Changes Of The PAR-2-AP Induced Relaxation Of The Colonic Muscle Strips And The Sensitivity Of The Afferent Nerve Of Rats Following Bacillary Dysentery And The Underlying Mechanism

PartⅠ:Change of the PAR-2-AP Induced Relaxation of the Colonic Muscle Strips of Rats following Bacillary Dysentery and the Underlying MechanismObjectiveAlthough it is well known that the gastrointestinal motility is altered in animal models of post infectious irritable bowel syndrome (PI-IBS), the mechanism was unknown. Protease-activated receptor-2 (PAR-2), a subfamily of seven transmembrane G-protein coupled receptors, is involved in the hypersensitivity of IBS patients, little is know about its effect on the dysmotility following gastrointestinal infection. We hypothesized that expression of PAR-2 might be downregulated and that is one of the reasons of the high contractility of the colon following infection. In order to test this hypothesis, the response of colonic smooth muscle to an agonist of PAR-2, PAR-2-activating peptide (PAR-2-AP) in rats following Shigella flexneri infection at different time was detected. The expression of PAR-2 is detected by Western blot. The location of PAR-2 on rat distal colon is examined by immunohistochemisty. MethodsBacteria preparationClinical isolates of Shigella flexneri were obtained from Clinical Laboratory, Qi Lu hospital, Ji Nan, China. The strains were routinely grown in blood plate at 37℃, overnight.Intestinal infection and tissue preparationHealthy male Wistar rats (200-220 g) were divided into control and Shigella flexneri-treated (SF-treated) groups at random. After an overnight fast (water ad libitum), rats received intragastric administration of inocula of 108 colony forming units (CFU) in 1 ml of sterile normal saline. The rats in control group were treated with lml sterile normal saline.On days 4,7,11,18,25 and 35 following the Shigella Flexneri treatment, rats were sacrificed by cervical dislocation and distal (1cm from rectum,2-3 cm length) colon was excised. The colonic tissue isolated from control or SF-treated rats was prepared for Hematoxylin and Eosin (H & E) staining, Myeloperoxidase (MPO) assay, tension recording or Western blot analysis.MPO assay and histological analysisThe colonic tissue removed from the distal colon was homogenized, and the homogenate was used to measure MPO activity by using a kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). One MPO activity unit was defined as per gram tissue decomposes 1μM H2O2 in reaction system at 37℃.Full-thickness paraffin-embedded sections (4μm) of distal colon from control and SF-treated Wistar rat were stained with Hematoxylin and Eosin (H & E) for evaluation of the degree of inflammation.Preparation of smooth muscle strips A segment (2-3 cm length) of distal colon (1 cm from rectum) was immediately removed from control and SF-treated rats and opened along the mesenteric border then pinned flat in a dish filled with oxygenated Krebs solution. Two kind of full thickness muscle strips (4 mm wide,10 mm long) were prepared:strips parallel to the long axis of the circular muscle layer of colon were designated circular muscle (CM), strips parallel to the long axis of the longitudinal muscle layer of colon were designated longitudinal muscle (LM). Strips were suspended in an organ bath which was filled with 5 ml Krebs solution, continuously perfused with 95% O2 and 5% CO2 and maintained at 37℃.Experimental protocolAfter equilibration for at least 45 min with flushing every 15 min under a preload of 1 g, the mechanical response to the PAR-2-AP, PAR-2-RP were examined. In some experiments, muscle strips were pretreated with tetrodotoxin (TTX,10μM) for 30 min before administration of PAR-2-AP or PAR-2-RP.ImmunohistochemistryFrozen sections (4μm) were washed three times in phosphate buffered saline (PBS) and incubated with 3% hydrogen peroxide for 10 min to block the activity of endogenous peroxidase. The sections were washed with PBS again and treated with 5% normal rabbit non-immune serum for 15 min followed by incubation with primary goat polyclonal antibody for PAR-2 overnight at 4℃.The sections were rinsed and incubated with HRP conjugated rabbit anti goat secondary antibody for 30 min at room temperature followed by washing with PBS. The reaction products were visualized with a 3,3'-diaminobenzidine tetrahydrochloride substrate kit. For the sections of negative controls, the primary antibody was replaced with PBS.Western blot analysisTissue from distal colon was homogenized, centrifuged at 12000 g for 10 