RESUMO
Despite all efforts to combat the pandemic of COVID-19, we are still living with high numbers of infected persons, an overburdened health care system, and the lack of an effective and definitive treatment. Understanding the pathophysiology of the disease is crucial for the development of new technologies and therapies for the best clinical management of patients. Since the manipulation of the whole virus requires a structure with an adequate level of biosafety, the development of alternative technologies, such as the synthesis of peptides from viral proteins, is a possible solution to circumvent this problem. In addition, the use and validation of animal models is of extreme importance to screen new drugs and to compress the organism's response to the disease. Peptides derived from recombinant S protein from SARS-CoV-2 were synthesized and validated by in silico, in vitro and in vivo methodologies. Macrophages and neutrophils were challenged with the peptides and the production of inflammatory mediators and activation profile were evaluated. These peptides were also inoculated into the swim bladder of transgenic zebrafish larvae at 6 days post fertilization (dpf) to mimic the inflammatory process triggered by the virus, which was evaluated by confocal microscopy. In addition, toxicity and oxidative stress assays were also developed. In silico and molecular dynamics assays revealed that the peptides bind to the ACE2 receptor stably and interact with receptors and adhesion molecules, such as MHC and TCR, from humans and zebrafish. Macrophages stimulated with one of the peptides showed increased production of NO, TNF-α and CXCL2. Inoculation of the peptides in zebrafish larvae triggered an inflammatory process marked by macrophage recruitment and increased mortality, as well as histopathological changes, similarly to what is observed in individuals with COVID-19. The use of peptides is a valuable alternative for the study of host immune response in the context of COVID-19. The use of zebrafish as an animal model also proved to be appropriate and effective in evaluating the inflammatory process, comparable to humans.
Assuntos
COVID-19 , SARS-CoV-2 , Animais , Humanos , Peixe-Zebra , Macrófagos , PeptídeosRESUMO
Although the exact mechanism of the pathogenesis of coronavirus SARS-CoV-2 (COVID-19) is not fully understood, oxidative stress and the release of pro-inflammatory cytokines have been highlighted as playing a vital role in the pathogenesis of the disease. In this sense, alternative treatments are needed to reduce the level of inflammation caused by COVID-19. Therefore, this study aimed to investigate the potential effect of red photobiomodulation (PBM) as an attractive therapy to downregulate the cytokine storm caused by COVID-19 in a zebrafish model. RT-qPCR analyses and protein-protein interaction prediction among SARS-CoV-2 and Danio rerio proteins showed that recombinant Spike protein (rSpike) was responsible for generating systemic inflammatory processes with significantly increased levels of pro-inflammatory (il1b, il6, tnfa, and nfkbiab), oxidative stress (romo1) and energy metabolism (slc2a1a and coa1) mRNA markers, with a pattern similar to those observed in COVID-19 cases in humans. On the other hand, PBM treatment was able to decrease the mRNA levels of these pro-inflammatory and oxidative stress markers compared with rSpike in various tissues, promoting an anti-inflammatory response. Conversely, PBM promotes cellular and tissue repair of injured tissues and significantly increases the survival rate of rSpike-inoculated individuals. Additionally, metabolomics analysis showed that the most-impacted metabolic pathways between PBM and the rSpike treated groups were related to steroid metabolism, immune system, and lipid metabolism. Together, our findings suggest that the inflammatory process is an incisive feature of COVID-19 and red PBM can be used as a novel therapeutic agent for COVID-19 by regulating the inflammatory response. Nevertheless, the need for more clinical trials remains, and there is a significant gap to overcome before clinical trials can commence.
Assuntos
COVID-19 , Animais , Humanos , Peixe-Zebra/metabolismo , SARS-CoV-2/metabolismo , Síndrome da Liberação de Citocina , Citocinas/metabolismo , RNA Mensageiro , Proteínas de Membrana , Proteínas MitocondriaisRESUMO
Regulation of inflammation is a critical process for maintaining physiological homeostasis. The λ-carrageenan (λ-CGN) is a mucopolysaccharide extracted from the cell wall of red algae (Chondrus crispus) capable of inducing acute intestinal inflammation, which is translated into the production of acute phase reactants secreted into the blood circulation. However, the associated mechanisms in vertebrates are not well understood. Here, we investigated the crucial factors behind the inflammatory milieu of λ-CGN-mediated inflammation administered at 0, 1.75, and 3.5% (v/w) by i.p. injection into the peritoneal cavity of adult zebrafish (ZF) (Danio rerio). We found that polymorphonuclear leukocytes (neutrophils) and lymphocytes infiltrating the ZF peritoneal cavity had short-term persistence. Nevertheless, they generate a strong pattern of inflammation that affects systemically and is enough to produce edema in the cavity. Consistent with these findings, cell infiltration, which causes notable tissue changes, resulted in the overexpression of several acute inflammatory markers at the protein level. Using reversed-phase high-performance liquid chromatography followed by a hybrid linear ion-trap mass spectrometry shotgun proteomic approach, we identified 2938 plasma proteins among the animals injected with PBS and 3.5% λ-CGN. First, the bioinformatic analysis revealed the composition of the plasma proteome. Interestingly, 72 commonly expressed proteins were recorded among the treated and control groups, but, surprisingly, 2830 novel proteins were differentially expressed exclusively in the λ-CGN-induced group. Furthermore, from the commonly expressed proteins, compared to the control group 62 proteins got a significant (p < 0.05) upregulation in the λ-CGN-treated group, while the remaining ten proteins were downregulated. Next, we obtained the major protein-protein interaction networks between hub protein clusters in the blood plasma of the λ-CGN induced group. Moreover, to understand the molecular underpinnings of these effects based on the unveiled protein sets, we performed a bioinformatic structural similarity analysis and generated overlapping 3D reconstructions between ZF and humans during acute inflammation. Biological pathway analysis pointed to the activation and abundance of diverse classical immune and acute phase reactants, several catalytic enzymes, and varied proteins supporting the immune response. Together, this information can be used for testing and finding novel pharmacological targets to treat human intestinal inflammatory diseases.
