biomedicines Article Genetic and Pharmacological Blockade of Sigma-1 Receptors Attenuates Inflammation-Associated Hypersensitivity during Acute Colitis in CD1 Mice Sergio López-Estévez 1,2 , Mònica Aguilera 1,2 , Georgia Gris 3 , Beatriz de la Puente 3 , Alicia Carceller 3 and Vicente Martínez 1,2,4, * 1 Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain; sergio.lopez.estevez@gmail.com (S.L.-E.); monica.aguilerap@gmail.com (M.A.) 2 Neuroscience Institute, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain 3 Department of Pharmacology, Welab Barcelona, 08028 Barcelona, Spain; ggris@welab.barcelona (G.G.); bdelapuente@welab.barcelona (B.d.l.P.); acarceller@welab.barcelona (A.C.) 4 Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, 28049 Madrid, Spain * Correspondence: vicente.martinez@uab.es; Tel.: +34-93-5813834 Abstract: Sigma-1 receptors (σ1 Rs) are implicated in nociception, including pain sensitization, and inflammation. We assessed the role of σ1 Rs on acute colitis-associated hypersensitivity using both genetic (constitutive knockout) and pharmacological blockade of the receptor. Colitis was induced in CD1 wild-type (WT) and σ1 R KO mice (exposure to dextran sodium sulfate, 3%). A von Frey test was used to assess referred mechanosensitivity (abdominal and plantar withdrawal responses). The effects of the selective σ1 R antagonists BD1063 and E-52862 were also assessed in WT animals. The expression of immune and sensory-related markers (RT-qPCR, Western blot) was assessed in the colon and lumbosacral spinal cord. The genetic ablation or pharmacological blockade of σ1 Rs attenuated acute colonic inflammation in a similar manner. Mechanosensitivity was similar in WT and σ1 R KO mice before colitis. In WT mice, but not in σ1 R KO, colitis was associated with the development of referred Citation: López-Estévez, S.; Aguilera, mechanical hypersensitivity, manifested as a reduction in the withdrawal thresholds to mechanical M.; Gris, G.; de la Puente, B.; probing (paw and abdominal wall). In WT mice, BD1063 and E-52862 blocked colitis-associated Carceller, A.; Martínez, V. Genetic and Pharmacological Blockade of hypersensitivity. A genotype- and treatment-related differential regulation of sensory-related markers Sigma-1 Receptors Attenuates was detected locally (colon) and within the spinal cord. σ1 Rs are involved in the development of Inflammation-Associated acute intestinal inflammation and its associated referred mechanical hypersensitivity. The selective Hypersensitivity during Acute Colitis modulation of sensory-related pathways within the colon and spinal cord might be part of the in CD1 Mice. Biomedicines 2023, 11, underlying mechanisms. These observations support the pharmacological use of σ1 R antagonists for 2758. https://doi.org/10.3390/ the treatment of intestinal inflammation-induced hypersensitivity. biomedicines11102758 Academic Editor: Shao-Ming Wang Keywords: BD1063; colitis; E-52862; hypersensitivity; intestinal inflammation; pain; sigma 1 receptor; visceral pain Received: 30 August 2023 Revised: 3 October 2023 Accepted: 5 October 2023 Published: 12 October 2023 1. Introduction Visceral pain is a common symptom in inflammatory and functional gastrointesti- nal (GI) disorders [1–3]. In these conditions, multiple factors, including the release of Copyright: © 2023 by the authors. inflammatory mediators combined with a disturbed epithelial barrier function, contribute Licensee MDPI, Basel, Switzerland. to the sensitization of peripheral nerve endings within the gut wall, thus resulting in an This article is an open access article altered visceral sensory perception and abdominal pain [2,4]. Moreover, visceral pain distributed under the terms and typically refers to non-visceral somatic structures due to the convergence of visceral and conditions of the Creative Commons somatic nerve fibers in the same second-order neurons within the dorsal horn of the spinal Attribution (CC BY) license (https:// cord [5,6]. Therefore, states of visceral pain/hypersensitivity are frequently associated with creativecommons.org/licenses/by/ referred somatic hypersensitivity. Taking into account the differences in their anatomical 4.0/). Biomedicines 2023, 11, 2758. https://doi.org/10.3390/biomedicines11102758 https://www.mdpi.com/journal/biomedicines Biomedicines 2023, 11, 2758 2 of 22 origin and the neurobiological mechanisms that mediate the sensory process, visceral and somatic pain are considered different entities. A comprehensive review of the visceral pain neurobiological substrata, including the pathways and receptors/mediators involved, can be found in [3,5,6]. So far, specific pharmacological treatments against visceral pain, such as pain arising from the GI tract, have not been approved, and clinicians often use the same medications as for somatic pain [3]. In this respect, several studies suggest sigma-1 