Animals ; Rats ; Neuroprotective Agents/pharmacology* ; Amyloid beta-Peptides ; PC12 Cells ; Polyunsaturated Alkamides/pharmacology* ; Apoptosis ; Cell Survival ; Peptide Fragments

OBJECTIVE: To elucidate the renoprotective effect of resveratrol (RSV) on sphingosine kinase 1 (SphK1) signaling pathway and expression of its downstream molecules including activator protein 1 (AP-1) and transformation growth factor-β1 (TGF-β1) in lipopolysaccharide (LPS)-induced glomerular mesangial cells (GMCs). METHODS: The rat GMCs line (HBZY-1) were cultured and randomly divided into 5 groups, including control, LPS (100 ng/mL), and 5, 10, 20 µmol/L RSV-treated groups. In addition, SphK1 inhibitor (SK-II) was used as positive control. GMCs were pretreated with RSV for 2 h and treated with LPS for another 24 h. GMCs proliferation was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. The proteins expression of SphK1, p-c-Jun and TGF-β1 in GMCs were detected by Western blot, and DNA-binding activity of AP-1 was performed by electrophoretic mobility shift assay (EMSA). The binding activity between RSV and SphK1 protein was detected by AutoDock Vina and visualized by Discovery Studio 2016. RESULTS: LPS could obviously stimulate GMCs proliferation, elevate SphK1, p-c-Jun and TGF-β1 expression levels and increase the DNA-binding activity of AP-1 (P<0.05 or P<0.01), whereas these effects were significantly blocked by RSV pretreatment. It was also suggested that the effect of RSV was similar to SK-II (P>0.05). Moreover, RSV exhibited good binding affinity towards SphK1, with docking scores of -8.1 kcal/moL and formed hydrogen bonds with ASP-178 and LEU-268 in SphK1. CONCLUSION RSV inhibited LPS-induced GMCs proliferation and TGF-β1 expression, which may be independent of its hypoglycemic effect on preventing the development of mesangial cell fibrosis and closely related to the direct inhibition of SphK1 pathway.
Animals ; Rats ; Lipopolysaccharides/pharmacology* ; Mesangial Cells ; Resveratrol/pharmacology* ; Transcription Factor AP-1 ; Transforming Growth Factor beta1 ; Intercellular Signaling Peptides and Proteins ; Cell Proliferation ; DNA ; Cells, Cultured

OBJECTIVE: To explore the specific pharmacological molecular mechanisms of Kai Xin San (KXS) on treating Alzheimer's disease (AD) based on network pharmacology and experimental validation. METHODS: The chemical compounds of KXS and their corresponding targets were screened using the Encyclopedia of Traditional Chinese Medicine (ETCM) database. AD-related target proteins were obtained from MalaCards database and DisGeNET databases. Key compounds and targets were identified from the compound-target-disease network and protein-protein interaction (PPI) network analysis. Functional enrichment analysis predicted the potential key signaling pathways involved in the treatment of AD with KXS. The binding affinities between key ingredients and targets were further verified using molecular docking. Finally, the predicted key signaling pathway was validated experimentally. Positioning navigation and space search experiments were conducted to evaluate the cognitive improvement effect of KXS on AD rats. Western blot was used to further examine and investigate the expression of the key target proteins related to the predicted pathway. RESULTS: In total, 38 active compounds and 469 corresponding targets of KXS were screened, and 264 target proteins associated with AD were identified. The compound-target-disease and PPI networks identified key active ingredients and protein targets. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis suggested a potential effect of KXS in the treatment of AD via the amyloid beta (A β)-glycogen synthase kinase-3 beta (GSK3 β)-Tau pathway. Molecular docking revealed a high binding affinity between the key ingredients and targets. In vivo, KXS treatment significantly improved cognitive deficits in AD rats induced by Aβ_1-42, decreased the levels of Aβ, p-GSK3β, p-Tau and cyclin-dependent kinase 5, and increased the expressions of protein phosphatase 1 alpha (PP1A) and PP2A (P<0.05 or P<0.01). CONCLUSION KXS exerted neuroprotective effects by regulating the Aβ -GSK3β-Tau signaling pathway, which provides novel insights into the therapeutic mechanism of KXS and a feasible pharmacological strategy for the treatment of AD.
Rats ; Animals ; Alzheimer Disease/drug therapy* ; Amyloid beta-Peptides ; Glycogen Synthase Kinase 3 beta ; Network Pharmacology ; Molecular Docking Simulation ; Glycogen Synthase Kinase 3/therapeutic use* ; Drugs, Chinese Herbal/therapeutic use*

