You Min Lin

Pathophysiology of motility dysfunction in bowel obstruction: role of stretch-induced COX-2

In gastrointestinal conditions such as bowel obstruction, pseudo-obstruction, and idiopathic megacolon, the lumen of affected bowel segments is distended and its motility function impaired. Our hypothesis is that mechanical stretch of the distended segments alters gene expression of cyclooxygenase-2 (COX-2), which impairs motility function. Partial obstruction was induced with a silicon band in the distal colon of rats for up to 7 days, and wild-type and COX-2 gene-deficient mice for 4 days. Mechanical stretch was mimicked in vitro in colonic circular muscle strips and in primary culture of colonic circular smooth muscle cells (SMC) with a Flexercell system. The rat colonic circular muscle contractility was significantly decreased in the distended segment oral to obstruction, but not in the aboral segment. This change started as early as day 1 and persisted for at least 7 days after obstruction. The expression of COX-2 mRNA and protein increased dramatically also in the oral, but not aboral, segment. The upregulation of COX-2 expression started at 12 h and the effect persisted for 7 days. At 24 h after obstruction, the COX-2 mRNA level in the oral segment increased 26-fold compared with controls. This was not accompanied by any significant increase of myeloperoxidase or inflammatory cytokines. Immunohistochemical studies showed that COX-2 was selectively induced in the colonic SMC. In vitro stretch of colonic muscle strips or cultured SMC drastically induced COX-2 expression. Incubation of circular muscle strips from obstructed segment with COX-2 inhibitor NS-398 restored the contractility. The impairment of muscle contractility in obstructed colon was attenuated in the COX-2 gene-deficient mice. In conclusion, mechanical stretch in obstruction induces marked expression of COX-2 in the colonic SMC, and stretch-induced COX-2 plays a critical role in the suppression of smooth muscle contractility in bowel obstruction.

Prophylactic and therapeutic benefits of COX-2 inhibitor on motility dysfunction in bowel obstruction: roles of PGE2 and EP receptors

We reported previously that mechanical stretch in rat colonic obstruction induces cyclooxygenase (COX)-2 expression in smooth muscle cells. The aims of the present study were to investigate whether in vivo treatment with COX-2 inhibitor has prophylactic and therapeutic effects on motility dysfunction in colon obstruction, and if so what are the underlying mechanisms. Partial colon obstruction was induced with a silicon band in the distal colon of 6-8-wk-old Sprague-Dawley rats; obstruction was maintained for 3 days or 7 days. Daily administration of COX-2 inhibitor NS-398 (5 mg/kg) or vehicle was started before or after the induction of obstruction to study its prophylactic and therapeutic effects, respectively. The smooth muscle contractility was significantly suppressed, and colonic transit rate was slower in colonic obstruction. Prophylactic treatment with NS-398 significantly prevented the impairments of colonic transit and smooth muscle contractility and attenuated fecal collection in the occluded colons. When NS-398 was administered therapeutically 3 days after the initiation of obstruction, the muscle contractility and colonic transit still improved on day 7. Obstruction led to marked increase of COX-2 expression and prostaglandin E(2) (PGE(2)) synthesis. Exogenous PGE(2) decreased colonic smooth muscle contractility. All four PGE(2) E-prostanoid receptor types (EP1 to EP4) were detected in rat colonic muscularis externa. Treatments with EP1 and EP3 antagonists suppressed muscle contractility in control tissue but did not improve contractility in obstruction tissue. On the contrary, the EP2 and EP4 antagonists did not affect control tissue but significantly restored muscle contractility in obstruction. We concluded that our study shows that COX-2 inhibitor has prophylactic and therapeutic benefits for motility dysfunction in bowel obstruction. PGE(2) and its receptors EP2 and EP4 are involved in the motility dysfunction in obstruction, whereas EP1 and EP3 mediate PGE(2) regulation of colonic smooth muscle contractile function in normal state.

Pathogenesis of abdominal pain in bowel obstruction: Role of mechanical stress-induced upregulation of nerve growth factor in gut smooth muscle cells.

