Growth hormone releasing peptide-6 (GHRP-6) prevents doxorubicin-induced myocardial and extra-myocardial damages by activating prosurvival mechanisms.

Introduction: Dilated cardiomyopathy (DCM) is a fatal myocardial condition with ventricular structural changes and functional deficits, leading to systolic dysfunction and heart failure (HF). DCM is a frequent complication in oncologic patients receiving Doxorubicin (Dox). Dox is a highly cardiotoxic drug, whereas its damaging spectrum affects most of the organs by multiple pathogenic cascades. Experimentally reproduced DCM/HF through Dox administrations has shed light on the pathogenic drivers of cardiotoxicity. Growth hormone (GH) releasing peptide 6 (GHRP-6) is a GH secretagogue with expanding and promising cardioprotective pharmacological properties. Here we examined whether GHRP-6 administration concomitant to Dox prevented the onset of DCM/HF and multiple organs damages in otherwise healthy rats. Methods: Myocardial changes were sequentially evaluated by transthoracic echocardiography. Autopsy was conducted at the end of the administration period when ventricular dilation was established. Semiquantitative histopathologic study included heart and other internal organs samples. Myocardial tissue fragments were also addressed for electron microscopy study, and characterization of the transcriptional expression ratio between Bcl-2 and Bax. Serum samples were destined for REDOX system balance assessment. Results and discussion: GHRP-6 administration in parallel to Dox prevented myocardial fibers consumption and ventricular dilation, accounting for an effective preservation of the LV systolic function. GHRP-6 also attenuated extracardiac toxicity preserving epithelial organs integrity, inhibiting interstitial fibrosis, and ultimately reducing morbidity and mortality. Mechanistically, GHRP-6 proved to sustain cellular antioxidant defense, upregulate prosurvival gene Bcl-2, and preserve cardiomyocyte mitochondrial integrity. These evidences contribute to pave potential avenues for the clinical use of GHRP-6 in Dox-treated subjects.

The effects of Phycocyanobilin on experimental arthritis involve the reduction in nociception and synovial neutrophil infiltration, inhibition of cytokine production, and modulation of the neuronal proteome.

Introduction: The antinociceptive and pharmacological activities of C-Phycocyanin (C-PC) and Phycocyanobilin (PCB) in the context of inflammatory arthritis remain unexplored so far. In the present study, we aimed to assess the protective actions of these compounds in an experimental mice model that replicates key aspects of human rheumatoid arthritis. Methods: Antigen-induced arthritis (AIA) was established by intradermal injection of methylated bovine serum albumin in C57BL/6 mice, and one hour before the antigen challenge, either C-PC (2, 4, or 8 mg/kg) or PCB (0.1 or 1 mg/kg) were administered intraperitoneally. Proteome profiling was also conducted on glutamate-exposed SH-SY5Y neuronal cells to evaluate the PCB impact on this key signaling pathway associated with nociceptive neuronal sensitization. Results and discussion: C-PC and PCB notably ameliorated hypernociception, synovial neutrophil infiltration, myeloperoxidase activity, and the periarticular cytokine concentration of IFN-γ, TNF-α, IL-17A, and IL-4 dose-dependently in AIA mice. In addition, 1 mg/kg PCB downregulated the gene expression for T-bet, RORγ, and IFN-γ in the popliteal lymph nodes, accompanied by a significant reduction in the pathological arthritic index of AIA mice. Noteworthy, neuronal proteome analysis revealed that PCB modulated biological processes such as pain, inflammation, and glutamatergic transmission, all of which are involved in arthritic pathology. Conclusions: These findings demonstrate the remarkable efficacy of PCB in alleviating the nociception and inflammation in the AIA mice model and shed new light on mechanisms underlying the PCB modulation of the neuronal proteome. This research work opens a new avenue to explore the translational potential of PCB in developing a therapeutic strategy for inflammation and pain in rheumatoid arthritis.

Evidence of SARS-CoV-2 infection in postmortem lung, kidney, and liver samples, revealing cellular targets involved in COVID-19 pathogenesis.

