Bioactivity of substances isolated from natural products on mollusks Biomphalaria glabrata (Say, 1818) (Planorbidae): a review.

Schistosomiasis is a neglected tropical disease caused by parasitic worms of several species of the genus Schistosoma. Transmission occurs by parasitic larvae that stay in freshwater snails of the genus Biomphalaria. Thus, the search for new products that are biodegradable has increased the interest in products of plant origin. The aim of this article is to review the isolated substances from natural products that showed molluscicidal activity against the species Biomphalaria glabrata in order to reevaluate the most promising prototypes and update the progress of research to obtain a new molluscicide. We perform searches using scientific databases, such as Scientific Electronic Library Online (SciELO), Google schoolar, PUBMED, Web of Science and Latin American and Caribbean Literature on Health Sciences (LILACS). From 2000 to 2022, using the keywords "isolated substances", "molluscicidal activity" and "Biomphalaria glabrata". In the present study, it was possible to observe 19 promising molluscicidal molecules with a lethal concentration below 20 µg/mL. Of these promising isolates, only 5 isolates had the CL90 calculated and within the value recommended by WHO: Benzoic acid, 2',4',6'-Trihydroxydihydrochalcone, Divaricatic acid, Piplartine and 2-hydroxy-1,4-naphthoquinone (Lapachol). We conclude that beyond a few results in the area, the researches don't follow the methodological pattern (exposure time and measure units, toxicity test), in this way, as they don't follow a pattern on the result's exposure (LC), not following, in sum, the recommended by WHO.

Comparative evaluation of plant extract effects on peritoneal, medullary and J774 cells. G8 macrophages.

The use of medicinal plants as raw material for extracts production and pure substances isolation and subsequence development of new drugs represents a constantly growing area. However, some stages are indispensable before pharmacologically evaluating natural products such as medicines. Toxicity tests in mammalian cells are essential to initiate new drugs development or verify the substance's biocompatibility. Thus, we verified the toxicity of crude extracts and fractions with different polarities obtained from the leaves and stems of eight plant species. The toxic effect was evaluated on macrophages obtained from the bone marrow and peritoneal cavity of a Swiss webster mouse and J774 macrophages. G8 cell lineage. These macrophages were cultured in a 96-well plate, and the compounds were added at a concentration of 100 µg/mL for 24 hours. After this time, the supernatant was removed. The toxicity was evaluated for lactate dehydrogenase (LDH) release assay and the resazurin assay, which uses an indicator dye to measure oxidation-reduction reactions. The results showed a difference in the percentage of toxicity when comparing the same extract in different types of macrophages. This outcome indicates that these cells from different origins may exhibit different responses when exposed to the same natural compounds.

Natural products as a control measure of the Achatina fulica (Gastropoda: Achatinidae).

Achatina fulica is a terrestrial mollusk known as the giant African snail that is related to environmental, economic, urban, and public health problems. As control measures for this mollusk, cooking salt (NaCl) and calcium oxide (CaO) are used, and baits are composed of metaldehyde. However, these measures have environmental toxicity and impact the soil. In this way, natural products have been tested on this mollusk to discover and develop a substance to combat this urban and agricultural pest. This article aims to evaluate studies involving natural products to control the population of Achatina fulica. Articles and works published in books were included in the present work. A total of 1,103 works were found during the search. Of these, 14 works met the objective of these review and were included in this article. The tests do not possess methodological standardization, do not have a maximum concentration to be considered active, or a maximum exposure time. A lack of standardization in the methodology of tests on A. fulica was observed. The performance of tests on other life stages of the mollusk, as well as tests that analyze other parameters, are essential. Only one article analyzed presented phytochemical analysis. No ecotoxicity tests were reported either. Some extracts showed promising results, highlighting the aqueous extract of Capsicum frutescens. More studies investigating the molluscicidal activity of natural products on A. fulica are needed. It is very relevant that the new studies present a phytochemical analysis of the tested extracts, as well as ecotoxicity studies.

Physalin pool from Physalis angulata L. leaves and physalin D inhibit P2X7 receptor function in vitro and acute lung injury in vivo.