Abstract Abdominal pain is one of the major symptoms in bowel obstruction (BO); its cellular mechanisms remain incompletely understood. We tested the hypothesis that mechanical stress in obstruction upregulates expression of nociception mediator nerve growth factor (NGF) in gut smooth muscle cells (SMCs), and NGF sensitizes primary sensory nerve to contribute to pain in BO. Partial colon obstruction was induced with a silicon band implanted in the distal bowel of Sprague-Dawley rats. Colon-projecting sensory neurons in the dorsal root ganglia (T13 to L2) were identified for patch-clamp and gene expression studies. Referred visceral sensitivity was assessed by measuring withdrawal response to stimulation by von Frey filaments in the lower abdomen. Membrane excitability of colon-projecting dorsal root ganglia neurons was significantly enhanced, and the withdrawal response to von Frey filament stimulation markedly increased in BO rats. The expression of NGF mRNA and protein was increased in a time-dependent manner (day 1-day 7) in colonic SMC but not in mucosa/submucosa of the obstructed colon. Mechanical stretch in vitro caused robust NGF mRNA and protein expression in colonic SMC. Treatment with anti-NGF antibody attenuated colon neuron hyperexcitability and referred hypersensitivity in BO rats. Obstruction led to significant increases of tetrodotoxin-resistant Na+ currents and mRNA expression of Nav1.8 but not Nav1.6 and Nav1.7 in colon neurons; these changes were abolished by anti-NGF treatment. In conclusion, mechanical stress-induced upregulation of NGF in colon SMC underlies the visceral hypersensitivity in BO through increased gene expression and activity of tetrodotoxin-resistant Na+ channels in sensory neurons.

Mechanical Stress Is a Pro-Inflammatory Stimulus in the Gut: In Vitro, In Vivo and Ex Vivo Evidence

Aims Inflammatory infiltrates and pro-inflammatory mediators are found increased in obstructive and functional bowel disorders, in which lumen distention is present. However, what caused the low level inflammation is not well known. We tested the hypothesis that lumen distention- associated mechanical stress may induce expression of specific inflammatory mediators in gut smooth muscle. Methods Static mechanical stretch (18% elongation) was applied in vitro in primary culture of rat colonic circular smooth muscle cells (RCCSMCs) with a Flexercell FX-4000 Tension Plus System. Mechanical distention in vivo was induced in rats with an obstruction band placed in the distal colon. Results In the primary culture of RCCSMCs, we found that static stretch significantly induced mRNA expression of iNOS, IL-6, and MCP-1 in 3 hours by 6.0(±1.4), 2.5(±0.5), and 2.2(±0.5) fold (n = 6∼8, p<0.05), respectively. However, gene expression of TNF-α, IL-1β, and IL-8 was not significantly affected by mechanical stretch. In the in vivo model of colon obstruction, we found that gene expression of iNOS, IL-6, and MCP-1 is also significantly increased in a time-dependent manner in the mechanically distended proximal segment, but not in the sham controls or distal segments. The conditioned medium from the muscle strips of the stretched proximal segment, but not the distal segment or control, significantly induced translocation and phosphorylation of NF-κB p65. This treatment further increased mRNA expression of inflammatory mediators in the naïve cells. However, treatment of the conditioned medium from the proximal segment with neutralizing antibody against rat IL-6 significantly attenuated the activation of NF-κB and gene expression of inflammatory mediators. Conclusions Our studies demonstrate that mechanical stress induces gene expression of inflammatory mediators i.e. iNOS, IL-6, and MCP-1 in colonic SMC. Further ex vivo study showed that mechanical stress functions as a pro-inflammatory stimulus in the gut.

