Breast cancer therapy: from the perspective of glucose metabolism and glycosylation.

Breast cancer is a leading cause of mortality and the most prevalent form of malignant tumor among women worldwide. Breast cancer cells exhibit an elevated glycolysis and altered glucose metabolism. Moreover, these cells display abnormal glycosylation patterns, influencing invasion, proliferation, metastasis, and drug resistance. Consequently, targeting glycolysis and mitigating abnormal glycosylation represent key therapeutic strategies for breast cancer. This review underscores the importance of protein glycosylation and glucose metabolism alterations in breast cancer. The current research efforts in developing effective interventions targeting glycolysis and glycosylation are further discussed.

The role of peroxisome proliferator-activated receptor γ in lipid metabolism and inflammation in atherosclerosis.

Cardiovascular disease events are the result of functional and structural abnormalities in the arteries and heart. Atherosclerosis is the main cause and pathological basis of cardiovascular diseases. Atherosclerosis is a multifactorial disease associated with dyslipidemia, inflammation, and oxidative stress, among which dyslipidemia and chronic inflammation occur in all processes. Under the influence of lipoproteins, the arterial intima causes inflammation, necrosis, fibrosis, and calcification, leading to plaque formation in specific parts of the artery, which further develops into plaque rupture and secondary thrombosis. Foam cell formation from macrophages is an early event in the development of atherosclerosis. Lipid uptake causes a vascular inflammatory response, and persistent inflammatory infiltration in the lesion area further promotes the development of the disease. Inhibition of macrophage differentiation into foam cell and reduction of the level of proinflammatory factors in macrophages can effectively alleviate the occurrence and development of atherosclerosis. Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated nuclear receptor that plays an important antiatherosclerotic role by regulating triglyceride metabolism, lipid uptake, cholesterol efflux, macrophage polarity, and inhibiting inflammatory signaling pathways. In addition, PPARγ shifts its binding to ligands and co-activators or co-repressors of transcription of target genes through posttranslational modification, thereby affecting the regulation of its downstream target genes. Many ligand agonists have also been developed targeting PPARγ. In this review, we summarized the role of PPARγ in lipid metabolism and inflammation in development of atherosclerosis, the posttranslational regulatory mechanism of PPARγ, and further discusses the value of PPARγ as an antiatherosclerosis target.

The antimicrobial peptide LK2(6)A(L) exhibits anti-inflammatory activity by binding to the myeloid differentiation 2 domain and protects against LPS-induced acute lung injury in mice.

Acute lung injury (ALI) is a life-threatening disease that is generally attributable to an uncontrolled inflammatory response in the lung, but there is a lack of effective treatments. At present, regulating the inflammatory response has become an important strategy for treating ALI. In the present study, LK2(6)A(L), a peptide derived from the natural antimicrobial peptide temporin-1CEa, inhibited lipopolysaccharide (LPS)-induced expression of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and NO in RAW264.7 cells. Herein, the anti-inflammatory mechanism of LK2(6)A(L) was investigated. The RNA-sequencing (RNA-seq) results showed that LK2(6)A(L) significantly inhibited the TLR4-mediated NF-κB and MAPK signaling pathways in LPS-induced RAW264.7 cells. The results of co-immunoprecipitation (Co-IP), pull-down experiment, confocal laser scanning microscopy, and surface plasmon resonance (SPR) suggested that MD2 was the direct target of LK2(6)A(L). Chemical inhibition of MD2 and its knockdown abolished the anti-inflammatory effect of LK2(6)A(L). Molecular dynamic simulation indicated that LK2(6)A(L) could bind to the active domain of the MD2 hydrophobic pocket via six hydrogen bonds. The truncated peptides were designed based on analysis of the molecular docking of LK2(6)A(L) to MD2. The truncated peptide IS-7 showed strong affinity to MD2 and a remarkable inhibitory effect on pro-inflammatory factors that was comparable to the effect of LK2(6)A(L). Finally, LK2(6)A(L) and IS-7 relieved inflammatory symptoms and lung tissue destruction in the ALI mouse model. Overall, our study suggested that LK2(6)A(L) showed promising anti-inflammatory activity by targeting MD2, and the amino acid domain 7-13 was an important area that binds with MD2 and also an anti-inflammatory active region. LK2(6)A(L) and IS-7 may be potential new treatments for ALI and other acute inflammatory diseases.

