RESUMEN
Abdominal pain is common in the general population and, in patients with irritable bowel syndrome, is attributed to visceral hypersensitivity. We found that oral administration of specific Lactobacillus strains induced the expression of mu-opioid and cannabinoid receptors in intestinal epithelial cells, and mediated analgesic functions in the gut-similar to the effects of morphine. These results suggest that the microbiology of the intestinal tract influences our visceral perception, and suggest new approaches for the treatment of abdominal pain and irritable bowel syndrome.
Asunto(s)
Dolor Abdominal/fisiopatología , Intestinos/fisiopatología , Lactobacillus acidophilus/fisiología , Receptores de Cannabinoides/fisiología , Receptores Opioides/fisiología , Dolor Abdominal/prevención & control , Administración Oral , Analgésicos Opioides/administración & dosificación , Analgésicos Opioides/farmacología , Animales , Antagonistas de Receptores de Cannabinoides , Colon/efectos de los fármacos , Colon/microbiología , Colon/fisiopatología , Relación Dosis-Respuesta a Droga , Células HT29 , Humanos , Indoles/administración & dosificación , Indoles/farmacología , Intestinos/efectos de los fármacos , Intestinos/microbiología , Masculino , Ratones , Ratones Endogámicos BALB C , Morfina/administración & dosificación , Morfina/farmacología , Naloxona/administración & dosificación , Naloxona/farmacología , Antagonistas de Narcóticos/administración & dosificación , Antagonistas de Narcóticos/farmacología , Probióticos/administración & dosificación , Probióticos/farmacología , Ratas , Ratas Sprague-Dawley , Receptor Cannabinoide CB2/antagonistas & inhibidores , Receptor Cannabinoide CB2/biosíntesis , Receptor Cannabinoide CB2/fisiología , Receptores de Cannabinoides/biosíntesis , Receptores Opioides/biosíntesis , Receptores Opioides mu/antagonistas & inhibidores , Receptores Opioides mu/biosíntesis , Receptores Opioides mu/fisiología , Recto/efectos de los fármacos , Recto/microbiología , Recto/fisiopatologíaRESUMEN
The symptoms of irritable bowel syndrome (IBS) include significant abdominal pain and bloating. Current treatments are empirical and often poorly efficacious, and there is a need for the development of new and efficient analgesics aimed at IBS patients. T-type calcium channels have previously been validated as a potential target to treat certain neuropathic pain pathologies. Here we report that T-type calcium channels encoded by the Ca(V)3.2 isoform are expressed in colonic nociceptive primary afferent neurons and that they contribute to the exaggerated pain perception in a butyrate-mediated rodent model of IBS. Both the selective genetic inhibition of Ca(V)3.2 channels and pharmacological blockade with calcium channel antagonists attenuates IBS-like painful symptoms. Mechanistically, butyrate acts to promote the increased insertion of Ca(V)3.2 channels into primary sensory neuron membranes, likely via a posttranslational effect. The butyrate-mediated regulation can be recapitulated with recombinant Ca(V)3.2 channels expressed in HEK cells and may provide a convenient in vitro screening system for the identification of T-type channel blockers relevant to visceral pain. These results implicate T-type calcium channels in the pathophysiology of chronic visceral pain and suggest Ca(V)3.2 as a promising target for the development of efficient analgesics for the visceral discomfort and pain associated with IBS.