There is an urgent need to understand severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-host interactions involved in virus spread and pathogenesis, which might contribute to the identification of new therapeutic targets. In this study, we investigated the presence of SARS-CoV-2 in postmortem lung, kidney, and liver samples of patients who died with coronavirus disease (COVID-19) and its relationship with host factors involved in virus spread and pathogenesis, using microscopy-based methods. The cases analyzed showed advanced stages of diffuse acute alveolar damage and fibrosis. We identified the SARS-CoV-2 nucleocapsid (NC) in a variety of cells, colocalizing with mitochondrial proteins, lipid droplets (LDs), and key host proteins that have been implicated in inflammation, tissue repair, and the SARS-CoV-2 life cycle (vimentin, NLRP3, fibronectin, LC3B, DDX3X, and PPARγ), pointing to vimentin and LDs as platforms involved not only in the viral life cycle but also in inflammation and pathogenesis. SARS-CoV-2 isolated from a patient´s nasal swab was grown in cell culture and used to infect hamsters. Target cells identified in human tissue samples included lung epithelial and endothelial cells; lipogenic fibroblast-like cells (FLCs) showing features of lipofibroblasts such as activated PPARγ signaling and LDs; lung FLCs expressing fibronectin and vimentin and macrophages, both with evidence of NLRP3- and IL1ß-induced responses; regulatory cells expressing immune-checkpoint proteins involved in lung repair responses and contributing to inflammatory responses in the lung; CD34+ liver endothelial cells and hepatocytes expressing vimentin; renal interstitial cells; and the juxtaglomerular apparatus. This suggests that SARS-CoV-2 may directly interfere with critical lung, renal, and liver functions involved in COVID-19-pathogenesis.

Anti-inflammatory mechanisms and pharmacological actions of phycocyanobilin in a mouse model of experimental autoimmune encephalomyelitis: A therapeutic promise for multiple sclerosis.

Cytokines, demyelination and neuroaxonal degeneration in the central nervous system are pivotal elements implicated in the pathogenesis of multiple sclerosis (MS) and its nonclinical model of experimental autoimmune encephalomyelitis (EAE). Phycocyanobilin (PCB), a chromophore of the biliprotein C-Phycocyanin (C-PC) from Spirulina platensis, has antioxidant, immunoregulatory and anti-inflammatory effects in this disease, and it could complement the effect of other Disease Modifying Treatments (DMT), such as Interferon-ß (IFN-ß). Here, our main goal was to evaluate the potential PCB benefits and its mechanisms of action to counteract the chronic EAE in mice. MOG35-55-induced EAE was implemented in C57BL/6 female mice. Clinical signs, pro-inflammatory cytokines levels by ELISA, qPCR in the brain and immunohistochemistry using precursor/mature oligodendrocytes cells antibodies in the spinal cord, were assessed. PCB enhanced the neurological condition, and waned the brain concentrations of IL-17A and IL-6, pro-inflammatory cytokines, in a dose-dependent manner. A down- or up-regulating activity of PCB at 1 mg/kg was identified in the brain on three (LINGO1, NOTCH1, and TNF-α), and five genes (MAL, CXCL12, MOG, OLIG1, and NKX2-2), respectively. Interestingly, a reduction of demyelination, active microglia/macrophages density, and axonal damage was detected along with an increase in oligodendrocyte precursor cells and mature oligodendrocytes, when assessed the spinal cords of EAE mice that took up PCB. The studies in vitro in rodent encephalitogenic T cells and in vivo in the EAE mouse model with the PCB/IFN-ß combination, showed an enhanced positive effect of this combined therapy. Overall, these results demonstrate the anti-inflammatory activity and the protective properties of PCB on the myelin and support its use with IFN-ß as an improved DMT combination for MS.

Positive effects of Phycocyanobilin on gene expression in glutamate-induced excitotoxicity in SH-SY5Y cells and animal models of multiple sclerosis and cerebral ischemia.

Background: Oxidative stress has a predominant role in the pathogenesis of neurodegenerative diseases and therefore the modulation of genes and the identification of biological pathways associated with antioxidant therapies, have an impact on its treatment. Objective: The objective of this study was the comparison of 2 methods for the analysis of real-time PCR (qPCR) data, through the use of the evaluation of genes that mediate the effect of Phycocyanobilin (PCB) and its validation in animal models. Methods: We evaluated the effect of PCB:" in vitro" on gene modulation through qPCR analyzed by parametric ANOVA and multivariate principal component analysis (PCA) in a model of glutamate-induced excitotoxicity in the SH-SY5Y cell line and" in vivo"; in animal models of multiple sclerosis (MS) and cerebral ischemia (CI). Results: The results showed that PCA is a robust and powerful method that allows the assessment of gene expression profiles. We detected the significant down-regulation of the CYBB (NOX2), and HMOX1 by the action of PCB in SH-5YSH cell line insulted with Glutamate. The decrease in pro-inflammatory cytokines and markers related to apoptosis and innate immune response, mediated the effect of PCB in the animal models of MS and CI, respectively. Conclusion: We concluded that the mechanisms by which PCB protected cells included the reduction of oxidative stress damage, which could contribute to its clinical efficacy for the treatment of neurodegenerative diseases.