Visual LAMP method for the detection of Vibrio vulnificus in aquatic products and environmental water.

BACKGROUND: A visual, rapid, simple method was developed based on a loop-mediated isothermal amplification (LAMP) assay to detect Vibrio vulnificus in aquatic products and aquaculture waters. RESULTS: Genomic DNA was extracted from Vibrio vulnificus using the boiling method, and optimized primers were used to detect the gyrB gene using a visual LAMP method. The sensitivity of the assay was 10 fg/µL, and the obtained results were stable and reliable. Out of 655 aquatic product samples and 558 aquaculture water samples, the positive rates of Vibrio vulnificus detection were 9.01% and 8.60%, respectively, which are markedly higher than those of the traditional culture identification methods. CONCLUSION: The relatively simple technical requirements, low equipment cost, and rapid detection make the visual LAMP method for the detection of Vibrio vulnificus a convenient choice for field detection in the aquaculture industry.

Membrane mechanism of temporin-1CEc, an antimicrobial peptide isolated from the skin secretions of Rana chensinensis, and its systemic analogs.

Antimicrobial peptides (AMPs) are new and powerful target molecules in the development of new antibacterial agents. Temporin-1CEc, a natural peptide isolated and purified from the skin secretions of the Chinese brown frog Rana chensinensis, exhibits low or no antibacterial activity against gram-negative and gram-positive bacteria, which limits its potential therapeutic use; however, it displays low hemolysis to human erythrocytes. Here, a series of temporin-1CEc analogs was designed and synthesized by amino acid residue substitutions based on cationicity, hydrophobicity, amphipathicity and secondary structure to understand the structure-activity relationships of this peptide in depth. The results showed that all of the analogs, except for 2K and 4K, had significantly improved antibacterial activity against the tested standard bacterial strains and multidrug-resistant bacterial strains compared to temporin-1CEc. 2K2L and 2K4L, but not 4K2L and 4K4L, showed the strongest antibacterial activity compared with their parent peptides 2K and 4K, suggesting that peptide hydrophobicity plays a more important role in antibacterial activity than cationicity for this series of AMPs. However, the antibacterial activity of the 6 Trp-containing analogs of 2K4L decreased with a further increase in hydrophobicity based on the results of 2K4L, indicating that it is more important to balance cationicity and hydrophobicity. Moreover, an increase in AMP hydrophobicity led to hemolysis. Notably, all of the peptides adopted α-helical structures in 50% trifluoroethanol/water and 30 mM SDS solutions. 2K2L and 2K4L displayed broad-spectrum antibacterial activity against sensitive and multidrug-resistant bacteria, effectively killing the tested multidrug resistant strain Staphylococcus epidermidis (MRSE1208). 2K2L and 2K4L were able to increase the permeability of the outer and inner membranes by depolarization and disturb the integration of the cytoplasmic membrane of MRSE1208 cells, leading to leakage of its cellular contents. In addition, 2K2L and 2K4L at low concentrations inhibited biofilm formation and degraded mature 1-day-old MRSE1208 biofilms. Notably, 2K2L and 2K4L inhibited the formation of MRSE1208 biofilms at concentrations below its MIC value, suggesting that the peptide may exert an inhibitory effect through not only direct antimicrobial activity but also a biofilm-specific mechanism. Collectively, these results suggest that 2K2L and 2K4L could be effective antibiotics against multidrug-resistant bacterial strains.

Binding Properties of DNA and Antimicrobial Peptide Chensinin-1b Containing Lipophilic Alkyl Tails.

