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. 2025 Jun 23;99(10):4281–4289. doi: 10.1007/s00204-025-04109-w

Crotalus durissus terrificus snake venom activates 3T3-L1 preadipocytes to release PGE2, which depends on COX-2 expression and is modulated by PGE2 EP2 receptor

Danilo Santos Teixeira 1,#, Rodrigo Maia-Marques 1,✉,#, Catarina Teixeira 1,✉
PMCID: PMC12454571  PMID: 40550992

Abstract

Snakebites are a worldwide public health problem with high-cost treatment in many countries. Crotalus genus snakes, commonly known as rattlesnakes, cause the most lethal snakebites in Brazil. Crotalus ssp envenomation is characterised by systemic neurotoxicity, myotoxicity, renal failure and minor local effects. Despite the knowledge of envenomation’s pathogenesis, the impact of Crotalus venom on adipose tissue (AT) cells is unknown. AT is an endocrine organ capable of releasing diverse immunomodulatory molecules, including prostaglandin E2 (PGE2). Herein, we investigated the effects of Crotalus durissus terrificus venom (CdtV) on preadipocytes in vitro, focussing on the release of PGE2 and mechanisms involved. CdtV (5 and 10 μg/mL) induced a marked release of PGE2 by preadipocytes (3–24 h) compared to controls. Pre-treatment of cells with SC-560 or NS-398, selective inhibitors for cyclooxygenase (COX)-1 and COX-2 enzymes, respectively, decreased CdtV-induced PGE2 release after 24 h. CdtV (1 and 10 μg/mL) did not change COX-1 protein expression by preadipocytes, but induced COX-2 protein expression at all time intervals evaluated. Pre-treating preadipocytes with AH6809, an inhibitor of the PGE2 EP2 receptor, significantly increased the CdtV-induced PGE2 release. EP1, EP3, or EP4 receptor antagonists did not change the CdtV-induced PGE2 release. Additionally, CdtV did not alter the EP2 receptor protein expression in preadipocytes. These findings demonstrate that CdtV activates preadipocytes to produce PGE2 via COX-1 activation and COX-2 protein expression and PGE2 triggers a negative feedback loop via EP2 for its own production. These results highlight AT as another target for CdtV that may contribute to envenomation.

Keywords: Crotalus durissus snake venom, Preadipocyte, Prostaglandin E2, PGE2 receptors

Introduction

Snakebites are a worldwide public health problem with high-cost treatment in many countries (Gutiérrez et al. 2011). The Viperidae and Elapidae families comprise clinically important groups of venomous snakes (Bucaretchi et al. 2016; da Silva et al. 2024). The Viperidae family is responsible for the majority of envenomation, whereas the genus Crotalus causes the highest number of deaths from snakebites in Brazil (Gutiérrez et al. 2009, 2010). The most important effects of envenomation by the Crotalus species are systemic neurotoxicity, myotoxicity, coagulopathy, and acute renal failure associated with myotoxicity, culminating in failure of end organs and death (Frare et al. 2019). Moreover, researchers have reported that Crotalus venom induces hypotension and bradycardia (Simões et al. 2021). In contrast to the Bothrops genus venoms (Viperidae family), which are proinflammatory, Crotalus durissus terrificus venom has unique features that does not induce an inflammatory response at the bite site (Baudou et al. 2023). Reports on clinical observations of CdtV envenomation victims indicate minor inflammatory signs and symptoms at the bite site, such as slight oedema and erythema as well as mild pain and local or regional paraesthesia that may persist for different periods of time (Rosenfeld 1971; De Carvalho et al. 2019). Systemic neuro- and myotoxicity induced by C. durissus ssp. bites have been associated with a high concentration of crotoxin, CdtV’s major component that can induce peripheral neuromuscular paralysis and cardiorespiratory failure, which are some of its main lethal effects (D Vaz de Melo et al. 2020). In addition to neurotoxicity, crotoxin induces the biosynthesis of the eicosanoids, such as lipoxin A4 (LXA4) via the lipoxygenase pathway as well as the prostaglandins E2, D2 and 15-d-PGJ2 by macrophages via the cyclooxygenase (COX)-1 pathway as well as cross talk with the endogenous Ca2+-independent PLA2 (Sampaio et al. 2006; Giannotti et al. 2017). The release of PGE2 by macrophages is also induced by the crude venom of CdtV (Sampaio et al. 2006).