receptors (σ1 Rs) as an effective phar- macological target for pain treatment, including visceral pain [7–9]. The σ1 R is a ligand- regulated molecular chaperone with a wide central and peripheral distribution, which has been implicated in a variety of physiological and pathological conditions, including immuno- and neuromodulatory effects related to the modulation of pain mechanisms at either the central or peripheral levels [7–9]. Interestingly, several pieces of evidence suggest that the pharmacological agonism or antagonism of σ1 R does not interfere with the per- ception of several stimuli in basal conditions [10,11] and, consequently, with normal pain responses. However, under pathological conditions with states of altered pain signaling, such as in some chemical and neuropathic somatic pain models, the genetic or pharmaco- logic blockade of σ1 Rs could have a positive impact on sensory mechanisms, leading to the modulation of pain behavior and hypersensitivity [10,12,13]. Following these observations, further studies have validated the pharmacological blockade of σ1 R as an effective option to treat central and peripheral inflammatory pain [9,14–16]. Although these evidences, similar studies related to visceral pain are scarce. In a model of visceral pain induced by the intracolonic injection of capsaicin, and in a model of cyclophosphamide-induced cystitis, the constitutive absence of σ1 Rs (σ1 R knockout (KO) mice) or the blockade of the receptor with selective antagonisms resulted in a reduction in the number of pain-related behav- iors [17,18]. Following this, we have shown the implications of σ1 Rs on the development of intestinal inflammation-associated referred hypersensitivity in C57BL76 σ1 R KO mice [19]. To further expand these observations, the present work aims to assess the potential role of σ1 Rs in the development of acute intestinal inflammation and inflammation-related hypersensitivity in CD1 mice with dextran sodium sulfate (DSS)-induced acute colitis. For this purpose, two approaches were followed: (i) the genetic blockade of σ1 Rs based on the constitutive absence of the receptor, using KO mice; and (ii) the pharmacological blockade of σ1 Rs, using the selective σ1 R antagonists BD1063 and E-52862. Moreover, to gain in- sight into the underlying mechanisms associated with σ1 R-mediated effects, changes (gene and protein expression) in the peripheral (colon) and central (spinal cord) sensory-related markers involved in pain processing and sensitization mechanisms were also characterized. 2. Materials and Methods 2.1. Animals Young adult male CD1 mice (n = 96; Crl/CD1 (ICR); Charles River, France) and σ1 receptor knockout CD1 mice (n = 16) [20], 6–7 weeks old at the beginning of the studies, were used. The animals were generated and maintained by Charles River under a contract with Esteve Pharmaceuticals S.A. (Barcelona, Spain). To obtain CD-1 KO mice, homozygous KO mice [18] were backcrossed for 10 generations onto the CD-1 background to reduce to less than 1% the genetic material remaining from the original background [21]. The animals were group-housed in standard polycarbonate cages (4–6 animals per cage, with direct bedding of wood fibrillated fibers) and maintained in conventional conditions in an environmentally controlled room (20–22 ◦ C, 12 h light/dark cycle), with food and water ad libitum, except when receiving DSS. The mice were allowed to acclimatize to the animal facility for at least 1 week before starting the studies. All experiments were performed in accordance with the EU and local regulations and were approved by the Ethical Committee of the Universitat Autònoma de Barcelona (protocols 3039 and 3957) and the Generalitat de Catalunya (protocols 8823 and 9915). Biomedicines 2023, 11, 2758 3 of 22 2.2. Colitis Induction A solution of DSS (45 kDa; 3% concentration in normal tap water; TdB Consultancy AB, Uppsala, Sweden) was used to induce colitis. Fresh DSS solutions were prepared daily during the 5-day treatment period. Following this protocol, CD1 mice develop a flare of acute colitis that peaks 7–8 days after starting the exposure to DSS. Similar protocols have been used in previous studies in mice to induce colitis [22,23]. The control mice received normal tap water. 2.3. Drugs The selective σ1R antagonists BD1063 (1-[2-(3,4-dichlorophenyl) ethyl]-4-methylpiperazine di- hydrochloride) [24] and E-52862 (also named S1RA or MR309; 4-[2-[[5-methyl-1-(2-naphthalenyl)- 1H-pyrazol-3-yl]oxy]ethyl] morpholine) [25] were used (Laboratorios Dr. Esteve S.A., Barcelona, Biomedicines 2023, 11, x FOR PEER REVIEW 4 of 22 Spain). 