Cytoprotective Compound Research Articles

Glycyl-l-histidyl-l-lysine prevents copper- and zinc-induced protein aggregation and central nervous system cell death in vitro.

Common features of neurodegenerative diseases are oxidative and inflammatory imbalances as well as the misfolding of proteins. An excess of free metal ions can be pathological and contribute to cell death, but only copper and zinc strongly promote protein aggregation. Herein we demonstrate that the endogenous copper-binding tripeptide glycyl-l-histidyl-l-lysine (GHK) has the ability to bind to and reduce copper redox activity and to prevent copper- and zinc-induced cell death in vitro. In addition, GHK prevents copper- and zinc-induced bovine serum albumin aggregation and reverses aggregation through resolubilizing the protein. We further demonstrate the enhanced toxicity of copper during inflammation and the ability of GHK to attenuate this toxicity. Finally, we investigated the effects of copper on enhancing paraquat toxicity and report a protective effect of GHK. We therefore conclude that GHK has potential as a cytoprotective compound with regard to copper and zinc toxicity, with positive effects on protein solubility and aggregation that warrant further investigation in the treatment of neurodegenerative diseases.

Curcumin coating: a novel solution to mitigate inherent carbon nanotube toxicity

Multi-walled Carbon Nanotubes (MWCNTs) are inert structures with high aspect ratios that are widely used as vehicles for targeted drug delivery in cancer and many other diseases. They are largely non-toxic in nature however, when cells are exposed to these nanotubes for prolonged durations or at high concentrations, they show certain adverse effects. These include cytotoxicity, inflammation, generation of oxidative stress, and genotoxicity among others. To combat such adverse effects, various moieties can be attached to the surface of these nanotubes. Curcumin is a known anti-inflammatory, antioxidant and cytoprotective compound derived from a medicinal plant called Curcuma longa. In this study, we have synthesized and characterized Curcumin coated-lysine functionalized MWCNTs and further evaluated the cytoprotective, anti-inflammatory, antioxidant and antiapoptotic effect of Curcumin coating on the surface of MWCNTs. The results show a significant decrease in the level of inflammatory molecules like IL-6, IL-8, IL-1β, TNFα and NFκB in cells exposed to Curcumin-coated MWCNTs as compared to the uncoated ones at both transcript and protein levels. Further, compared to the uncoated samples, there is a reduction in ROS production and upregulation of antioxidant enzyme-Catalase in the cells treated with Curcumin-coated MWCNTs. Curcumin coating also helped in recovery of mitochondrial membrane potential in the cells exposed to MWCNTs. Lastly, cells exposed to Curcumin-coated MWCNTs showed reduced cell death as compared to the ones exposed to uncoated MWCNTs. Our findings suggest that coating of Curcumin on the surface of MWCNTs reduces its ability to cause inflammation, oxidative stress, and cell death.Graphical (a) Synthesis of Curcumin-coated-Lysine-functionalized MWCNTs. (b) Flow of research depicting experimental groups and studies performed along with the underlying techniques used.

Melatonin as a chronobiotic/cytoprotective agent in bone. Doses involved

AbstractBecause the chronobiotic and cytoprotective molecule melatonin diminishes with age, its involvement in postmenopausal and senescence pathology has been considered since long. One relevant melatonin target site in aging individuals is bone where melatonin chronobiotic effects mediated by MT1 and MT2 receptors are demonstrable. Precursors of bone cells located in bone marrow are exposed to high quantities of melatonin and the possibility arises that melatonin acts a cytoprotective compound via an autacoid effect. Proteins that are incorporated into the bone matrix, like procollagen type I c‐peptide, augment after melatonin exposure. Melatonin augments osteoprotegerin, an osteoblastic protein that inhibits the differentiation of osteoclasts. Osteoclasts are target cells for melatonin as they degrade bone partly by generating free radicals. Osteoclast activity and bone resorption are impaired via the free radical scavenger properties of melatonin. The administration of melatonin in chronobiotic doses (less than 10 mg daily) is commonly used in clinical studies on melatonin effect on bone. However, human equivalent doses allometrically derived from animal studies are in the 1–1.5 mg/kg/day range for a 75 kg human adult, a dose rarely used clinically. In view of the absence of toxicity of melatonin in phase 1 pharmacological studies with doses up to 100 mg in normal volunteers, further investigation is needed to determine whether high melatonin doses have higher therapeutic efficacy in preventing bone loss.