min at 4℃.Total proteins were fractionated on a 5% to 10% gradient sodium dodecyl sulfatepolyacrylamide gel. They were transferred to 0.45μm Polyvinylidene-Fluoride (PVDF) membranes. Membranes were blocked in blocking buffer (5% non-fat dry milk, TTBS) for 90 min at room temperature, incubated with primary goat anti-PAR-2 antibody and P-actin primary antibody respectively at 4℃overnight. After washing 5 min for three times, the PVDF membranes were incubated for 1h at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies respectively. Finally, immunoreactive proteins were detected by ECL plus.Quantification of each band was performed using Scion Image densitometry analysis software.Statistical analyses1. In the experiments on muscle strips, mean value of the average tension for 3-min period before treatment with PAR-2-AP/RP was taken as the baseline. In the groups pretreated with blockers, the baselinewas the average tension after the blocker were administrated. The average tension at different time after each PAR-2-AP/RP treatment was normalized to a standardized ratio where the baseline for each experiment was equal to 100.2. In the western blot analysis, the statistical value is the value of the gray scale division between rats treated with SF and the control rats, in which the gray scale was equal to 100%.3. All the values in these experiments were presented as mean±SEM. One way analysis of variance (ANOVA) and the student's t-test was used to analyze. P< 0.05 was considered to be a significant difference.Results1. Diarrhea or stool with liquor uris appeared 3-4 days in rats following intragastric administration of Shigella flexner, became most severe at 7-11 days, and gradually disappeared at 18 days later. Neutrophils appeared at the mucosa of the distal colon at 4-11 days, and disappeared at 18 days. The increase of the body weight of the rat in SF-treated groups significantly lower at 11 days. The MPO activity of colon increased at 7 days, reached the highest level at 11 days, and returned to normal at 18 days.2. Exogenous PAR-2-AP (1-10μM) dose dependently inhibited the spontaneous contraction of circular and longitudinal muscle strips of distal colon, the inhibitory effect reached the maximal at 2 min. PAR-2-RP had no apparent effect on the contraction of muscle strips.At 2 minute following treatment of three doses of PAR-2-AP (1,5,10μM), the average tension of the longitudinal muscle strips decreased by 8.52±1.37%(P< 0.05 vs. RP),13.77±2.91%(P< 0.05 vs. RP) and 15.27±4.04%(P< 0.05 vs. RP), while the average tension of the circular muscle strips decreased by 10.29±2.71%(P < 0.05 vs. RP),12.95±3.04%(P< 0.05 vs. RP) and 16.11±3.93%(P< 0.05 vs. RP).Pretreatment of TTX did not influence the inhibitory effect of PAR-2-AP on the contraction of longitudinal and circular muscle strips of distal colon.3. The inhibitory effect of PAR-2-AP on the contraction of longitudinal muscle strips did not change, but that on the circular muscle strips decreased following intragastric administration of Shigella flexner. Compared to that of the control group, the response of circular muscle strips of the SF-treated rats to PAR-2-AP significantely decreased at 11-35 days following intestinal infection.4. PAR-2 immunopositive cells were located at the longitudinal and circular smooth muscle and myenteric nerve plexus of the distal colon. The number of the PAR-2 immunopositive cells decreased at 18 day in SF-treated rat.5. From 11 and 35 days following intestinal infection, the amount of PAR-2 expressed on colon was significantly decreased.ConclusionsPAR-2-AP induced an relaxation of the colonic smooth muscle which is mediated by the PAR-2 expressed on the smooth muscle.The enteric nerverous system did not contribute to this effect.The relaxant effect of PAR-2-AP on rat colonic smooth muscle decreased at 11-35 days following the treatment of SF. The long-term downregulation of the inhibitory effect of PAR-2-AP on colonic muscle strips was partly dued to the decrease of PAR-2 expression. PartⅡChange of the Sensitization of Jejunal Mesenteric Afferent Nerve in Rats following Bacillary DysenteryObjectiveIBS (Irritable bowl syndrome) is one of the most common disorders of gastrointestinal tract, although it is widely recognized that the paresthesia is the key physiopathological basis of this syndrome, the mechanisms are unknown. About one third of these patients are post-infectious IBS (PI-IBS). They experienced intestinal infection before the appearance of gastrointestinal disorders. The classical animal model