6-Thioguanine (6-TG) (Sigma-Aldrich, St. Louis, MO, USA) and 5-aminosalicylic acid (mesalazine, 5-ASA) (Cayman Chemical, Ann Arbor, MI, USA) were used as positive controls, given their demonstrated anti-inflammatory activity within the GI tract [26,27]. All drugs were day 3), andimmediately dissolved at the peak of inflammation before (immediately use in a 0.5% before euthanasia, experimental solution of hydroxypropylmethyl day cellulose (HPMC; 7)Sigma-Aldrich) (Figure 1). in distilled water. The doses were selected based on previously published data and/or pilot studies in our experimental conditions. Table 1. Summary of experimental groups. 2.4. Evaluation of Referred Mechanical Hypersensitivity: Von Frey Test Genotype Colitis Induction Treatment n The mechanical sensitivity was determined using the classical von Frey test, following No (Tap water) - 6 Wild-typeprotocols [19]. The animals were placed in compartment enclosures previously published Yes (3% DSS) - 6 Study 1 chamber with a framed metal mesh floor through which von Frey monofilaments in a test No (Tap water) - sensitivity was assessed 8 (North Coast σ1RMedical, knockout Inc., Gilroy, CA, USA) were applied. Pain Yes (3% DSS) - after a 30 min habituation period to the testing environment. Referred pain was determined 8 in two separate body regions, the hind paws and Vehicle (5 mL/kg,wall. the abdominal po, BID) 15 the When testing sensitivity of the abdominal wall, the perianal and BD1063 (20 mg/Kg, external genitaliapo, BID)were avoided, areas 6 concentrating the stimulation Noon(Tap thewater) lower and E-52862 (20 mg/Kg, mid-abdomen, aspo, BID) commonly 9 reported 6-TG (2 mg/Kg, in the literature [17,18,28]. Paw sensitivity was quantified po, SID) the hind by measuring 6 paw withdrawal response to punctate mechanical stimulation,5-ASA (50as mg/Kg, po, BID) described elsewhere 6[10,11]. Study 2 Wild-type Pain thresholds were determined using the up-down Vehicle (5 mL/kg, po, BID)and represent method paradigm 15 the mechanical stimulus that produces 50% of BD1063 the maximal response (20 mg/Kg, [29,30]. The po, BID) 6 data were normalized to a baseline measurement (taken as 1), obtained 24 h before starting Yes (3% DSS) E-52862 (20 mg/Kg, po, BID) 9 the experimental procedures (Figure 1). All measurements were performed twice, with a 6-TG (2 mg/Kg, po, SID) 6 30–40 min recovery period in between, by two treatment-blinded independent investigators. 5-ASA (50 mg/Kg, po, BID) 6 For each animal, the mean value of the two observations was taken as the measure of pain Abbreviations: po: oral; BID: twice a day; SID: once a day. sensitivity. Figure 1. Details of the experimental protocols followed. See Table 1 for details on the treatments Figure 1. Details applied. VF: vonofFrey the test. experimental protocols followed. See Table 1 for details on the treatments applied. VF: von Frey test. In the second study, the WT mice were randomly divided into five experimental groups according to the treatment received (Table 1): vehicle (0.5% HPMC in distilled wa- ter, 5 mL/kg, po), BD1063 (20 mg/Kg, po, BID), E-52862 (20 mg/Kg, po, BID), 6-TG (2 mg/kg, po, SID), or 5-ASA (50 mg/kg, po, BID). The treatments were applied in a preven- Biomedicines 2023, 11, 2758 4 of 22 Table 1. Summary of experimental groups. Colitis Genotype Treatment n Induction No (Tap water) - 6 Wild-type Yes (3% DSS) - 6 Study 1 No (Tap water) - 8 σ1 R knockout Yes (3% DSS) - 8 Vehicle (5 mL/kg, po, BID) 15 BD1063 (20 mg/Kg, po, BID) 6 No (Tap water) E-52862 (20 mg/Kg, po, BID) 9 6-TG (2 mg/Kg, po, SID) 6 5-ASA (50 mg/Kg, po, BID) 6 Study 2 Wild-type Vehicle (5 mL/kg, po, BID) 15 BD1063 (20 mg/Kg, po, BID) 6 Yes (3% DSS) E-52862 (20 mg/Kg, po, BID) 9 6-TG (2 mg/Kg, po, SID) 6 5-ASA (50 mg/Kg, po, BID) 6 Abbreviations: po: oral; BID: twice a day; SID: once a day. 2.5. Experimental Protocols In the first study, the WT and σ1 R KO mice were randomly divided into 2 experimental groups per genotype, receiving either tap water or a solution of 3% DSS during a 5-day period (days 0–5) (Table 1). The animals were euthanized after a 2-day recovery period following DSS exposure (experimental day 7) for the assessment of colitis and the obtention of samples (Figure 1), coinciding with the peak of inflammation [23,31,32]. Throughout the study, their individual body weight, general state, and the presence of clinical signs associated with the development of colitis were assessed on a daily basis. Mechanical sensitivity (von Frey test) was assessed at the beginning of the studies (experimental day −1, as a baseline measure of sensitivity), during DSS exposure (experimental day 3), and at the peak of inflammation (immediately before euthanasia, experimental day 7) (Figure 1). In the second study, the WT mice were randomly divided into five experimental groups according to the treatment received (Table 1): vehicle (0.5% HPMC in distilled water, 5 mL/kg, po), BD1063 (20 mg/Kg, po, BID), E-52862 (20 mg/Kg, po, BID), 6-TG (2 mg/kg, po, SID), or 5-ASA (50 mg/kg, po, BID). The treatments were applied in a preventive manner starting 1 day before the initiation of the colitis induction protocol and after the baseline