Epidermal Growth Factor in Healing Diabetic Foot Ulcers: From Gene Expression to Tissue Healing and Systemic Biomarker Circulation.

Lower-extremity diabetic ulcers are responsible for 80% of annual worldwide nontraumatic amputations. Epidermal growth factor (EGF) reduction is one of the molecular pillars of diabetic ulcer chronicity, thus EGF administration may be considered a type of replacement therapy. Topical EGF ad-ministration to improve and speed wound healing began in 1989 on burn patients as part of an acute-healing therapy. Further clinical studies based on topically administering EGF to different chronic wounds resulted in disappointing out-comes. An analysis of the literature on unsuccessful clinical trials identifi ed a lack of knowledge concerning: (I) molecular and cellular foundations of wound chronicity and (II) the phar-macodynamic requisites governing EGF interaction with its receptor to promote cell response. Yet, EGF intra- and perile-sional infi ltration were shown to circumvent the pharmacody-namic limitations of topical application. Since the fi rst studies, the following decades of basic and clinical research on EGF therapy for problem wounds have shed light on potential uses of growth factors in regenerative medicine. EGF's molecular and biochemical effects at both local and systemic levels are diverse: (1) downregulation of genes encoding infl ammation mediators and increased expression of genes involved in cell proliferation, angiogenesis and matrix secretion; (2) EGF in-tervention positively impacts both mesenchymal and epithelial cells, reducing infl ammation and stimulating the recruitment of precursor circulating cells that promote the formation of new blood vessels; (3) at the subcellular level, upregulation of the EGF receptor with subsequent intracellular traffi cking, includ-ing mitochondrial allocation along with restored morphology of multiple organelles; and (4) local EGF infi ltration resulting in a systemic, organismal repercussion, thus contributing to attenuation of circulating infl ammatory and catabolic reac-tants, restored reduction-oxidation balance, and decreased toxic glycation products and soluble apoptogenic effectors. It is likely that EGF treatment may rearrange critical epigenetic drivers of diabetic metabolic memory. KEYWORDS Epidermal Growth Factor, diabetes, diabetes complications, wound healing, diabetic foot, amputation, ulcer, Cuba.

Phycocyanobilin reduces brain injury after endothelin-1- induced focal cerebral ischaemia.

Pharmacological therapies for interrupting biochemical events of the ischaemic cascade and protecting against stroke in humans are as yet unavailable. Up to now, the neuroprotective activity in cerebral ischaemia of phycocyanobilin (PCB), a tetrapyrrolic natural antioxidant, has not been fully examined. Here, we evaluated if PCB protects PC12 neuronal cells against oxygen and glucose deprivation plus reperfusion, and its protective effects in a rat model of endothelin-1-induced focal brain ischaemia. PCB was purified from the cyanobacteria Spirulina platensis and characterized by spectrophotometric, liquid and gas chromatography and mass spectrometry techniques. In Wistar rats, PCB at 50, 100 and 200 µg/kg or phosphate-buffered saline (vehicle) was administered intraperitoneally at equal subdoses in a therapeutic schedule (30 minutes, 1, 3 and 6 hours after the surgery). Brain expression of myelin basic protein (MBP) and the enzyme CNPase was determined by immunoelectron microscopy. PCB was obtained with high purity (>95%) and the absence of solvent contaminants and was able to ameliorate PC12 cell ischaemic injury. PCB treatment significantly decreased brain infarct volume, limited the exploratory behaviour impairment and preserved viable cortical neurons in ischaemic rats in a dose-dependent manner, compared to the vehicle group. Furthermore, PCB at high doses restored the MBP and CNPase expression levels in ischaemic rats. An improved PCB purification method from its natural source is reported, obtaining PCB that is suitable for pharmacological trials showing neuroprotective effects against experimental ischaemic stroke. Therefore, PCB could be a therapeutic pharmacological alternative for ischaemic stroke patients.

