 2 (RUNX2), and thus significantly promoted osteogenesis in MC3T3-E1 cells. E0924G also significantly decreased osteoclast differentiation and inhibited bone resorption and F-actin ring formation in receptor activator of NF-κB ligand (RANKL)-induced osteoclasts from RAW264.7 macrophages. Importantly, oral administration of E0924G in both ovariectomized (OVX) rats and SAMP6 senile mice significantly increased bone mineral density and decreased bone loss compared to OVX controls or SAMR1 mice. Further mechanistic studies showed that E0924G could bind to and then activate peroxisome proliferator-activated receptor delta (PPARδ), and the pro-osteoblast effect and the inhibition of osteoclast differentiation induced by E0924G were significantly abolished when PPARδ was knocked down or inhibited. In conclusion, these data strongly suggest that E0924G has the potential to prevent OVX-induced and age-related osteoporosis by dual regulation of bone formation and bone resorption through activation of the PPARδ signaling pathway.

A pan-PPAR agonist E17241 ameliorates hyperglycemia and diabetic dyslipidemia in KKAy mice via up-regulating ABCA1 in islet, liver, and white adipose tissue.

OBJECTIVE: Type 2 diabetes mellitus (T2DM) is a common chronic metabolic disease. Peroxisome proliferator-activated receptors (PPARs) play crucial roles in regulating glucolipid metabolism. Previous studies showed that E17241 could ameliorate atherosclerosis and lower fasting blood glucose levels in ApoE-/- mice. In this work, we investigated the role of E17241 in glycolipid metabolism in diabetic KKAy mice. APPROACH AND RESULTS: We confirmed that E17241 is a powerful pan-PPAR agonist with a potent agonistic activity on PPARγ, a high activity on PPARα, and a moderate activity on PPARδ. E17241 also significantly increased the protein expression of ATP-binding cassette transporter 1 (ABCA1), a crucial downstream target gene for PPARs. E17241 clearly lowered plasma glucose levels, improved OGTT and ITT, decreased islet cholesterol content, improved ß-cell function, and promoted insulin secretion in KKAy mice. Moreover, E17241 could significantly lower plasma total cholesterol and triglyceride levels, reduce liver lipid deposition, and improve the adipocyte hypertrophy and the inflammatory response in epididymal white adipose tissue. Further mechanistic studies indicated that E17241 boosts cholesterol efflux and insulin secretion in an ABCA1 dependent manner. RNA-seq and qRT-PCR analysis demonstrated that E17241 induced different expression of PPAR target genes in liver and adipose tissue differently from the PPARγ agonist rosiglitazone. In addition, E17241 treatment was also demonstrated to have an exhilarating cardiorenal benefits. CONCLUSIONS: Our results demonstrate that E17241 regulates glucolipid metabolism in KKAy diabetic mice while having cardiorenal benefits without inducing weight gain. It is a promising drug candidate for the treatment of T2DM.

Visualization of the Ferroptosis in Atherosclerotic Plaques with Nanoprobe Engineered by Macrophage Cell Membranes.

Atherosclerosis (AS) is the root cause of cardiovascular diseases. Ferroptosis is characterized by highly iron-dependent lipid peroxidation and has been reported to play an important role in the pathogenesis of AS. Visualization of the ferroptosis process in atherosclerotic plaques is of great importance for diagnosing and treating AS. In this work, the rationally designed fluorescent probe FAS1 exhibited excellent advantages including large Stokes shift, sensitivity to environmental viscosity, good photostability, and improved water solubility. It also could co-locate with commercial lipid droplets (LDs) probes (BODIPY 493/503) well in RAW264.7 cells treated by the ferroptosis inducer. After self-assembly into nanoparticles and then encapsulation with macrophage membranes, the engineered FAS1@MM NPs could successfully target the atherosclerotic plaques in Western diet-induced apolipoprotein E knockout (ApoE-/-) mice and reveal the association of ferroptosis with AS through fluorescence imaging in vivo. This study may provide additional insights into the roles of ferroptosis in the diagnosis and treatment of AS.

SIRT1 Activator E1231 Alleviates Nonalcoholic Fatty Liver Disease by Regulating Lipid Metabolism.

Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases. Silencing information regulator 1 (SIRT1) was demonstrated to modulate cholesterol and lipid metabolism in NAFLD. Here, a novel SIRT1 activator, E1231, was studied for its potential improvement effects on NAFLD. C57BL/6J mice were fed a high-fat and high-cholesterol diet (HFHC) for 40 weeks to create a NAFLD mouse model, and E1231 was administered by oral gavage (50 mg/kg body weight, once/day) for 4 weeks. Liver-related plasma biochemistry parameter tests, Oil Red O staining, and hematoxylin-eosin staining results showed that E1231 treatment ameliorated plasma dyslipidemia, plasma marker levels of liver damage (alanine aminotransferase (ALT) and aspartate aminotransferase (AST)), liver total cholesterol (TC) and triglycerides (TG) contents, and obviously decreased hepatic steatosis score and NAFLD Activity Score (NAS) in the NAFLD mouse model. Western blot results showed that E1231 treatment significantly regulated lipid-metabolism-related protein expression. In particular, E1231 treatment increased SIRT1, PGC-1α, and p-AMPKα protein expression but decreased ACC and SCD-1 protein expression. Additionally, in vitro studies demonstrated that E1231 inhibited lipid accumulation and improved mitochondrial function in free-fatty-acid-challenged hepatocytes, and required SIRT1 activation. In conclusion, this study illustrated that the SIRT1 activator E1231 alleviated HFHC-induced NAFLD development and improved liver injury by regulating the SIRT1-AMPKα pathway, and might be a promising candidate compound for NAFLD treatment.

Moldavica acid A, a new salicylic acid derivative from Dracocephalum moldavica.

In this paper, we present the discovery of a novel salicylic acid derivative, moldavica acid A (1), and a new natural dibenzo[b,f]oxepin, moldavica acid B (2), together with four known phenylpropionic acids (3-6) and protocatechuic acid (7) that were isolated from Dracocephalum moldavica L. Their structures were elucidated by comprehensive spectroscopic methods, including infrared and nuclear magnetic resonance. Compound 1 is the first example of salicylic acid linking a carboxylated α-pyrone via an ethyl bridge. Beyond expanding the knowledge of the chemical diversity of D. moldavica, both compounds 1 and 2 were shown to upregulate the expression of Kruppel-like factor 2, which could serve as a prospective therapeutic target for the treatment of atherosclerosis.

Trichostatin D as a Novel KLF2 Activator Attenuates TNFα-Induced Endothelial Inflammation.

Krüppel-like factor 2 (KLF2) is an atherosclerotic protective transcription factor that maintains endothelial cell homeostasis through its anti-inflammatory, anti-oxidant, and antithrombotic properties. The aim of this study was to discover KLF2 activators from microbial secondary metabolites and explore their potential molecular mechanisms. By using a high-throughput screening model based on a KLF2 promoter luciferase reporter assay, column chromatography, electrospray ionization mass spectrometry (ESI-MS), and nuclear magnetic resonance (NMR) spectra, trichostatin D (TSD) was isolated from the rice fermentation of Streptomyces sp. CPCC203909 and identified as a novel KLF2 activator. Real-time-quantitative polymerase chain reaction (RT-qPCR) results showed that TSD upregulated the mRNA level of KLF2 in endothelial cells. Functional assays showed that TSD attenuated monocyte adhesion to endothelial cells, decreased vascular cell adhesion protein 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) expression, and exhibited an anti-inflammatory effect in tumor necrosis factor alpha (TNFα)-induced endothelial cells. We further demonstrated through siRNA and western blot assays that the effects of TSD on monocyte adhesion and inflammation in endothelial cells were partly dependent on upregulating KLF2 expression and then inhibiting the NOD-like receptor protein 3 (NLRP3)/Caspase-1/interleukin-1beta (IL-1ß) signaling pathway. Furthermore, histone deacetylase (HDAC) overexpression and molecular docking analysis results showed that TSD upregulated KLF2 expression by inhibiting HDAC 4, 5, and 7 activities. Taken together, TSD was isolated from the fermentation of Streptomyces sp. CPCC203909 and first reported as a potential activator of KLF2 in this study. Furthermore, TSD upregulated KLF2 expression by inhibiting HDAC 4, 5, and 7 and attenuated endothelial inflammation via regulation of the KLF2/NLRP3/Caspase-1/IL-1ß signaling pathway.

Synthesis of N-methylpyridine-chlorofuranformamide analogs as novel OPG up-regulators and inhibitors of RANKL-induced osteoclastogenesis.

The OPG/RANKL/RANK pathway is a promising target for the design of therapeutic agents used in the treatment of osteoporosis. E09241 with an N-methylpyridine-chlorofuranformamide structural skeleton was previously identified to decrease bone loss and thus protect against osteoporosis in ovariectomized rats through increasing osteoprotegerin (OPG) expression. In this study, 36 derivatives of E09241 (3a) were prepared. The synthesis, up-regulation of OPG activities, SAR (structure-activity relationship), and cytotoxicity of these compounds are presented. Compounds with good up-regulating OPG activities could inhibit RANKL (the receptor activator of nuclear factor-kappa B ligand)-induced osteoclastogenesis in RAW264.7 cells. Particularly, compounds 3c and 3i1 significantly reduced NFATc1 and MMP-9 protein expression through inhibition of the NF-κB and MAPK pathways in RANKL induced RAW264.7 cells. In addition, compounds 3c and 3v significantly promoted osteoblast differentiation in MC3T3-E1 cells in osteogenic medium, and compounds 3c, 3v, and 3i1 obviously increased OPG protein expression and secretion in MC3T3-E1 cells. Furthermore, the pharmacokinetic profiles, acute toxicity, and hERG K+ channel effects of compounds 3a, 3c, 3e, 3v, and 3i1 were investigated. Taken together, these results indicate that N-methylpyridine-chlorofuranformamide analog 3i1 could serve as a promising lead for the development of new agents for treating osteoporosis.