measurement of sensitivity. The animals were administered orally once (9:00–10:00 AM) or twice daily (9:00–10:00 AM and 18:00–19:00 PM), as indicated (Table 1). The doses and treatment protocols were based on pilot studies or previous reports showing efficacy in similar experimental conditions. For each treatment group, the animals were randomly divided into 2 subgroups, receiving either tap water or 3% DSS during a 5-day period (experimental days 0–5) for the induction of colitis (Figure 1; Table 1). As in the previous study, the animals were euthanized after a 2-day recovery period following DSS exposure (experimental day 7) for the assessment of colitis and the obtention of samples (see below). Throughout the study, their individual body weight, general state, and the presence of clinical signs associated with the development of colitis were assessed on a daily basis. Mechanical sensitivity (von Frey test) was assessed at the beginning of the studies (experimental day −1, as a baseline measure of sensitivity), during DSS exposure (experimental day 3), and at the peak of inflammation (immediately before euthanasia, experimental day 7) (Figure 1). 2.6. Sample Collection Immediately after the last von Frey test (experimental day 7), the mice were deeply anesthetized with isoflurane (Isoflo; Esteve, Barcelona, Spain) and euthanatized by exsan- guination through intracardiac puncture, followed by cervical dislocation. Thereafter, a Biomedicines 2023, 11, 2758 5 of 22 medial laparotomy was performed, the ceco-colonic region was localized, and the cecum and colon were dissected. Two tissue samples from the proximal-middle colon (about 1.5 cm each) were collected. A sample was frozen immediately in liquid nitrogen. A second sample was fixed in 4% paraformaldehyde. After an overnight fixing, the tissues were paraffin-embedded and 5 µm-thick sections were obtained. Lumbosacral (L3-S2) spinal cord samples were also collected and frozen immediately in liquid nitrogen. The frozen samples were stored at −80 ◦ C until analysis. During the necropsy, the liver, adrenal glands, thymus, and spleen were dissected and weighed. 2.7. Clinical and Macroscopic Assessment of Inflammation The clinical assessment of inflammation included daily monitoring of the body weight, appearance of feces, and general health condition [23]. A score (0–8) was assigned to the health condition (including hunched posture, piloerection, fecal consistency and anal inflammation), where 0 indicates normal activity/fur/fecal content/no anal inflamma- tion, 1 indicates abnormal gait/bristly fur/wet anus/loose fecal content, and 2 indicates prostrated animal/dirty fur/watery or bloody rest on anus/watery diarrhea. During the necropsy, the macroscopic appearance of the colon was scored (macroscopic inflammatory score, 0–15) according to procedures established elsewhere [23]. In brief, the consistency of the fecal contents (score 0–3), the presence of visible fecal blood (score 0–3), the evidence and extent of edema (0–3), the wall thickness (0–3), tissue stiffness (0–2), and presence of ulcerations (0–1) were assessed. 2.8. Histological Studies For the histological examinations, hematoxylin–eosin-stained sections from the colon were obtained following standard procedures. A histopathological score (ranging from 0: normal, to 12: maximal alterations) was assigned to each animal [19,33]. The parameters scored included: epithelial structure (0: normal; 1: mild alterations of the villi; 2: local villi destruction and/or fusion; 3: generalized villi destruction and/or fusion), structure of the crypts (0: normal; 1: mild alterations of the crypts; 2: local destruction of the crypts; 3: generalized destruction of the crypts), presence of edema (0: normal; 1: mild local edema in submucosa and/or lamina propria; 2: moderate diffuse edema in submucosa and/or lamina propria; 3: severe generalized edema in submucosa and/or lamina propria), and presence of inflammatory infiltrate (0: normal; 1: mild localized infiltrate; 2: mild generalized infiltrate; 3: severe generalized infiltrate). Scoring was performed on coded slides by two independent researchers, and the mean value of the two scores was taken as the final score per animal. 