Biomedicine & Pharmacotherapy 143 (2021) 112126 Contents lists available at ScienceDirect Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha Intestinal inflammation-associated hypersensitivity is attenuated in a DSS model of colitis in Sigma-1 knockout C57BL/6 mice Sergio López-Estévez a, b, Georgia Gris c, Beatriz de la Puente c, Alicia Carceller c, Vicente Martínez a, b, d, * a Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain b Neuroscience Institute, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain c Department of Pharmacology, Drug Discovery & Preclinical Development, ESTEVE, 08028 Barcelona, Spain d Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain A R T I C L E I N F O A B S T R A C T Keywords: Sigma-1 receptors (σ1R) have been implicated in several pain pathways. We assessed the implication of σ1Rs in Colitis the development of intestinal inflammation and inflammation-associated referred hypersensitivity in a model of Hyperalgesia colitis in σ1R knockout (KO) mice. Colitis was induced with dextran sulfate sodium (DSS) in wild type (WT) and Hypersensitivity σ1R KO mice. The development of referred mechanical hypersensitivity (von Frey test) was assessed. Colonic and Intestinal inflammation Pain spinal changes in expression of immune- and sensory-related markers were also investigated (RT-qPCR/Western Sigma 1 receptor blot). Absence of σ1Rs had little impact in colitis generation and progression, although during the chronic phase a reduction in edema and a down-regulation of iNOS gene expression was observed. In σ1R KO mice, inflammation- associated hypersensitivity was significantly attenuated (paw) or completely prevented (abdomen). During co­ litis, in WT mice, changes in the colonic expression of nociceptive markers were observed during the acute and chronic phases of inflammation. Although σ1R KO mice showed similar regulation in the acute phase, an attenuated response was observed during the chronic phase of colitis. These differences were especially relevant for CB2 and TRPV1 receptors, which could play an important role in σ1-mediated regulation of sensitivity. No changes were detected on ERK phosphorylation at the level of the lumbosacral spinal cord. In summary, intes­ tinal inflammation-associated referred hyperalgesia was reduced (paw) or absent (abdomen) in σ1R KO mice, thus confirming an important role for σ1R in the development of colitis-associated hypersensitivity. These results identify σ1Rs as a possible therapeutic target for the treatment of hypersensitivity associated to intestinal inflammation. 1. Introduction inflammation and has altered colonic sensitivity with increased perception as key manifestations [3,4]. Additionally, changes in somatic Evidences suggest that inflammation within the gastrointestinal sensitivity, characterized by referred hypersensitivity, have also been tract, even at a low degree, and the presence of visceral and somatic observed in states of intestinal inflammation [5,6]. In this context, in­ hypersensitivity are associated phenomena. Indeed, inflammatory con­ testinal inflammation and the associated state of hypersensitivity are ditions of the gastrointestinal tract, such as inflammatory bowel disease still areas of medical needs, without fully effective therapeutic (IBD), are associated to both somatic and visceral hypersensitivity [1,2]. approaches. Moreover, irritable bowel syndrome (IBS), the main functional gastro­ During the last years, several targets have been explored as potential intestinal disorder, has been associated to a low degree of intestinal treatments of visceral pain arising from the gut. However, at the current Abbreviations: β2m, β-2-microglobulin; σ1Rs, sigma-1 receptors; CB1, Cannabinoid receptor 1; CB2, Cannabinoid receptor 2; COX-2, Cyclooxygenase 2; DSS, Dextran sulfate sodium; IBD, Inflammatory bowel disease; IBS, Irritable bowel syndrome; INF-γ, Interferon γ; IL-1β, Interleukin 1β; IL-6, Interleukin 6; IL-10, Interleukin 10; IL-12p40, Interleukin 12 subunit p40; iNOS, Nitric oxide synthase 2 (inducible); MOR, µ-Opioid receptor; NGF, Nerve growth factor; PAR2, Protease activated receptor 2; SERT, Serotonin transporter; TPH1, Tryptophan hydroxylase 1; TRPV1/3, Transient receptor potential vanilloid 1 and 3; VF, von Frey. * Correspondence to: Unit of Physiology, Veterinary School, Campus UAB, Bellaterra, 080193 Barcelona, Spain. E-mail address: Vicente.martinez@uab.es (V. Martínez). https://doi.org/10.1016/j.biopha.2021.112126 Received 9 July 2021; Received in revised form 20 August 2021; Accepted 24 August 2021 Available online 30 August 2021 0753-3322/© 2021 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). S. López-Estévez et al. Biomedicine & Pharmacotherapy 143 (2021) 112126 time there are no effective treatments for this type of pain. Some efforts evaluated after a 30 min habituation period to the testing environment. have been directed towards the validation of analgesic treatment posi­ Referred pain was assessed in two separate regions, the abdominal wall tively validated against somatic pain, although the differences between and the hind paw. When assessing sensitivity of the abdominal wall the these pain modalities might be relevant in this respect. In recent years, perianal and external genitalia areas were avoided, concentrating the sigma-1 receptors (σ1Rs) have been implicated in pain mechanisms and mechanical stimulation on the lower and mid abdomen, as previously suggested as potential pharmacological target for the treatment of so­ reported [17,23]. Paw sensitivity was quantified by measuring the hind matic pain. σ1R is a neuromodulatory, ligand-regulated membrane paw withdrawal response to punctate mechanical stimulation, as pre­ protein chaperone that exerts its functions through multiprotein com­ viously described [24,25]. In all cases, pain thresholds were determined plex assembly [7,8]. The relation of σ1 Rs and pain was first suggested by using the up-down method paradigm and represent the mechanical studies showing a relationship between σR systems and opioid-mediated stimulus that produces 50% of maximal response [26]. Data obtained analgesia [9,10]. Evidences indicate that σ1R ligands fail to modify were normalized to a baseline measurement (taken as 1), taken 24 h normal pain responses by themselves, as demonstrated in classical before starting the experimental procedures (Fig. 1). All measurements models of thermal and mechanical acute nociception [11,12]. However, were performed twice, with a 30-min recovery period in between, by σ1R ligands seem to play a key role in modulating pain behavior in states two independent investigators. The mean values of the two observations of sensitization and chronic pain conditions [13]. In this respect, recent were taken, for each animal, as the measure of pain sensitivity. studies show that both central and peripheral pharmacological blockade of σ1Rs could be an effective option to treat inflammatory pain [13–16]. 2.4. Experimental protocol As it relates to visceral pain responses arising from the gut, evidences indicate a potential modulatory role for σ1Rs. In this sense, σ1Rs selec­ WT and σ1R KO mice were randomly divided into 2 experimental tive antagonists were effective preventing visceral pain-related re­ groups per genotype (n = 12–19 per group). In a random assignment, sponses elicited by intracolonic capsaicin in mice [17]. the experimental groups received tap water or a solution of 2% DSS Taking into account these considerations, the aims of the present during a 5-day period (days 0–5). After DSS/water exposure, all animals study were to assess the potential modulatory role of σ1Rs on intestinal received normal water and were allowed to recover for a 2-day (acute inflammation and the development of inflammation-associated hyper­ inflammatory phase) or a 16-day period (chronic inflammatory phase) sensitivity in a murine model of dextran sulfate sodium (DSS)-induced before euthanasia. Individual body weight, general state and the pres­ colitis. With this objective, we assessed the development of colitis and ence of clinical signs were assessed on a daily basis throughout the inflammation-associated visceral and somatic hypersensitivity in σ1R study. Von Frey test was performed 4 times during the experimental knockout (KO) mice compared to wild-type (WT) animals. Moreover, procedure: the day before starting the administration of DSS (taken as a colitis-associated changes in peripheral (colon) and central (spinal cord) basal measure of sensitivity, day − 1), at approximately half of time of sensory-related markers implicated in pain processing and sensitization DSS exposure (day 3), at the end of acute inflammatory phase (day 7) were also assessed. and at the end of the experiment (chronic inflammatory phase, day 21). Animals were euthanized for the collection of samples immediately after 2. Materials and methods the last von Frey test evaluation (see below). See Fig. 1 for details of the experimental protocol. 2.1. Animals 2.5. Samples collection Male WT C57BL/6J mice (n = 28, Charles River Laboratories, Lyon, France) and σ1R KO C57BL/6J mice (n = 31, Esteve Pharmaceuticals S. Immediately after the last von Frey test (days 7 or 21, for the acute A., Barcelona, Spain), both aged 6-weeks at the time of starting the and chronic phase, respectively), mice were deeply anesthetized with studies, were used. Mice were group-housed in standard cages (four-six isoflurane (Isoflo; Esteve, Barcelona, Spain) and euthanatized by mice per cage) and maintained under standard condition of photoperiod exsanguination through intracardiac puncture followed by cervical (12:12 h light-dark cycle) and climate (20–22 ºC, 40–70% humidity), dislocation. Thereafter, a medial laparotomy was performed, the ceco- with ad libitum access to a standard diet and tap water, except when colonic region localized and the cecum and colon dissected. After­ receiving