P2X7 receptor promotes inflammatory response and neuropathic pain. New drugs capable of impairing inflammation and pain-reducing adverse effects extracted from plant extracts have been studied. Physalis angulate L. possesses traditional uses and exhibits antiparasitic, anti-inflammatory, antimicrobial, antinociceptive, antimalarial, antileishmanial, immunosuppressive, antiasthmatic. diuretic, and antitumor activities. The most representative phytochemical constituents identified with medicinal importance are the physalins and withanolides. However, the mechanism of anti-inflammatory action is scarce. Although some physalins and withanolides subtypes have anti-inflammatory activity, only four physalins subtypes (B, D, F, and G) have further studies. Therefore, we evaluated the crude ethanolic extract enriched with physalins B, D, F, and G from P. angulata leaves, a pool containing the physalins B, D, F, G, and the physalins individually, as P2X7 receptor antagonists. For this purpose, we evaluated ATP-induced dye uptake, macroscopic currents, and interleukin 1-ß (IL-1ß) in vitro. The crude extract and pool dose-dependently inhibited P2X7 receptor function. Thus, physalin B, D, F, and G individually evaluated for 5'-triphosphate (ATP)-induced dye uptake assay, whole-cell patch-clamp, and cytokine release showed distinct antagonist levels. Physalin D displayed higher potency and efficacy than physalin B, F, and G for all these parameters. In vivo mice model as ATP-induced paw edema was potently inhibited for physalin D, in contrast to physalin B, F, and G. ATP and lipopolysaccharide (LPS)-induced pleurisy in mice were reversed for physalin D treatment. Molecular modeling and computational simulation predicted the intermolecular interactions between the P2X7 receptor and physalin derivatives. In silico results indicated physalin D and F as a potent allosteric P2X7 receptor antagonist. These data confirm physalin D as a promisor source for developing a new P2X7 receptor antagonist with anti-inflammatory action.

8-Hydroxy-2-(1H-1,2,3-triazol-1-yl)-1,4-naphtoquinone derivatives inhibited P2X7 Receptor-Induced dye uptake into murine Macrophages.

Extracellular adenosine 5'-triphosphate (ATP) triggers the P2X7 receptor (P2X7R) ionic channel to stimulate the release of the interleukin-IL-1ß cytokine into macrophages. The current study explored the reaction of six structurally diverse triazole derivatives on P2X7-mediated dye uptake into murine peritoneal macrophages. P2X7R activity determined by ATP-evoked fluorescent dye uptake. Triazole derivatives toxicity measured using dextran rhodamine exclusion based colorimetric assay. A740004 and BBG, both P2X7R antagonist, inhibited ATP-induced dye uptake. In contrast, the derivatives 5a, 5b, 5e, and 5f did not diminish P2X7R activity in concentrations until 100 µM. 5c and 5d analogs caused a potent inhibitory activity on P2X7-induced dye uptake. Dextran Rhodamine exclusion measurements after 24 h of continuous treatment with triazole derivatives indicated a moderated toxicity for all molecules. In conclusion, this study showed that a series of new hybrid 1,2,3-triazolic naphthoquinones reduces P2X7R-induced dye uptake into murine macrophages. In silico analysis indicates a good pharmacokinetic profile and molecular docking results of these analogs indicate the potential to bind into an allosteric site located into the P2X7R pore and juxtaposed with the ATP binding pocket. In this manner, the compounds 5c and 5d may be used as a scaffold for new P2X7R inhibitors with reduced toxicity, and good anti-inflammatory activity.

1,4-Naphthoquinones potently inhibiting P2X7 receptor activity.

P2X7 receptor (P2X7R) is an ATP-gated ion-channel with potential therapeutic applications. In this study, we prepared and searched a series of 1,4-naphthoquinones derivatives to evaluate their antagonistic effect on both human and murine P2X7 receptors. We explored the structure-activity relationship and binding mode of the most active compounds using a molecular modeling approach. Biological analysis of this series (eight analogues and two compounds) revealed significant in vitro inhibition against both human and murine P2X7R. Further characterization revealed that AN-03 and AN-04 had greater potency than BBG and A740003 in inhibiting dye uptake, IL-1ß release, and carrageenan-induced paw edema in vivo. Moreover, we used electrophysiology and molecular docking analysis for characterizing AN-03 and AN-04 action mechanism. These results suggest 1,4-napthoquinones, mainly AN-04, as potential leads to design new P2X7R blockers and anti-inflammatory drugs.

TRPing on the pore phenomenon: what do we know about transient receptor potential ion channel-related pore dilation up to now?

Ion channels allow for rapid ion diffusion through the plasma membrane. In some conditions, ion channels induce changes in the critical plasma membrane permeability that permit 900-Da solutes to enter cells. This process is known as the pore phenomenon. Some transient receptor potential (TRP) channel subtypes have been highlighted such as the P2X7 receptor, plasma membrane VDAC-1 channel, and pannexin hemichannels. The TRP ion channels are considered multimodal transducers that respond to several kinds of stimuli. In addition, many TRP channel subtypes are involved in physiological and pathophysiological processes such as inflammation, pain, and cancer. The TRPA1, TRPM8, and TRPV1-4 subtypes have been shown to promote large-molecular-weight solute uptake, including impermeable fluorescent dyes, QX-314 hydrophilic lidocaine derivative, gabapentin, and antineoplastic drugs. This review discusses the current knowledge of TRP-associated pores and encourages scientists to study their features and explore them as novel therapeutic tools.