2.9. Gene Expression: Quantitative Reverse Transcription-PCR The total RNA was extracted from frozen tissue of the colon and spinal cord samples using TRI reagent with a Ribopure Kit (Ambion/Applied Biosystems, Foster City, CA, USA). Later, a two-step quantitative real-time PCR (RT-qPCR) was performed. The RNA samples were converted into cDNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The PCR reaction mixture was incubated on the Bio-Rad CFX384 Touch Real-Time PCR Detection System (Bio-Rad). All samples were assayed in tripli- cate. The cycle thresholds for each sample were obtained, and the data were analyzed using the comparative Ct method (2−∆∆Ct ) with the WT control (for the analysis of the samples originated in the first experiment, including the σ1 R KO mice) or the vehicle group (for the analysis of the samples generated in the second experiment pharmaco- logical treatments in WT mice) serving as the calibrator [34]. TaqMan® gene expression assays (hydrolysis probes, Applied Biosystems) for interferon γ (INF-γ) (Mm01168134_m1), interleukin 1β (IL-1β) (Mm00434228_m1), interleukin 6 (IL-6) (Mm00446190_m1), inter- leukin 10 (IL-10) (Mm00439614_m1), interleukin 12 (IL-12p40) (Mm00434174_m1), cannabi- noid receptors 1 (CB1) (Mm01212171_s1) and 2 (CB2) (Mm00438286_m1), µ-opioid re- ceptor (MOR) (Mm01188089_m1), tryptophan hydroxylase 1 (TPH1) (Mm00493794_m1), Biomedicines 2023, 11, 2758 6 of 22 transient receptor potential vanilloid 1 (TRPV1) (Mm01246302_m1), nerve growth factor (NGF) (Mm00443039_m1), metabotropic glutamate receptor 2 (GluR2) (Mm01235831_m1), tachykinin receptor 1 (NK1r) (Mm00436892_m1), neuronal nitric oxide synthase 1 (nos 1, nNOS) (Mm01208059_m1), glutamate receptor ionotropic, NMDA 2B (Grin2b, NR2B) (Mm00433820_m1), and σ1 receptor (σ1 R) (Mm00448086_m1) were used. β-2-microglobulin (Mm00437762_m1) was used as an endogenous reference gene. 2.10. Protein Expression: Western Blot Lumbosacral spinal cord samples were homogenized by sonication in radioimmuno- precipitation assay (RIPA) buffer, and the supernatant was obtained. Equal amounts of protein (30 µg) were fractionated by 10% (w/v) SDS–PAGE and transferred onto a polyvinylidene difluoride membrane, blocked with 5% non-fat dry milk in Tris-Tween 20-buffered Saline (T-TBS) for 1 h. The membranes were then incubated in 1% non-fat dry milk in T-TBS overnight at 4 ◦ C with primary antibodies against the protein targets of interest (Table 2). Mouse or rabbit anti-GAPDH antibody or anti-β-tubulin antibody was used as a loading control depending on the protein of interest, and the origin of the primary antibody used. After washing with T-TBS, the blots were incubated for 1 h with secondary peroxidase-conjugated antibodies (see Table 2). Immunoreactive bands were detected by a peroxidase reaction using an enhanced chemiluminescence method (WesternSure® PREMIUM Chemiluminescent Substrate, Li-cor) and the CDiGit ® Blot Scanner (LI-COR). Quantification of the Western blots was carried out with Image Studio™ Lite Software, version 5.2.5. Table 2. Details of antibodies used for Western blot. Type Reactivity Host Dilution Source Primary β-tubulin Goat polyclonal 1:1000 Santa Cruz Biotech. (#sc-9935) Primary CaMKII Mouse monoclonal 1:2000 Invitrogen (#MA1-048) Primary pCaMKII Mouse monoclonal 1:1000 Invitrogen (#MA1-047) Primary GFAP Mouse monoclonal 1:10,000 Cell Signaling (#3670) Primary GAPDH Mouse monoclonal 1:80,000 Sigma-Aldrich (#G8795) Primary GAPDH Rabbit polyclonal 1:20,000 Sigma-Aldrich (#G9545) Primary tERK Rabbit polyclonal 1:30,000 Sigma-Aldrich (#M5670) Primary pERK Mouse monoclonal 1:1000 Sigma-Aldrich (#M8159) Primary p38 Rabbit polyclonal 1:1000 Invitrogen (#AHO1202) Primary pp38 Rabbit monoclonal 1:1000 Invitrogen (#MA5-15177) Secondary Anti-Mouse IgG Goat polyclonal 1:2000 Sigma-Aldrich (#A5278) Secondary Anti-Rabbit IgG Goat polyclonal 1:4000 Sigma-Aldrich (#A9169) Secondary Anti-Goat IgG Donkey polyclonal 1:2000 Abcam (#ab97110) 2.11. Statistical Analysis The data are expressed as the mean ± SEM. A robust analysis (one iteration) was used to obtain the mean ± SEM for the RT-qPCR data. The data were analyzed by one-, two-, or three-way ANOVA, as appropriate, followed, when necessary, by a Bonferroni’s multiple comparisons test. The data were considered statistically significant when p < 0.05. Statistical analyses were performed using GraphPad Prism 9 (GraphPad Software, La Jolla, CA, USA) or SPSS program (version 17 for Windows, IBM, Madrid, Spain). 3. Results 3.1. σ1 R KO CD1 Mice Develop an Attenuated Acute Colitis WT CD1 mice exposed to 3% DSS for 5 consecutive days showed clinical signs consis- tent with the development of acute colitis, including body weight loss, piloerection, loose feces/watery diarrhea, and the presence of fecal blood. These changes were particularly ev- ident at experimental days 6 and 7. Clinical signs and body weight loss were attenuated in the σ1 R KO animals exposed to DSS (Figure 2A). A three-way ANOVA revealed significant effects of time (p < 0.05), genotype (p < 0.05), and DSS exposure (p < 0.05) and significant effect interactions among the three factors (p < 0.05), thus indicating a different response to DSS over time in both genotypes. Water intake was similar across the experimental groups. loose feces/watery diarrhea, and the presence of fecal blood. These changes were particu- larly evident at experimental days 6 and 7. Clinical signs and body weight loss were at- tenuated in the σ1R KO animals exposed to DSS (Figure 2A). A three-way ANOVA re- vealed significant