DSS. Mice were allowed to acclimatize to the animal facility ward, two tissue samples from the proximal-middle colon (about 1.5 cm for at least 1 week before starting the studies. All procedures were each) were collected. A sample was frozen immediately in liquid nitro­ approved by the Ethical Committee of the Universitat Autònoma de gen and a second sample was fixed in 4% paraformaldehyde. After an Barcelona (protocols 3039 and 3957) and the Generalitat de Catalunya overnight fixing, tissues were paraffin embedded and 5-µm-thick sec­ (protocols 8823 and 9915). tions were obtained. The lumbar enlargement (L3-S2) of spinal cord was dissected and frozen immediately in liquid nitrogen. Frozen samples 2.2. Colitis induction were stored at − 80 ºC until analysis. In addition, the liver, the adrenal glands, the thymus, and the spleen were dissected and weighed. Serum A solution of DSS (45 kDa; 2% concentration in water; TdB Consul­ was obtained by centrifugation of blood samples (15 min, 10,000×g, tancy AB, Uppsala, Sweden) was used to induce colitis. Fresh DSS so­ 4 ºC) and maintained at − 80 ºC until analysis. lutions were prepared daily during the 5-day treatment period (from experimental day 0 to day 5). Following this protocol animals develop 2.6. Clinical and macroscopic assessment of inflammation an acute colitis (7–8 days after starting DSS exposure) that progresses to chronicity. Similar protocols have been used in previous studies in mice Clinical assessment of inflammation included daily monitoring of to induce colitis [18–22]. Normal tap water was used as the control body weight, appearance of feces and general health condition [19]. A treatment. score (0− 8) was assigned for health condition (including hunch posture, piloerection, fecal consistency and aspect of the anus); where 0 indicates 2.3. Evaluation of referred mechanical hypersensitivity: von Frey test normal activity/fur/normal feces/normal anus, 1 indicates abnormal gait/bristly fur/wet/watery feces/wet anus and 2 indicates prostrated Animals were placed into compartment enclosures in a test chamber animal/dirty fur/watery diarrhea/bloody rest on anus. At necropsy, the with a framed metal mesh floor through which von Frey monofilaments macroscopic appearance of the colon was scored following previously (bending force range from 0.04 to 2 g; North Coast Medical, Inc.; Gilroy, published procedures [19]. Briefly, the presence of inflammatory signs CA, USA) were applied for pain assessment. Pain sensitivity was (inflammatory score): consistency of fecal contents (score 0–3); presence 2 S. López-Estévez et al. Biomedicine & Pharmacotherapy 143 (2021) 112126 Fig. 1. Schematic representation of the experimental protocols followed in the study. VF: von Frey test. of visible fecal blood (score 0–3); evidence and extent of edema (0− 3); (Mm00443039_m1), nitric oxide synthase 2 (inducible, iNOS) wall thickness (0− 3); tissue stiffness (0− 2) and presence of ulcerations (Mm00440502_m1), prostaglandin-endoperoxide synthase (Cyclo­ (0− 1) were assessed; resulting in a maximum total score of 15. oxygenase 2, COX-2) (Mm00478374_m1), protease activated receptor 2 (PAR2) (Mm00433160_m1), serotonin transporter (SERT) 2.7. Histological studies (Mm00439391_m1), transient receptor potential vanilloid 1 (TRPV1) (Mm01246302_m1) and 3 (TRPV3) (Mm00455003_m1), tryptophan For histological examination, hematoxylin-eosin-stained sections hydroxylase 1 (TPH1) (Mm00493794_m1) and σ1 receptor (σ1R) from the colon were obtained following standard procedures. A histo­ (Mm00448086_m1). β-2-microglobulin (β2m) (Mm00437762_m1) was pathological score (ranging from 0, normal, to 12, maximal alterations) used as endogenous reference gene. was assigned to each animal [27]. Specifically, parameters scored included: epithelial structure (0: normal; 1: mild alterations of the villi; 2.10. Protein expression using Western blot 2: local villi destruction and/or fusion; 3: generalized villi destruction and/or fusion), structure of the crypts (0: normal; 1: mild alterations of Dissected spinal cord samples were homogenized by sonication in the crypts; 2: local destruction of the crypts; 3: generalized destruction radioimmunoprecipitation assay (RIPA) buffer and the supernatant was of the crypts), presence of edema (0: normal; 1: mild local edema in obtained. Equal amounts of protein (30 µg) were fractionated by 10% submucosa and/or lamina propria; 2: moderate diffuse edema in sub­ (w/v) SDS–PAGE and transferred onto a polyvinylidene difluoride mucosa and/or lamina propria; 3: severe generalized edema in submu­ membrane, blocked with 5% non-fat dry milk in Tris–Tween 20-buffered cosa and/or lamina propria), and presence of inflammatory infiltrate (0: Saline (T–TBS) for 1 h. Membranes were then incubated overnight at 4 normal; 1: mild localized infiltrate; 2: mild generalized infiltrate; 3: ºC in 1% non-fat dry milk in T–TBS with rabbit primary polyclonal an­ severe generalized