PGE2 is an important mediator that modulates a series of physiological processes, including vascular tonus, blood pressure (Stock et al. 2001), kidney pressure (Wang et al. 2019), and gastric homeostasis (Takeuchi and Amagase 2018). PGE2 synthesis is regulated by key enzymes in the COX system. Moreover, PGE2’s wide-range effects are mediated by four receptor subtypes (EP1–4) (Sugimoto and Narumiya 2007), which differ in tissue distribution, ligand-binding affinity, and coupling to distinct intracellular signalling pathways. In pathophysiological conditions, however, systemic levels of PGE2 drastically increase. In this condition, PGE2 has been described as an important lipid mediator with dual effects during the inflammatory response. Notably, this mediator can positively or negatively modulate the inflammatory reaction depending on the microenvironment. On the inflammatory side, PGE2 potentiates the increase of vascular permeability induced by vasoactive amines and other mediators, thus leading to oedema formation. Regarding anti-inflammatory effects, researchers have demonstrated that PGE2 stimulates the release of the anti-inflammatory mediators interleukin-10 and LXA4 (MacKenzie et al. 2013; Loynes et al. 2018). In this sense CdtV inhibits the macrophages’ phagocytotic activity in vitro via production of LXA4 by these cells (Sampaio et al. 2006). Additionally, PGE2 has been associated with the nephrotoxic effect of Crotalus durissus ssp venom: C. durissus cascavella has been associated to kidney cell production of prostaglandins and other mediators. However, Crotalus venom’s ability to stimulate other PGE2 sources remains unknown. Regarding production of PGE2, the adipose tissue (AT) is largely known to produce and be regulated by this lipid mediator.

AT is widely distributed throughout the human body, producing and secreting an array of inflammatory mediators that contribute to the development and regulation of diverse inflammatory diseases. This is evidenced by AT’s participation in obesity, rheumatoid arthritis, and COVID-19 (Giles et al. 2018; Kawai et al. 2021; Yu et al. 2022). AT primarily comprises mature adipocytes and the stromal vascular fraction (SVF), which generates many of the mediators secreted by this tissue. Within the SVF, preadipocytes (the precursors of mature adipocytes), account for up to 50% of human AT cells (Dordevic et al. 2014). These cells are more active than mature adipocytes due to the high metabolic activity which is needed to support proliferation and differentiation into mature adipocytes as well as adaptation to the microenvironment. Moreover, preadipocytes have important immune and haemostatic functions (Charrière et al. 2006; Dashty et al. 2012). Despite the AT’s importance to physiological homeostasis and pathophysiological conditions, no evidence indicates that AT cells are activated by Crotalus venom; therefore, we hypothesise that CdtV activates AT to produce pro and/or anti-inflammatory mediators. Furthermore, adipocytes can produce and release mediators which modulate the inflammatory response. In this study we investigated the effects of CdtV on preadipocytes in culture, focussing on the release of PGE2 and the mechanisms involved in this effect. The findings reported herein demonstrate that CdtV activates preadipocytes to produce PGE2 by a mechanism dependent on COX-1 activation and COX-2 protein expression. Additionally, the PGE2 released by this venom stimulates a negative feedback loop via EP2 for its own production.

Material and methods

Venom, chemicals and reagents

Crotalus durissus terrificus venom (CdtV) was collected, lyophilized, identified and provided by the Herpetology Laboratory of Instituto Butantan. The venom batches used were tested for endotoxin contamination using the quantitative limulus amoebocyte lysate test (Takayama et al. 1994), which revealed undetectable levels of endotoxin (< 0.125 EU/mL). The venom was reconstituted in sterile PBS and filtered immediately before use. L-glutamine was purchased from USB (Cleveland, OH, USA). Dulbecco’s Modified Eagle Medium (DMEM) and Foetal Bovine Serum (FBS) were purchased from Life Technologies (São Paulo, SP, Brazil); gentamicin was purchased from Schering-Plough (Whitehouse Station, NJ, USA); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO) and mouse anti-β-actin monoclonal antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA); and polyclonal antibody against COX-1, the PGE2 enzyme immunoassay kit and compounds SC-560, NS-398, SC-19220, AH6890, L-798106 and GW 627368X were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Polyclonal antibody against COX-2, HRP-conjugated anti-mouse secondary antibody were purchased from Thermo Fisher (Waltham, Massachusetts, USA). HRP-conjugated anti-rabbit secondary antibody and nitrocellulose membrane were purchased from GE Healthcare (Buckinghamshire, UK).

3T3-L1 cell culture

3T3-L1 murine preadipocytes obtained from the American Type Culture Collection were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% (v/v) FBS until confluence. Before stimulation with CdtV, FBS in the media was replaced by 0.2% Bovine Serum Albumin (BSA).

Quantification of PGE2

Quantification of PGE2 was performed in the supernatants collected from cell cultures by enzyme immunoassay (EIA) using a commercially available kit (Cayman Chemicals, ThermoFisher). The tests were performed according to the supplier’s specifications. Concentrations were estimated from the standard curve and represented in pg/mL.