Cellular mechanism of mechanotranscription in colonic smooth muscle cells

Mechanical stretch in obstruction induces expression of cyclooxygenase-2 (COX-2) in gut smooth muscle cells (SMCs). The stretch-induced COX-2 plays a critical role in motility dysfunction in obstructive bowel disorders (OBDs). The aims of the present study were to investigate the intracellular mechanism of mechanotranscription of COX-2 in colonic SMCs and to determine whether inhibition of mechanotranscription has therapeutic benefits in OBDs. Static stretch was mimicked in vitro in primary culture of rat colonic circular SMCs (RCCSMCs) and in colonic circular muscle strips. Partial obstruction was surgically induced with a silicon band in the distal colon of rats and COX-2-deficient mice. Static stretch of RCCSMCs significantly induced expression of COX-2 mRNA and protein and activated MAP kinases ERKs, p38, and JNKs. ERKs inhibitor PD98059, p38 inhibitor SB203580, and JNKs inhibitor SP600125 significantly blocked stretch-induced COX-2 expression. Pharmacological and molecular inhibition of stretch-activated ion channels (SACs) and integrins significantly suppressed stretch-induced expression of COX-2. SAC blockers inhibited stretch-activated ERKs, p38, and JNKs, but inhibition of integrins attenuated p38 activation only. In colonic circular muscle strips, stretch led to activation of MAPKs, induction of COX-2, and suppression of contractility. Inhibition of p38 with SB203580 blocked COX-2 expression and restored muscle contractility. Administration of SB203580 in vivo inhibited obstruction-induced COX-2 and improved motility function. Stretch-induced expression of COX-2 in RCCSMCs depends on mechanosensors, SACs, and integrins and an intracellular signaling mechanism involving MAPKs ERKs, p38, and JNKs. Inhibitors of the mechanotranscription pathway have therapeutic potentials for OBDs.

Are Interstitial Cells of Cajal Involved in Mechanical Stress-Induced Gene Expression and Impairment of Smooth Muscle Contractility in Bowel Obstruction?

Background and Aims The network of interstitial cells of Cajal (ICC) is altered in obstructive bowel disorders (OBD). However, whether alteration in ICC network is a cause or consequence of OBD remains unknown. This study tested the hypothesis that mechanical dilation in obstruction disrupts the ICC network and that ICC do not mediate mechanotranscription of COX-2 and impairment of smooth muscle contractility in obstruction. Methods Medical-grade silicon bands were wrapped around the distal colon to induce partial obstruction in wild-type and ICC deficient (W/Wv) mice. Results In wild-type mice, colon obstruction led to time-dependent alterations of the ICC network in the proximal colon segment. Although unaffected on days 1 and 3, the ICC density decreased markedly and the network was disrupted on day 7 of obstruction. COX-2 expression increased, and circular muscle contractility decreased significantly in the segment proximal to obstruction. In W/Wv control mice, COX-2 mRNA level was 4.0 (±1.1)-fold higher (n=4) and circular muscle contractility was lower than in wild-type control mice. Obstruction further increased COX-2 mRNA level in W/Wv mice to 7.2 (±1.0)-fold vs. W/Wv controls [28.8 (±4.1)-fold vs. wild-type controls] on day 3. Obstruction further suppressed smooth muscle contractility in W/Wv mice. However, daily administration of COX-2 inhibitor NS-398 significantly improved muscle contractility in both W/Wv sham and obstruction mice. Conclusions Lumen dilation disrupts the ICC network. ICC deficiency has limited effect on stretch-induced expression of COX-2 and suppression of smooth muscle contractility in obstruction. Rather, stretch-induced COX-2 plays a critical role in motility dysfunction in partial colon obstruction.

Microsomal Prostaglandin e Synthase-1 Plays a Critical Role in Long-term Motility Dysfunction after Bowel Obstruction

Motility dysfunction is present not only during bowel obstruction (BO), but after obstruction is resolved. Previous studies found that lumen distension associated mechano-transcription of COX-2 and production of PGE2 in gut smooth muscle cells (SMC) account for motility dysfunction during obstruction. We hypothesized that PGE2 may exert autocrine effect in SMC to induce microsomal prostaglandin E synthase-1 (mPGES-1), which contributes to motility dysfunction after obstruction is resolved. Partial colon obstruction was induced in rats with an obstruction band, which was released 7 days later. Rats were further studied in the post-BO state. Circular muscle contractility of the mid colon (previously distended during obstruction) remained suppressed, and colon transit was impaired in the post-BO state. The COX-2, mPGES-1, and PGE2 levels were all increased in the distended bowel during obstruction. However, after obstruction was resolved, COX-2 expression returned to normal, whereas mPGES-1 and PGE2 levels remained increased. Expression of mPGES-1 in colon SMC was inducible by stretch or PGE2. Administration of mPGES-1 inhibitor Cay 10526 either before or after the release of obstruction normalized PGE2 levels and improved motility in the post-BO rats. In conclusion, mPGES-1 plays a critical role in the continuous suppression of motor function in the post-BO state.