infiltrate). Scoring was performed on coded slides by tibodies recognizing the mitogen-activated protein kinase (MAPK, total two independent researchers and the mean value of the two scores was ERK ½, 1:30000) or mouse monoclonal antibodies recognizing the taken as the final score per animal. activated MAPK (diphosphorylated MAPK, pERK ½, 1:1000). Rabbit polyclonal anti-GAPDH antibody (1:20000) or mouse monoclonal anti- 2.8. Serum haptoglobin GAPDH antibody (1:80000) were used as a loading control, respec­ tively. After washing with T–TBS, the blots were incubated for 1 h with Serum concentrations of haptoglobin were determined using a horseradish peroxidase–conjugated goat anti-rabbit IgG (1:4000) or commercial ELISA kit, following manufacturer’s instructions (sensi­ goat anti-mouse IgG (1:2000). The immunoreactive bands were detected tivity; 0.005 mg/ml; intraassay variability: 5.3–6.3%; interassay vari­ by a peroxidase reaction using an enhanced chemiluminescence method ability: 4.1–5.7%; “PHASE” TM Haptoglobin Assay; Tridelta (WesternSure® PREMIUM Chemiluminescent Substrate, Li-cor) and Development Limited, Maynooth, County Kildare, Ireland). CDiGit ® Blot Scanner (Li-cor). All antibodies were obtained from Sig­ ma–Aldrich Co. (Madrid, Spain). Quantification was realized with Image Studio™ Lite Software. 2.9. Gene expression using Quantitative Reverse Transcription-PCR Total RNA was extracted from frozen tissue samples using TRI re­ 2.11. Statistical analysis agent with Ribopure Kit (Ambion/Applied biosystems, Foster City, CA, USA). RNA was purified by via precipitation with lithium chloride [28]. Data are expressed as mean ± SEM. A robust analysis (one iteration) Later, a two-step quantitative real-time PCR (RT-qPCR) was performed. was used to obtain mean ± SEM for RT-qPCR data. Data were analyzed RNA samples were converted into cDNA using a High Capacity cDNA by one-, two or three-way ANOVA, as appropriate, followed, when Reverse Transcription Kit (Applied Biosystems). The PCR reaction necessary, by a Bonferroni’s multiple comparisons test. Data were mixture was incubated on the Bio-Rad CFX384 Touch Real-Time PCR considered statistically significant when P < 0.05. Statistical analyses Detection System (Bio-Rad). All samples were assayed in triplicate. The were performed using GraphPad Prism version 7.0 (GraphPad Software, cycle thresholds for each sample were obtained and data were analyzed La Jolla, CA, USA) or SPSS program (version 17 for Windows, IBM, using the comparative Ct method (2-ΔΔCt) with the WT vehicle group Madrid, Spain). serving as the calibrator [29]. TaqMan® gene expression assays (hy­ drolysis probes, Applied Biosystems) used included: cannabinoid re­ 3. Results ceptors 1 (CB1) (Mm01212171_s1) and 2 (CB2) (Mm00438286_m1), interferon γ (INF-γ) (Mm01168134_m1), interleukin 1β (IL-1β) 3.1. Colitis development in WT and σ1R KO mice (Mm00434228_m1), 6 (IL-6) (Mm00446190_m1), 10 (IL-10) (Mm00439614_m1) and 12 (IL-12p40) (Mm00434174_m1), µ-opioid Regardless the genotype considered, mice receiving water showed a receptor (MOR) (Mm01188089_m1), nerve growth factor (NGF) steady and linear increase in body weight, without clinical signs 3 S. López-Estévez et al. Biomedicine & Pharmacotherapy 143 (2021) 112126 throughout the experimental period (Fig. 2). Conversely, in animals phase). The improvement observed in σ1R KO mice was mainly associ­ exposed to DSS, body weight loss was observed from experimental day ated to a reduction in the presence of edema (statistically significant 6, with a peak reduction between days 9 and 10, and a progressive re­ interaction by genotype and time on the presence of edema -P < 0.05-, covery up to day 21, although without reaching the body weight of Fig. 3B). Regardless of the phenotype considered, no significant changes control animals not exposed to DSS. No differences in this pattern were were observed for the relative weight of body organs (data not shown). observed between σ1R KO and WT mice. In animals exposed to DSS, The acute phase protein haptoglobin showed a similar increase in clinical signs (mainly bristly fur, wet/watery feces and wet anus) WT and σ1R KO animals during acute inflammation (P < 0.05 vs. consistent with the development of a colitic state appeared with similar respective control). Haptoglobin levels showed a tendency towards temporal pattern to that described for body weight changes, reaching a normalization during the chronic phase, although they remained maximum at day 9 and disappearing completely by the end of the significantly increased when compared to non-inflamed animals experimental time (Fig. 2). No genotype-related differences were (Fig. 3C). observed in the incidence and severity of clinical signs. Water intake was similar across experimental groups (data not shown). At necropsy, WT mice receiving DSS showed macroscopic signs of 3.2. Inflammatory markers are differentially regulated in WT and σ1R colonic inflammation, both at