Specific sublingual immunotherapy in children with perennial rhinitis: a systemic review and meta-analysis.

BACKGROUND: Allergic rhinitis (AR) is a major public health problem and is increasing worldwide. AR affects children's learning efficiency, sleep quality, and other major aspects of life. Sublingual immunotherapy (SLIT) is effective and safe for children with seasonal AR, but for children with perennial allergic rhinitis (PAR) caused by house dust mites (HDM), its effectiveness and safety is less convincing. METHODS: Medical literature databases up to 2019 were searched for published and unpublished pieces of evidence. Studies were individually screened by 2 reviewers against the eligibility criteria. Primary outcomes were total nasal symptoms scores (TNSS) and total medication scores (TMS). The secondary outcomes were total ocular symptoms scores and adverse events (AEs). Random effect models and fixed-effect models were used to calculate the standard mean difference (SMD) and risk ratio (RR), respectively. RESULTS: We identified 3772 abstracts, of which only 16 studies met our established criteria. SLIT significantly reduced TNSS (SMD -1.73; 95% CI, -2.62 to -0.84; p = 0.0001) and TMS (SMD -1.21; 95% CI, -1.75 to -0.67; p < 0.00001). Compared with children taking placebo, children taking SLIT were 1.08 (95% CI, 1 to 1.17; p = 0.05), 1.15 (95% CI, 0.87 to 1.51; p = 0.32), and 1.68 (95% CI, 0.68 to 4.11; p = 0.26) times more likely to develop mild, moderate, and severe AEs, respectively. CONCLUSION: HDM SLIT can effectively alleviate TNSS and TMS in children with PAR, but care should be taken to avoid harm due to possible adverse drug reactions.

Tau Protein and Zebrafish Models for Tau-Induced Neurodegeneration.

Tauopathies are a specific type of slow and progressive neurodegeneration, which involves intracellular deposition of fibrillar material composed of abnormal hyperphosphorylation of the microtubule associated protein (MAP) tau. Despite many years of intensive research, our understanding of the molecular events that lead to neurodegeneration is far from complete. No effective therapeutic treatments have been defined, and questions surround the validity and utility of existing animal models. It is an urgent need to develop a novel animal model to study the underlying neurodegenerative mechanisms of tauopathies. Zebrafish models of tauopathies could complement existing models by providing an in vivo platform for genetic and chemical screens in order to identify new therapeutic targets and compounds, meanwhile zebrafish models have permitted discovery of unique characteristics of these genes that could have been difficultly observed in other models. Novel transgenic zebrafish models expressing wild-type or mutant forms of human 4R-tau in neurons have recently been reported. These studies show disease-relevant changes including tau hyperphosphorylation, aggregation and somato-dendritic relocalization. This review highlights the availability of transgenic tau zebrafish models that allow more detailed biochemical studies of tau in the zebrafish CNS to characterize solubility, fibril morphology and further clarify phosphorylation proceedings. Furthermore, a deeper knowledge of the zebrafish brain and a better characterization of tau caused by alterations in neurodegenerative disorders are needed.

Effects of microtopographic patterns on platelet adhesion and activation on titanium oxide surfaces.

This study systematically investigated the effects of microtopographic patterns of titanium oxide on platelet adhesion and activation in order to reveal the mechanisms of interactions between platelet and surface topography. Periodic arrays of groove and pillar patterns with dimensions ranging from submicron to several micrometers were fabricated by photolithography and deep reactive-ion etching on silicon substrates, followed by the sputter deposition of titanium oxide (TiO(2)). Platelet adhesion and activation on TiO(2) patterned surfaces were evaluated by lactate dehydrogenase (LDH) and GMP-140 assays, respectively. The morphology of adherent platelets was examined by scanning electron microscope (SEM). The results showed that the microtopographic patterns were able to effectively manipulate the platelet response by varying geometry and size of patterns. A groove pattern resulted in much higher levels of platelet adhesion and activation than a pillar pattern. The study revealed that a difference in pattern size led to two distinctive modes of platelet adhesion: the "bridging" mode in which platelets can bridge over spacing between adjacent patterns when spacing size is smaller than 1.5 µm; and the "full-contact" mode in which platelets cannot bridge but fully contact the entire surface when spacing size is larger than 3 µm. Our analysis indicates good correlations between platelet behavior and hydrophobicity/wetting anisotropy in "bridging" mode, and effective surface contact area in "full-contact" mode.