Microbiota dysbiosis and its pathophysiological significance in bowel obstruction

Bowel obstruction (OB) causes local and systemic dysfunctions. Here we investigated whether obstruction leads to alterations in microbiota community composition and total abundance, and if so whether these changes contribute to dysfunctions in OB. Partial colon obstruction was maintained in rats for 7 days. The mid colon and its intraluminal feces - proximal to the obstruction - were studied. OB did not cause bacterial overgrowth or mucosa inflammation, but induced profound changes in fecal microbiota composition and diversity. At the phylum level, the 16S rRNA sequencing showed a significant decrease in the relative abundance of Firmicutes with corresponding increases in Proteobacteria and Bacteroidetes in OB compared with sham controls. Daily treatment using broad spectrum antibiotics dramatically reduced total bacterial abundance, but increased the relative presence of Proteobacteria. Antibiotics eliminated viable bacteria in the spleen and liver, but not in the mesentery lymph node in OB. Although antibiotic treatment decreased muscle contractility in sham rats, it had little effect on OB-associated suppression of muscle contractility or inflammatory changes in the muscle layer. In conclusion, obstruction leads to marked dysbiosis in the colon. Antibiotic eradication of microbiota had limited effects on obstruction-associated changes in inflammation, motility, or bacterial translocation.

Mechano-Regulation of Gene Expression is a Common Pathophysiological Mechanism Involved in Obstructive Disorders Throughout the Gastrointestinal Tract

G A A b st ra ct s may induce abnormal gastric slow wave patterns, however these patterns are poorly understood, because of the incomplete detail provided by previous recording methods (EGG / sparse-electrodes). In this study, we describe gastroparetic slow wave patterns in highresolution (HR) spatiotemporal detail. Methods: Patients with diabetic (n=10) and idiopathic (n=2) gastroparesis undergoing gastric stimulator implantation were enrolled (median age 41 yrs, mean ‘total symptom score’ 17±2 out of 20, mean 4-hr gastric emptying 28±19%). HR electrical mapping was performed using flexible arrays (256 electrodes; 4 mm spacing) placed over the anterior corpus and/or corpus-antrum border immediately after laparotomy. Propagation patterns were quantified by isochronal mapping. Results were compared to data from 12 patients with no known stomach pathology undergoing routine surgery, who were assessed in the same manner. Mean ± SEM are reported. Results: Slow wave propagation was exclusively normal in all controls (29 recordings; mean duration 4.65 min; freq. 2.8±0.3 c/min), whereas a range of abnormal propagation patterns were observed in 11/12 gastroparetics (21 recordings; mean duration 7.3 min). Diabetics: Abnormalities comprised incomplete conduction blocks (n=6 recordings; 3.0±0.2 c/min), corpus ectopic pacemakers (n=6; 3.5±0.3 c/min), antral focal events (n=2), and complete conduction block with escape (n=1; 2.2 c/ min). There was also one instance each of regular antral tachygastria (4 c/min), and disorganized / fibrillation-type behavior in the corpus with multiple wavelets propagating (n=1; 4.9 c/min). Wavefront collisions and retrograde propagation were each recorded in 5 patients. Idiopathics: Both patients demonstrated highly disorganized propagation with incomplete conduction blocks, competing ectopics, retrograde propagation, and colliding wavefronts. Conclusion: A diverse range of slow wave initiation and conduction abnormalities were revealed by HR mapping in patients with gastroparesis. Several dysrhythmic mechanisms are described here for the first time in humans. These abnormalities were spatially complex, meaning they might go undetected by lower resolution tests such as EGG. The clinical significance of these findings needs to be determined.