Candida glycerinogenes-Promoted α-Pinene and Squalene Co-production Strategy Based on α-Pinene Stress.

α-Pinene is a naturally occurring monoterpene, which is widely used in fragrances, cosmetics, and foods. Due to the high cellular toxicity of α-pinene, this work considered the application of Candida glycerinogenes, an effective industrial strain with high resistance, in α-pinene synthesis. It was found that α-pinene-induced stress resulted in an intracellular accumulation of reactive oxygen species with an increased formation of squalene as a cytoprotective compound. As squalene is a downstream product in the mevalonate (MVA) pathway for α-pinene synthesis, a strategy based on the promotion of α-pinene and squalene co-production under α-pinene stress is proposed. By introducing the α-pinene synthesis pathway and enhancing the MVA pathway, the production of both α-pinene and squalene is increased. We have demonstrated that intracellular synthesis of α-pinene is effective in promoting squalene synthesis. The generation of intercellular reactive oxygen that accompanies α-pinene synthesis promotes squalene synthesis with a resultant cellular protection and upregulation of MVA pathway genes that facilitate α-pinene production. In addition, we have overexpressed phosphatase and introduced NPP as a substrate to synthesize α-pinene, where co-dependent fermentation yielded 208 mg/L squalene and 12.8 mg/L α-pinene. This work establishes a viable strategy to promote terpene-co-dependent fermentation based on stress.

FC091: Changes in the Gut Microbiome and Systemic Metabolome in an In Vivo Model of Peritoneal Dialysis Supplemented with Alanyl-Glutamine

Abstract BACKGROUND AND AIMS Long-term usage of peritoneal dialysis (PD) leads to morphological and functional changes—such as progressive thickening, vascularization and vasculopathy—of the peritoneum, thereby limiting PD efficiency. Vasculopathy induces changes in permeability of the endothelial barrier, which has been suggested to influence the gut microbiome (gMB). Microbial dysbiosis in turn can potentiate inflammation, a risk factor for cardiovascular disease (CVD). The complex interplay of molecules associated with CKD and PD with the gMB is hypothesized to be in causal relationship with occurrences of cardiovascular events and progression of CVD. Novel PD fluids that slow or prevent these processes are in need. The immunomodulatory and cytoprotective compound alanyl-glutamine (AG) has been shown in other model systems to improve the endothelial barrier function, supposedly protecting the gMB from the extraintestinal environment. Our aim is to identify changes of the gMB and the systemic metabolome caused by the addition of AG to PD under conditions of CKD. METHOD Mice (C57/Bl6 NCrl) underwent a 5/6 nephrectomy to induce uraemia. Via a subcutaneously implanted catheter the animals received five times per week a daily injection of 2 mL PD fluid (3.86% glucose with/without 8 mM AG) for six weeks to a total of 30 injections. During the experiment, mice received standard chow and tap water ad libitum. At the end of the experiment, fecal samples from three different intestinal sites and stool were collected and a serum sample was drawn from each mouse. Tissue samples from parietal and visceral peritoneum and relevant organs were preserved for paraffin and cryo sections. Peritoneal effluent cells were spun down and snap-frozen in liquid nitrogen and the supernatant was stored on −80°C. Bacterial ribosomal RNA (rRNA) was isolated from each fecal sample and the V4 region of the 16S rRNA was amplified. Assessment of the plasma metabolome was performed by targeted mass spectrometry (MxP Quant 500, Biocrates, Austria). The experiment was approved by the institutional animal ethics committee. RESULTS Elevated creatinine levels after subtotal nephrectomy confirmed the uremic status of the mice. Descriptive analysis of the gMB showed decreased Shannon diversity, indicating reduced bacterial diversity, in uremic and PD fluid receiving mice over time, whereas AG receiving and control mice retained a stable Shannon diversity. Furthermore, the gMB of mice exposed to PD fluid with AG showed five bacterial classes and 13 bacterial orders to be differently abundant versus mice exposed to PD fluid without AG. In the colon of uremic mice Clostridia was decreased versus control. Serum concentrations of 630 metabolites were assessed by our metabolomics approach. A significant increase in alanine and glutamine serum concentrations was seen in mice exposed to AG supplemented PD fluid. Further, 17 amino acids and four biogenic amines showed to be differently abundant in mice exposed to AG supplemented PD fluid versus without AG. Lastly, plasma of uremic mice showed a significant increase of the toxic amino acid symmetric dimethyl arginine (SDMA) and citrulline. CONCLUSION Exposure to PD fluid and having chronic kidney disease both affect the gut microbiome and systemic metabolome. Supplementation of AG to PD fluid showed changes at overall microbiome diversity and serum metabolites. Further analysis of these changes in the context of tissue alterations will clarify functional differences of the cytoprotective additive AG. Preservation of the mesothelial and endothelial barrier function may represent a mechanistic explanation for the observed improvement in protein loss and systemic inflammation in a recent phase II clinical trial in PD patients. Better understanding of the gut-peritoneal barrier may open up new therapeutic targets for improving clinical outcome of PD treatment and its long-term efficacy.

