Safety Assessment of Plant-Derived Proteins and Peptides as Used in Cosmetics.

The Expert Panel for Cosmetic Ingredient Safety (Panel) reviewed the safety of 19 plant-derived proteins and peptides, which function mainly as skin and/or hair conditioning agents in personal care products. The Panel concluded that 18 plant-derived proteins and peptides are safe as used in the present practices of use and concentration as described in this safety assessment, while the data on Hydrolyzed Maple Sycamore Protein are insufficient to determine safety.

BmK86-P1, a New Degradation Peptide with Desirable Thermostability and Kv1.2 Channel-Specific Activity from Traditional Chinese Scorpion Medicinal Material.

Thermally processed Buthus martensii Karsch scorpions are a traditional Chinese medical material for treating various diseases. However, their pharmacological foundation remains unclear. Here, a new degraded peptide of scorpion toxin was identified in Chinese scorpion medicinal material by proteomics. It was named BmK86-P1 and has six conserved cysteine residues. Homology modeling and circular dichroism spectra experiments revealed that BmK86-P1 not only contained representative disulfide bond-stabilized α-helical and ß-sheet motifs but also showed remarkable stability at test temperatures from 20-95 °C. Electrophysiology experiments indicated that BmK86-P1 was a highly potent and selective inhibitor of the hKv1.2 channel with IC50 values of 28.5 ± 6.3 nM. Structural and functional dissection revealed that two residues of BmK86-P1 (i.e., Lys19 and Ile21) were the key residues that interacted with the hKv1.2 channel. In addition, channel chimeras and mutagenesis experiments revealed that three amino acids (i.e., Gln357, Val381 and Thr383) of the hKv1.2 channel were responsible for BmK86-P1 selectivity. This research uncovered a new bioactive peptide from traditional Chinese scorpion medicinal material that has desirable thermostability and Kv1.2 channel-specific activity, which strongly suggests that thermally processed scorpions are novel peptide resources for new drug discovery for the Kv1.2 channel-related ataxia and epilepsy diseases.

Oral Administration of Resveratrol-Selenium-Peptide Nanocomposites Alleviates Alzheimer's Disease-like Pathogenesis by Inhibiting Aß Aggregation and Regulating Gut Microbiota.

Alzheimer's disease (AD) is a neurodegenerative disease associated with amyloid-ß (Aß) deposition, leading to neurotoxicity (oxidative stress and neuroinflammation) and gut microbiota imbalance. Resveratrol (Res) has neuroprotective properties, but its bioavailability in vivo is very low. Herein, we developed a small Res-selenium-peptide nanocomposite to enable the application of Res for eliminating Aß aggregate-induced neurotoxicity and mitigating gut microbiota disorder in aluminum chloride (AlCl3) and d-galactose(d-gal)-induced AD model mice. Res functional selenium nanoparticles (Res@SeNPs) (8 ± 0.34 nm) were prepared first, after which the surface of Res@SeNPs was decorated with a blood-brain barrier transport peptide (TGN peptide) to generate Res-selenium-peptide nanocomposites (TGN-Res@SeNPs) (14 ± 0.12 nm). Oral administration of TGN-Res@SeNPs improves cognitive disorder through (1) interacting with Aß and decreasing Aß aggregation, effectively inhibiting Aß deposition in the hippocampus; (2) decreasing Aß-induced reactive oxygen species (ROS) and increasing activity of antioxidation enzymes in PC12 cells and in vivo; (3) down-regulating Aß-induced neuroinflammation via the nuclear factor kappa B/mitogen-activated protein kinase/Akt signal pathway in BV-2 cells and in vivo; and (4) alleviating gut microbiota disorder, particularly with respect to oxidative stress and inflammatory-related bacteria such as Alistipes, Helicobacter, Rikenella, Desulfovibrio, and Faecalibaculum. Thus, we anticipate that Res-selenium-peptide nanocomposites will offer a new potential strategy for the treatment of AD.

Transcriptome analysis reveals the hepatoprotective mechanism of soybean meal peptides against alcohol-induced acute liver injury mice.