C-Phycocyanin and Phycocyanobilin as Remyelination Therapies for Enhancing Recovery in Multiple Sclerosis and Ischemic Stroke: A Preclinical Perspective.

Myelin loss has a crucial impact on behavior disabilities associated to Multiple Sclerosis (MS) and Ischemic Stroke (IS). Although several MS therapies are approved, none of them promote remyelination in patients, limiting their ability for chronic recovery. With no available therapeutic options, enhanced demyelination in stroke survivors is correlated with a poorer behavioral recovery. Here, we show the experimental findings of our group and others supporting the remyelinating effects of C-Phycocyanin (C-PC), the main biliprotein of Spirulina platensis and its linked tetrapyrrole Phycocyanobilin (PCB), in models of these illnesses. C-PC promoted white matter regeneration in rats and mice affected by experimental autoimmune encephalomyelitis. Electron microscopy analysis in cerebral cortex from ischemic rats revealed a potent remyelinating action of PCB treatment after stroke. Among others biological processes, we discussed the role of regulatory T cell induction, the control of oxidative stress and pro-inflammatory mediators, gene expression modulation and COX-2 inhibition as potential mechanisms involved in the C-PC and PCB effects on the recruitment, differentiation and maturation of oligodendrocyte precursor cells in demyelinated lesions. The assembled evidence supports the implementation of clinical trials to demonstrate the recovery effects of C-PC and PCB in these diseases.

Beneficial effects of oral administration of C-Phycocyanin and Phycocyanobilin in rodent models of experimental autoimmune encephalomyelitis.

The only three oral treatments currently available for multiple sclerosis (MS) target the relapsing forms of the disease and concerns regarding efficacy, safety and tolerability limit their use. Identifying novel oral disease-modifying therapies for MS, targeting both its inflammatory and neurodegenerative components is still a major goal. AIM: The scope of this study was to provide evidence that the oral administration of C-Phycocyanin (C-PC), the main biliprotein of the Spirulina platensis cyanobacteria and its tetrapyrrolic prosthetic group, Phycocyanobilin (PCB), exert ameliorating actions on rodent models of experimental autoimmune encephalomyelitis (EAE). MAIN METHODS: EAE was induced in Lewis rats using the spinal cord encephalitogen from Sprague Dawley rats and in C57BL6 mice with MOG35-55 peptide. Clinical signs, motor function, oxidative stress markers, cytokine levels by ELISA and transmission electron microscopy analysis were assessed. KEY FINDINGS: Either prophylactic or early therapeutic administration of C-PC to Lewis rats with EAE, significantly improved clinical signs and restored the motor function of the animals. Furthermore, C-PC positively modulated oxidative stress markers measured in brain homogenate and serum and protected the integrity of cerebral myelin sheaths as shown by transmission electron microscopy analysis. In C57BL/6 mice with EAE, PCB orally improved clinical status of the animals and reduced the expression levels of brain IL-6 and IFN-γ proinflammatory cytokines. SIGNIFICANCE: These results, for the first time, support the fact that both C-PC and PCB administered orally could potentially improve neuroinflammation, protect from demyelination and axonal loss, which may be translated into an improved quality of life for MS patients.

Diabetic Foot Ulcers and Epidermal Growth Factor: Revisiting the Local Delivery Route for a Successful Outcome.

Soon after epidermal growth factor (EGF) discovery, some in vivo models appeared demonstrating its property to enhance cutaneous wound healing. EGF was the first growth factor (GF) introduced in the clinical arena as a healing enhancer, exerting its mitogenic effects on epithelial, fibroblastoid, and endothelial cells via a tyrosine kinase membrane receptor. Compelling evidences from the 90s documented that, for EGF, locally prolonged bioavailability and hourly interaction with the receptor were necessary for a successful tissue response. Eventually, the enthusiasm on the clinical use of EGF to steer the healing process was wiped out as the topical route to deliver proteins started to be questioned. The simultaneous in vivo experiments, emphasizing the impact of the parenterally administered EGF on epithelial and nonepithelial organs in terms of mitogenesis and cytoprotection, rendered the theoretical fundamentals for the injectable use of EGF and shaped the hypothesis that locally infiltrating the diabetic ulcers would lead to an effective healing. Although the diabetic chronic wounds microenvironment is hostile for local GFs bioavailability, EGF local infiltration circumvented the limitations of its topical application, thus expanding its therapeutic prospect. Our clinical pharmacovigilance and basic studies attest the significance of the GF local infiltration for chronic wounds healing.