Pharmacological interventions

To evaluate the participation of COX-1, COX-2 and each PGE2 receptor subtype in the CdtV-induced effects, pharmacological interventions were performed with selective inhibitors or antagonists in concentrations described in the literature (Choi et al. 2008; Lin et al. 2008, 2009; Chen et al. 2012): 1 μM SC-560 (COX-1 inhibitor, 1 h before CdtV); 1 μM NS-398 (COX-2 inhibitor, 1 h before CdtV); 10 μM SC-19220 (EP1 receptor antagonist, 1 h before CdtV); 10 μM AH 6809 (EP2 receptor antagonist, 1 h before CdtV); 1 μM L-798,106 (EP3 receptor antagonist, 1 h before CdtV); 10 μM GW 627368X (EP4 receptor antagonist, 1 h before CdtV). Some of the used compounds were prepared in DMSO at concentration lower than 1%. Cells treated with the inhibitors were analysed for viability by the MTT colorimetric assay. No significant changes in cell viability were registered with any of the above agents or vehicles at the concentrations used.

Western blotting

The protein content of COX-1, COX-2 and EP2 receptor was determined in cell lysates by western blotting. For this purpose, the cells incubated or not with CdtV were lysed by adding 100 μL/well of Laemmli buffer (0.5 M Tris–HCl, 20% SDS, 1% glycerol, 1 M β-mercaptoethanol, 0.1% bromophenol blue) and boiled at 100 °C for 10 min. Samples were resolved by SDS-PAGE (12% bis-acrylamide gel) electrophoresis. The proteins were transferred to a nitrocellulose membrane with a Mini Trans-Blot (Bio-Rad Laboratories, Richmond, CA, USA). The membranes were blocked for 1 h with 5% non-fat dry milk in Tris-buffered saline Tween 20 (20 mM Tris, 100 mM NaCl and 0.5% Tween 20, pH 7.2) and incubated overnight at 4 °C with COX-1, COX-2 or EP2 primary antibodies (1:1000 dilution) and for 1 h at room temperature with the β-actin primary antibody (1:3000 dilution). Next, the membranes were washed and incubated with the appropriate secondary antibody conjugated to horseradish peroxidase. Immunoreactive bands were detected using an entry-level peroxidase substrate for enhanced chemiluminescence (Pierce ECL Western Blotting Substrate) according to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA). Band images were captured with an ImageQuant LAS 4000 mini biomolecular imager (GE Healthcare) and analyzed with ImageQuant TL software (GE Healthcare).

Statistical analysis

The results were expressed as mean + standard error of the mean (S.E.M.). One-way or Two-way analysis of variance (ANOVA) were used, followed by multiple comparisons with the Bonferroni post-test. The normality and homoscedasticity of all samples were checked previously. The data were analysed with GraphPad Prism 8.0.1 (GraphPad, San Diego, CA, USA). A significance level of p < 0.05 was adopted.

Results

CdtV stimulates the release of PGE2 by preadipocytes

PGE2 is a mediator relevant to both physiological and pathological processes. In AT, it is released by constitutive cells to mediate preadipocyte differentiation. However, to date, researchers do not know whether CdtV can activate this tissue to synthesise PGE2. Our preliminary results indicate that CdtV doses up to 10 μg/mL do not affect preadipocyte cell culture viability (Fig. 1). Hence, 10 μg/mL is defined as the maximum non-cytotoxic concentration and is used in later steps. To investigate whether CdtV promotes PGE2 release by preadipocytes, this venom was added (1, 5, or 10 μg/mL) to the cell culture. At selected time intervals, PGE2 concentrations were evaluated in the cell culture supernatants. Figure 2 demonstrates that CdtV at concentrations of 5 and 10 μg/mL induced a marked release of PGE2 by preadipocytes at 3, 24, and 48 h after incubation compared to the respective negative control group. This result demonstrates that CdtV can activate AT to release PGE2.

Fig. 1.

Fig. 1

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Time course of CdtV effect on viability of preadipocyte cell culture. 3T3-L1 preadipocytes were incubated with CdtV (1–20 μg/mL), DMEM (negative control), or 10% Triton (positive control) for 1 up to 48 h. Metabolic activity was assessed by the MTT assay. Results are expressed as mean + S.E.M. of 3 independent assays (n = 4). * p < 0.05 vs DMEM group of the respective experimental time (ANOVA, Bonferroni’s post-test)

Fig. 2.