the acute and chronic phase, character­ KO mice during colitis ized by shortening in length and an increase in its relative weight (P < 0.05 vs. WT mice receiving water; Fig. 3A). Similar changes were In control conditions, independently of the genotype and time of observed in σ1R KO mice. A slight attenuation in inflammation-related measurement, expression of the cytokines assessed was detectable in all parameters was observed in σ1R KO mice when compared to WT, colonic samples. In WT mice, colitis was associated to an up-regulation although statistical significance was not achieved (Fig. 3A). of the expression of the pro-inflammatory cytokines INF-γ, IL-1β and IL- Microscopic analysis of colonic tissue samples showed a normal 6, which was particularly evident during the acute phase of inflamma­ histological structure in control animals. Regardless the genotype tion and persisted during the chronic phase, although relatively atten­ considered, exposure to DSS led to a similar significant increase in his­ uated (in all cases P < 0.05 vs WT control mice, Fig. 4). No significant topathological scores (Fig. 3B). Colonic alterations were attenuated changes were observed in the local expression of IL-12p40 and IL-10. during the chronic phase, however statistical significance was only In σ1R KO mice an up-regulation of INF-γ, IL-1β and IL-6 was achieved for σ1R KO mice (P < 0.05 vs. σ1R KO mice during the acute observed during the acute phase of inflammation (all P < 0.05 vs. non- inflamed animals), but expression levels were basically normalized Fig. 2. Time-related changes in relative body weight (% change from day 0, taken as 100%, upper panel) and clinical signs (lower panel) in the different experi­ mental groups. Data are mean ± SEM (n = 12–18). The DSS-treatment period is indicated by the grey area. 4 S. López-Estévez et al. Biomedicine & Pharmacotherapy 143 (2021) 112126 Fig. 3. Assessment of colonic inflammation at the time of necropsy in the different experimental groups. (A) Macroscopic scores. (B) Histopathological scores: inflammatory infiltrate, edema, state of the crypts and epithelial structure. (C) Plasma concentrations of the acute phase protein haptoglobin. Data are mean ± SEM of 6–10 animals per group. *P < 0.05 vs. respective control group; #P < 0.05 vs. respective acute DSS-treated group. during the chronic phase (all P > 0.05 vs. non-inflamed animals, Fig. 4). 3.3. Colitis-associated mechanical hypersensitivity is attenuated in σ 1R A significant up-regulation of IL-10 expression was detected in σ1R KO KO mice mice during the chronic phase (P < 0.05 vs. other experimental groups). Similarly to that observed in WT animals, no changes were observed in Baseline (experimental day − 1) abdominal and paw withdrawal the expression of IL12p-40. thresholds during the von Frey test were similar in WT and σ1R KO mice Colonic expression of iNOS was up-regulated in acute colitic WT (Fig. 6). mice, although with relatively high variability (P < 0.05 vs control WT In healthy WT mice, abdominal and somatic mechanical sensitivity group). This up-regulation persisted during the chronic phase, although was stable throughout the experimental time (Fig. 7). However, in WT with some attenuation, (Fig. 5). In σ1R KO mice, iNOS expression was animals receiving DSS a reduction in the withdrawal thresholds was up-regulated during acute colitis, while returning during the chronic observed from experimental day 3 throughout experimental day 21, phase to the levels detected in non-inflamed animals (Fig. 5). indicating the development of mechanical hypersensitivity (in all cases In colitic WT mice, 3 out of 5 animals showed COX-2 expression P < 0.05 vs. control group). levels above the mean expression of the control group, during either the As it relates to σ1R KO mice, mechanical thresholds were stable in acute or the chronic phase, thus suggesting a tendency for COX-2 up- colitic animals and showed only a transitory reduction in experimental regulation during colitis. However, no statistical significance was ach­ day 7 for abdominal sensitivity (P < 0.05 vs. control group), with a re­ ieved, probably because of the high interindividual variability observed. turn towards basal sensitivity on experimental day 21; whereas no No expression changes were observed in σ1R KO mice, regardless the changes were observed for somatic sensitivity (Fig. 7). inflammatory phase considered (Fig. 5). Fig. 4. Heat map of the relative mRNA expression of colonic pro- (INF-γ, IL-1β, IL-6 and IL-12p40) and anti-inflammatory cytokines (IL-10) in the different experimental groups. Each vertical line, with a number, corresponds to an individual animal within the corresponding experimental group. 5 S. López-Estévez et al. Biomedicine & Pharmacotherapy 143 (2021) 112126 Fig. 5. Colonic expression of inflammatory markers: iNOS, and COX-2. Each point represents an individual animal; the horizontal bar with errors represents the mean ± SEM. *P < 0.05 vs. respective control group; +P < 0.05 vs. respective WT DSS-treated group. 