effects of time (p < 0.05), genotype (p < 0.05), and DSS exposure (p < 0.05) and significant effect interactions among the three factors (p < 0.05), thus indicating a dif- 7 of 22 Biomedicines 2023, 11, 2758 ferent response to DSS over time in both genotypes. Water intake was similar across the experimental groups. In the current experimental conditions, no mortality was observed In the current experimental conditions, no mortality was observed associated with DSS associated with DSS exposure. exposure. A B C Figure 2. Assessment of DSS-induced Figure 2. Assessment of acute colitis in DSS-induced WTcolitis acute andinσ1WT R KO and CD1 σ1 R KOmice. CD1 (A) mice.Changes in in rela- (A) Changes relative body weighttive (%body change from weight day 0,from (% change taken dayas0, 100%) taken asand 100%) clinical signs. and clinical TheThe signs. DSS-treatment DSS-treatment period period is indicated by the greybyarea. is indicated (B)area. the grey Assessment of inflammation-related (B) Assessment parameters of inflammation-related parameters (macro- scores, (macroscopic scopic scores, colonic length, and colon relative weight) as determined during necropsy. (C) Histo- colonic length, and colon relative weight) as determined during necropsy. (C) Histopathological pathological scores of the colon scores for the of the colon for different experimental the different experimental groups. Data groups. Data areare mean mean ± SEM ± SEM ofanimals of 6–8 6–8 per animals per group. *:group. p < 0.05 *: pvs. respective < 0.05 control vs. respective group; control group;#:#:p p< <0.05 0.05vs. vs. WT miceexposed WT mice exposed to DSS. to DSS. During the necropsy, macroscopic signs of inflammation, together with a shortening and increased relative weight of the colon, was observed in WT mice receiving DSS (all p < 0.05 vs. non-colitic animals; Figure 2B). These inflammation-related parameters were significantly attenuated in the σ1 R KO mice (p < 0.05 vs. DSS-exposed WT mice for the three parameters assessed; Figure 2B). significantly attenuated in the σ1R KO mice (p < 0.05 vs. DSS-exposed WT mice for the three parameters assessed; Figure 2B). Microscopic analysis of the colonic tissue samples showed a normal histological structure Biomedicines 2023, 11, in 2758the control animals. Significant histopathological alterations were observed 8 of 22 in the WT mice exposed to DSS, reaching a total score of 10.04 ± 1.44 (p < 0.05 vs. non- colitic WT mice: 0.63 ± 0.21; Figures 2C and S1). In the healthy σ1R KO mice, an essentially normal histological structure was observed. Microscopic analysis of Significant histopathological the colonic tissue samples showedalterations were a normal histological observed in the σ1R KO mice exposed to DSS (histopathological score; 5.91 ± 0.92; p < 0.05 in structure in the control animals. Significant histopathological alterations were observed the WT mice exposed to DSS, reaching a total score of 10.04 ± 1.44 (p < 0.05 vs. non-colitic vs. healthy σ1R KO mice: WT mice:1.57 ± 0.34), 0.63 ± 0.21; although Figure 2C and a significant Figure S1). Inattenuation the healthy σ1was R KOobserved vs. mice, an essentially the WT animals exposed normal tohistological DSS (p < structure 0.05, Figures 2C andSignificant was observed. S1). Thishistopathological attenuation was partic-were alterations ularly evident, as it relates to the presence observed in the σ 1 R KO miceof submucosal edema (σ1R KO mice: 0.5 ± 0.16; exposed to DSS (histopathological score; 5.91 ± 0.92; p < 0.05 healthy σ1 R KO mice: 1.57 ± 0.34), although a significant attenuation was observed p < 0.05 vs. WT mice:vs.2.54 ± 0.37). vs. the WT animals exposed to DSS (p < 0.05, Figure 2C and Figure S1). This attenuation The mRNA of was all particularly the analyzed cytokines evident, was as it relates detectable to the presence ofand quantifiable submucosal edema (σby RT- 1 R KO mice: qPCR in the colonic tissues analyzed. 0.5 ± 0.16; p < 0.05 vs.TheWT basal expression mice: 2.54 ± 0.37). was similar in the WT and σ1R KO mice, except for the pro-inflammatory cytokine IL-12p40, The mRNA of all the analyzed cytokines which was was detectable upregulated and quantifiable by by RT-qPCR 4-fold in the σ1R KO animals (p < 0.05 vs. WT animals; Figure 3). Regardless of the geno- in the colonic tissues analyzed. The basal expression was similar in the WT and σ1 R KO mice, except for the pro-inflammatory cytokine IL-12p40, which was upregulated by 4-fold type considered, theinexpressionthe σ1 R KO of the pro-inflammatory animals (p < 0.05 vs. WT animals;cytokines Figure INF-γ, IL-1β,ofand 3). Regardless IL-6 the genotype was similarly upregulated in the considered, theDSS-treated expression of the mice (p < 0.05 vs. control pro-inflammatory WT cytokines or σIL-1β, INF-γ, 1R KO andmice; IL-6 was Figure 3). The pro-inflammatory cytokine similarly upregulated IL-12p40 in the wasmice DSS-treated upregulated (p < 0.05 vs.only in the control WT colitic or σ1 R KOWTmice; Figure 3). The pro-inflammatory cytokine IL-12p40 was upregulated only in the colitic WT mice (p < 0.05 vs. control WT mice). No changes were observed in the expression of the mice (p < 0.05 vs. control WT mice). No changes were observed in the expression of the anti-inflammatory cytokine IL-10, regardless anti-inflammatory cytokine IL-10, of the genotype regardless of the considered (Figure genotype considered 3). 