Fig. 2

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Release of PGE2 by preadipocytes stimulated with CdtV. 3T3-L1 preadipocytes were incubated with CdtV (1, 5 or 10 μg/mL) or DMEM for 3, 24 or 48 h. Concentration of PGE2 in cell culture supernatants was evaluated by EIA. Results represent the time course of PGE2 release induced by CdtV and are expressed as mean + S.E.M. of 3 independent assays (n = 4). * p < 0.05 vs DMEM (ANOVA, Bonferroni’s post-test)

The COX enzymatic system participates in the CdtV-induced PGE2 release by preadipocytes

Prostanoid production is determined by a cascade of key enzymes; the first essential step is the modification of arachidonic acid by the COX system, which comprises at least four isotypes of COXs; COX-1(constitutive isoform) and COX-2 (constitutive and inducible isoform) are the most studied. We investigated whether COX-1 and -2 enzymes are involved in the PGE2 release, which is induced by CdtV in preadipocytes. For this, preadipocytes were preincubated (1 h) with SC-560 or NS-398, which are selective inhibitors of the isoforms COX-1 and COX-2, respectively, or with inhibitors vehicle (DMSO < 1%), followed by a 24-h incubation with CdtV. Then, PGE2 was quantified in cell culture supernatants. Figure 3 demonstrates that CdtV induced a marked release of PGE2 from preadipocytes in the vehicle group compared to the negative control group (DMEM plus vehicle). Cells that were pre-treated with pharmacological COX enzyme inhibitors and incubated with DMEM did not release PGE2. However, cells that were pre-treated with NS-398, SC-560, or both, demonstrated a decrease of PGE2 release after 24 h of incubation with CdtV compared to the CdtV plus vehicle group. These data indicate that both COX-1 and -2 contribute to PGE2 production induced by CdtV. On these bases, we examined the effects of CdtV on the protein expression of these enzymes. Immunoreactive bands showed in Fig. 4A demonstrate that CdtV did not change COX-1 protein expression by preadipocytes. The densitometric analysis of these bands, seen in Fig. 4B, confirms Fig. 4A, demonstrating that CdtV did not alter COX-1 protein expression in any concentration (1 or 10 μg/mL) or time interval studied, as expected. Conversely, Figs. 4A and C demonstrate that CdtV at the lower concentration (1 μg/mL) stimulated COX-2 protein expression only after 48 h of incubation, although the higher concentration (10 μg/mL) caused this effect at all time intervals observed. Together, these data indicate that COX-1 and COX-2 mediate PGE2 production, which is induced by CdtV and that this venom promotes an early COX-2 protein expression, which can contribute to the mechanism involved in the increased release of PGE2 provoked by this venom.

Fig. 3.

Fig. 3

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Participation of COX-1 and -2 in PGE2 release induced by CdtV in preadipocytes. 3T3-L1 preadipocytes were pretreated with the COX-1 and COX-2 inhibitors SC-560 and NS-398, respectively, or vehicle (DMSO < 1%) for 1 h. After this, the cells were stimulated with CdtV (10 μg/mL) or DMEM for 24 h. Levels of PGE2 present in cell culture supernatants were quantified by EIA. Results are expressed as mean + S.E.M. (n = 4). # p < 0.05 vs negative control (vehicle + DMEM); * p < 0.05 vs positive control (vehicle + CdtV) (ANOVA, Bonferroni’s post-test)

Fig. 4.

Fig. 4

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Effect of CdtV on COX-1 and COX-2 protein expression in preadipocytes. 3T3-L1 preadipocytes were stimulated with CdtV (1 or 10 μg/mL) or DMEM for 1, 6, 24 or 48 h. COXs protein expression was assessed by western blotting. A Immunoreactive bands of COX-2, COX-1 and β-actin (loading control). Representative blots were cropped from the full-length blots. B Densitometric analysis of immunoreactive bands of COX-1 and C COX-2. Results are expressed as mean + S.E.M (n = 4). * p < 0.05 vs control (ANOVA, Bonferroni’s post-test)

EP2 receptor regulates the PGE2 release induced by CdtV

The effects initiated by PGE2 are due to its binding to a specific receptor with four subtypes: EP1–4. In addition to transducing PGE2 effects, these receptors modulate PGE2 release depending on the cell microenvironment and stimulus. Since CdtV induces PGE2 release by preadipocytes, we investigated EP receptors participation in this event. Figure 5 demonstrates that preadipocytes pretreated with the vehicle and incubated with CdtV displayed a marked PGE2 release. Furthermore, the EP receptor antagonists per se did not induce PGE2 release by these cells. The cells that were pretreated with AH6809, an inhibitor of the EP2 receptor, and then incubated with CdtV released more PGE2 compared to the vehicle plus venom group. However, pretreatment with EP1, EP3, or EP4 antagonists did not change the PGE2 release induced by CdtV. We further investigated whether CdtV could promote changes in EP2 protein expression. Figure 6 indicates that the venom did not alter the EP2 protein expression in preadipocytes. Together, these results indicate that the EP2 receptor modulates the release of PGE2 induced by CdtV in preadipocytes, thereby triggering a negative feedback loop for production of this mediator. This effect is not dependent on the increase of EP2 protein expression.

Fig. 5.