To each their own: Disparate abilities of Nrf2 activators to protect against various electrophilic and oxidative insults

Electrophilic and oxidative stress contribute to major diseases such as cancer and diabetes. Small‐molecule activators of the Nrf2 cytoprotective pathway show significant promise for prevention and amelioration of chronic diseases by combating these stressors through upregulation of detoxification and antioxidant enzymes. An understudied area is whether distinct small molecular Nrf2 activators can protect cells from all electrophilic and oxidative stressors, or whether certain molecules are better suited to protect against a particular type of stress.In this study, human keratinocyte HaCaT cells were preincubated for 24 h with one of four Nrf2 activators before different stressors were added for an additional 24 h and cell death was assessed. Three electrophiles were tested: sulforaphane (SFN), the subject of over 70 clinical trials, the multiple sclerosis drug Tecfidera (dimethyl fumarate, DMF), and bardoxolone methyl (BARD), in clinical trials for treatment of chronic kidney diseases and other conditions including COVID‐19. SFN, DMF, and BARD target C151 of the Keap1 protein, a negative regulator of Nrf2. Also tested was KI696, an inhibitor of the Keap1‐Nrf2 interaction. Three chemically distinct stressors—hydrogen peroxide, menadione (a quinone that rapidly generates ROS through one‐electron redox cycling), or chlorambucil (an alkylating chemotherapy agent)—were tested. Stressors were added to the cells at their LC50 concentration after pretreatment with a range of cytoprotective compound concentrations. Unexpectedly, SFN pretreatment showed little to no rescue for all stressors. However, DMF rescued the viability of cells stressed with hydrogen peroxide, menadione, and chlorambucil. BARD showed no rescue for hydrogen peroxide‐treated cells. KI696 improved the cell’s ability to survive chlorambucil. The ability of a given Nrf2 activator (or lack thereof) to protect against a given stressor points to a need to better understand the cytoprotective environment (specific enzymes upregulated, glutathione levels, etc.) that best protect against specific stressors.

Efficacy and Tolerability of a New Formulation in Rectal Ointment Based on Zn-L-Carnosine (Proctilor®) in the Treatment of Haemorrhoidal Disease.

Haemorrhoidal disease (HD) shows high prevalence in western countries, reaching 4.4% per year in the US. Topical preparations are the first-line treatments, which are readily available as “over-the-counter” (OTC) products, often containing a nonstandardised mixture of “natural” remedies, or anaesthetics or cortisol;those latter are not free from undesirable effects. The Zinc-L-Carnosine is a cytoprotective compound, promoting mucosal repair in the gastrointestinal tract and also in mucosal repair, following radiation injuries to the rectum as well as in ulcerative colitis. Our aim was to study the efficacy of Zinc-L-Carnosine in relieving acute symptoms of HD, testing a preparation in the rectal ointment, Proctilor®, in patients complaining of bleeding or thrombosed piles. In a multicentre open trial, 21 patients older than 18 years of age were enrolled. The symptoms of HD were graded according to the Haemorrhoidal Disease Symptoms Score (HDSS) in association with the Short Health Scale (SHS) to assess the influence of HD on quality of life. The pain was assessed with the VAS score, bowel habit by means of the Bristol scale. The patients were evaluated at enrolment (T0) and 2 (T1) and 4 (T2) weeks of treatment with Proctilor® rectal ointment. There were 10 men and 11 women; mean age, 49 years. Pain, bleeding, and thrombosis were all significantly reduced after treatment; the mean VAS score decreased from 4.71 ± 3.05 at T0 to.52 ± 0.87 and.05 ± 0.22 at T1 and T2, respectively; (mean ± SD; p < 0.001 in both cases). Similarly, the HDSS score showed to be significantly reduced between T0, T1 (8.05 ± 4.55 vs. 1.14 ± 1.01), and T2 (8.05 ± 4.55 vs. 24 ± 0.44) (mean ± SD; p < 0.001 in both cases). Quality of life showed to be improved as the SHS score decreased significantly with treatment (7.90 ± 4.17 at T0 vs. 4.24 ± 0.44 at T1 vs. 4.05 ± 0.22 at T2; mean ± SD; p < 0.001 in both cases). The Bristol score of defecation remained substantially unchanged. No side effects or discontinuation of treatment were reported. Results of our investigation suggest a role of Proctilor® rectal ointment in treating symptomatic HD with good results and an excellent safety profile. However, our preliminary results encourage further studies on a larger number of patients to confirm the role of Zinc-L-Carnosine in the rectal ointment for the topical treatment of HD.