of PI-IBS was made by infusion of Chemicals (TNBS) into colon or infection of parasite, which is different from that of the experience of PI-IBS patients. The most common cause of the PI-IBS was the bacillary dysentery. We hypothesized that the intestinal infection of Shigella flexneri might develop a long-term of hyperalgesis in gut, which is similar to that of the PI-IBS. In order to test this hypothesis, we record the spikes of the jejunal afferent fibers and examined the mechanical and chemical sensitivity.MethodsThe rat model of Shigella flexneri infection was the same with that of PartⅠThe jejunal tissue isolated from control or SF-treated rats was prepared for MPO assay and histological analysis, which were the same with that of PartⅠ.Mesenteric nerve preparationAt different time after the treatment of SF or normal saline, rats were deeply anesthetized with 2% pentobarbital sodium and a segment of proximal jejunum with a length of 2 cm and the mesentery attached was excised carefully. After cleared with ice Krebs, the segment was placed in the perfusion chamber of the organ bath and superfused with Krebs buffer saturated with a 95% O2 and 5% CO2- mixture.The lumen of the prepared segment was continuously perfused with Krebs buffer from the proximal side of the jejunum (10 mL h-1), and the outside of the segments was also perfused by the same solute with a speed of 10ml min-1. While the intestinal segment remained in the perfusion chamber, the mesenteric nerve was dissociated out of the neurovascular mesenteric bundle under a viewing microscope. The distal end of the nerve was put on the electrode to record the spontaneous and induced discharge.The recording of mesenteric afferent nerve dischargeThe electrodes were connected to a single channel preamplifier/filter [1902, Cambridge Electronic Design (CED), Cambridge, UK]. The signal was amplified 10 000 times and filtered with a bandwidth of 100 Hz to 1 kHz. Signals from the pressure transducer recording the intraluminal intestinal pressure were transmitted to another CED single channel 1902 preamplifier/filter. The output from the 1902, together with the signals from the pressure transducer were sent to a interface system (Micro 1401, CED, Cambridge, UK), saved on the hard drive of a laptop computer, and viewed online using SPIKE 2 software (version 5.20; CED, Cambridge, UK).Experimental protocolsFor the afferent response to chemical stimuli, the outsid perfusion was stopped 5 min before the treatment of drug and started again 5 min after the administration of drug. For continuous ramp distension, the outlet cannula in the intestinal lumen was closed, while perfusion with Krebs buffer was continued at 30 mL h" to distend the gut segment.Data analysisThe discharge frequency during the 2 min recording period prior to administration of test stimuli was taken as the baseline.The mean increase in peak impulse frequency per second above baseline discharge frequency during a 3 s period of maximum afferent firing was evaluated as the afferent nerve response to chemical stimulation. The response to ramp distension was evaluated by quantifying the peak impulse frequency per second over a 3 s period at 10 mmHg increments of intraluminal pressure until 60 mmHg was reached.All the values in these experiments were presented as mean±SEM. One way analysis of variance (ANOVA) and the student's t-test was used to analyze the data. P< 0.05 was considered to be a significant difference. Results1. The mean frequency of the mesenteric afferent fiber was 8±2 spikes/sec.2. No obvious mucosal inflammation was observed in jejunum of rats in SF-treated group. The MPO activity of jejunum in SF-treated rats was not changed compared with that of the control group,.3. Administration of 5-HT (250μM) significantly increased the afferent discharge. Treatment of SF did not influence the excitatory effect of 5-HT on the afferent spikes.4. A pressure-dependent increase in afferent nerve discharge occurred in both treatments during mechanical stimulation by ramp distension, and the mechanical sensitivity increased in SF-treated rats at 25 days after the intragastrical infection.5.Afferent nerve discharge increased significantly after the administration of Bradykinin. Compared to that of the control group, the sensitivity of the afferent nerve to Bradykinin increased at 25 days following the treatment of SF.ConclusionsThe sensitivity of the jejunal mesenteric afferent nerve to Bradykinin and mechanical stimuli were increased significantly at 25 days after the treatment of SF.