RESUMO
Small RNAs (sRNAs) and microRNAs (miRNAs) are small endogenous noncoding single-stranded RNAs that regulate gene expression in eukaryotes. Experiments in mice and humans have revealed that a typical small RNA can affect the expression of a wide range of genes, implying that small RNAs function as global regulators. Here, we used small RNA deep sequencing to investigate how jararhagin, a metalloproteinase toxin produced from the venom of Bothrops jararaca, affected mmu-miRNAs expression in mice 2 hours (Jar 2hrs) and 24 hours (Jar 24hrs) after injection compared to PBS control. The findings revealed that seven mmu-miRNAs were substantially differentially expressed (p value (p (Corr) cut-off 0.05, fold change ≥ 2) at 2 hrs after jararhagin exposure and that the majority of them were upregulated when compared to PBS. In contrast to these findings, a comparison of Jar 24hrs vs. PBS 24hrs demonstrated that the majority of identified mmu-miRNAs were downregulated. Furthermore, the studies demonstrated that mmu-miRNAs can target the expression of several genes involved in the MAPK signaling pathway. The steady antithetical regulation of mmu-miRNAs may correlate with the expression of genes that trigger apoptosis via MAPK in the early stages, and this effect intensifies with time. The findings expand our understanding of the effects of jararhagin on local tissue lesions at the molecular level.
Assuntos
Bothrops , Venenos de Crotalídeos , MicroRNAs , Animais , Bothrops/metabolismo , Venenos de Crotalídeos/metabolismo , Humanos , Metaloendopeptidases/metabolismo , Metaloproteases/metabolismo , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Músculo Esquelético/metabolismo , Veneno de Bothrops jararacaRESUMO
Jararhagin is a hyperalgesic metalloproteinase from Bothrops jararaca venom. In rodents, jararhagin induces nociceptive behaviors that correlate with an increase in peripheral cytokine levels. However, the role of the spinal cord glia in pain processing after peripheral stimulus of jararhagin has not been investigated. Aiming to explore this proposal, mice received intraplantar (i.pl.) injection of jararhagin and the following parameters were evaluated: hyperalgesia, spinal cord TNF-α, IL-1ß levels, and CX3CR1, GFAP and p-NFκB activation. The effects of intrathecal (i.t.) injection of TNF-α soluble receptor (etanercept), IL-1 receptor antagonist (IL-1Ra), and inhibitors of NFκB (PDTC), microglia (minocycline) and astrocytes (α-aminoadipate) were investigated. Jararhagin inoculation induced cytokine production (TNF-α and IL-1ß) in the spinal cord, which was reduced by treatment with PDTC (40% and 50%, respectively). Jararhagin mechanical hyperalgesia and cytokine production were inhibited by treatment with etanercept (67%), IL-1Ra (60%), PDTC (70%), minocycline (60%) and α-aminoadipate (45%). Furthermore, jararhagin induced an increase in p-NFκB, CX3CR1 and GFAP detection in the spinal cord indicating activation of NFκB, microglia and astrocytes. These results demonstrate for the first time that jararhagin-induced mechanical hyperalgesia is dependent on spinal cord activation of glial cells, consequent NFκB activation, and cytokine production in mice.
Assuntos
Astrócitos/efeitos dos fármacos , Venenos de Crotalídeos/toxicidade , Hiperalgesia , Metaloendopeptidases/toxicidade , Microglia/efeitos dos fármacos , Dor , Medula Espinal/efeitos dos fármacos , Animais , Bothrops/metabolismo , Citocinas/metabolismo , Hiperalgesia/induzido quimicamente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Dor/induzido quimicamente , Veneno de Bothrops jararacaRESUMO
Snakebite envenoming affects millions of people worldwide, being officially considered a neglected tropical disease by the World Health Organization. The antivenom is effective in neutralizing the systemic effects of envenomation, but local effects are poorly neutralized, often leading to permanent disability. The natural resistance of the South American pit viper Bothrops jararaca to its venom is partly attributed to BJ46a, a natural snake venom metalloendopeptidase inhibitor. Upon complex formation, BJ46a binds non-covalently to the metalloendopeptidase, rendering it unable to exert its proteolytic activity. However, the structural features that govern this interaction are largely unknown. In this work, we applied structural mass spectrometry techniques (cross-linking-MS and hydrogen-deuterium exchange MS) and in silico analyses (molecular modeling, docking, and dynamics simulations) to understand the interaction between BJ46a and jararhagin, a metalloendopeptidase from B. jararaca venom. We explored the distance restraints generated from XL-MS experiments to guide the modeling of BJ46a and jararhagin, as well as the protein-protein docking simulations. HDX-MS data pinpointed regions of protection/deprotection at the interface of the BJ46a-jararhagin complex which, in addition to the molecular dynamics simulation data, reinforced our proposed interaction model. Ultimately, the structural understanding of snake venom metalloendopeptidases inhibition by BJ46a could lead to the rational design of drugs to improve anti-snake venom therapeutics, alleviating the high morbidity rates currently observed.