Fig. 5

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Participation of EP receptors in the release of PGE2 induced by CdtV in preadipocytes. 3T3-L1 preadipocytes were treated with 10 μM SC-19220 (EP1 receptor antagonist), 10 μM AH6809 (EP2 receptor antagonist), 1 μM L-798106 (EP3 receptor antagonist), 10 μM AH23848 (EP4 receptor antagonist) or vehicle (DMSO < 1%) for 1 h, and then stimulated with CdtV (10 μg/mL) for 3 h. Concentrations of PGE2 in cell culture supernatants was quantified by EIA. Results are expressed as mean + S.E.M. (n = 4). * p < 0.05 vs negative control (vehicle + DMEM); # p < 0.05 vs positive control (vehicle + CdtV) (ANOVA, Bonferroni’s post-test)

Fig. 6.

Fig. 6

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Effect of CdtV on EP2 protein expression in preadipocytes. 3T3-L1 preadipocytes were stimulated with CdtV (10 μg/mL) or DMEM for 3 or 48 h. EP2 protein expression was assessed by western blotting. A Immunoreactive bands of EP2 and β-actin (loading control). Representative blots were cropped from the full-length blots. B Densitometric analysis of immunoreactive bands of EP2. Results are expressed as mean + S.E.M (n = 4)

Discussion

CdtV induces systemic neurotoxic, myotoxic, nephrotoxic and cardiovascular effects but lacks relevant local effects. The clinical importance and gravity of Crotalus envenomation in Latin America and the considerable number of studies on envenomation pathophysiology have not translated to a complete characterisation of the tissues targeted by crotalic venoms. Herein, we demonstrate for the first time that CdtV stimulates preadipocytes, leading to the release of PGE2, an important mediator of AT physiology that also fulfils dual roles during inflammatory responses. This result evidences a new source of this mediator upon CdtV stimulus and points to AT as another target for crotalic venom. Despite the critical differences between venoms from Crotalus and Bothrops genera in their components and biological actions, our results align with previous data that demonstrates the ability of Bothrops snake venom to activate AT (Maia-Marques et al. 2022); this reinforces the importance of AT in the response to snake envenomation and suggests AT as a common target for venoms of the Viperidae family.

AT is an endocrine organ that influences distant tissues and other organs; consequently, it is plausible to suggest that CdtV activation of this tissue impacts the systemic effects of this venom. In this context, the PGE2 release demonstrated here aligns with previous data showing that PGE2 contributes to CdtV’s systemic effects. Indeed, PGE2’s participation in the anti-inflammatory effect of crotoxin was demonstrated in an experimental model of acute intestinal inflammation (De Almeida et al. 2015). However, PGE2 is also associated with increased vascular permeability induced by the same toxin in lung parenchyma (Sartim et al. 2020). PGE2 is an important lipid mediator implicated in the development and regulation of inflammation and adipocyte differentiation. An essential step in PGE2 synthesis involves metabolising free arachidonic acid from cell membranes by COX enzymes, which generate intermediate products that are then processed by tissue-specific terminal synthases, leading to PGE2 formation. COX-1 and COX-2 are the most studied isoforms of this enzyme group; while COX-1 is constitutively expressed in many tissues, COX-2 is constitutively expressed in some tissues but is inducible in inflammatory processes. Both isoforms are responsible for producing prostaglandins in several physiological and pathological conditions.

To understand the mechanisms involved in CdtV-induced PGE2 production, we used pharmacological approaches. Our results demonstrating that the pharmacological inhibition of COX-1 and COX-2 isoforms diminished the preadipocyte release of PGE2 that was stimulated by CdtV, indicate that both isoforms contribute to this effect. In this context, the finding that CdtV induces COX-2 expression in preadipocytes adds important information regarding the molecular mechanism triggered by CdtV that leads to PGE2 production in preadipocytes. This is the first time that researchers have demonstrated that the crude venom of C. d. terrificus induces COX-2 protein expression. Our results agree with reports that CB, a major phospholipase A2 of CdtV, increases COX-2 gene expression in the lung tissue of mice (Sartim et al. 2020) and COX-2 protein expression in myoblasts in culture (Silva et al. 2021). Conversely, Moreira et al. (2007) demonstrated that CdtV did not induce COX-2 expression, but increased COX-1 activity in macrophages in culture, thus suggesting that CdtV's activation of PGE2’s synthetic pathways is associated with the cell type and/or function. Hence, our results highlight the preadipocytes as another cell target for this venom.