Multidrug-resistant bacteria present an important threat to human health. In this study, due to the weak antimicrobial activity of chensinin-1b against multidrug-resistant (MDR) bacteria, three lipo-chensinin-1b peptides, including OA-C1b, LA-C1b and PA-C1b, were designed and their activities against MDR bacteria were examined. Both the OA-C1b and LA-C1b peptides exhibited potent antimicrobial activity against selected multidrug-resistant bacterial strains. In addition to the direct disruption of bacterial membranes by antimicrobial peptides, it has also been proposed that DNA is a superior intracellular target for antimicrobial peptides. ctDNA was used as a model to investigate the binding properties of DNA and lipo-chensinin-1b peptides using a variety of biophysical methods. The kinetics results of both UV-Vis and CD spectroscopy suggested that the interaction between lipo-chensinin-1b peptides and ctDNA was concentration-dependent and resulted in an increase in polynucleotide helicity. Viscosity measurements, Trp fluorescence and iodide quenching experiments indicated that nonclassical groove binding and electrostatic binding interaction modes were utilized when the peptides interacted with the ctDNA. In addition, the formation of peptide-ctDNA complexes was monitored using dynamic light scattering experiments, during which the peptide exhibited the ability to neutralize the negative charges on the surface of the ctDNA. These results promote the possibility of designing peptide-based antibiotics targeted to DNA.

Potential role of a series of lysine-/leucine-rich antimicrobial peptide in inhibiting lipopolysaccharide-induced inflammation.

Antimicrobial peptides have broad-spectrum killing activities against bacteria, enveloped viruses, fungi and several parasites via cell membrane permeation and exhibit primarily immunomodulatory and anti-infective functions in their interactions with host cells. However, the mechanism underlying their anti-inflammatory activity remains to be elucidated. L-K6, an analog of temporin-1CEb isolated from the skin secretion of Rana chensinensis, has demonstrated a wide range of antimicrobial activities against gram-negative and gram-positive bacteria. In this study, the potent anti-inflammatory mechanism of L-K6 and its analogs in lipopolysaccharide (LPS)-stimulated human macrophage U937 cells were evaluated. We found that L-K6 suppressed the expression of inflammatory factors by two downstream signaling components in the MyD88-dependent pathway, including the mitogen-activated protein kinases (MAPKs) and the NF (nuclear factor)-κB signaling pathway, but its analog L-K5, which had the same amino acid sequence as L-K6 but no Lys residue at the -COOH terminal, only inhibited the phosphorylation of I-κB and NF-κB. Importantly, L-K6 and L-K5 were actively taken up by U937 cells through an independent cell membrane disruption mechanism and were eventually localized to the perinuclear region. The L-K6 uptake process was mediated by endocytosis, but L-K5 was specifically taken up by U937 cells via TLR4 endocytosis. Our results demonstrated that L-K6 can neutralize LPS and diassociate LPS micelles to inhibit LPS from triggering the proinflammatory signaling pathway, and by partially inhibiting inflammatory responses by the intracellular target. However, L-K5 may mainly inhibit proinflammatory responses by intracellular reporters to modulate the NF-κB signaling pathway.

Xylarianins A-D from the endophytic fungus Xylaria sp. SYPF 8246 as natural inhibitors of human carboxylesterase 2.

Eighteen secondary metabolites were isolated from the fermentation broth of the endophytic fungus Xylaria sp. SYPF 8246, including four new compounds, xylarianins A-D (1-4), three new natural products, 6-methoxycarbonyl-2'-methyl-3,5,4',6'-tetramethoxy-diphenyl ether (5), 2-chlor-6-methoxycarbonyl-2'-rnethyl-3,5,4',6'-tetramethoxy-diphenyl ether (6), and 2-chlor-4'-hydroxy-6-methoxy carbonyl-2'-methyl-3,5,6'-trimethoxy-diphenyl ether (7), and eleven known compounds (8-18). Their structural elucidations were conducted by using 1D and 2D NMR, HRESIMS, and Rh2(OCOCF3)4-induced electronic circular dichroism (ECD) spectra analyses. The integrated 1H and 13C NMR data of three new natural products 5-7 were reported for the first time. All the isolated compounds were assayed for their inhibitory activities against human carboxylesterase 2 (hCE 2). Compounds 1, 5-9, and 18 displayed significant inhibitory activities against hCE 2 with IC50 values of 10.43 ±â€¯0.51, 6.69 ±â€¯0.85, 12.36 ±â€¯1.27, 18.25 ±â€¯1.78, 29.78 ±â€¯0.48, 18.86 ±â€¯1.87, and 20.72 ±â€¯1.51 µM, respectively. The interactions between compounds 1 and 5 with hCE 2 were anaylzed by molecular docking.