Asunto(s)
Canales de Calcio Tipo T/fisiología , Colon/inervación , Colon/fisiopatología , Síndrome del Colon Irritable/fisiopatología , Animales , Secuencia de Bases , Butiratos/toxicidad , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo T/deficiencia , Canales de Calcio Tipo T/genética , Modelos Animales de Enfermedad , Fenómenos Electrofisiológicos , Técnicas de Silenciamiento del Gen , Síndrome del Colon Irritable/inducido químicamente , Síndrome del Colon Irritable/tratamiento farmacológico , Masculino , Neuralgia/tratamiento farmacológico , Neuralgia/fisiopatología , Nociceptores/fisiología , Percepción del Dolor/fisiología , ARN Interferente Pequeño/genética , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Chronic abdominal pain is the most common cause for gastroenterology consultation and is frequently associated with functional gastrointestinal disorders including irritable bowel syndrome and inflammatory bowel disease. These disorders present similar brain/gut/microbiota trialogue alterations, associated with abnormal intestinal permeability, intestinal dysbiosis and colonic hypersensitivity (CHS). Intestinal dysbiosis can alter colon homeostasis leading to abnormal activation of the innate immunity that promotes CHS, perhaps involving the toll-like receptors (TLRs), which play a central role in innate immunity. AIM: To understand the mechanisms between early life event paradigm on intestinal permeability, fecal microbiota composition and CHS development in mice with TLRs expression in colonocytes. METHODS: Maternal separation model (NMS) CHS model, which mimics deleterious events in childhood that can induce a wide range of chronic disorders during adulthood were used. Colonic sensitivity of NMS mice was evaluated by colorectal distension (CRD) coupled with intracolonic pressure variation (IPV) measurement. Fecal microbiota composition was analyzed by 16S rRNA sequencing from weaning to CRD periods. TLR mRNA expression was evaluated in colonocytes. Additionally, the effect of acute intrarectal instillation of the TLR5 agonist flagellin (FliC) on CHS in adult naive wildtype mice was analyzed. RESULTS: Around 50% of NMS mice exhibited increased intestinal permeability and CHS associated with intestinal dysbiosis, characterized by a significant decrease of species richness, an alteration of the core fecal microbiota and a specific increased relative abundance of flagellated bacteria. Only TLR5 mRNA expression was increased in colonocytes of NMS mice with CHS. Acute intrarectal instillation of FliC induced transient increase of IPV, reflecting transient CHS appearance. CONCLUSION: Altogether, these data suggest a pathophysiological continuum between intestinal dysbiosis and CHS, with a role for TLR5.
Asunto(s)
Disbiosis , Receptor Toll-Like 5 , Animales , Colon , Modelos Animales de Enfermedad , Disbiosis/metabolismo , Flagelina/metabolismo , Flagelina/farmacología , Privación Materna , Ratones , ARN Mensajero/metabolismo , ARN Ribosómico 16S , Receptor Toll-Like 5/genética , Receptor Toll-Like 5/metabolismo , Receptores Toll-Like/metabolismoRESUMEN
BACKGROUND: Infectious gastroenteritis is a risk factor for the development of post-infectious Irritable Bowel Syndrome (PI-IBS). Recent clinical studies reported a higher prevalence of the intestinal parasite Blastocystis in IBS patients. Using a rat model, we investigated the possible association between Blastocystis infection, colonic hypersensitivity (CHS), behavioral disturbances and gut microbiota changes. METHODS: Rats were orally infected with Blastocystis subtype 4 (ST4) cysts, isolated from human stool samples. Colonic sensitivity was assessed by colorectal distension and animal behavior with an automatic behavior recognition system (PhenoTyper), the Elevated Plus Maze test and the Forced Swimming tests. Feces were collected at different time points after infection to study microbiota composition by 16 S rRNA amplicon sequencing and for short-chain fatty acid (SFCA) analysis. RESULTS: Blastocystis-infected animals had non-inflammatory CHS with increased serine protease activity. Infection was also associated with anxiety- and depressive-like behaviors. Analysis of fecal microbiota composition showed an increase in bacterial richness associated with altered microbiota composition. These changes included an increase in the relative abundance of Oscillospira and a decrease in Clostridium, which seem to be associated with lower levels of SCFAs in the feces from infected rats. CONCLUSIONS: Our findings suggest that experimental infection of rats with Blastocystis mimics IBS symptoms with the establishment of CHS related to microbiota and metabolic shifts.