Pathogenic Microenvironment from Diabetic-Obese Visceral and Subcutaneous Adipocytes Activating Differentiation of Human Healthy Preadipocytes Increases Intracellular Fat, Effect of the Apocarotenoid Crocetin.

In diabetes mellitus type 2 (DM2), developed obesity is referred to as diabesity. Implementation of a healthy diet, such as the Mediterranean, prevents diabesity. Saffron is frequently used in this diet because of its bioactive components, such as crocetin (CCT), exhibit healthful properties. It is well known that obesity, defined as an excessive accumulation of fat, leads to cardiometabolic pathology through adiposopathy or hypertrophic growth of adipose tissue (AT).This is related to an impaired adipogenic process or death of adipocytes by obesogenic signals. We aimed to evaluate the effect of the pathogenic microenvironment and CCT, activating differentiation of healthy preadipocytes (PA). For this, we used human cryopreserved PA from visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) depots obtained from healthy and obese-DM2 donors. We studied the effect of a metabolically detrimental (diabesogenic) environment, generated by obese-DM2 adipocytes from VAT (VdDM) or SAT (SdDM), on the viability and accumulation of intracellular fat of adipocytes differentiated from healthy PA, in the presence or absence of CCT (1 or 10 μM). Intracellular fat was quantified by Oil Red O staining. Cytotoxicity was measured using the MTT assay. Our results showed that diabesogenic conditions induce cytotoxicity and provide a proadipogenic environment only for visceral PA. CCT at 10 μM acted as an antiadipogenic and cytoprotective compound.

Bisindolylpyrrole triggers transient mitochondrial permeability transitions to cause apoptosis in a VDAC1/2 and cyclophilin D-dependent manner via the ANT-associated pore

Bisindolylpyrrole at 0.1 μM is cytoprotective in 2% FBS that is counteracted by cyclosporin-A (CsA), an inhibitor of cyclophilin-D (CypD). We hypothesized that the cytoprotective effect might be due to transient mitochondrial permeability transition (tPT). This study tested the hypothesis that bisindolylpyrrole can trigger tPT extensively, thereby leading to cell death under certain conditions. Indeed, CsA-sensitive tPT-mediated apoptosis could be induced by bisindolylpyrrole at > 5 μM in HeLa cells cultured in 0.1% FBS, depending on CypD and VDAC1/2, as shown by siRNA knockdown experiments. Rat liver mitochondria also underwent swelling in response to bisindolylpyrrole, which proceeded at a slower rate than Ca2+-induced swelling, and which was blocked by the VDAC inhibitor tubulin and the ANT inhibitor bongkrekate, indicating the involvement of the ANT-associated, smaller pore. We examined why 0.1% FBS is a prerequisite for apoptosis and found that apoptosis is blocked by PKC activation, which is counteracted by the overexpressed defective PKCε. In mitochondrial suspensions, bisindolylpyrrole triggered CsA-sensitive swelling, which was suppressed selectively by pretreatment with PKCε, but not in the co-presence of tubulin. These data suggest that upon PKC inactivation the cytoprotective compound bisindolylpyrrole can induce prolonged tPT causing apoptosis in a CypD-dependent manner through the VDAC1/2-regulated ANT-associated pore.

Diallyl Trisulfide (DATS) Suppresses AGE-Induced Cardiomyocyte Apoptosis by Targeting ROS-Mediated PKCδ Activation.