Resveratrol (RES), a natural polyphenolic phytochemical, has been suggested as a putative anti-aging molecule for the prevention and treatment of Alzheimer's disease (AD) by the activation of sirtuin 1 (Sirt1/Sir2). In this study, we tested the effects of RES and Sirt1/Sir2 on sleep and courtship memory in a Drosophila model by overexpression of amyloid precursor protein (APP), whose duplications and mutations cause familial AD. We found a mild but significant transcriptional increase of Drosophila Sir2 (dSir2) by RES supplementation for up to 17 days in APP flies, but not for 7 days. RES and dSir2 almost completely reversed the sleep and memory deficits in APP flies. We further demonstrated that dSir2 acts as a sleep promotor in Drosophila neurons. Interestingly, RES increased sleep in the absence of dSir2 in dSir2-null mutants, and RES further enhanced sleep when dSir2 was either overexpressed or knocked down in APP flies. Finally, we showed that Aβ aggregates in APP flies were reduced by RES and dSir2, probably via inhibiting Drosophila β-secretase (dBACE). Our data suggest that RES rescues the APP-induced behavioral deficits and Aβ burden largely, but not exclusively, via dSir2.
Animals ; Alzheimer Disease/metabolism* ; Amyloid beta-Peptides ; Amyloid beta-Protein Precursor/metabolism* ; Drosophila/physiology* ; Drosophila Proteins/metabolism* ; Resveratrol/pharmacology* ; Sirtuin 1 ; Sleep

Candida albicans is one of the major causes of invasive fungal infections and a serious opportunistic pathogen in immunocompromised individuals. The antimicrobial peptide AMP-17 has prominent anti-Candida activity, and proteomic analysis revealed significant differences in the expression of cell wall (XOG1) and oxidative stress (SRR1) genes upon the action of AMP-17 on C. albicans, suggesting that AMP-17 may exert anti-C. albicans effects by affecting the expression of XOG1 and SRR1 genes. To further investigate whether XOG1 and SRR1 genes were the targets of AMP-17, C. albicans xog1Δ/Δ and srr1Δ/Δ mutants were constructed using the clustered regulatory interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) system. Phenotypic observations revealed that deletion of two genes had no significant effect on C. albicans growth and biofilm formation, whereas XOG1 gene deletion affected in vitro stress response and mycelium formation of C. albicans. Drug sensitivity assay showed that the MIC₈₀ values of AMP-17 against xog1Δ/Δ and srr1Δ/Δ mutants increased from 8 μg/mL (for the wild type C. albicans SC5314) to 16 μg/mL, while the MIC₈₀ values against srr1Δ/Δ: : srr1 revertants decreased to the level of the wild type SC5314. In addition, the ability of AMP-17 to inhibit biofilm formation of both deletion strains was significantly reduced compared to that of wild type SC5314, indicating that the susceptibility of the deletion mutants to AMP-17 was reduced in both the yeast state and during biofilm formation. These results suggest that XOG1 and SRR1 genes are likely two of the potential targets for AMP-17 to exert anti-C. albicans effects, which may facilitate further exploration of the antibacterial mechanism of novel peptide antifungal drugs.
Humans ; Candida albicans ; Antimicrobial Peptides ; Proteomics ; Peptides/pharmacology* ; Transcription Factors/metabolism* ; Antifungal Agents/pharmacology*