3.4. Expression of colonic sensory-related markers is modulated during 3.5. ERK expression within the spinal cord is not affected by colitis DSS-induced colitis ERK protein was detected in all spinal cord samples, regardless the Expression of the sensory-related markers assessed was detected in experimental group considered. Similar levels of tERK and pERK were all colonic samples, except for σ1R, that, as expected, was only found in detected in WT and σ1R KO mice in control conditions. During colitis, WT animals. In healthy animals, expression levels of the markers regardless the phase considered, no changes were observed in tERK or assessed were comparable, regardless the genotype considered. pERK content or the ratio pERK/tERK (Fig. 9). During colitis in WT mice, there was an overall down-regulation of all sensory markers analyzed when compared with non-inflamed ani­ 4. Discussion mals. This was particularly evident during the acute phase, with a ten­ dency towards normalization during the chronic phase (Fig. 8). In In the present study, we assessed the potential role of σ1Rs in the particular, except for CB2 and PAR-2 which showed no changes, all development of colitis and inflammation-associated changes in referred analyzed markers were down-regulated during both the acute and mechanical sensitivity using a murine model KO for σ1Rs. Results ob­ chronic phases of colitis in WT mice (in all cases P < 0.05 vs. respective tained indicate that σ1Rs only marginally affected the development of control group). In these animals, expression of σ1R was down-regulated intestinal (colonic) inflammation or its progression from acute to during acute colitis (P < 0.05 vs. control group), returning to basal chronic, but they seem to play an important role in the development of expression levels during the chronic phase. inflammation-associated hypersensitivity. Regarding σ1R KO mice, an overall down regulation was also In WT animals, exposure to DSS led to the development of colitis with observed during the acute phase, but a trend towards baseline levels was similar clinical, histopathological and molecular alterations to those observed in the chronic phase of colitis. Nevertheless, CB1, MOR and previously described [18,19,30]. Moreover, we also observed that the TPH1 showed a persistent down-regulation during both the acute and inflammatory condition showed a chronification, characterized by the the chronic phase of inflammation (in all cases P < 0.05 vs. respective persistence over time, although with some attenuation, of the structural, control group). On the other hand, expression of NGF, SERT, TRPV1 and molecular and biochemical alterations observed during the acute phase TRPV3 in σ1R KO mice was down-regulated only during the acute phase and a remission of the clinical signs. This chronification process co­ of colitis (in all cases P < 0.05 vs. respective control group), while incides with the evolution of DSS-induced colitis previously described during the chronic phase a slight down-regulation was detected, for the same strain of mice [18] and shares similarities with the quies­ although statistical significance was not achieved (Fig. 8). In σ1R KO cent phases of inflammatory bowel disease in humans [30]. mice, expression of CB2 was significantly up-regulated in the chronic In σ1R KO animals, exposure to DSS led to the induction of colitis phase of colitis. with, essentially, the same characteristics as those discussed above for Fig. 6. Baseline abdominal (A) and paw (B) withdrawal thresholds in the von Frey test in WT and σ1R KO mice. Data are mean ± SEM of n = 6–10 mice per group. 6 S. López-Estévez et al. Biomedicine & Pharmacotherapy 143 (2021) 112126 Fig. 7. Sensitivity thresholds to mechanical stimulation during DSS-induced colitis in WT and σ1R KO mice. (A) abdominal and (B) paw withdrawal thresholds in WT and σ1R KO mice. In all cases, mechanical sensitivity was determined in basal conditions (day − 1, d-1) and at experimental days 3 (d3),7 (d7) and 21 (d21). Data show normalized values (relative force) with respect to basal measurements at experimental day − 1, taken as a relative force of 1. Reductions in relative force indicate the development of hypersensitivity. Data are mean ± SEM of n = 6–10 mice per group. *P < 0.05 vs. d-1 of respective experimental group. WT mice. Overall, these observations suggest that σ1Rs play a minor role with colitis, showed similar expression levels as those detected in in the development of intestinal inflammation. Nevertheless, some dif­ non-inflamed controls. This suggests that σ1Rs might regulate vascular ferences were observed between WT and σ1R mice, particularly as it permeability and extravasation, at least partially throughout relates to structural and molecular parameters during the chronic phase NO-dependent mechanisms, and thus, exert some modulatory effects on of