Chronic high-glucose exposure results in the production of advanced glycation end-products (AGEs) leading to reactive oxygen species (ROS) generation, which contributes to the development of diabetic cardiomyopathy. PKCδ activation leading to ROS production and mitochondrial dysfunction involved in AGE-induced cardiomyocyte apoptosis was reported in our previous study. Diallyl trisulfide (DATS) is a natural cytoprotective compound under various stress conditions. In this study, the cardioprotective effect of DATS against rat streptozotocin (STZ)-induced diabetic mellitus (DM) and AGE-induced H9c2 cardiomyoblast cell/neonatal rat ventricular myocyte (NRVM) damage was assessed. We observed that DATS treatment led to a dose-dependent increase in cell viability and decreased levels of ROS, inhibition of PKCδ activation, and recuded apoptosis-related proteins. Most importantly, DATS reduced PKCδ mitochondrial translocation induced by AGE. However, apoptosis was not inhibited by DATS in cells transfected with PKCδ-wild type (WT). Inhibition of PKCδ by PKCδ-kinase-deficient (KD) or rottlerin not only inhibited cardiac PKCδ activation but also attenuated cardiac cell apoptosis. Interestingly, overexpression of PKCδ-WT plasmids reversed the inhibitory effects of DATS on PKCδ activation and apoptosis in cardiac cells exposed to AGE, indicating that DATS may inhibit AGE-induced apoptosis by downregulating PKCδ activation. Similar results were observed in AGE-induced NRVM cells and STZ-treated DM rats following DATS administration. Taken together, our results suggested that DATS reduced AGE-induced cardiomyocyte apoptosis by eliminating ROS and downstream PKCδ signaling, suggesting that DATS has potential in diabetic cardiomyopathy (DCM) treatment.

The antibiotic robenidine exhibits guanabenz-like cytoprotective properties by a mechanism independent of protein phosphatase PP1:PPP1R15A

The aminoguanidine compound robenidine is widely used as an antibiotic for the control of coccidiosis, a protozoal infection in poultry and rabbits. Interestingly, robenidine is structurally similar to guanabenz (analogs), which are currently undergoing clinical trials as cytoprotective agents for the management of neurodegenerative diseases. Here we show that robenidine and guanabenz protect cells from a tunicamycin-induced unfolded protein response to a similar degree. Both compounds also reduced the tumor necrosis factor α-induced activation of NF-κB. The cytoprotective effects of guanabenz (analogs) have been explained previously by their ability to maintain eIF2α phosphorylation by allosterically inhibiting protein phosphatase PP1:PPP1R15A. However, using a novel split-luciferase-based protein-protein interaction assay, we demonstrate here that neither robenidine nor guanabenz disrupt the interaction between PPP1R15A and either PP1 or eIF2α in intact cells. Moreover, both drugs also inhibited the unfolded protein response in cells that expressed a nonphosphorylatable mutant (S51A) of eIF2α. Our results identify robenidine as a PP1:PPP1R15A-independent cytoprotective compound that holds potential for the management of protein misfolding-associated diseases.

The Differences in the Proteome Profile of Cannabidiol-Treated Skin Fibroblasts following UVA or UVB Irradiation in 2D and 3D Cell Cultures.

Cannabidiol (CBD), as the only phytocannabinoid that has no psychoactive effect, has both antioxidant and anti-inflammatory effects, and thus might be suggested as a cytoprotective compound against UV-induced metabolic changes in skin cells. Therefore, the aim of this study was to investigate the level of protective CBD activity by evaluating the proteomic profile of 2D and 3D cultured skin fibroblasts models following exposure to UVA and UVB radiation. The CBD cytoprotective effect against UV-induced damage in 2D and 3D cultured fibroblasts were different. The main alterations focus on the range of cell reaction and involved different proteins associated with various molecular functions. In the 2D cultured cells, following UV radiation, the major changes were associated with proteins involved in antioxidant response and inflammation, while, in the 3D cultured fibroblasts, CBD action against UV induced changes were mainly associated with the activation of signalling pathways. Therefore, the knowledge of the CBD action in a multilayer skin cells model allowed for the prediction of changes in cell-cell interactions and skin cell metabolism. Knowledge about the lower protective effect of CBD in 3D cultured fibroblasts should be taken into account during the design of UV light protection.