Objective: To fabricate TiO₂ nanotube material functionalized by antimicrobial peptide LL-37, and to explore its effects on biological behaviors such as adhesion and migration of human keratinocytes (HaCaT) and its antibacterial properties. Methods: The TiO₂ nanotube array (NT) was constructed on the surface of polished titanium (PT) by anodization, and the antimicrobial peptide LL-37 was loaded on the surface of TiO₂ nanotube (LL-37/NT) by physical adsorption. Three samples were selected by simple random sampling in each group. Surface morphology, roughness, hydrophilicity and release characteristics of LL-37 of the samples were analyzed with a field emission scanning electron microscope, an atomic force microscope, a contact angle measuring device and a microplate absorbance reader. HaCaT cells were respectively cultured on the surface of three groups of titanium samples. Each group had 3 replicates. The morphology of cell was observed by field emission scanning electron microscope. The number of cell adhesion was observed by cellular immunofluorescence staining. Cell counting kit-8 (CCK-8) assay was used to detect cell proliferation. Wound scratch assay was used to observe the migration of HaCaT. The above experiments were used to evaluate the effect of each group on the biological behavior of HaCaT cells. To evaluate their antibacterial effects, Porphyromonas gingivalis (Pg) was respectively inoculated on the surface of three groups of titanium samples. Each group had 3 replicates. The morphology of bacteria was observed by field emission scanning electron microscope. Bacterial viability was determined by live/dead bacterial staining. Results: A uniform array of nanotubes could be seen on the surface of titanium samples in LL-37/NT group, and the top of the tube was covered with granular LL-37. Compared with PT group [the roughness was (2.30±0.18) nm, the contact angle was 71.8°±1.7°], the roughness [(20.40±3.10) and (19.10±4.11) nm] and hydrophilicity (the contact angles were 22.4°±3.1° and 25.3°±2.2°, respectively) of titanium samples increased in NT and LL-37/NT group (P<0.001). The results of in vitro release test showed that the release of antimicrobial peptide LL-37 was characterized by early sudden release (1-4 h) and long-term (1-7 d) slow release. With the immunofluorescence, more cell attachment was found on NT and LL-37/NT than that on PT at the first 0.5 and 2.0 h of culture (P<0.05). The results of CCK-8 showed that there was no significant difference in the proliferation of cells among groups at 1, 3 and 5 days after culture. Wound scratch assay showed that compared with PT and NT group, the cell moved fastest on the surface of titanium samples in LL-37/NT group at 24 h of culture [(96.4±4.9)%] (F=35.55, P<0.001). A monolayer cells could be formed and filled with the scratch in 24 h at LL-37/NT group. The results of bacterial test in vitro showed that compared with the PT group, the bacterial morphology in the NT and LL-37/NT groups was significantly wrinkled, and obvious bacterial rupture could be seen on the surface of titanium samples in LL-37/NT group. The results of bacteria staining showed that the green fluorescence intensity of titanium samples in LL-37/NT group was the lowest in all groups (F=66.54,P<0.001). Conclusions: LL-37/NT is beneficial to the adhesion and migration of HaCaT cells and has excellent antibacterial properties, this provides a new strategy for the optimal design of implant neck materials.
Humans ; Titanium/chemistry* ; Antimicrobial Peptides ; Cathelicidins ; Sincalide ; Anti-Bacterial Agents/pharmacology* ; Nanotubes/chemistry* ; Dental Materials ; Bacteria ; Keratinocytes ; Surface Properties

Evolution and natural selection have endowed animal venoms, including scorpion venoms, with a wide range of pharmacological properties. Consequently, scorpions, their venoms, and/or their body parts have been used since time immemorial in traditional medicines, especially in Africa and Asia. With respect to their pharmacological potential, bioactive peptides from scorpion venoms have become an important source of scientific research. With the rapid increase in the characterization of various components from scorpion venoms, a large number of peptides are identified with an aim of combating a myriad of emerging global health problems. Moreover, some scorpion venom-derived peptides have been established as potential scaffolds helpful for drug development. In this review, we summarize the promising scorpion venoms-derived peptides as drug candidates. Accordingly, we highlight the data and knowledge needed for continuous characterization and development of additional natural peptides from scorpion venoms, as potential drugs that can treat related diseases.
Animals ; Scorpion Venoms/pharmacology* ; Peptides/pharmacology* ; Scorpions ; Drug Development ; Medicine, Traditional

Spinal cord stimulation (SCS)-induced analgesia was characterized, and its underlying mechanisms were examined in a spared nerve injury model of neuropathic pain in rats. The analgesic effect of SCS with moderate mechanical hypersensitivity was increased with increasing stimulation intensity between the 20% and 80% motor thresholds. Various frequencies (2, 15, 50, 100, 10000 Hz, and 2/100 Hz dense-dispersed) of SCS were similarly effective. SCS-induced analgesia was maintained without tolerance within 24 h of continuous stimulation. SCS at 2 Hz significantly increased methionine enkephalin content in the cerebrospinal fluid. The analgesic effect of 2 Hz was abolished by μ or κ opioid receptor antagonist. The effect of 100 Hz was prevented by a κ antagonist, and that of 10 kHz was blocked by any of the μ, δ, or κ receptor antagonists, suggesting that the analgesic effect of SCS at different frequencies is mediated by different endorphins and opioid receptors.
Analgesics ; Animals ; Narcotic Antagonists/pharmacology* ; Neuralgia/therapy* ; Opioid Peptides ; Rats ; Receptors, Opioid/physiology* ; Receptors, Opioid, kappa ; Spinal Cord ; Spinal Cord Stimulation