This study aimed was to explore the hepatoprotective potential of soybean meal peptides (SPs) against alcohol-induced liver injury and investigate the underlying mechanisms through transcriptome analysis. The chemical antioxidant analysis of SPs exhibited potent ABTS radical scavenging capacity (11.94 ± 0.41 mg TE/100 mg peptide), ferric reducing antioxidant power (6.42 ± 0.32 mmol Fe2+/100 mg peptide), and oxygen radical absorption capacity (14.78 ± 0.01 mg TE/100 mg peptide). Moreover, SPs increased cell viability and reduced intracellular reactive oxygen species levels in Caco-2 cells by H2O2-induced, and without cytotoxicity. In the mice model, preintervention with SPs reduced the levels of aspartate transaminase/alanine transaminase, total cholesterol, triglyceride and malondialdehyde by alcohol-induced, meanwhile, increased the levels of total superoxide dismutase, glutathione and catalase by alcohol-induced. Histological analysis showed that SPs alleviated the liver injury by alcohol-induced and no toxic effects on the kidneys. According to transcriptome analysis, 1737 genes were significantly differentially expressed (1076 up-regulated and 661 down-regulated) after SPs pretreatment. The main functions of these genes were related to inflammation, lipid metabolism and oxidation. The findings from the present study suggested that SPs produced positive hepatoprotection and showed potential to be used as a dietary supplement or an ingredient of functional food.

A novel peptide identified from skin secretions of Bombina maxima possesses LPS-neutralizing activity.

Lipopolysaccharide (LPS) is a major pathogenic factor in endotoxin shock or sepsis. Most antibiotics have little clinical anti-endotoxin activity, but some antimicrobial peptides (AMPs) have been shown to be effective in blocking LPS. We identified a novel peptide from the skin secretions of Bombina maxima (B. _maxima) by challenging the skin of frogs with an LPS solution. Peptide 2 has an amino acid sequence of LVGKLLKGAVGDVCGLLPIC. Peptide 2 possesses low hemolytic activity, low cytotoxicity against RAW 264.7 cells, and strong anti-inflammatory activity. Moreover, peptide 2 plays an anti-inflammatory role by inhibiting inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). A biolayer interferometry (BLI) assay indicated that peptide 2 binds to LPS with strong affinity and that this interaction has an affinity constant (KD) value of 1.05 × 10-9 M. A survival study showed that peptide 2 possesses potent LPS-neutralizing activity to protect LPS-treated mice from death. In conclusion, we have identified a potent peptide with LPS neutralizing activity, which lays a foundation for future research and development.

Anti-Acne Action of Peptides Isolated from Burdock Root-Preliminary Studies and Pilot Testing.

This work aimed to study the anti-bacterial, anti-biofilm and anti-oxidant potential effects of low molecular weight (LMW) peptides (Br-p) isolated from burdock (Arctium lappa L.) roots. We conducted a preliminary study to exclude or confirm the antibiotic activity of the LMW peptides fraction of this plant. Br-p were isolated using gel filtration and a 10 kDa cut-off membrane. The obtained peptides were identified by MALDI TOF/TOF. Antibacterial activity was tested against acne strains using diffusion tests, MIC and MBC. The fibroblast cytotoxicity of Br-p was tested, and the selectivity index (SI) value was determined. The fraction of 46 Br-p peptides isolated from burdock root with a molecular weight below 5000 Da and theoretic pI (isoelectric point) of 3.67-11.83 showed a narrow spectrum of activity against Gram-positive acne bacterial strains. One of the Br-p peptides assessed on MALDI RapidDeNovo was LRCDYGRFFASKSLYDPLKKRR cationic peptide. It was analogous to that contained in A. lappa protein, and theoretically it was matched as a peptide with antibiotic nature. Br-p did not show toxicity to fibroblasts in the tested concentration up to 10 mg/mL, obtaining CC50 10 mg/mL. The SI value for the tested Propionibacterium strains ranged from 160 to 320. Finally, an active dressing based on chitosan/alginate/genipin was prepared using freeze-drying. The formed dressing was evaluated for its anti-acne activity. To sum up: preliminary biological studies confirmed the anti-acne properties of the isolated peptide fraction from burdock root and pointed to the possibility of using it to create an active dressing on the skin.

LVFFARK-PEG-Stabilized Black Phosphorus Nanosheets Potently Inhibit Amyloid-ß Fibrillogenesis.