The physiological and pathophysiological effects initiated by PGE2 occur through its binding to four subtypes of G protein-coupled receptors, known as EP1–4. These receptors also regulate PGE2 release depending on the tissue and cell microenvironment (Golden et al. 2020). The results, which portray that pharmacologically inhibiting EP2 receptors increases CdtV-induced PGE2 production, point out the participation of this receptor subtype as a negative modulator of CdtV-induced PGE2 release. This regulation was not observed when using EP1, EP3, or EP4 receptor inhibitors. Thus, under CdtV’s effect, the autocrine action of PGE2 released by preadipocytes activates a negative feedback loop on its own production. It is worth noting that this negative modulation occurred during the initial venom action period (3 h) when a substantial release of this mediator was seen, which was not sustained after 48 h. However, we previously demonstrated that Bothrops venom can induce positive regulation of PGE2 production via EP receptors. Maia-Marques et al. (2022) showed that PGE2 released by preadipocytes that were stimulated with B. moojeni snake venom amplifies its own release by launching a positive feedback loop via the EP1 receptor. Additionally, stimulating preadipocytes with a PLA2 isolated from B. asper snake venom led to a similar PGE2 positive feedback mechanism by engaging the EP4 receptor (Leiguez et al. 2020). A PGE2 positive feedback loop was also caused by a snake venom metalloproteinase, BaP1, via the EP4 receptor in murine synoviocytes (Viana et al. 2020). Notably, EP receptor engagement by Bothrops venom or its toxins begins positive feedback for PGE2 release, whereas EP receptor activation by Crotalus venom results in negative feedback for the release of this mediator. In sum, our data suggest that EP receptors’ modulation of PGE2 release is related to the venom composition and origin (genus and species). However, we cannot eliminate the importance of the cell type for different PGE2 synthesis modulation that is initiated by EP receptors on different venoms.

Together, the data presented point out that CdtV stimulates preadipocytes to produce lipid mediators, such as PGE2. This effect depends on COX-1 activation and COX-2 expression. Additionally, in preadipocytes stimulated by CdtV, the activation of the EP2 receptor by autocrine PGE2 action begins a negative feedback loop. This is the first demonstration that CdtV activates AT cells. Therefore, our findings led to the discovery of another target cell and, consequently, another target tissue for CdtV. Since AT modulates many physiological and pathophysiological processes in the organism, our findings reveal that this tissue can be a source of lipid mediators during Crotalus envenomation.

Author contributions

C.T., D.S.T. and R.M. designed the study; D.S.T. performed the experiments; D.S.T. collected and analysed the data; C.T. provided reagents and venom; R.M. and C.T. wrote and revised the manuscript. All the authors discussed the results and implications and commented on the manuscript at all stages.

Funding

Fundação Butantan (https://www.fundacaobutantan.org.br/), Brazil, funded this research. R. M. is a recipient of a fellowship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, https://www.gov.br/capes/pt-br), grant no. 88887.655304/2021-00, Brazil. D. S. T. is a recipient of a fellowship from CAPES (https://www.gov.br/capes/pt-br), grant no. 88887.569469/2020-00, Brazil. C.T. is a recipient of a Conselho Nacional de Desenvolvimento Científico e Tecnológico fellowship PQ-CNPq, Brazil grant no. 310930/2019-7 (https://www.gov.br/cnpq/pt br). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data availability

Access to the data is available on request.

Declarations

Conflict of interest

The authors have declared that no competing interests exist.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Danilo Santos Teixeira and Rodrigo Maia-Marques contributed equally to this work.

Contributor Information

Rodrigo Maia-Marques, Email: rodrigomaiamarques@gmail.com.

Catarina Teixeira, Email: catarina.teixeira@butantan.gov.br.