Psidium guajava bioactive product chemical analysis and heavy metal toxicity reduction

The present study had as its objective to verify the Psidium guajava var. Pomifera L. chelating, antioxidant and cytoprotective effects against mercury and aluminum. The ethanolic extract, tannic and flavonoid fractions were subjected to LC-MS analysis. The Ferric Reducing Antioxidant Power (FRAP) and ferric ion reduction demonstrated a present antioxidant activity. The fungicidal and bactericidal activity of these metals were established. After determining the sub-allelopathic doses, germination tests using Lactuca sativa were performed. Quercetin and its derivatives were the main compounds identified in the extract and the fractions. Mercury chloride significantly reduced the bactericidal effect of the flavonoid fraction (p < 0.001). None of the fractions were cytoprotective against mercury or aluminum in the fungal model assays. Using a sub-allelopathic concentration (64 μg/mL), the ethanolic extract, flavonoid and tannic fractions were found to be cytoprotective against aluminum for radicles, however only the tannic fraction was cytoprotective for caulicles. These data suggest that natural P. guajava products are promising cytoprotective compound sources. This activity may be related to the antioxidant effect of secondary metabolites, mainly flavonoids. Our results point to a potential for environmental intervention product and technique development aimed at mitigating contamination by toxic metals such as mercury and aluminum.

Proteins involved in the antioxidant and inflammatory response in rutin-treated human skin fibroblasts exposed to UVA or UVB irradiation

BackgroundRutin, due to its polyphenolic structure, has antioxidant properties and can be used as a cytoprotective compound against UV-induced effects on skin cells. ObjectiveThe aim of this study was to examine the effect of rutin on proteomic profile in human skin fibroblasts irradiated with UV dose that induces apoptosis. MethodsProteome analysis based on the results obtained by the QExactive OrbiTrap mass spectrometer. ResultsResults show that rutin treatment more strongly protects against UVA-induced rather than UVB-induced increases in the total expression of proteins involved in antioxidant (such as SOD, TrxR, and Prxs 1/2) and inflammatory response (e.g., IL-17F, PAK2, and YWHAZ). However, in the case of UVB-irradiated cells, rutin additionally enhances the levels of disulfide-isomerase − an enzyme that is responsible for the formation and breakage of disulfide bonds. Moreover, UVB radiation promotes rutin-Keap1 adduct formation, which leads to the activation of Nrf2, a factor that is responsible for the synthesis of cytoprotective proteins. Furthermore, rutin partially prevents UV-induced apoptosis by restoring the physiological levels of p53, cytochrome c, and cell cycle and apoptosis regulator protein 2 that were increased following irradiation. ConclusionIn conclusion, our results show that rutin effectively prevents UV-induced damages associated with proinflammatory and prooxidative activity and protects cells against apoptosis.

Oxaliplatin-induced enteric neuronal loss and intestinal dysfunction is prevented by co-treatment with BGP-15.

Gastrointestinal side effects of chemotherapy are an under-recognized clinical problem, leading to dose reduction, delays and cessation of treatment, presenting a constant challenge for efficient and tolerated anti-cancer treatment. We have found that oxaliplatin treatment results in intestinal dysfunction, oxidative stress and loss of enteric neurons. BGP-15 is a novel cytoprotective compound with potential HSP72 co-inducing and PARP inhibiting properties. In this study, we investigated the potential of BGP-15 to alleviate oxaliplatin-induced enteric neuropathy and intestinal dysfunction. Balb/c mice received oxaliplatin (3mg·kg-1 ·day-1 ) with and without BGP-15 (15mg·kg-1 ·day-1 : i.p.) tri-weekly for 14days. Gastrointestinal transit was analysed via in vivo X-ray imaging, before and after treatment. Colons were collected to assess ex vivo motility, neuronal mitochondrial superoxide and cytochrome c levels and for immunohistochemical analysis of myenteric neurons. Oxaliplatin-induced neuronal loss increased the proportion of neuronal NO synthase-immunoreactive neurons and increased levels of mitochondrial superoxide and cytochrome c in the myenteric plexus. These changes were correlated with an increase in PARP-2 immunoreactivity in the colonic mucosa and were attenuated by BGP-15 co-treatment. Significant delays in gastrointestinal transit, intestinal emptying and pellet formation, impaired colonic motor activity, reduced faecal water content and lack of weight gain associated with oxaliplatin treatment were restored to sham levels in mice co-treated with BGP-15. Our results showed that BGP-15 ameliorated oxidative stress, increased enteric neuronal survival and alleviated oxaliplatin-induced intestinal dysfunction, suggesting that BGP-15 may relieve the gastrointestinal side effects of chemotherapy.