Deposition of amyloid-ß (Aß) aggregates in the brain is a main pathological hallmark of Alzheimer's disease (AD), so inhibition of Aß aggregation has been considered as a promising strategy for AD prevention and treatment. Black phosphorus (BP) is a 2D nanomaterial with high biocompatibility and unique biodegradability, but its potential application in biomedicine suffers from the rapid degradability and unfunctionability. To overcome the drawbacks and broaden its application, we have herein designed an Aß inhibitor (LK7)-coupled and polyethylene glycol (PEG)-stabilized BP-based nanosystem. The PEGylated-LK7-BP nanosheets (PEG-LK7@BP) not only exhibited a good stability but also demonstrated a significantly enhanced inhibitory potency on Aß42 fibrillogenesis in comparison with its counterparts. This elaborately designed PEG-LK7@BP stopped the conformational transition and suppressed the fibrillization of Aß42, so it could completely rescue cultured cells from the toxicity of Aß42 (by increasing the cell viability from 72 to 100%) at 100 µg/mL. It is considered that PEG-LK7@BP could bind Aß species by enhanced electrostatic and hydrophobic interactions and thus efficiently alleviated Aß-Aß interactions. Meanwhile, the coupled LK7 on the BP surface formed a high local concentration that enhanced the affinity between the nanosystem and Aß species. Finally, PEG could improve the stability and dispersibility of the nanoplatform to make it show an increased inhibitory effect on the amyloid formation. Hence, this work proved that PEG-LK7@BP is a promising nanosystem for the development of amyloid inhibitors fighting against AD.

Evaluation of peptide-rich root extracts of Calliandria portoriscensis (Jacq.) Benth (Mimosaceae) for in vitro antimicrobial activity and brine shrimp lethality.

BACKGROUND: Several Host defence peptides (HDPs) are low molecular weight (< 50 amino acids residues) peptides detected in several ethnomedicinal plants and have particularly gained research interest in recent times. Due to their wide range of bioactivity, occurrence, abundance and ability to induce very little resistance, they hold promising potentials in drug development. This study investigated the presence of bioactive peptides in the roots of Calliandra portoricensis (CPr) (Mimosaceae) and evaluated its antimicrobial activity against gram-negative and gram-positive bacteria. METHODS: The crude peptide extract was obtained and pre-purified on pre-loaded tube of RP-C18 solid phase cartridges (strata giga tube C18-E; 5 g, 20 mL, Phenomenex, Germany). Peptide enriched fraction was chemically analysed for arginine-rich/aromatic amino acid-rich peptides using a modified G-250 analytical stain and ninhydrin on thin layer chromatography (TLC) for a preliminary screening. Furthermore, MALDI TOF/TOF peptidomics was used to detect the presence and masses of the peptides. Extracts from CPr were used to test the ability to inhibit microbial growth using p-INT (Para-iodonitrotetrazolium violet) dye, with 0.1% gentamycin as positive control. The concentration that inhibits the growth of microorganisms by 50% (IC50) were determined. Toxicity of the two extracts was accessed using freshly hatched nauplii of Artemia salina. Data analysis were evaluated using Microsoft excel and GraphPad Prism5. RESULTS: Low molecular weight (LMW) peptides were detected in CPr using TLC and MALDI-TOF MS. Generally, the extracts exhibited good inhibition (70-95%) against the gram-negative and gram-positive bacteria, except MRSA6 typed strain. Enhanced activity was observed in the pre-purified peptide fraction than in the methanol crude, except on MRSA6. The greatest antimicrobial inhibition by pre-purified peptide fraction was against MRSA22 (IC50 = 0.69 ± 0.33 µg/mL). The crude methanol extract (LC50 = 5.13 µg/mL) was slightly more toxic than the peptide extract (LC50 = 6.12 µg/mL). CONCLUSIONS: This is the first report on detection of bioactive LMW peptides in Mimosaceae family. These peptides appear to be rich in arginine and aromatic amino acids. The peptide extract, in its pre-purified form showed a lower Brine shrimp cytotoxicity and an enhanced antimicrobial activity against the tested gram-negative and gram-positive bacteria.

The Traditional Formula Kai-Xin-San Alleviates Polyglutamine-Mediated Neurotoxicity by Modulating Proteostasis Network in Caenorhabditis elegans.