Synthetic Growth Hormone-Releasing Peptides (GHRPs): A Historical Appraisal of the Evidences Supporting Their Cytoprotective Effects.

BACKGROUND: Growth hormone-releasing peptides (GHRPs) constitute a group of small synthetic peptides that stimulate the growth hormone secretion and the downstream axis activity. Mounting evidences since the early 1980s delineated unexpected pharmacological cardioprotective and cytoprotective properties for the GHRPs. However, despite intense basic pharmacological research, alternatives to prevent cell and tissue demise before lethal insults have remained as an empty niche in the clinical armamentarium. Here, we have rigorously reviewed the investigational development of GHRPs and their clinical niching perspectives. METHODOLOGY: PubMed/MEDLINE databases, including original research and review articles, were explored. The search design was date escalated from 1980 and included articles in English only. RESULTS AND CONCLUSIONS: GHRPs bind to two different receptors (GHS-R1a and CD36), which redundantly or independently exert relevant biological effects. GHRPs' binding to CD36 activates prosurvival pathways such as PI-3K/AKT1, thus reducing cellular death. Furthermore, GHRPs decrease reactive oxygen species (ROS) spillover, enhance the antioxidant defenses, and reduce inflammation. These cytoprotective abilities have been revealed in cardiac, neuronal, gastrointestinal, and hepatic cells, representing a comprehensive spectrum of protection of parenchymal organs. Antifibrotic effects have been attributed to some of the GHRPs by counteracting fibrogenic cytokines. In addition, GHRP family members have shown a potent myotropic effect by promoting anabolia and inhibiting catabolia. Finally, GHRPs exhibit a broad safety profile in preclinical and clinical settings. Despite these fragmented lines incite to envision multiple pharmacological uses for GHRPs, especially as a myocardial reperfusion damage-attenuating candidate, this family of "drugable" peptides awaits for a definitive clinical niche.

Lipofundin-induced hyperlipidemia promotes oxidative stress and atherosclerotic lesions in new zealand white rabbits.

Atherosclerosis represents a major cause of death in the world. It is known that Lipofundin 20% induces atherosclerotic lesions in rabbits, but its effects on serum lipids behaviour and redox environment have not been addressed. In this study, New Zealand rabbits were treated with 2 mL/kg of Lipofundin for 8 days. Then, redox biomarkers and serum lipids were determined spectrophotometrically. On the other hand, the development of atherosclerotic lesions was confirmed by eosin/hematoxylin staining and electron microscopy. At the end of the experiment, total cholesterol, triglycerides, cholesterol-LDL, and cholesterol-HDL levels were significantly increased. Also, a high index of biomolecules damage, a disruption of both enzymatic and nonenzymatic defenses, and a reduction of nitric oxide were observed. Our data demonstrated that Lipofundin 20% induces hyperlipidemia, which promotes an oxidative stress state. Due to the importance of these phenomena as risk factors for atherogenesis, we suggest that Lipofundin induces atherosclerosis mainly through these mechanisms.

C-Phycocyanin ameliorates experimental autoimmune encephalomyelitis and induces regulatory T cells.

For decades Experimental Autoimmune Encephalitis (EAE) has remained as an unsurpassed multiple sclerosis (MS) animal model. C-Phycocyanin (C-Pc) has been reported to exhibit pharmacological properties that may be expected to symptomatically improve EAE and MS. However, in this paper we reveal a basic underlying mechanism that may provide a new approach to the rationale of the overall beneficial effect of this natural antioxidant. We demonstrate that C-Pc is able to trigger mechanisms preventing or downgrading EAE expression and induces a regulatory T cell (Treg) response, in peripheral blood mononuclear cells (PBMC) from MS patients. These results agree with reports suggesting that Treg limit acute MS attacks and that C-Pc may act as a neuroprotector and thereby reverts the organic and functional damage in neurodegenerative disorders of the central nervous system (CNS). Moreover, evidence is provided on the antioxidant activity of C-Pc within the CNS, intended to improve the myelin and axonal damage of EAE induced Lewis rats. Our results indicate that specific Treg activation may represent a central and essential mechanism in supporting the therapeutic potential of C-Pc for MS and may lead to new and more effective therapies; this property would then complement and enhance other proven active principles such as interferons (IFN), giving rise to combined therapies.