OBJECTIVE: To investigate the effect of moxibustion on autophagy and amyloid β-peptide_1-42 (Aβ_1-42) protein expression in amyloid precursor protein/presenilin 1 (APP/PS1) double-transgenic mice with Alzheimer's disease (AD). METHODS: After 2-month adaptive feeding, fifty-six 6-month-old APP/PS1 double transgenic AD mice were randomly divided into a model group, a moxibustion group, a rapamycin group and an inhibitor group, 14 mice in each group. Another 14 C57BL/6J mice with the same age were used as a normal group. The mice in the moxibustion group were treated with monkshood cake-separated moxibustion at "Baihui"(GV 20), "Fengfu" (GV 16) and "Dazhui" (GV 14) for 20 min; the mice in the rapamycin group were intraperitoneally injected with rapamycin (2 mg/kg); the mice in the inhibitor group were treated with moxibustion and injection of 1.5 mg/kg 3-methyladenine (3-MA). All the treatments were given once a day for consecutive 2 weeks. The morphology of hippocampal tissue was observed by HE staining; the ultrastructure of hippocampal tissue was observed by transmission electron microscopy; the expression of Aβ_1-42 protein in frontal cortex and hippocampal tissue was detected by immunohistochemistry; the expressions of mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), p70 ribosomal protein S6 kinase (p70S6K) and phosphorylated p70S6K (p-p70S6K) protein in hippocampus were detected by Western blot method. RESULTS: Compared with the normal group, the number of neuron cells was decreased, cells were necrotic and deformed, and autophagy vesicle and lysosome were decreased in the model group. Compared with the model group, the number of neuron cells was increased, cell necrosis was decreased, and autophagy vesicle and lysosome were increased in the moxibustion group and the rapamycin group. Compared with the normal group, the protein expressions of Aβ_1-42, mTOR, p-mTOR, p70S6K and p-p70S6K in the model group were increased (P<0.05); compared with the model group, the protein expressions of Aβ_1-42, mTOR, p-mTOR, p70S6K and p-p70S6K in the moxibustion group, rapamycin group and inhibitor group were decreased (P<0.05); compared with the inhibitor group, the protein expressions of Aβ_1-42, mTOR, p-mTOR, p70S6K and p-p70S6K in the moxibustion group and rapamycin group were decreased (P<0.05); compared with the rapamycin group, the protein expressions of mTOR, p-mTOR, p70S6K and p-p70S6K in the moxibustion group were decreased (P<0.05). CONCLUSION Moxibustion could enhance autophagy in hippocampal tissue of APP/PS1 double transgenic AD mice and reduce abnormal Aβ aggregation in brain tissue, the mechanism may be related to the inhibition of mTOR/p70S6K signaling pathway.
Alzheimer Disease/therapy* ; Amyloid beta-Peptides/genetics* ; Animals ; Autophagy ; Disease Models, Animal ; Hippocampus/metabolism* ; Mammals/metabolism* ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Moxibustion ; Ribosomal Protein S6 Kinases, 70-kDa/pharmacology* ; Signal Transduction ; Sirolimus/pharmacology* ; TOR Serine-Threonine Kinases/metabolism*

Natural antimicrobial peptides have strong bactericidal activities. An obstacle of the development of antimicrobial peptides resides in the difficulty of developing peptides with high biocompatibility. In this study, molecular dynamics analysis was employed to assess the structural characteristics and biological activities of peptides. A (RXKY)₂(YRY)₂ structure was used as a template to design an antimicrobial peptide RIKL of high-efficiency and low-toxicity, where X represents Ile and Y represents Leu. The secondary structure of the antimicrobial peptide was detected by circular dichroism (CD), and the structures of RIKL in water and in POPC/POPG membrane environment were measured using molecular dynamics. The biological activity of RIKL was further studied by assessing its antimicrobial activity, hemolytic activity, eukaryotic cytotoxicity, and salt ion stability. CD results showed that RIKL presented an α-helical structure in a simulated bacterial membrane environment. Molecular dynamics simulation predicted that the secondary structure of RIKL could be partly retained in water and POPG environment, while this secondary structure was weakened in the POPC environment. Antimicrobial test suggested that RIKL had high antimicrobial activities, and the geometric mean of the Minimum Inhibitory Concentration (MIC) was 3.1 μmol/L. The hemolysis indicated that RIKL had no hemolytic activity within the detection range, and cytotoxicity test suggested the cytotoxicity of RIKL was low. Stability test showed that RIKL maintained antimicrobial activities under different pH, serum concentrations and salt environments. Based on the above results, RIKL has high cell selectivity and has the potential as a highly effective antibacterial drug.
Amino Acid Sequence ; Antimicrobial Peptides/pharmacology* ; Microbial Sensitivity Tests ; Protein Structure, Secondary