The inherited polyglutamine (polyQ) expansion diseases are characterized by progressive accumulation of aggregation-prone polyQ proteins, which may provoke proteostasis imbalance and result in significant neurotoxicity. Using polyQ transgenic Caenorhabditis elegans models, we find that Kai-Xin-San (KXS), a well-known herbal formula traditionally used to treat mental disorders in China, can alleviate polyQ-mediated neuronal death and associated chemosensory deficiency. Intriguingly, KXS does not reduce polyQ aggregation in vitro as demonstrated by Thioflavin-T test, but does inhibit polyQ aggregation in C. elegans models, indicating an indirect aggregation-inhibitory mechanism. Further investigation reveals that KXS can modulate two key arms of the protein quality control system, that is, heat shock response and autophagy, to clear polyQ aggregates, but has little effect on proteasome activity. In addition, KXS is able to reduce oxidative stress, which is involved in proteostasis and neurodegeneration, but has no effect on life span or dietary restriction response. To examine potential interaction of the four component herbs of KXS, a dissection strategy was used to study the effects of differential herbal combinations in C. elegans polyQ models. While the four herbs do contribute additively to KXS function, Panax ginseng is found to be the most effective constituent. Taken together, these findings not only demonstrate the neuroprotective ability of KXS but also suggest its potential as a proteostasis regulator in protein aggregation disorders and provide an insight into the mechanism studies of traditionally used complex prescriptions and their rationality.

Cardiovascular and respiratory safety evaluation of Musca Domestica larvae low molecular weight peptide in beagle dogs.

Background: Many studies have demonstrated that the water extracts and low-molecular-weight peptide (LMWP) of the Musca domestica larvae contain significant biological activity. However, the cardiovascular and respiratory safety evaluations of LMWP are yet to be sufficiently investigated. Aim: This study focused on the cardiovascular and respiratory safety evaluations of the M. domestica larvae LMWP in beagle dogs. Methods: Direct cardiovascular and respiratory effects of three different doses of the M. domestica larvae LMWP were investigated following only once oral administration in conscious telemetered dogs, whereby ECG, arterial pressure, and respiratory data were collected using the Data Science International telemetric system. Results: The PR, QT, and QTcf intervals were significantly shortened in the medium-dose LMWP treatment group at 3 h after drug administration. Furthermore, no significant differences were observed in any of the corresponding indexes of other treatment groups at different time points compared to those of the control group. P wave, ST segment, R wave, systolic pressure, diastolic pressure, and mean pressure were significantly different, although these differences had no significant dose-effect relationship. Respiratory frequency significantly increased in the medium-dose LMWP treatment group at 8 h after drug administration compared to that of the control group. Respiratory rate and tidal volume showed no significant differences at varying time points among all LMWP treatment groups. Conclusions: No toxicological effects related to cardiovascular and respiratory safety in beagle dogs were observed at any dose level of the M. domestica larvae LMWP.

Comparison of neuroprotective efficacy of poly-arginine R18 and R18D (D-enantiomer) peptides following permanent middle cerebral artery occlusion in the Wistar rat and in vitro toxicity studies.

We have previously demonstrated that arginine-rich and poly-arginine peptides possess potent neuroprotective properties, with poly-arginine peptide R18 identified as being highly effective at reducing infarct volume following middle cerebral artery occlusion (MCAO) in the Sprague Dawley rat. Since peptides synthesised using D-isoform amino acids have greater stability than L-isoform peptides due to increased resistance to proteolytic degradation, they represent potentially more effective peptide therapeutics. Therefore we compared the neuroprotective efficacy of R18 and its D-enantiomer R18D following permanent MCAO in the Wistar rat. Furthermore, as increased peptide stability may also increase peptide toxicity, we examined the effects of R18 and R18D on cultured cortical neurons, astrocytes, brain endothelial cells (bEND.3), and embryonic kidney cells (HEK293) following a 10-minute or 24-hour peptide exposure duration. The in vivo studies demonstrated that R18D resulted in a greater reduction in mean infarct volume compared to R18 (33%, p = 0.004 vs 12%, p = 0.27) after intravenous administration at 300 nmol/kg 30 minutes after MCAO. Both R18D and R18 reduced cerebral hemisphere swelling to a comparable degree (27%, p = 0.03 and 30%, p = 0.02), and improved neurological assessment scores (1.5, p = 0.02 and 2, p = 0.058 vs 3 for vehicle). No abnormal histological findings specific to peptide treatments were observed in hematoxylin and eosin stained sections of kidney, liver, spleen, lung and heart. In vitro studies demonstrated that R18 and R18D were most toxic to neurons, followed by astrocytes, HEK293 and bEND.3 cells, but only at high concentrations and/or following 24-hour exposure. These findings further highlight the neuroprotective properties of poly-arginine peptides, and indicate that R18D at the dose examined is more potent than R18 in Wistar rats, and justify continued investigation of the R18 peptide as a novel neuroprotective agent for stroke.