Interactions between chensinin-1, a natural antimicrobial peptide derived from Rana chensinensis, and lipopolysaccharide.

Lipopolysaccharide (LPS) plays a critical role in the pathogenesis of sepsis caused by gram-negative bacterial infections. Therefore, LPS-neutralizing molecules would have important clinical applications. Chensinin-1, a novel antimicrobial peptide with atypical structural features, was found in the skin secretions of the Chinese brown frog Rana chensinensis. To understand the role of LPS in the bacterial susceptibility to chensinin-1 and to investigate its anti-endotoxin effects, the interactions of chensinin-1 with LPS were investigated in this study using circular dichroism, in situ IR, isothermal titration calorimetry, and zeta potential. This study is the first to use in situ IR spectroscopy to evaluate the secondary structural changes of this peptide. The capacity of chensinin-1 to block the LPS-dependent cytokine secretion of macrophages was also investigated. Our results show that chensinin-1 can form α-helical structures in LPS suspensions. LPS can affect the antimicrobial activity of chensinin-1, and chensinin-1 was able to mitigate the effects of LPS. These data may facilitate the development of antimicrobial peptides with potent antimicrobial and anti-endotoxin activities.

Inhibitory Effects of Antimicrobial Peptides on Lipopolysaccharide-Induced Inflammation.

Antimicrobial peptides (AMPs) are usually small molecule peptides, which display broad-spectrum antimicrobial activity, high efficiency, and stability. For the multiple-antibiotic-resistant strains, AMPs play a significant role in the development of novel antibiotics because of their broad-spectrum antimicrobial activities and specific antimicrobial mechanism. Besides broad-spectrum antibacterial activity, AMPs also have anti-inflammatory activity. The neutralization of lipopolysaccharides (LPS) plays a key role in anti-inflammatory action of AMPs. On the one hand, AMPs can readily penetrate the cell wall barrier by neutralizing LPS to remove Gram-negative bacteria that can lead to infection. On the contrary, AMPs can also inhibit the production of biological inflammatory cytokines to reduce the inflammatory response through neutralizing circulating LPS. In addition, AMPs also modulate the host immune system by chemotaxis of leukocytes, to promote immune cell proliferation, epithelialization, and angiogenesis and thus play a protective role. This review summarizes some recent researches about anti-inflammatory AMPs, with a focus on the interaction of AMPs and LPS on the past decade.

Free radical scavenging activity and neuroprotective potentials of D138, one Cu(II)/Zn(II) Schiff-base complex derived from N,N'-bis(2-hydroxynaphthylmethylidene)-1,3-propanediamine.

There is increasing evidence that free radicals play an important role in neuronal damages induced by diabetes mellitus or cerebral ischemia insults. Antioxidants with free radical scavenging activities have been shown to be beneficial and neuroprotective for these pathological conditions. Here, we report free radical scavenging activity and neuroprotective potential of D138, one copper(II)/zinc(II) Schiff-base complex derived from N,N'-2(2-hydroxynaphthylmethylidene)-1,3-propanediamine. The data from three in vitro assays, 2,2-diphenyl-1-picrylhydrazyl assay, nitro blue tetrazolium assay and hydroxyl radical scavenging assay, indicated that D138 presented a potent free radical scavenging activity. The neuroprotective and antioxidative effects of D138 were further evaluated in vivo using bilateral common carotid artery occlusion (BCCAO) mouse model and streptozotocin (STZ) diabetic mouse model. Our results indicated that treatment of D138 significantly ameliorated the hippocampal neuronal damage and the oxidative stress levels in these animal models. Moreover, D138 also reversed the behavioral deficiencies induced by BCCAO or STZ, as assessed by Y-maze test and fear conditioning test. In conclusion, all these findings support that D138 exerts free radical scavenging and neuroprotective activities and has the potentials to be a potent therapeutic candidate for brain oxidative damage induced by cerebral ischemia or diabetes mellitus.