Is pannexin the pore associated with the P2X7 receptor?

The P2X7 receptor (P2X7R), an ATP-gated cation channel, is expressed predominantly in leukocytes. Activation of P2X7R has been implicated in the formation of a cytolytic pore (i.e., a large conductance channel) that allows the passage of molecules up to 900 Da in macrophages. At least two hypotheses have been presented to explain the conversion of a nonselective cation channel to a cytolytic pore. One hypothesis suggests that the pore is a separate molecular structure activated by P2X7R, and the second asserts that this is an intrinsic property of P2X7R (pore dilation). Based on connexin knockout and hemichannel antagonist studies, some groups have concluded that connexins and pannexins, the hemichannel-forming proteins in vertebrates, are fundamental components of the large conductance channel associated with P2X7R. Dye uptake and electrophysiology experiments were used to evaluate the efficacy and specificity of some hemichannel antagonists under conditions known to open the large conductance channel associated with P2X7R. Hemichannel antagonists and interference RNA (RNAi) targeting pannexin-1 did not affect P2X7R macroscopic currents [ATP, 1,570±189 pA; ATP+100 µM carbenoxolone (CBX), 1,498±100 pA; ATP+1 mM probenecid (Prob), 1,522±9 pA] or dye uptake in a FACS assay (ATP, 63±5%; ATP+100 µM CBX, 51.51±8.4%; ATP+1 mM Prob, 57.7±4.3%) in mouse macrophages. These findings strongly suggest that the high-permeability pore evident after prolonged P2X7R activation does not occur through connexin or pannexin hemichannels in murine macrophages. Another membrane protein may be involved in P2X7R pore formation.

Large-conductance channel formation mediated by P2X7 receptor activation is regulated through distinct intracellular signaling pathways in peritoneal macrophages and 2BH4 cells.

The P2X(7) receptor (P2X7R) is a ligand-gated ATP receptor that acts as a low- and large-conductance channel (pore) and is known to be coupled to several downstream effectors. Recently, we demonstrated that the formation of a large-conductance channel associated with the P2X(7) receptor is induced by increasing the intracellular Ca(2+) concentration (Faria et al., Am J Physiol Cell Physiol 297:C28-C42, 2005). Here, we investigated the intracellular signaling pathways associated with P2X(7) large-conductance channel formation using the patch clamp technique in conjunction with fluorescent imaging and flow cytometry assays in 2BH4 cells and peritoneal macrophages. Different antagonists were applied to investigate the following pathways: Ca(2+)-calmodulin, phospholipase A, phospholipase D, phospholipase C, protein kinase C (PKC), mitogen-activated protein kinase (MAPK), MAPK/extracellular signal-regulated kinase, phosphoinositide 3-kinase (PI3K), and cytoskeletal proteins. Macroscopic ionic currents induced by 1 mM ATP were reduced by 85% in the presence of PKC antagonists. The addition of antagonists for MAPK, PI3K, and the cytoskeleton (actin, intermediary filament, and microtubule) blocked 92%, 83%, and 95% of the ionic currents induced by 1 mM ATP, respectively. Our results show that PKC, MAPK, PI3K, and cytoskeletal components are involved in P2X(7) receptor large-channel formation in 2BH4 cells and peritoneal macrophages.

Pharmacological properties of a pore induced by raising intracellular Ca2+.

Recent studies on the P2X(7) receptor in 2BH4 cells and peritoneal macrophages have demonstrated that the raise in intracellular Ca(2+) concentration induces a pore opening similar to P2X(7) receptor pore. Herein, we have investigated whether the pore activated by the elevation of intracellular Ca(2+) concentration is associated to P2X(7) receptor. Using patch clamp in cell attached, whole cell configuration, and dye uptake, we measured the pore opening in cell types that express the P2X(7) receptor (2BH4 cells and peritoneal macrophages) and in cells that do not express this receptor (HEK-293 and IT45-RI cells). In 2BH4 cells, the stimulation with ionomycin (5-10 microM) increased intracellular free Ca(2+) concentration and induced pore formation with conductance of 421 +/- 14 pS, half-time (t(1/2)) for ethidium bromide uptake of 118 +/- 17 s, and t(1/2) for Lucifer yellow of 122 +/- 11 s. P2X(7) receptor antagonists did not block these effects. Stimulation of HEK-293 and IT45-RI cells resulted in pore formation with properties similar to those found for 2BH4 cells. Connexin hemichannel inhibitors (carbenoxolone and heptanol) also did not inhibit the pore-induced effect following the increase in intracellular Ca(2+) concentration. However, 5-(N,N-hexamethylene)-amiloride, a P2X(7) receptor pore blocker, inhibited the induced pore. Moreover, intracellular signaling modulators, such as calmodulin, phospholipase C, mitogen-activated protein kinase, and cytoskeleton components were important for the pore formation. Additionally, we confirmed the results obtained for electrophysiology by using the flow cytometry, and we discarded the possibility of cellular death induced by raising intracellular Ca(2+) at the doses used by using lactate dehydrogenase release assay. In conclusion, increased concentration in intracellular Ca(+2) induces a novel membrane pore pharmacologically different from the P2X(7) associated pore and hemigap-junction pore.