colitis. Firstly, the presence of submucosal edema was significantly inflammation. Additionally, in σ1R KO mice, expression of reduced in σ1R KO vs. WT mice during chronic colitis. This finding pro-inflammatory cytokines, as well as iNOS and COX-2, was normalized agrees with previous data showing a reduction in subepithelial edema in during the chronic phase of colitis, while remaining up-regulated in WT σ1R KO mice in a model of cyclophosphamide-induce cystitis [31] or the animals. Altogether, these data suggest that σ1Rs might exert a positive reduction in paw edema, elicited by the intraplantar injection of carra­ immunomodulatory action, as previously suggested in other models [14, geenan, associated to the blockade of σ1Rs with specific antagonists [14, 31,32], likely facilitating the recovery in chronic conditions, at least as it 15]. Moreover, the attenuation of paw edema might implicate relates to intestinal inflammation. NOS-dependent mechanisms affecting vascular permeability and Compelling evidences implicate σ1Rs in pain mechanisms [12]. In extravasation [14,15]. Interestingly, in σ1R KO mice treated with DSS, our experimental conditions, σ1R KO and WT mice showed similar iNOS expression, which was moderately up-regulated in WT animals mechanosensitivity, as determined by assessing withdrawal thresholds 7 S. López-Estévez et al. Biomedicine & Pharmacotherapy 143 (2021) 112126 Fig. 8. (A) Heat map of the relative mRNA expression of sensory-related markers in the different experimental groups. Note that, as expected, no σ1R expression was detected in σ1R KO animals. Each vertical line, with a number, corresponds to an individual animal within the corresponding experimental group. “X” denotes samples with no expression detected. (B) Detail of the expression changes for the mean genes modified, according to A. Each point represents an individual animal; the horizontal bar with errors represents the mean ± SEM. *P < 0.05 vs. respective control group; #P < 0.05 vs. respective acute control group; +P < 0.05 vs. respective WT group. to the mechanical stimulation of the lower abdominal wall, likely abdominal level only a transitory state of hypersensitivity was observed reflecting responses associated to the mechanical stimulation of the during the acute phase of colitis, with a clear tendency towards normali­ abdominal wall and the underlying viscera (mainly intestine), or the zation during the chronic phase. These observations are in agreement with hind limbs. These observations agree with previous data showing that previous data showing a reduction in behavioral responses to visceral pain σ1R KO mice perceived and responded normally to acute somatic me­ in σ1R KO mice after intracolonic administration of capsaicin [17] or chanical nociceptive stimuli [17,24] and with pharmacological obser­ during cystitis [31] and the reduction of nociceptive responses associated to vations showing that σ1R ligands, either agonists or antagonists, have no neuropathic pain [24,36,37]. Moreover, these data in σ1R KO mice further effects by themselves on somatic mechanosensitivity in basal conditions confirm pharmacological observations showing blockade of somatic pain [11,33,34]. Altogether, these results support the view that σ1Rs are not responses by selective σ1R antagonists [14,15,25,34,38]. Altogether, these involved in normal pain responses. Alternatively, we cannot discard that data strongly suggest a key involvement of σ1Rs in the development of compensatory mechanisms, associated to the constitutive absence of inflammation-dependent referred hypersensitivity, consistent with previ­ σ1Rs, lead to normal basal pain responses in these animals. ous reports. Moreover, taking into account the fact that the stimulation of Intestinal inflammation has been associated to the development of the abdominal wall is likely to implicate somatic and visceral pain-related visceral hypersensitivity as well as referred hyperalgesia in several body responses, our observations also support an implication of σ1Rs in visceral regions, including the abdominal wall, tail and hind paws [1,5,6,35]. In sensitivity. agreement with this, results obtained here show that colitic WT mice To further understand the mechanisms implicated in these changes showed mechanical hypersensitivity, manifested as a persistent reduction we assessed the local (colon) expression of different sensory-related in the withdrawal threshold to the mechanical stimulation of the abdom­ markers implicated in viscerosensitivity. During colitis, a general inal wall (likely reflecting a combination of somatic hypersensitivity -from down-regulation of sensory markers was observed, regardless the ge­ the abdominal wall per se- and visceral hypersensitivity of the underlying notype considered. Thus, supporting the development of nociceptive viscera -intestine-) and the hind limbs. Interestingly, the sensitizing effects alterations, at least at a molecular level, during inflammation. It