P 103 - Proteomic analysis of rutin effect on human skin fibroblasts exposed to UVA or UVB irradiation

Rutin, due to its polyphenolic structure reveals antioxidant properties and may be used as a cytoprotective compound against UV-induced effects on skin cells. Therefore, the aim of this study was to examine the rutin effects on UV-induced changes in proteome profile of human skin fibroblasts cultured in vitro. PCA analysis of results obtained by QExactive OrbiTrap mass spectrometer show clear differentiation only between control and UVA treated cells, although significant changes in the individual proteins level are also observed between the rest of tested fibroblast groups. Rutin treatment prevents UVA-induced, even 2-fold, increase in the total expression of proteins involved in antioxidant (e.g. superoxide dismutases, disulfide isomerase) and inflammatory response (e.g. IL-17, PAK2, YWHAZ), what is not observed in fibroblasts after rutin treatment following UVB irradiation. However, rutin treatment of fibroblasts after UVB radiation promotes rutin-Keap1 adducts formation thus activation of Nrf2 - responsible for cytoprotective proteins synthesis. Rutin also prevents UV-induced apoptosis, through restoring the pro-apoptotic proteins level (e.g. p53 and cytochrome c) that is enhanced after the irradiation. In conclusion, our results show that rutin effectively prevents UV-induced damages associated with proinflammatory/prooxidative and proapoptotic activity.

Cytoprotective Drug-Tissue Plasminogen Activator Protease Interaction Assays: Screening of Two Novel Cytoprotective Chromones.

Tissue plasminogen activator (tPA) is currently used in combination with endovascular procedures to enhance recanalization and cerebral reperfusion and is also currently administered as standard-of-care thrombolytic therapy to patients within 3-4.5h of an ischemic stroke. Since tPA is not neuroprotective or cytoprotective, adjuvant therapy with a neuroprotective or an optimized cytoprotective compound is required to provide the best care to stroke victims to maximally promote clinical recovery. In this article, we describe the use of a sensitive standardized protease assay with CH3SO2-D-hexahydrotyrosine-Gly-Arg-p-nitroanilide•AcOH, a chromogenic protease substrate that is cleaved to 4-nitroaniline (p-nitroaniline) and measured spectrophotometrically at 405nm (OD405nm), and how the assay can be used as an effective screening assay to study drug-tPA interactions. While we focus on two compounds of interest in our drug development pipeline, the assay is broadly applicable to all small molecule neuroprotective or cytoprotective compounds currently being discovered and developed worldwide. In this present study, we found that the specific tPA inhibitor, plasminogen activator inhibitor-1 (PAI-1; 0.25μM), significantly (p<0.0001) inhibited 4-nitroaniline release, by 97.74% during the 10-min duration of the assay, which is indicative of tPA protease inhibition. In addition, two lead chromone cytoprotective candidates, 2-(3',4',5'-trihydroxyphenyl)chromen-4-one (3',4',5'-trihydroxyflavone) (CSMC-19) and 3-hydroxy-2-[3-hydroxy-4-(pyrrolidin-1-yl)phenyl]benzo[h]chromen-4-one (CSMC-140), also significantly (p<0.05) reduced 4-nitroaniline accumulation, but to a lesser extent. The reduction was 68 and 45%, respectively, at 10μM, and extrapolated IC50 values were 4.37 and >10μM for CSMC-19 and CSMC-140, respectively. Using bonafide 4-nitroaniline, we then demonstrated that the reduction of 4-nitroaniline detection was not due to drug-4-nitroaniline quenching of signal detection at OD405nm. In conclusion, the results suggest that high concentrations of both cytoprotectives reduced 4-nitroaniline production in vitro, but the inhibition only occurs with concentrations 104-1025-fold that of EC50 values in an efficacy assay. Thus, CSMC-19 and CSMC-140 should be further developed and evaluated in embolic stroke models in the absence or presence of a thrombolytic. If necessary, they could be administered once effective tPA thrombolysis has been confirmed to avoid the possibility that the chromone will reduce the efficacy of tPA in patients. Stroke investigator developing new cytoprotective small molecules should consider adding this sensitive assay to their development and screening repertoire to assess possible drug-tPA interactions in vitro as a de-risking step.

Harmful Effects of Neonatal Epileptic Seizures on Cerebrovascular Functions: Does Sex Matter?