Asunto(s)
Conducta Animal/fisiología , Infecciones por Blastocystis/patología , Blastocystis/patogenicidad , Enfermedades del Colon/complicaciones , Disbiosis/etiología , Animales , Área Bajo la Curva , Infecciones por Blastocystis/complicaciones , Enfermedades del Colon/patología , Modelos Animales de Enfermedad , Ácidos Grasos Volátiles/análisis , Heces/microbiología , Heces/parasitología , Microbiota , Curva ROC , Ratas , Ratas Wistar , Serina Proteasas/metabolismoRESUMEN
Irritable Bowel Syndrome (IBS) and Inflammatory Bowel Disease (IBD) are related gastrointestinal disorders characterized by abdominal pain associated with colonic hypersensitivity (CHS). Studies in humans have reported an abnormal colonization of Adherent-Invasive E. coli (AIEC) in the ileum of Crohn's disease (CD) patients associated with overexpression of the bacterial colonizing receptor CEACAM6. The aim of the present study was to investigate whether AIEC reference strain LF82 could induce intestinal impairment during infectious and/or post-infectious periods and subsequently the development of CHS. Transgenic mice overexpressing human CEACAM6 protein (TG) and their wild-type littermates were gavaged by CD-associated AIEC bacteria (reference strain LF82) or PBS for 3 d. Colonic hypersensitivity was assessed by colorectal distension (CRD) test during infectious (D4) and post-infectious periods (D21). Several markers of intestinal inflammation were monitored and the colonic expression of purinergic P2X receptors was quantified. At D4, an increased visceromotor response (VMR) to the CRD test was observed in TG mice infected with CD-associated AIEC LF82 in comparison with non-infected TG mice and persisted in a subgroup of infected animals at D21 after bacteria clearance. Increased VMR was associated with low-grade intestinal inflammation, increased intestinal permeability and expression of P2X 3, 4 and 7. This study shows that certain susceptible hosts infected with CD-associated AIEC bacteria can develop persistent CHS associated with low-grade inflammation and increased P2X receptors expression. Thus, CD-associated AIEC infection in CEACAM6 transgenic mice could be used as a novel post-infectious mouse model mimicking quiescent IBD with IBS-like symptoms such as visceral pain.
Asunto(s)
Colitis/patología , Enfermedad de Crohn/microbiología , Infecciones por Escherichia coli/fisiopatología , Escherichia coli/patogenicidad , Inflamación/microbiología , Receptores Purinérgicos P2X/genética , Regulación hacia Arriba , Animales , Antígenos CD/genética , Moléculas de Adhesión Celular/genética , Colitis/genética , Colitis/metabolismo , Colitis/microbiología , Colon/metabolismo , Colon/microbiología , Colon/patología , Escherichia coli/aislamiento & purificación , Infecciones por Escherichia coli/genética , Infecciones por Escherichia coli/metabolismo , Infecciones por Escherichia coli/microbiología , Femenino , Proteínas Ligadas a GPI/genética , Íleon/microbiología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , PermeabilidadRESUMEN
AIM: To investigate anti-hypersensitive effects of α2δ-1 ligands in non-inflammatory and inflammation-associated colonic hypersensitivity (CHS) mouse models. METHODS: To induce an inflammation-associated CHS, 1% dextran sulfate sodium (DSS) was administered to C57Bl/6J male mice, in drinking water, for 14 d. Regarding the non-inflammatory neonatal maternal separation (NMS) -induced CHS model, wild-type C57BI/6J pups were isolated from their mother from day 2 to day 14 (P2 to P14), three hours per day (from 9:00 a.m. to 12:00 p.m.). Colorectal distension was performed by inflating distension probe from 20 µL to 100 µL