3). (Figure Figure 3. Colonic expression Figure 3.ofColonic pro- (interferon-γ expression of pro-(INF-γ), IL-1β,(INF-γ), (interferon-γ IL-6, and IL-12p40) IL-1β, and anti-in- IL-6, and IL-12p40) and anti- flammatory cytokines inflammatory (IL-10) in WT and σ cytokines 1R KO (IL-10) CD1 in WT andmice. Each σ1 R KO CD1point represents mice. Each an individual point represents an individual animal; the horizontal animal; bar with errors represents the horizontal the represents bar with errors mean ± SEM. *: p± 0.05) and paw withdrawal thresholds (WT: 1.19 ± 0.08 g; σ1 R The WT and σ1Rσ1KO mice showed similar baseline abdominal (WT: 1.48 ± 0.02 g; σ1R KO: 1.43 ± 0.04 g; p > 0.05) and paw withdrawal thresholds (WT: 1.19 ± 0.08 g; σ1R KO: 1.21 Biomedicines 2023, 11, x FOR PEER REVIEW Biomedicines 2023, 11, 2758 9 of 22 ± 0.09 g; p > 0.05), as assessed with the von Frey test immediately before coli (experimental day −1). During the development of acute colitis, the WT mic KO: 1.21 ± 0.09 g; p > 0.05), as assessed with the von Frey test immediately before colitis referred hyperalgesia, manifested as a time-related, progressive reduction in induction (experimental day −1). During the development of acute colitis, the WT mice abdominal withdrawal developed referred thresholds hyperalgesia, manifested(both p < 0.05 vs. as a time-related, respective progressive sensitivity reduction in t basal conditions). In the control animals not exposed to DSS, paw and abd the paw and abdominal withdrawal thresholds (both p < 0.05 vs. respective sensitivity thresholds in basal conditions). In the control animals not exposed to DSS, paw and drawal thresholds remained stable over time (Figure 4). abdominal withdrawal thresholds remained stable over time (Figure 4). Figure 4. Time-course changes of sensitivity thresholds to mechanical probing of the abdominal wall Figure 4. Time-course changes of sensitivity thresholds to mechanical probing of t and hind paw during DSS-induced colitis in WT and σ1 R KO CD1 mice. The data represent abdominal wall and hind withdrawal paw (A) and pawduring withdrawalDSS-induced thresholds (B)colitis in WT in WT and and σ1 R KO σ1R KO animals CD1 mice. (normalized for The abdominal measurementswithdrawal at experimental (A) day −and paw 1 (d − 1) in withdrawal thresholds each experimental group). Data(B) are in WT mean andofσ1R KO ± SEM malized forgroup. 6 animals per measurements *: p < 0.05 vs. at d −experimental day(basal 1 of respective group −1 (dresponse) −1) in each experimental and the same time-pointgroup). ±inSEM of 6 animals the corresponding pergroup. control group. *: p < 0.05 vs. d −1 of respective group (basal response time-point in the corresponding control group. Similar to that observed in the WT animals, the σ R KO mice not exposed to DSS 1 showed a stable pain sensitivity throughout the experimental time (revealed by the absence Similar of changes in thetowithdrawal that observed in the thresholds). WT animals, Moreover, no changesthe σ1R in pain KO mice sensitivity werenot exp showed a stable pain sensitivity throughout the experimental time (reveale detected in the σ1 R KO mice exposed to DSS. In these animals, paw and abdominal withdrawal thresholds remained stable over time and were similar to the basal values sence of changes (determined in animals in the same the withdrawal before colitisthresholds). induction) or toMoreover, nothresholds the withdrawal changes in pa were detected determined in the in the σ animals 1R KO without mice(Figure colitis exposed 4). to DSS. In these animals, paw an withdrawal thresholds remained stable over time and were similar to the (determined in the same animals before colitis induction) or to the withdraw determined in the animals without colitis (Figure 4). mRNA for the sensory-related markers assessed was detected in all colo Biomedicines 2023, 11, 2758 10 of 22 mRNA for the sensory-related markers assessed was detected in all colonic samples, with the exception of σ1 Rs, which, as expected, was not detected in the KO mice. A dif- ferential gene expression regulation was observed during colitis. The basal expression iomedicines 2023, 11, x FOR PEER REVIEW 10 of 22 of the anti-nociceptive (CB1 and MOR) and pro-nociceptive markers (NGF, TPH1) was similar in the WT and σ1 R KO animals. However, a significant upregulation of the CB2 receptors (anti-nociceptive) and a