Coffee extract and caffeine enhance the heat shock response and promote proteostasis in an HSF-1-dependent manner in Caenorhabditis elegans.

As the population ages, there is a critical need to uncover strategies to combat diseases of aging. Studies in the soil-dwelling nematode Caenorhabditis elegans have demonstrated the protective effects of coffee extract and caffeine in promoting the induction of conserved longevity pathways including the insulin-like signaling pathway and the oxidative stress response. We were interested in determining the effects of coffee and caffeine treatment on the regulation of the heat shock response. The heat shock response is a highly conserved cellular response that functions as a cytoprotective mechanism during stress, mediated by the heat shock transcription factor HSF-1. In the worm, HSF-1 not only promotes protection against stress but is also essential for development and longevity. Induction of the heat shock response has been suggested to be beneficial for diseases of protein conformation by preventing protein misfolding and aggregation, and as such has been proposed as a therapeutic target for age-associated neurodegenerative disorders. In this study, we demonstrate that coffee is a potent, dose-dependent, inducer of the heat shock response. Treatment with a moderate dose of pure caffeine was also able to induce the heat shock response, indicating caffeine as an important component within coffee for producing this response. The effects that we observe with both coffee and pure caffeine on the heat shock response are both dependent on HSF-1. In a C. elegans Huntington's disease model, worms treated with caffeine were protected from polyglutamine aggregates and toxicity, an effect that was also HSF-1-dependent. In conclusion, these results demonstrate caffeinated coffee, and pure caffeine, as protective substances that promote proteostasis through induction of the heat shock response.

Shear-sensitive nanocapsule drug release for site-specific inhibition of occlusive thrombus formation.

Essentials Vessel stenosis due to large thrombus formation increases local shear 1-2 orders of magnitude. High shear at stenotic sites was exploited to trigger eptifibatide release from nanocapsules. Local delivery of eptifibatide prevented vessel occlusion without increased tail bleeding times. Local nanocapsule delivery of eptifibatide may be safer than systemic antiplatelet therapies. SUMMARY: Background Myocardial infarction and stroke remain the leading causes of mortality and morbidity. The major limitation of current antiplatelet therapy is that the effective concentrations are limited because of bleeding complications. Targeted delivery of antiplatelet drug to sites of thrombosis would overcome these limitations. Objectives Here, we have exploited a key biomechanical feature specific to thrombosis, i.e. significantly increased blood shear stress resulting from a reduction in the lumen of the vessel, to achieve site-directed delivery of the clinically used antiplatelet agent eptifibatide by using shear-sensitive phosphatidylcholine (PC)-based nanocapsules. Methods PC-based nanocapsules (2.8 × 1012 ) with high-dose encapsulated eptifibatide were introduced into microfluidic blood perfusion assays and into in vivo models of thrombosis and tail bleeding. Results Shear-triggered nanocapsule delivery of eptifibatide inhibited in vitro thrombus formation selectively under stenotic and high shear flow conditions above a shear rate of 1000 s-1 while leaving thrombus formation under physiologic shear rates unaffected. Thrombosis was effectively prevented in in vivo models of vessel wall damage. Importantly, mice infused with shear-sensitive antiplatelet nanocapsules did not show prolonged bleeding times. Conclusions Targeted delivery of eptifibatide by shear-sensitive nanocapsules offers site-specific antiplatelet potential, and may form a basis for developing more potent and safer antiplatelet drugs.

Antioxidant and neuroprotective effects of Dictyophora indusiata polysaccharide in Caenorhabditis elegans.