References

  1. Baudou FG, Gutiérrez JM, Rodríguez JP (2023) Immune response to neurotoxic South American snake venoms. Toxicon. 10.1016/j.toxicon.2023.107300 [DOI] [PubMed] [Google Scholar]
  2. Bucaretchi F, De Capitani EM, Vieira RJ et al (2016) Coral snake bites (Micrurus spp.) in Brazil: a review of literature reports. Clin Toxicol 54:222–234. 10.3109/15563650.2015.1135337 [DOI] [PubMed] [Google Scholar]
  3. Charrière GM, Cousin B, Arnaud E et al (2006) Macrophage characteristics of stem cells revealed by transcriptome profiling. Exp Cell Res 312:3205–3214. 10.1016/j.yexcr.2006.06.034 [DOI] [PubMed] [Google Scholar]
  4. Chen L, Miao Y, Zhang Y et al (2012) Inactivation of the E-prostanoid 3 receptor attenuates the angiotensin II pressor response via decreasing arterial contractility. Arterioscler Thromb Vasc Biol 32:3024–3032. 10.1161/ATVBAHA.112.254052 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Choi HC, Kim HS, Lee KY et al (2008) NS-398, a selective COX-2 inhibitor, inhibits proliferation of IL-1β-stimulated vascular smooth muscle cells by induction of ΗΟ-1. Biochem Biophys Res Commun 376:753–757. 10.1016/j.bbrc.2008.09.056 [DOI] [PubMed] [Google Scholar]
  6. D Vaz de Melo P, de Almeida Lima S, Araújo P et al (2020) Immunoprotection against lethal effects of Crotalus durissus snake venom elicited by synthetic epitopes trapped in liposomes. Int J Biol Macromol 161:299–307. 10.1016/j.ijbiomac.2020.05.171 [DOI] [PubMed] [Google Scholar]
  7. da Silva FFB, de Andrade MT, Siqueira-Silva T et al (2024) Predicting the drivers of Bothrops snakebite incidence across Brazil: a spatial analysis. Toxicon. 10.1016/j.toxicon.2024.108107 [DOI] [PubMed] [Google Scholar]
  8. Dashty M, Akbarkhanzadeh V, Zeebregts CJ et al (2012) Characterization of coagulation factor synthesis in nine human primary cell types. Sci Rep 2:1–9. 10.1038/srep00787 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. De Almeida CS, Andrade-Oliveira V, Câmara NOS et al (2015) Crotoxin from Crotalus durissus terrificus is able to down-modulate the acute intestinal inflammation in mice. PLoS ONE 10:1–19. 10.1371/journal.pone.0121427 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. De Carvalho LH, Teixeira LF, Zaqueo KD et al (2019) Local and systemic effects caused by Crotalus durissus terrificus, Crotalus durissus collilineatus, and Crotalus durissus cascavella snake venoms in swiss mice. Rev Soc Bras Med Trop. 10.1590/0037-8682-0526-2018 [DOI] [PubMed] [Google Scholar]
  11. Dordevic AL, Konstantopoulos N, Cameron-Smith D (2014) 3T3-L1 preadipocytes exhibit heightened monocyte-chemoattractant protein-1 response to acute fatty acid exposure. PLoS ONE 9:1–8. 10.1371/journal.pone.0099382 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Frare BT, Resende YKS, Dornelas BDC et al (2019) Clinical, laboratory, and therapeutic aspects of Crotalus durissus (South American Rattlesnake) victims: a literature review. Biomed Res Int. 10.1155/2019/1345923 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Giannotti KC, Leiguez E, De CAEZ et al (2017) A snake venom group IIA PLA2 with immunomodulatory activity induces formation of lipid droplets containing 15-d-PGJ2 in macrophages. Sci Rep 7:1–15. 10.1038/s41598-017-04498-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Giles JT, Ferrante AW, Broderick R et al (2018) Adipose tissue macrophages in rheumatoid arthritis: prevalence, disease-related indicators, and associations with cardiometabolic risk factors. Arthritis Care Res 70:175–184. 10.1002/acr.23253 [DOI] [PubMed] [Google Scholar]
  15. Golden J, Illingworth L, Kavarian P et al (2020) EP2 receptor blockade attenuates COX-2 upregulation during intestinal inflammation. Shock 54:394–401. 10.1097/SHK.0000000000001444 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gutiérrez JM, Lomonte B, León G et al (2009) Snake venomics and antivenomics: proteomic tools in the design and control of antivenoms for the treatment of snakebite envenoming. J Proteomics 72:165–182. 10.1016/j.jprot.2009.01.008 [DOI] [PubMed] [Google Scholar]
  17. Gutiérrez JM, Williams D, Fan HW, Warrell DA (2010) Snakebite envenoming from a global perspective: towards an integrated approach. Toxicon 56:1223–1235. 10.1016/j.toxicon.2009.11.020 [DOI] [PubMed] [Google Scholar]
  18. Gutiérrez JM, León G, Lomonte B, Angulo Y (2011) Antivenoms for snakebite envenomings. Inflamm Allergy Drug Targets 10:369–380. 10.2174/187152811797200669 [DOI] [PubMed] [Google Scholar]