significant downregulation of TRPV1 (pro-nociceptive) observed in both genotypes, were detected in the σ1 R KOwhere controlthe miceNGF (bothmRNA p < 0.05 levels were downregulated vs. expression by colitis levels in WT animals; Figure 5). During colitis, similar modulation in the expression of sensory-related mark- in both genotypes (p < 0.05 vs. healthy WT and σ1R KO mice, respectively; Figure 5). The ers was observed in both genotypes, where the NGF mRNA levels were downregulated anti-nociceptive markers by colitis in both showed genotypes (p 0.5 vs. the same relative structural weight alterations in non-colitic and without animals), histopatholog- ical improving scores as those of the vehicle-treated animals the histopathological alterations (Figure 7). also exposed to DSS (Figure 7C). The reference compounds, 6-TG and 5-ASA, attenuated the macroscopical scores of inflammation (although statistical significance was not achieved) and the increase in the relative colonic weight (p > 0.5 vs. relative weight in non-colitic animals), without improv- ing the histopathological alterations (Figure 7). mRNA expression of all the analyzed cytokines was detectable and quantifiable by Biomedicines 2023, 11, 2758 12 of 22 mRNA expression of all the analyzed cytokines was detectable and quantifiable by RT-qPCR in all samples. In the vehicle-treated animals, DSS exposure led to expression changes equal to those observed in the first study, comparing WT and σ1 R KO animals. However, due to the variability observed, statistical significance was not achieved (Figure 8). In animals exposed to DSS, BD1063 showed a tendency to normalize the expression of the pro-inflammatory cytokines INF-γ, IL-1β, and IL-6, without affecting the expression of IL-12p40. On the contrary, treatment with E-52862 resulted in a significant increase Biomedicines 2023, 11, x FOR PEER REVIEW 12 of 22 in the expression of all the pro-inflammatory cytokines assessed, although with high inter-individual variability (Figure 8). 6-TG and 5-ASA showed moderate effects, with a tendency to reduce the expression of pro-inflammatory cytokines, with the exception γ, INF-γ, of which, which, in the 5-ASA-DSS-treated animals,animals, in the 5-ASA-DSS-treated showedshowed an upregulation with high an upregulation interin- with high dividual variability interindividual (Figure(Figure variability 8). No8). changes were observed No changes in theinexpression were observed of theofanti- the expression the inflammatory cytokine anti-inflammatory IL-10, cytokine regardless IL-10, of the regardless of treatment considered. the treatment considered. A B C Figure 7. Figure 7. Assessment Assessment of of DSS-induced DSS-induced acute acute colitis colitis in in WT WT CD1 CD1 mice mice and and effects effects of of pharmacological pharmacological treatments. (A) Changes in relative body weight (% change from day 0, taken as treatments. (A) Changes in relative body weight (% change from day 0, taken as 100%) and 100%) and clinical clinical signs associated with exposure to DSS and pharmacological treatments (σ 1R antagonists, BD1063 or signs associated with exposure to DSS and pharmacological treatments (σ1 R antagonists, BD1063 or E- E-52862, 52862, 6-TG 6-TG or 5-ASA) or 5-ASA) at experimental at experimental dayday 7. (B) 7. (B) Assessment Assessment of colonic of colonic inflammation inflammation (inflamma- (inflammatory tory score, colon length, and colon relative weight) at the time of necropsy in the different experi- score, colon length, and colon relative weight) at the time of necropsy in the different experimental mental groups. (C) Histopathological scores in the different experimental groups. Data are mean ± groups. (C) Histopathological scores in the different experimental groups. Data are mean ± SEM SEM of 6–15 animals per group. *: p < 0.05 vs. respective control group; #: p < 0.05 vs. DSS-vehicle- of animals per group. *: p < 0.05 vs. respective control group; #: p < 0.05 vs. DSS-vehicle- 6–15 group. treated treated group. Biomedicines 2023, 11, x FOR PEER REVIEW 13 of 22 Biomedicines 2023, 11, 2758 13 of 22 Figure 8. Colonic Figure expression 8. Colonic of pro-(interferon-γ expression (INFγ), of pro-(interferon-γ IL-1β, (INFγ), IL-6,IL-6, IL-1β, andand IL-12p40) andand IL-12p40) anti-inflam- anti- matory cytokines (IL-10) in animals with DSS-induced colitis treated with the selective σ1R σantago- inflammatory cytokines (IL-10) in animals with DSS-induced colitis treated with the selective 1R nists (BD1063 or E-52862), 6-TG, or 5-ASA. Each point represents an individual animal; the horizon- antagonists (BD1063 or E-52862), 6-TG, or 5-ASA. Each point represents an individual animal; the tal bar with errors horizontal represents bar with the meanthe errors represents ± SEM. mean ± *: SEM. p < 0.05 *: p vs. respective < 0.05 non DSS-exposed vs. respective non DSS-exposed control group; #: p