There is a growing interest in the potential contribution of sex‐related variables to cerebrovascular functions in healthy and sick neonates. Epileptic seizures, the most common adverse neurological event in human newborns, may lead to cerebrovascular injury and abnormalities in cerebral blood flow regulation. Clinical studies suggest that male neonates are more susceptible to seizures, with a higher incidence of neurological complications. Cerebral vascular health is an important contributing factor to brain homeostasis and neuronal health. Newborn piglets provide a valuable and translationally relevant animal model for studying the cerebrovascular effects of epileptic seizures in the neonatal brain. This study in newborn pigs (2–5 days old) was designed to investigate whether sex‐related differences contribute to systemic parameters and cerebrovascular functions in: 1) control healthy animals and 2) piglets subjected to bicuculline‐induced epileptic seizures leading to prolonged cerebral vascular dysfunction during the delayed postictal period. After a 48‐h recovery period from epileptic seizures, postictal cerebrovascular functions were tested using intravital microscopy via the closed cranial window technique. We recorded the responses of small‐ and medium‐sized pial cerebral arterioles (50–100 μm) to a battery of vasodilator stimuli including: A) endothelium‐dependent hypercapnia and bradykinin; B) dual endothelium/astrocyte‐dependent stimuli glutamate, ionotropic glutamate receptor agonist quisqualic acid, and hemin; C) astrocyte‐dependent vasodilator adenosine diphosphate, and D) endothelium/astrocyte‐independent vasodilator sodium nitroprusside (SNP). We found no sex‐related differences in cardiovascular parameters (mean arterial blood pressure, heart rate), arterial blood gases, or core body temperature in either control or postictal groups of newborn piglets. A significant reduction (50–70%) in responses of pial arterioles to endothelium‐ and/or astrocyte‐dependent vasodilators was observed 48 h after seizures (delayed postictal period) in both male and female piglets. In piglets of both sexes, vasodilation to SNP was preserved during the delayed postictal period, suggesting that vascular smooth muscle contractility was not affected by seizures. An antioxidant and cytoprotective compound, the carbon‐monoxide (CO)‐releasing molecule CORM‐A1 (3 mg/kg i.p.) that was administered shortly before and/or during seizures, prevented postictal loss of cerebrovascular functions in both male and female piglets. Overall, we observed no sex‐specific differences in selected systemic cardiovascular parameters and cerebrovascular dilator functions in control and epileptic newborn pigs. In both male and female piglets, epileptic seizures lead to severe cerebral vascular dysfunction during the delayed postictal period. Antioxidant therapy with CORM‐A1 is neuroprotective in both male and female newborn pigs because it effectively prevents deleterious effects of epileptic seizures on cerebral blood flow regulation in a sex‐independent manner.Support or Funding InformationThe study was supported by NIH/NHLBI/NINDS.

The High Cost of Stroke and Stroke Cytoprotection Research.

Acute ischemic stroke is inadequately treated in the USA and worldwide due to a lengthy history of neuroprotective drug failures in clinical trials. The majority of victims must endure life-long disabilities that not only affect their livelihood, but also have an enormous societal economic impact. The rapid development of a neuroprotective or cytoprotective compound would allow future stroke victims to receive a treatment to reduce disabilities and further promote recovery of function. This opinion article reviews in detail the enormous costs associated with developing a small molecule to treat stroke, as well as providing a timely overview of the cell-death time-course and relationship to the ischemic cascade. Distinct temporal patterns of cell-death of neurovascular unit components provide opportunities to intervene and optimize new cytoprotective strategies. However, adequate research funding is mandatory to allow stroke researchers to develop and test their novel therapeutic approach to treat stroke victims.

Identification of cytoprotective constituents of the flower buds of Tussilago farfara against glucose oxidase-induced oxidative stress in mouse fibroblast NIH3T3 cells and human keratinocyte HaCaT cells

A new cytoprotective compound, 1-[(4S)-3,4-dihydro-4-hydroxy-2,2-dimethyl-2H-1-benzopyran-6-yl]-ethanone (1) was isolated from the flower buds of Tussilago farfara L. (Compositae), together with eight known compounds, 3,4-dicaffeoyl isoquinic acid (2), trans-cinnamic acid (3), 4-hydroxyacetophenone (4), 4,5-dicaffeoylquinic acid methyl ester (5), 3,5-dicaffeoylquinic acid methyl ester (6), 4-hydroxybenzoic acid (7), isoquercetrin (8), and ligucyperonol (9). Compounds 2-4 were found in this plant for the first time. The isolates 1-9, were tested for their cytoprotective activities against glucose oxidase-induced oxidative stress in mouse fibroblast NIH3T3 cells and human keratinocyte HaCaT cells. Among them, 1 and 3 showed significant cytoprotective activities as determined by MTT assay and lactate dehydrogenase leakage, indicating their possibility as the potent cytoprotective agents. The structure of 1 was determined by spectroscopic data analysis including 1D- and 2D-NMR experiments, and its absolute configuration was elucidated by a circular dichroism.