Effects of antimicrobial peptide L-K6, a temporin-1CEb analog on oral pathogen growth, Streptococcus mutans biofilm formation, and anti-inflammatory activity.

Dental caries and periodontitis are common bacterial mouth infections. As a potentially attractive substitute for conventional antibiotics, antimicrobial peptides have been widely tested and used for controlling bacterial infections. In this study, we tested the efficacy of the peptides from the skin secretions of Rana chensinensis for killing several major cariogenic and periodontic pathogens as well as Candida albicans. L-K6, a temporin-1CEb analog, exhibited high antimicrobial activity against the tested oral pathogens and was able to inhibit Streptococcus mutans biofilm formation and reduce 1-day-old S. mutans biofilms with a minimum biofilm inhibitory concentration and reducing concentration of 3.13 and 6.25 µM, respectively. The results of confocal laser scanning microscopy demonstrated that the peptide significantly reduced cell viability within oral biofilms. Furthermore, as little as 5 µM L-K6 significantly inhibited lipopolysaccharide (LPS)- and interleukin-1ß-induced productions of interleukin-8 and tumor necrosis factor-α from THP-1 monocytic cells. This anti-inflammatory activity is associated with the binding of L-K6 to LPS and neutralizing LPS-induced proinflammatory responses in THP-1 cells, as well as dissociating LPS aggregates. Our results suggest that L-K6 may have potential clinical applications in treating dental caries by killing S. mutans within dental plaque and acting as anti-inflammatory agents in infected tissues.

Antimicrobial peptide 2K4L inhibits the inflammatory response in macrophages and Caenorhabditis elegans and protects against LPS-induced septic shock in mice.

2K4L is a rationally designed analog of the short α-helical peptide temporin-1CEc, a natural peptide isolated and purified from the skin secretions of the Chinese brown frog Rana chensinensis by substituting amino acid residues. 2K4L displayed improved and broad-spectrum antibacterial activity than temporin-1CEc in vitro. Here, the antibacterial and anti-inflammatory activities of 2K4L in macrophages, C. elegans and mice were investigated. The results demonstrated that 2K4L could enter THP-1 cells to kill a multidrug-resistant Acinetobacter baumannii strain (MRAB 0227) and a sensitive A. baumannii strain (AB 22933), as well as reduce proinflammatory responses induced by MRAB 0227 by inhibiting NF-κB signaling pathway. Similarly, 2K4L exhibited strong bactericidal activity against A. baumannii uptake into C. elegans, extending the lifespan and healthspan of the nematodes. Meanwhile, 2K4L alleviated the oxidative stress response by inhibiting the expression of core genes in the p38 MAPK/PMK-1 signaling pathway and downregulating the phosphorylation level of p38, thereby protecting the nematodes from damage by A. baumannii. Finally, in an LPS-induced septic model, 2K4L enhanced the survival of septic mice and decreased the production of proinflammatory cytokines by inhibiting the signaling protein expression of the MAPK and NF-κB signaling pathways and protecting LPS-induced septic mice from a lethal inflammatory response. In conclusion, 2K4L ameliorated LPS-induced inflammation both in vitro and in vivo.

The antibacterial activity and mechanisms of Trp-containing peptides against multidrug-resistant Pseudomonas aeruginosa persisters.