  19. Kawai T, Autieri MV, Scalia R (2021) Adipose tissue inflammation and metabolic dysfunction in obesity. Am J Physiol Cell Physiol 320:C375–C391. 10.1152/ajpcell.00379.2020 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Leiguez E, Motta P, Maia Marques R et al (2020) A representative GIIA phospholipase A2 activates preadipocytes to produce inflammatory mediators implicated in obesity development. Biomolecules 10:1593. 10.3390/biom10121593 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lin YS, Hsieh M, Lee YJ et al (2008) AH23848 accelerates inducible nitric oxide synthase degradation through attenuation of cAMP signaling in glomerular mesangial cells. Nitric Oxide Biol Chem 18:93–104. 10.1016/j.niox.2007.10.005 [DOI] [PubMed] [Google Scholar]
  22. Lin CC, Lin WN, Wang WJ et al (2009) Functional coupling expression of COX-2 and cPLA2 induced by ATP in rat vascular smooth muscle cells: role of ERK1/2, p38 MAPK, and NF-κB. Cardiovasc Res 82:522–531. 10.1093/cvr/cvp069 [DOI] [PubMed] [Google Scholar]
  23. Loynes CA, Lee JA, Robertson AL et al (2018) PGE2 production at sites of tissue injury promotes an anti-inflammatory neutrophil phenotype and determines the outcome of inflammation resolution in vivo. Sci Adv. 10.1126/sciadv.aar8320 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. MacKenzie KF, Clark K, Naqvi S et al (2013) PGE 2 induces macrophage IL-10 production and a regulatory-like phenotype via a protein kinase A-SIK–CRTC3 pathway. J Immunol 190:565–577. 10.4049/jimmunol.1202462 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Maia-Marques R, Teixeira DS, Janovits PM et al (2022) Bothrops moojeni snake venom induces an inflammatory response in preadipocytes: insights into a new aspect of envenomation. PLoS Negl Trop Dis 16:e0010658. 10.1371/journal.pntd.0010658 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Moreira V, Zamuner SR, Wallace JL, Teixeira C de FP (2007) Bothrops jararaca and Crotalus durissus terrificus venoms elicit distinct responses regarding to production of prostaglandins E2 and D2, and expression of cyclooxygenases. Toxicon 49:615–624. 10.1016/j.toxicon.2006.09.006 [DOI] [PubMed]
  27. Rosenfeld G (1971) Symptomatology, pathology, and treatment of snake bites in South America. In: Venomous animals and their venoms. Elsevier. pp 345–384
  28. Sampaio SC, Alba-Loureiro TC, Brigatte P et al (2006) Lipoxygenase-derived eicosanoids are involved in the inhibitory effect of Crotalus durissus terrificus venom or crotoxin on rat macrophage phagocytosis. Toxicon 47:313–321. 10.1016/j.toxicon.2005.11.008 [DOI] [PubMed] [Google Scholar]
  29. Sartim MA, Souza COS, Diniz CRAF et al (2020) Crotoxin-induced mice lung impairment: role of nicotinic acetylcholine receptors and cox-derived prostanoids. Biomolecules. 10.3390/biom10050794 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Silva NC, Alvarez AM, DeOcesano-Pereira C et al (2021) Catalytically active phospholipase A2 myotoxin from Crotalus durissus terrificus induces proliferation and differentiation of myoblasts dependent on prostaglandins produced by both COX-1 and COX-2 pathways. Int J Biol Macromol 187:603–613. 10.1016/j.ijbiomac.2021.07.121 [DOI] [PubMed] [Google Scholar]
  31. Simões LO, Alves QL, Camargo SB et al (2021) Cardiac effect induced by Crotalus durissus cascavella venom: morphofunctional evidence and mechanism of action. Toxicol Lett 337:121–133. 10.1016/j.toxlet.2020.11.019 [DOI] [PubMed] [Google Scholar]
  32. Stock JL, Shinjo K, Burkhardt J et al (2001) The prostaglandin E2 EP1 receptor mediates pain perception and regulates blood pressure. J Clin Invest 107:325–331. 10.1172/JCI6749 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sugimoto Y, Narumiya S (2007) Prostaglandin E receptors. J Biol Chem 282:11613–11617. 10.1074/jbc.R600038200 [DOI] [PubMed] [Google Scholar]
  34. Takayama K, Mitchell DH, Din ZZ et al (1994) Monomeric Re lipopolysaccharide from Escherichia coli is more active than the aggregated form in the Limulus amebocyte lysate assay and in inducing Egr-1 mRNA in murine peritoneal macrophages. J Biol Chem 269:2241–2244 [PubMed] [Google Scholar]
  35. Takeuchi K, Amagase K (2018) Roles of cyclooxygenase, prostaglandin E2 and EP receptors in mucosal protection and ulcer healing in the gastrointestinal tract. Curr Pharm des 24:2002–2011. 10.2174/1381612824666180629111227 [DOI] [PubMed] [Google Scholar]
  36. Viana MN, Leiguez E, Gutiérrez JM et al (2020) A representative metalloprotease induces PGE2 synthesis in fibroblast-like synoviocytes via the NF-$κ$B/COX-2 pathway with amplification by IL-1$β$ and the EP4 receptor. Sci Rep 10:1–15. 10.1038/s41598-020-59095-z [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wang T, Fu X, Chen Q et al (2019) Arachidonic acid metabolism and kidney inflammation. Int J Mol Sci 20:1–28. 10.3390/ijms20153683 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Yu L, Zhang X, Ye S et al (2022) Obesity and COVID-19: mechanistic insights from adipose tissue. J Clin Endocrinol Metab 107:1799–1811. 10.1210/clinem/dgac137 [DOI] [PMC free article] [PubMed] [Google Scholar]

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