by 20 µL increment step every 10 s. After a first colorectal distension (CRD), drugs were administered subcutaneously, in a cumulative manner, (Gabapentin at 30 mg/kg and 100 mg/kg; Pregabalin at 10 mg/kg and 30 mg/kg; Carbamazepine at 10 mg/kg and 30 mg/kg) and a second CRD was performed one hour after each injection. RESULTS: The visceromotor response (VMR) to CRD was increased by our NMS paradigm protocol in comparison to non-handled (NH) mice, considering the highest distension volumes (80 µL: 0.783 ± 0.056 mV/s vs 0.531 ± 0.034 mV/s, P < 0.05 and 100 µL: 1.087 ± 0.056 mV/s vs 0.634 ± 0.038 mV/s, P < 0.05 for NMS and NH mice, respectively). In the inflammation-associated CHS, DSS-treated mice showed a dramatic and significant increase in VMR at 60 and 80 µL distension volumes when compared to control mice (60 µL: 0.920 ± 0.079 mV/s vs 0.426 ± 0.100 mV/s P < 0.05 and 80 µL: 1.193 ± 0.097 mV/s vs 0.681 ± 0.094 mV/s P < 0.05 for DSS- and Water-treated mice, respectively). Carbamazepine failed to significantly reduce CHS in both models. Gabapentin significantly reduced CHS in the DSS-induced model for both subcutaneous injections at 30 or 100 mg/kg. Pregabalin significantly reduced VMR to CRD in the non-inflammatory NMS-induced CHS model for the acute subcutaneous administration of the highest cumulative dose (30 mg/kg) and significantly reduced CHS in low-dose DSS-treated mice in a dose-dependent manner. Finally, the percent decrease of AUC induced by acute GBP or Pregabalin treatment were higher in the inflammatory DSS-induced CHS model in comparison to the non-inflammatory NMS-induced CHS model. CONCLUSION: This preclinical study demonstrates α2δ-1 ligands efficacy on inflammation-associated CHS, highlighting their potential clinical interest in patients with chronic abdominal pain and moderate intestinal inflammation.
Asunto(s)
Canales de Calcio/fisiología , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Aminas/uso terapéutico , Animales , Ácidos Ciclohexanocarboxílicos/uso terapéutico , Sulfato de Dextran , Modelos Animales de Enfermedad , Gabapentina , Ligandos , Masculino , Ratones , Ratones Endogámicos C57BL , Pregabalina/uso terapéutico , Ácido gamma-Aminobutírico/uso terapéuticoRESUMEN
T-type calcium channels encoded by the Ca(V)3.2 isoform are expressed in nociceptive primary afferent neurons where they contribute to hyperalgesia and thus are considered as a potential therapeutic target to treat pathological pain. Here we report that the small organic state-dependent T-type channel antagonist TTA-A2 efficiently inhibits recombinant and native Ca(V)3.2 currents. Although TTA-A2 is a pan Ca(V)3 blocker, it demonstrates a higher potency for Ca(V)3.2 compared to Ca(V)3.1. TTA-A2 selectivity for T-type currents was demonstrated in sensory neurons where it lowered cell excitability uniquely on neurons expressing T-type channels. In vivo pharmacology in Ca(V)3.2 knockout and wild type mice reveal that TTA-A2-mediated antinociception critically depends on Ca(V)3.2 expression. The pathophysiology of irritable bowel syndrome (IBS) was recently demonstrated to involve Ca(V)3.2 in a rat model of this disease. Oral administration of TTA-A2 produced a dose-dependent reduction of hypersensitivity in an IBS model, demonstrating its therapeutic potential for the treatment of pathological pain. Overall, our results suggest that the high potency of TTA-A2 in the depolarized state strengthen its analgesic efficacy and selectivity toward pathological pain syndromes. This characteristic would be beneficial for the development of analgesics targeting T-type channels, in particular for the treatment of pain associated with IBS.