Bacterial persisters avoid antibiotic-mediated death by entering a dormant state and are considered a major cause of antibiotic treatment failure. Antimicrobial peptides (AMPs) with membrane-disrupting activity are promising drugs to eradicate persister cells. In this study, carbonyl cyanide m-chlorophenylhydrazone (CCCP), ciprofloxacin (CIP), and rifampicin (RFP) were applied to induce the formation of multidrug-resistant Pseudomonas aeruginosa (MRPA0108) persisters, and the antibacterial activity and mechanisms of I1W and L12W (two Trp-containing peptides designed in our lab) against MRPA0108 persisters were investigated. The results showed that I1W and L12W displayed potent antibacterial activity against MRPA0108 persisters. Both Trp-containing peptides disturbed the inner and outer membrane of MRPA0108 persisters. In addition, I1W and L12W revealed novel antibacterial mechanisms by decreasing the enzymatic activities of superoxide dismutase (SOD) and catalase (CAT), increasing reactive oxygen species (ROS) and malondialdehyde (MDA) levels, consequently leading to oxidative stress. The transcriptome profile of I1W-treated MRPA0108 persisters revealed that the genes involved in carbon metabolism, biosynthesis of amino acids, and the TCA cycle were downregulated, indicating that I1W interfered with metabolism and energy synthesis processes. Furthermore, both Trp-containing peptides displayed synergistic activities with antibiotic tobramycin and showed additive activities with cefepime or ciprofloxacin, which revealed a potential therapeutic strategy for the eradication of MRPA0108 persisters.

Chensinin-1b Alleviates DSS-Induced Inflammatory Bowel Disease by Inducing Macrophage Switching from the M1 to the M2 Phenotype.

Inflammatory bowel disease (IBD) is a chronic relapsing inflammatory disorder with an increasing prevalence worldwide. Macrophage polarization is involved in the pathogenesis of IBD. Repolarization of macrophage has thus emerged as a novel therapeutic approach for managing IBD. Chensinin-1b, derived from the skin of Rana chensinensis, is a derivative of a native antimicrobial peptide (AMP). It shows anti-inflammatory effects in sepsis models and can potentially modulate macrophage polarization. The objective of this research was to study the role of chensinin-1b in macrophage polarization and dextran sulfate sodium (DSS)-induced colitis. RAW264.7 macrophages were polarized to the M1 phenotype using lipopolysaccharide (LPS) and simultaneously administered chensinin-1b at various concentrations. The ability of chenisnin-1b to reorient macrophage polarization was assessed by ELISA, qRT-PCR, and flow cytometry analysis. The addition of chensinin-1b significantly restrained the expression of M1-associated proinflammatory cytokines and surface markers, including TNF-α, IL-6, NO, and CD86, and exaggerated the expression of M2-associated anti-inflammatory cytokines and surface markers, including IL-10, TGF-ß1, Arg-1, Fizz1, Chil3, and CD206. Mechanistically, via Western Blotting, we revealed that chensinin-1b induces macrophage polarization from the M1 to the M2 phenotype by inhibiting the phosphorylation of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK). In mouse models of colitis, intraperitoneal administration of chensinin-1b alleviated symptoms induced by DSS, including weight loss, elevated disease activity index (DAI) scores, colon shortening, colonic tissue damage, and splenomegaly. Consistent with our in vitro data, chensinin-1b induced significant decreases in the expression of M1 phenotype biomarkers and increases in the expression of M2 phenotype biomarkers in the mouse colitis model. Furthermore, chensinin-1b treatment repressesed NF-κB phosphorylation in vivo. Overall, our data showed that chensinin-1b attenuates IBD by repolarizing macrophages from the M1 to the M2 phenotype, suggesting its potential as a therapeutic candidate for IBD.

Antimicrobial peptide 2K4L disrupts the membrane of multidrug-resistant Acinetobacter baumannii and protects mice against sepsis.

Antimicrobial peptides represent a promising therapeutic alternative for the treatment of antibiotic-resistant bacterial infections. 2K4L is a rationally-designed analog of a short peptide temporin-1CEc, a natural peptide isolated and purified from the skin secretions of the Chinese brown frog Rana chensinensis by substituting amino acid residues. 2K4L adopt an α-helical confirm in a membrane-mimetic environment and displayed an improved and broad-spectrum antibacterial activity against sensitive and multidrug-resistant Gram-negative and Gram-positive bacterial strains. Here, the action mechanism of 2K4L on multidrug resistant Acinetobacter baumannii (MRAB) and protection on MRAB-infected mice was investigated. The results demonstrated high bactericidal activity of 2K4L against both a multidrug resistant A. baumannii 0227 strain (MRAB 0227) and a sensitive A. baumannii strain (AB 22934), indicating a potential therapeutic advantage of this peptide. Strong positively-charged residues significantly promoted the electrostatic interaction on 2K4L with lipopolysaccharides (LPS) of the bacterial outer membrane. High hydrophobicity and an α-helical confirm endowed 2K4L remarkably increase the permeability of A. baumannii cytoplasmic membrane by depolarization of membrane potential and disruption of membrane integration, as well as leakage of fluorescein from the liposomes. Additionally, 2K4L at low concentrations inhibited biofilm formation and degraded mature 1-day-old MRAB 0227 biofilms by reducing the expression of biofilm-related genes. In an invasive A. baumannii infection model, 2K4L enhanced the survival of sepsis mice and decreased the production of the proinflammatory cytokines downregulating the phosphorylation level of signaling protein in MAPK and NF-κB signaling pathways, indicating that 2K4L represents a novel therapeutic antibiotic candidate against invasive multidrug-resistant bacterial strain infections.