ETHNOPHARMACOLOGICAL RELEVANCE: Dictyophora indusiata is a medicinal mushroom traditionally used in China for a variety of conditions, including inflammatory and neural diseases. D. indusiata polysaccharides (DiPS) are shown to have in vitro antioxidant activity but in vivo evidence is lacking. This study aimes to explore the antioxidant capacity and related neuroptotective activities of DiPS using wild-type and neurodegenerative Caenorhabditis elegans models. MATERIALS AND METHODS: The antioxidant capacities of DiPS were first determined using paraquat survival and Pgst-4::GFP expression assays in wild-type and transgenic C. elegans models, respectively, and then further investigated by determining reactive oxygen species (ROS) level, malondialdehyde (MDA) content and superoxide dismutase (SOD) activity as well as functional parameters of mitochondria. The activation of stress response transcription factors and neuroptotective activities were examined using nuclear localization and chemosensory behavioral assays in transgenic nematodes, respectively. RESULTS: DiPS was shown not only to increase survival rate and reduce stress level under paraquat-induced oxidative conditions but also to decrease ROS and MDA levels and increase SOD activity in C. elegans models. Moreover, DiPS was also able to restore the functional parameters of mitochondria, including membrane potential and ATP content, in paraquat-stressed nematodes. In addition, nuclear translocation assays demonstrate that the stress response transcription factor DAF-16/FOXO was involved in the antioxidant activity of the polysaccharide. Further experiments reveal that DiPS was capable of reducing ROS levels and alleviating chemosensory behavior dysfunction in transgenic nematode models of neurodegenerative diseases mediated by polyglutamine and amyloid-ß protein. CONCLUSIONS: These findings demonstrate the antioxidant and neuroprotective activities of the D. indusiata polysaccharide DiPS in wild-type and neurodegenerative C. elegans models, and thus provide an important pharmacological basis for the therapeutic potential of D. indusiata in neurodegeneration.

Effective Inhibition of Cellular ROS Production by MXCXXC-Type Peptides: Potential Therapeutic Applications in Copper-Homeostasis Disorders.

Cyclic and acyclic peptides with sequences derived from metallochaperone binding sites, but differing at position 2, were analyzed for their inhibitory reactivity towards cellular ROS (reactive oxygen species) formation and catalytic activity towards oxidation with H2 O2 , in comparison with three commercial drugs clinically employed in chelation therapy for Wilson's disease. Acyclic peptides were more effective inhibitors than the cyclic ones, with one leading peptide with threonine at position 2 systematically showing the highest efficiency in reducing cellular ROS levels and in inhibiting Cu oxidation. This peptide was more effective than all commercial drugs in all aspects analyzed, and showed no toxicity towards human colon HT-29 cancer cells at concentrations 10-100 times higher than the IC50 of the commercial drugs, corroborating its high medicinal potential.

Antifungal Activity and Action Mechanism of Histatin 5-Halocidin Hybrid Peptides against Candida ssp.

The candidacidal activity of histatin 5 is initiated through cell wall binding, followed by translocation and intracellular targeting, while the halocidin peptide exerts its activity by attacking the Candida cell membrane. To improve antimicrobial activities and to understand the killing mechanism of two peptides, six hybrid peptides were designed by conjugating histatin 5 and halocidin. A comparative approach was established to study the activity, salt tolerance, cell wall glucan binding assay, cytotoxicity, generation of ROS and killing kinetics. CD spectrometry was conducted to evaluate secondary structures of these hybrid peptides. Furthermore the cellular localization of hybrid peptides was investigated by confocal fluorescence microscopy. Of the six hybrid congeners, di-PH2, di-WP2 and HHP1 had stronger activities than other hybrid peptides against all tested Candida strains. The MIC values of these peptides were 1-2, 2-4 and 2-4 µg/ml, respectively. Moreover, none of the hybrid peptides was cytotoxic in the hemolytic assay and cell-based cytotoxicity assay. Confocal laser microscopy showed that di-PH2 and HHP1 were translocated into cytoplasm whereas di-WP2 was accumulated on surface of C. albicans to exert their candidacidal activity. All translocated peptides (Hst 5, P113, di-PH2) were capable of generating intracellular ROS except HHP1. Additionally, the KFH residues at C-terminal end of these peptides were assumed for core sequence for active translocation.

Scorpion Venom Heat-Resistant Peptide Attenuates Glial Fibrillary Acidic Protein Expression via c-Jun/AP-1.