is of inflammation were significantly attenuated in σ1R KO mice. Indeed, in difficult to establish a direct correlation between gene expression σ1R KO animals, paw sensitivity was not affected during colitis, while at the changes of pain-related markers and pain-related responses since a 8 S. López-Estévez et al. Biomedicine & Pharmacotherapy 143 (2021) 112126 Fig. 9. (A) Representative Western blots showing tERK (left) and pERK (right) in the different experimental groups. (B) Relative spinal expression of tERK (left) and pERK (right). Data are mean ± SEM of n = 6 animals per group. down-regulation was detected for both pro- and anti-nociceptive Sensitization of pain mechanisms can occur at either peripheral and/ markers. Therefore, the final functional outcome is likely to depend or central levels. Our results, as discussed above, suggest that peripheral upon the balance between changes in expression that favor or counteract (colonic) changes might contribute to the sensitization processes asso­ pain-related mechanisms, as previously suggested for other experi­ ciated to inflammation. Nevertheless, to assess the potential participa­ mental conditions related to intestinal sensitivity [39,40]. Despite this, tion of central (spinal) sensitization, we also assessed ERK distinctive changes in some pain-related markers were observed in σ1R phosphorylation at the level of the lumbosacral spinal cord. Lower KO animals. Specifically, the up-regulation detected for CB2 during lumbar and upper sacral segments of the spinal cord represent the main chronic colitis might be of particular significance. Given the site of entry to the central nervous system for sensory afferents arising anti-nociceptive effects associated to the activation of CB2 [41–43], the from the colon implicated in pain responses in rodents [49]. Within the up-regulation observed might explain, at least in part, the attenuation of spinal cord, ERK phosphorylation is regarded as a key process involved pain-related responses observed in these animals during colitis. in pain processing and sensitization. Indeed, an increase in spinal The TRPV family of receptors has been related to inflammation and phosphorylated ERK (pERK) has been described in several models of pain. In particular, TRPV1 expression has been positively correlated somatic [24] and visceral pain [50]. Moreover, σ1Rs might be implicated with pain severity in patients with quiescent IBD [44] and an in this process since phosphorylation of spinal ERK was attenuated in up-regulation has also been observed during colonic inflammation [45], σ1R KO mice in a model of neuropathic pain where somatic hypersen­ likely contributing to the visceral hypersensitivity observed after colitis sitivity was induced by peripheral nerve injury [24,51]. Although these [46]. In the present studies, a down regulation of TRPV1 associated with evidences, in the present experimental conditions we did not detect inflammation, either acute or chronic, was observed in WT mice, even changes in ERK phosphorylation neither at the acute nor the chronic though the presence of mechanical hypersensitivity. These apparently phase of colitis, regardless the genotype considered. This might suggest contradictory observations reinforce the importance of the balance be­ the involvement of different mechanisms, with different involvement of tween pro- and anti-nociceptive mechanisms in the final outcome as it ERK and/or different kinetics in the phosphorylation process, as it re­ relates to pain, as discussed above. Alternatively, and given the lates to the development of sensitization during inflammatory and pro-nociceptive effects of TRPV1, a down-regulation of the receptor neuropathic pain. might be interpreted as a compensatory mechanism developed under In summary, the present data show that σ1Rs play a minor role in some conditions (such as acute inflammation) to avoid abnormal modulating intestinal (colonic) inflammation. Although some molecular excessive pain. Furthermore, recent studies have described interactions markers of inflammation were attenuated in σ1R KO mice, these changes between σ1R and TRPV1 receptors [47,48], thus indicating that both did not translate in an evident clinical improvement and only correlated receptors might interact during states of hypersensitivity facilitating with a moderate reduction in submucosal edema. As expected, inflam­ pain. Indeed, σ1R antagonism results in the negative regulation of the mation was associated to the development of hypersensitivity, likely of protein expression of TRPV1 in the plasma membrane of sensory neu­ both somatic and visceral origin. These pain-related alterations were rons and, consequently, a decrease in nociceptive responses [47]. attenuated in σ1R KO mice, thus confirming a role of σ1Rs in the Therefore, the lack of functional σ1Rs, leading to an altered TRPV1-σ1R development of hypersensitivity. 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