The antimicrobial peptide chensinin-1b alleviates the inflammatory response by targeting the TLR4/NF-κB signaling pathway and inhibits Pseudomonas aeruginosa infection and LPS-mediated sepsis.

Excessive inflammatory responses are a major contributor to the high mortality associated with sepsis, a prevalent global complication. Therefore, the potential therapeutic strategy for sepsis involves targeting macrophages and reducing proinflammatory cytokine release. Chensinin-1b, an analog of the natural antimicrobial peptide derived from Rana chensinensis skin secretion, exhibits broad-spectrum antibacterial activity and adopts a random coil conformation in both PBS and membrane solution. By efficiently neutralizing LPS, chensinin-1b holds promise in alleviating LPS-induced inflammatory responses. In this study, we established a mouse septic shock model by exposing mice to multiple-drug-resistant Pseudomonas aeruginosa, as well as an endotoxin-mediated sepsis model induced by LPS. Administering chensinin-1b significantly prolonged the survival of the experimental mice, concurrently mitigating inflammatory responses and reducing organ damage. Additionally, we investigated the anti-inflammatory mechanism of chensinin-1b using a constructed LPS-induced mouse macrophage RAW264.7 inflammatory model. Our findings demonstrated that chensinin-1b effectively mitigated the excessive activation of the TLR4/NF-κB signaling pathway by directly neutralizing extracellular LPS, thus ameliorating the inflammatory response. Moreover, upon blocking the TLR4 signaling pathway, chensinin-1b further reduced the release of proinflammatory cytokines induced by LPS, indicating alternative modes of regulation. Notably, chensinin-1b rapidly entered RAW264.7 cells within 30 min via endocytosis, diffusing into the cytoplasm while retaining its anti-inflammatory properties intracellularly. Although further investigations are warranted to comprehensively elucidate the intracellular anti-inflammatory mechanism of chensinin-1b, our findings substantiate its possession of anti-inflammatory properties both intracellularly and extracellularly. Thus, chensinin-1b emerges as a promising candidate for mitigating excessive inflammatory responses associated with sepsis.

Membrane-active and DNA binding related double-action antimycobacterial mechanism of antimicrobial peptide W3R6 and its synthetic analogs.

The emergence of multidrug- or extremely drug-resistant M. tuberculosis strains has made very few drugs available for current tuberculosis treatment. Antimicrobial peptides can be employed as a promising alternative strategy for TB treatment. Here, we designed and synthesized a series of peptide sequences based on the structure-activity relationships of natural sequences of antimicrobial peptides. The peptide W3R6 and its analogs were screened and found to have potent antimycobacterial activity against M. smegmatis, and no hemolytic activity against human erythrocytes. The evidence from the mechanism of action study indicated that W3R6 and its analogs can interact with the mycobacterial membrane in a lytic manner and form pores on the outer membrane of M. smegmatis. Significant colocalization of D-W3R6 with mycobacterial DNA was observed by confocal laser scanning microscopy and DNA retardation assays, which suggested that the antimycobacterial mechanism of action of the peptide was associated with the unprotected genomic DNA of M. smegmatis. In general, W3R6 and its analogs act on not only the mycobacterial membrane but also the genomic DNA in the cytoplasm, which makes it difficult for mycobacteria to generate resistance due to the peptides having two targets. In addition, the peptides can effectively eliminate M. smegmatis cells from infected macrophages. Our findings indicated that the antimicrobial peptide W3R6 could be a novel lead compound to overcome the threat from drug-resistant M. tuberculosis strains in the development of potent AMPs for TB therapeutic applications.