Scorpion venom has been used in the Orient to treat central nervous system diseases for many years, and the protein/peptide toxins in Buthus martensii Karsch (BmK) venom are believed to be the effective components. Scorpion venom heat-resistant peptide (SVHRP) is an active component of the scorpion venom extracted from BmK. In a previous study, we found that SVHRP could inhibit the formation of a glial scar, which is characterized by enhanced glial fibrillary acidic protein (GFAP) expression, in the epileptic hippocampus. However, the cellular and molecular mechanisms underlying this process remain to be clarified. The results of the present study indicate that endogenous GFAP expression in primary rat astrocytes was attenuated by SVHRP. We further demonstrate that the suppression of GFAP was primarily mediated by inhibiting both c-Jun expression and its binding with AP-1 DNA binding site and other factors at the GFAP promoter. These results support that SVHRP contributes to reducing GFAP at least in part by decreasing the activity of the transcription factor AP-1. In conclusion, the effects of SVHRP on astrocytes with respect to the c-Jun/AP-1 signaling pathway in vitro provide a practical basis for studying astrocyte activation and inhibition and a scientific basis for further studies of traditional medicine.

Genotoxicity testing of peptides: folate deprivation as a marker of exaggerated pharmacology.

The incidence of micronucleated-cells is considered to be a marker of a genotoxic event and can be caused by direct- or indirect-DNA reactive mechanisms. In particular, small increases in the incidence of micronuclei, which are not associated with toxicity in the target tissue or any structurally altering properties of the compound, trigger the suspicion that an indirect mechanism could be at play. In a bone marrow micronucleus test of a synthetic peptide (a dual agonist of the GLP-1 and GIP receptors) that had been integrated into a regulatory 13-week repeat-dose toxicity study in the rat, small increases in the incidence of micronuclei had been observed, together with pronounced reductions in food intake and body weight gain. Because it is well established that folate plays a crucial role in maintaining genomic integrity and pronounced reductions in food intake and body weight gain were observed, folate levels were determined from plasma samples initially collected for toxicokinetic analytics. A dose-dependent decrease in plasma folate levels was evident after 4 weeks of treatment at the mid and high dose levels, persisted until the end of the treatment duration of 13-weeks and returned to baseline levels during the recovery period of 4 weeks. Based on these properties, and the fact that the compound tested (peptide) per se is not expected to reach the nucleus and cause DNA damage, the rationale is supported that the elevated incidence of micronucleated polychromatic erythrocytes is directly linked to the exaggerated pharmacology of the compound resulting in a decreased folate level.

Activation of STAT3 is involved in neuroprotection by electroacupuncture pretreatment via cannabinoid CB1 receptors in rats.

Pretreatment with electroacupuncture (EA) attenuates cerebral ischemic injury through the endocannabinoid system, although the molecular mechanisms mediate this neuroprotection are unknown. It is well-known that signal transducer and activator of transcription 3 (STAT3) plays an essential role in cell survival and proliferation. Therefore, we investigated whether STAT3 is involved in EA pretreatment-induced neuroprotection via cannabinoid CB1 receptors (CB1R) after transient focal cerebral ischemia in rats. Two hours after EA pretreatment, focal cerebral ischemia was induced by middle cerebral artery occlusion (MACO) for 120 min. The expression of pSTAT3(Ser727), which is necessary for STAT3 activation, was examined in the ipsilateral ischemic penumbra. Infarct volumes and neurological scores were evaluated at 72 h after MACO in the presence or absence of the STAT3 inhibitor peptide (PpYLKTK). Neuronal apoptosis and the Bax/Bcl-2 ratio were also evaluated 24h after reperfusion. Our results showed that EA pretreatment significantly enhanced neuronal expression of pSTAT3(Ser727) in the ischemic penumbra 6h after reperfusion. Moreover, EA pretreatment reduced infarct volume, improved neurological outcome, inhibited neuronal apoptosis and decreased the Bax/Bcl-2 ratio following reperfusion. The beneficial effects of EA were attenuated by PpYLKTK administered 30 min before MACO, and PpYLKTK effectively reversed the increase in pSTAT3(Ser727) expression. Furthermore, CB1R antagonist or CB1R knockdown with siRNA blocked the elevation of pSTAT3(Ser727) expression by EA pretreatment, whereas the two CB1R agonists increased STAT3 activation. In conclusion, EA pretreatment enhances STAT3 activation via CB1R to protect against cerebral ischemia, suggesting that STAT3 activation may be a novel target for stroke intervention.