Assuntos
Leucócitos , Proteoma , Peixe-Zebra , Proteínas de Fase Aguda , Animais , Carragenina/metabolismo , Glicosaminoglicanos , Humanos , Inflamação/induzido quimicamente , Neutrófilos/metabolismo , Plasma/metabolismo , Proteômica , Peixe-Zebra/metabolismoRESUMO
The Spike protein (S protein) is a critical component in the infection of the new coronavirus (SARS-CoV-2). The objective of this work was to evaluate whether peptides from S protein could cause negative impact in the aquatic animals. The aquatic toxicity of SARS-CoV-2 Spike protein peptides derivatives has been evaluated in tadpoles (n = 50 tadpoles/5 replicates of 10 animals) from species Physalaemus cuvieri (Leptodactylidae). After synthesis, purification, and characterization of peptides (PSDP2001, PSDP2002, PSDP2003) an aquatic contamination has been simulated with these peptides during 24 h of exposure in two concentrations (100 and 500 ng/mL). The control group ("C") was composed of tadpoles kept in polyethylene containers containing de-chlorinated water. Oxidative stress, antioxidant biomarkers and AChE activity were assessed. In both concentrations, PSPD2002 and PSPD2003 increased catalase and superoxide dismutase antioxidants enzymes activities, as well as oxidative stress (nitrite levels, hydrogen peroxide and reactive oxygen species). All three peptides also increased acetylcholinesterase activity in the highest concentration. These peptides showed molecular interactions in silico with acetylcholinesterase and antioxidant enzymes. Aquatic particle contamination of SARS-CoV-2 has cholinesterasic effect in P. cuvieri tadpoles. These findings indicate that the COVID-19 can constitute environmental impact or biological damage potential.
Assuntos
COVID-19 , SARS-CoV-2 , Animais , Anuros , Humanos , Larva , Glicoproteína da Espícula de CoronavírusRESUMO
Since 1997, Poeciliopsis infans Woolman 1894 has been recognized as a new inhabitant of Lake Patzcuaro, Michoacan in Central Mexico. Between February 1997 and October 1998, nine fish samples were collected at Lake Patzcuaro. Morphometric and meristic counts were conducted on a random selection of 40 organisms of both sexes of Poeciliopsis infans. Males of these viviparous fish posses a modified anal fin called gonopodium. The characteristic hemal spine on the 18th caudal vertebra for this species is described herein and the bony components of the gonopodial structure and suspensoria that together comprise the gonopodial system, which is important for taxonomic studies at various levels of classification were reviewed. Poeciliopsis infans displays a high degree of sexual dimorphism in body shape and anal fin anatomy with the most conspicuous difference observed in anal fin height, which averages 40 percent of SI in males and 17 percent in females. Comparisons between male and female anal fins are described herein as well as the possible impacts of this species on Lake Patzcuaro fish fauna
Assuntos
Animais , Masculino , Feminino , Gônadas , Poecilia , Coluna Vertebral , Poecilia , Caracteres SexuaisRESUMO
Since 1997, Poeciliopsis infans Woolman 1894 has been recognized as a new inhabitant of Lake Patzcuaro, Michoacan in Central Mexico. Between February 1997 and October 1998, nine fish samples were collected at Lake Patzcuaro. Morphometric and meristic counts were conducted on a random selection of 40 organisms of both sexes of Poeciliopsis infans. Males of these viviparous fish posses a modified anal fin called gonopodium. The characteristic hemal spine on the 18th caudal vertebra for this species is described herein and the bony components of the gonopodial structure and suspensoria that together comprise the gonopodial system, which is important for taxonomic studies at various levels of classification were reviewed. Poeciliopsis infans displays a high degree of sexual dimorphism in body shape and anal fin anatomy with the most conspicuous difference observed in anal fin height, which averages 40% of SI in males and 17% in females. Comparisons between male and female anal fins are described herein as well as the possible impacts of this species on Lake Patzcuaro fish fauna.