Assuntos
Bothrops , Venenos de Crotalídeos , Animais , Espectrometria de Massas , Metaloendopeptidases , Veneno de Bothrops jararacaRESUMO
Jararhagin, a metalloprotease from Bothrops jararaca snake venom, is a toxin containing the metalloproteinase, disintegrin-like and cysteine-rich domains; it causes acute inflammation and damage to vascular tissue. However, the actions of these domains on key components of chronic inflammation have not been determined. Our aim was to investigate the effects of jararhagin (Jar), jararhagin-C (Jar-C) and o-phenantrolin-treated jararhagin (Jar-Phe), on inflammatory response, blood vessel formation and extracellular matrix deposition in the murine sponge model. The polyether-polyurethane sponge matrix was implanted into Balb/c mice and injected daily with Jar (400â¯ng), Jar-Phe (400â¯ng), Jar-C (200â¯ng) or saline (control). Nine days after implantation, the sponge discs were removed and processed. In the Jar-treated implants, some of inflammatory markers (N-acetyl-ß-d-glucosaminidase activity, CCL2 and TNF-α) and TGF-ß1 levels were higher compared with the control group. In the Jar-C group, the inflammatory markers myeloperoxidase activity and CXCL1 were higher compared with the control. In this group, VEGF levels and collagen deposition were also higher. Jar-Phe treatment was able to inhibit the activity and/or production of MPO, CXCL1, CCL2 and TGF-ß. The differential effects of these proteins in modulating the main components of fibrovascular tissue may be exploited in the management fibroproliferative diseases.
Assuntos
Venenos de Crotalídeos/química , Venenos de Crotalídeos/farmacologia , Metaloendopeptidases/química , Metaloendopeptidases/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Venenos de Serpentes/enzimologia , Animais , Biomarcadores/metabolismo , Fibrose/induzido quimicamente , Fibrose/metabolismo , Hemoglobinas/metabolismo , Inflamação/induzido quimicamente , Masculino , Camundongos , Domínios Proteicos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Veneno de Bothrops jararacaRESUMO
This article describes the data on the global expression profile of small RNA (smRNAs) molecules in mice gastrocnemius muscle exposed to jararhagin, snake venom metalloproteinase. The data include smRNAs in mice gastrocnemius muscle challenged with Jararhagin (Jar; n=4) in the right paw or phosphate-buffered saline (PBS; control; n=4) in the left paw. smRNA-Seq libraries were generated after 24 h of exposure to PBS or jararhagin. The expression profiles of smRNAs including microRNA and snoRNA were compared between both groups. The sequencing data from both groups have been uploaded to Zenodo http://dx.doi.org/10.5281/zenodo.56492.
RESUMO
Jararhagin is a hemorrhagic metalloprotease from Bothrops jararaca snake venom. The hyperalgesic mechanisms of jararhagin were investigated focusing on the role of proinflammatory cytokines (TNF-α and IL-1ß) and the transcription factor NFκB. Intraplantar administration of jararhagin (1, 10, 100 and 1000 ng/paw) induced mechanical hyperalgesia, and increased TNF-α levels at 1, 3 and 5 h, and IL-1ß levels at 0.5, 1 and 3 h after its injection in the paw tissue. Pre-treatment with morphine (2, 6, 12 µg/paw) inhibited jararhagin-induced mechanical hyperagesia. The systemic or local pre-treatment with etanercept (10 mg/kg and 100 µg/paw) and IL-1ra (30 mg/kg and 100 pg/paw) inhibited jararhagin-induced mechanical hyperalgesia. Co-administration of jararhagin (0.1 ng/paw) and TNF-α (0.1 pg/paw) or jararhagin (0.1 ng/paw) and IL-1ß (1 pg/paw) enhanced the mechanical hyperalgesia. The systemic or local pre-treatment with PDTC (NFκB inhibitor; 100 mg/kg and 100 µg/paw) inhibited jararhagin-induced mechanical hyperalgesia as well as PDTC decreased the jararhagin-induced production of TNF-α and IL-1ß. Thus, these data demonstrate the involvement of pro-inflammatory cytokines TNF-α and IL-1ß and nuclear transcription factor NFκB in jararhagin-induced mechanical hyperalgesia indicating that targeting these mechanisms might contribute to reduce the pain induced by B. jararaca snake venom.