Effects of frog skin peptide temporin-1CEa and its analogs on ox-LDL induced macrophage-derived foam cells.

Purpose: Atherosclerosis is one of the most important pathological foundations of cardiovascular and cerebrovascular diseases with high morbidity and mortality. Studies have shown that macrophages play important roles in lipid accumulation in the vascular wall and thrombosis formation in atherosclerotic plaques. This study aimed to explore the effect of frog skin antimicrobial peptides (AMPs) temporin-1CEa and its analogs on ox-LDL induced macrophage-derived foam cells. Methods: CCK-8, ORO staining, and intracellular cholesterol measurements were used to study cellular activity, lipid droplet formation and cholesterol levels, respectively. ELISA, real-time quantitative PCR, Western blotting and flow cytometry analysis were used to study the expression of inflammatory factors, mRNA and proteins associated with ox-LDL uptake and cholesterol efflux in macrophage-derived foam cells, respectively. Furthermore, the effects of AMPs on inflammation signaling pathways were studied. Results: Frog skin AMPs could significantly increase the cell viability of the ox-LDL-induced foaming macrophages and decrease the formation of intracellular lipid droplets and the levels of total cholesterol and cholesterol ester (CE). Frog skin AMPs inhibited foaming formation by reducing the protein expression of CD36, which regulates ox-LDL uptake but had no effect on the expression of efflux proteins ATP binding cassette subfamily A/G member 1 (ABCA1/ABCG1). Then, decreased mRNA expression of NF-κB and protein expression of p-NF-κB p65, p-IκB, p-JNK, p-ERK, p-p38 and the release of TNF-α and IL-6 occurred after exposure to the three frog skin AMPs. Conclusion: Frog skin peptide temporin-1CEa and its analogs can improve the ox-LDL induced formation of macrophage-derived foam cells, in addition, inhibit inflammatory cytokine release through inhibiting the NF-κB and MAPK signaling pathways, thereby inhibiting inflammatory responses in atherosclerosis.

Membrane interaction and antibacterial properties of chensinin-1, an antimicrobial peptide with atypical structural features from the skin of Rana chensinensis.

Many antimicrobial peptides from amphibian skin have been purified and structurally characterized and may be developed as therapeutic agents. Here we describe the antibacterial properties and membrane interaction of chensinin-1, a cationic arginine/histidine-rich antimicrobial peptide, from the skin secretions of Rana chensinensis. The amino acid composition, sequence, and atypical structure of chensinin-1 differ from other known antimicrobial peptides from amphibian skin. Chensinin-1 exhibited selective antimicrobial activity against Gram-positive bacteria, was inactive against Gram-negative bacteria, and had no hemolytic activity on human erythrocytes. The CD spectra for chensinin-1 indicated that the peptide adopted an aperiodic structure in water and a conformational structure with 20 % ß-strands, 8 % α-helices, and the remaining majority of random coils in the trifluoroethanol or SDS solutions. Time-kill kinetics against Gram-positive Bacillus cereus demonstrated that chensinin-1 was rapidly bactericidal at 2× MIC and PAE was found to be >5 h. Chensinin-1 caused rapid and large dye leakage from negatively charged model vesicles. Furthermore, membrane permeation assays on intact B. cereus indicated that chensinin-1 induced membrane depolarization in less than 1 min and followed to damage the integrity of the cytoplasmic membrane and resulted in efflux of molecules from cytoplasma. Hence, the primary target of chensinin-1 action was the cytoplasmic membrane of bacteria. Chensinin-1 was unable to overcome bacterial resistance imposed by the lipopolysaccharide leaflet, the major constituent of the outer membrane of Gram-negative bacteria. Lipopolysaccharide induced oligomerization of chensinin-1, thus preventing its translocation across the outer membrane.