Asunto(s)
Bencenoacetamidas/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo T/metabolismo , Hiperalgesia/tratamiento farmacológico , Neuronas/efectos de los fármacos , Piridinas/farmacología , Animales , Bloqueadores de los Canales de Calcio/uso terapéutico , Canales de Calcio Tipo T/genética , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Hiperalgesia/genética , Hiperalgesia/metabolismo , Masculino , Ratones , Ratones Noqueados , Neuronas/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Irritable bowel syndrome (IBS) is a common functional gastro-intestinal disorder characterized by intractable chronic abdominal pain. In this study, we examined the possible spinal mechanisms underlying colonic hypersensitivity (CHS) using a non-inflammatory rat model of IBS induced by rectal enemas of butyrate, a short-chain fatty acid. We hypothesized that spinal plasticity could be responsible for CHS and that ASIC channels, which are known to support pain-elicited currents in the spinal cord, could contribute to central sensitization in our model of IBS. First, in order to determine if visceral pain relies on changes in spinal activity, we analyzed Fos expression in the spinal cord of rats treated with butyrate following a challenge with repetitive noxious colorectal distension. We found that Fos immunoreactivity was increased in thoracic T10-11-12, lumbar L1-2-6 and sacral S1 spinal segments. In control rats treated with saline, noxious repetitive colorectal distensions evoked Fos expression only in L1-2-6 and S1 spinal segments. Secondly, intrathecal injection of PcTx1, a specific ASIC1A antagonist, in the lumbar spinal cord completely prevented the development of CHS induced by butyrate. ASIC1 and 2 mRNAs, especially ASIC1A, were upregulated in the lumbar spinal cord. ASIC1A could thus contribute to spinal sensitization in our model of IBS, as it is supported by spinal colocalization of ASIC1A and Fos proteins. The whole data pinpoint a potential critical role of thoracic spinal cord in non-inflammatory pain states such as IBS and suggest that ASIC channels are part of the molecular effectors of central sensitization leading to visceral pain.
Asunto(s)
Síndrome del Colon Irritable/fisiopatología , Proteínas del Tejido Nervioso/fisiología , Plasticidad Neuronal/fisiología , Canales de Sodio/fisiología , Médula Espinal/fisiopatología , Canales Iónicos Sensibles al Ácido , Anestesia por Inhalación , Anestésicos por Inhalación , Animales , Butiratos , Colon/fisiopatología , Cartilla de ADN , Enema , Expresión Génica/efectos de los fármacos , Genes fos/genética , Inmunohistoquímica , Síndrome del Colon Irritable/inducido químicamente , Isoflurano , Masculino , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Canales de Sodio/genéticaRESUMEN
BACKGROUND: Ileal lesions in Crohn's disease patients are colonized by pathogenic adherent-invasive Escherichia coli (AIEC) that harbor various virulence factors involved in adhesion to and invasion of intestinal epithelial cultured cells. We investigated in a mouse model of colonic inflammation the behavior of virulent AIEC reference bacteria LF82 compared to that of nonflagellated LF82 mutants. METHODS: BALBc/J mice with intact or dextran sulfate sodium (DSS)-injured colon were orally challenged daily with 10(8) bacteria. The severity of colitis was assessed by determining disease activity index, colonic histological score, and myeoloperoxidase activity. Flagellin receptor and cytokine expression was measured by reverse-transcriptase polymerase chain reaction (RT-PCR) in colonic tissue. RESULTS: In contrast to nonpathogenic E. coli, virulent LF82 bacteria exacerbated colitis in DSS-treated mice, substantially reducing survival rate, greatly lowering stool consistency, inducing marked weight loss and increased rectal bleeding, and significantly increasing erosive lesions and mucosal inflammation. Nonflagellated LF82 mutants behaved like nonpathogenic E. coli K-12. Interestingly, oral infection with LF82 virulent bacteria, but not with a nonvirulent LF82 mutant, induced a 7.0-fold increase in the levels of TLR5 and a 3.1-fold increase in those of ipaf mRNA, which encode respectively membrane and cytosolic receptors involved in the recognition of flagellin. Hence, a 5.6-fold increase in IL-1beta and a 5.3-fold increase in mRNA of IL-6 were observed in mice challenged with AIEC LF82 bacteria. CONCLUSIONS: Crohn's disease-associated virulent AIEC LF82 bacteria, via expression of flagella, are able to potentiate an inflammatory mucosal immune response involving increased expression of TLR5 and IPAF flagellin receptors.