Are second messengers crucial for opening the pore associated with P2X7 receptor?

Stimulation of the P2X7 receptor by ATP induces cell membrane depolarization, increase in intracellular Ca2+ concentration, and, in most cases, permeabilization of the cell membrane to molecules up to 900 Da. After the activation of P2X7, at least two phenomena occur: the opening of low-conductance (8 pS) cationic channels and pore formation. At least two conflicting hypotheses have been postulated to reconcile these findings: 1) the P2X7 pore is formed as a result of gradual permeability increase (dilation) of cationic channels, and 2) the P2X7 pore represents a distinct channel, possibly activated by a second messenger and not directly by extracellular nucleotides. In this study, we investigated whether second messengers are necessary to open the pore associated with the P2X7 receptor in cells that expressed the pore activity by using the patch-clamp technique in whole cell and cell-attached configurations in conjunction with fluorescent imaging. In peritoneal macrophages and 2BH4 cells, we detected permeabilization and single-channel currents in the cell-attached configuration when ATP was applied outside the membrane patch in a condition in which oxidized ATP and Lucifer yellow were maintained within the pipette. Our data support Ca2+ as a second messenger associated with pore formation because the permeabilization depended on the presence of intracellular Ca2+ and was blocked by BAPTA-AM. In addition, MAPK inhibitors (SB-203580 and PD-98059) blocked the permeabilization and single-channel currents in these cells. Together our data indicate that the P2X7 pore depends on second messengers such as Ca2+ and MAP kinases.

Hernioplastia diafragmática em cão com pericárdio bovino conservado em solução supersaturada de açúcar / Diaphragmatic hernioplasty in dogs with bovine pericardium preserved in supersaturated sugar solution

Foram utilizados sete cães adultos, três machos e quatro fêmeas, sem raça definida, com pesos entre 10 e 22kg, para avaliação do processo cicatricial do músculo diafragma, na presença do implante de pericárdio bovino conservado em solução supersaturada de açúcar a 300 por cento. Foi criado um defeito na porção muscular do hemidiafragma esquerdo de dimensões 8,0 x 5,0cm. Após a toracotomia no oitavo espaço intercostal esquerdo, o implante heterógeno foi fixado com fio poliamida nº 3-0 por meio de sutura simples contínua. Decorrido o período pré-estabelecido de pós-operatório, os animais foram submetidos a exames radiográficos simples e contrastado e a estudos macroscópico e histológico. Na avaliação radiográfica, foi verificada presença das silhuetas diafragmática e cardíaca, sem evidências de vísceras abdominais no interior do tórax. Macroscopicamente, notou-se a formação de tecido conjuntivo fibroso semi-transparente que ocluia o defeito diafragmático. O segmento de pericárdio bovino conservado em solução supersaturada de açúcar a 300 por cento, em temperatura ambiente, é substituído por uma fina camada de tecido conjuntivo fibroso e promove a restauração do defeito no músculo diafragma de cão

Reparaçäo do diafragma de cäes com segmento muscular homólogo ortotópico conservado em soluçäo supersaturada de açúcar / Diaphragm repair in dogs with muscular homologous ortothopic segment preserved in supersaturated sugar solution

0 uso de enxerto muscular homólogo conservado em soluçäo supersaturada de açúcar a 300 por cento foi pesquisado no músculo diafragma de cäes. Foram utilizados 12 cäes adultos, quatro machos e oito fêmeas, sem raça definida, com peso entre 9 e l8kg, para confecçäo de um defeito diafragmático na porçäo muscular, com dimensöes de 4,0 x 4,5cm, seguido da implantaçäo de um segmento de músculo diafragma homólogo. Seis cäes foram observados por um período de 30 dias de pós-operatório e seis por 60 dias, quando foram reoperados para observaçäo macroscópica e coleta de amostra para avaliaçäo histológica. Nos animais do grupo de 30 dias de pós-operatório verificou-se substituiçäo parcial e nos de 60 dias, substituiçäo total da porçäo muscular do diafragma por tecido de granulaçäo, o que permitiu o restabelecimento completo do diafragma por meio de firme inserçäo. 0 segmento de músculo diafragma homólogo conservado em soluçäo supersaturada de açúcar a 300 por cento, em temperatura ambiente, pode ser utilizado para reparaçäo de defeitos diafragmáticos, uma vez que é substituido por tecido conjuntivo fibroso, sem apresentar sinais clínicos nem histológico de rejeiçäo