摘要
第一部分：大鼠肠道痢疾杆菌感染对PAR-2-AP舒张结肠平滑肌作用的影响及其机制
目的
PAR-2(Protease-Activated Receptor 2)属于G-蛋白偶联受体家族,广泛分布于消化道,参与调控消化道的运动、分泌和炎症反应。在肠道急性炎症期,PAR-2激动剂抑制大鼠远端结肠运动的作用减弱,但炎症后PAR-2激动剂的这种作用是否恢复到正常还不清楚。在制作肠道感染和炎症模型时大多数实验室都采用化学物质刺激或是寄生虫感染的方法,而最与临床接近的大鼠肠道细菌感染模型鲜见报道。因此,本研究采用了胃内灌注痢疾杆菌的方式制作大鼠肠道感染模型,探讨了肠道感染后不同时间点PAR-2舒张结肠平滑肌作用的改变及其机制。
方法
细菌准备弗氏志贺痢疾杆菌(Shigella flexneri)由山东大学齐鲁医院检验科提供。
肠炎模型建立
健康雄性Wistar大鼠(200-220 g),随机分为对照组和痢疾杆菌感染组(SF -treated)。实验前禁食,自由饮水。对照组给予高压处理的生理盐水1 ml灌胃,感染组给予1×108 CFU ml-1痢疾杆菌菌液1 ml灌胃。观察大鼠肠道感染后大便性状的改变、进食及体重增长情况,并分别于灌胃后第4天、第7天、第11天、第18天、第25天和第35天断头处死动物,取远端结肠做石蜡切片,HE染色。取远端结肠组织做MPO测定、肌条张力测定及Western blot检测。
髓过氧化物酶活性测定
远端结肠组织匀浆后,利用MPO测定试剂盒(南京建成生物工程研究所)检测结肠全层组织中MPO活性。
HE染色
4%多聚甲醛固定新鲜结肠标本,石蜡包埋,切片厚4μm。常规脱蜡,水化。苏木素染色,水洗后盐酸酒精分化,氨水返蓝,伊红染色,脱水,透明,中性树胶封片。
结肠离体肌条张力记录
肌条制备
断头处死大鼠,快速取出2-3 cm远端结肠(距直肠1 cm)置于预冷的Krebs液中。沿肠系膜剪开肠腔,小心清理肠腔。沿肌纤维走向取结肠组织,制作纵行肌条和环形肌条(4 mm×10 mm)。将肌条分别置于盛有5 ml Krebs液的玻璃浴槽内,持续供给95%O2和5%CO2并恒温37℃,每15 min更换一次Krebs液。待肌条自发收缩稳定以后开始实验。
实验步骤
加入50μl不同浓度的PAR-2激动肽段(PAR-2 activating peptide, PAR-2-AP)或无活性的PAR-2反向肽段(PAR-2 reverse peptide, PAR-2-RP),使终浓度为1-10μM,观察PAR-2激活后结肠肌条自发收缩活动的改变。观察阻断剂TTX的作用时,先加入50μl TTX溶液使终浓度为10μM孵育30 min,再加入不同浓度的PAR-2-AP或PAR-2-RP。免疫组织化学
远端结肠冰冻切片用磷酸盐缓冲液冲洗3次,3%过氧化氢封闭10 min以封闭内源性过氧化物酶,PBS冲洗,5%正常兔血清封闭15 min后,滴加羊多克隆抗PAR-2抗体(1：50,50μl)后4℃环境中孵育过夜,然后用PBS冲洗,加入辣根酶(horseradish peroxidase, HRP)标记的抗山羊IgG工作液后室温孵育30 min,用DAB显色,苏木素复染,盐酸酒精分化,氨水返蓝,梯度酒精脱水,二甲苯透明,中性树胶封片。
Western blot
将结肠组织匀浆后,4℃离心,取上清液进行蛋白定量。与sample buffer按照1：1比例混匀,煮沸5 min使蛋白变性。10% SDS-PAGE (sodium dodecylsulfatepolyacrylamide gel)电泳,转膜,室温下5%脱脂奶粉封闭90 min,加入PAR-2一抗(sc-8205,1：2000)4℃孵育过夜。用TTBS冲洗3次,每次5 min后,在室温下加入辣根过氧化物酶标记的二抗(1：80000)孵育1hr,用TTBS冲洗3次后,ECL显影。
统计分析
在离体肌条实验中,以肌条的平均张力为观察指标,加PAR-2-AP或PAR-2-RP之前3 min的平均张力为基础值,标准化为100%,加入PAR-2-AP或PAR-2-RP之后不同时间点的平均张力为效应值,效应值与基础值的比值为统计指标。
在western blot实验中,对照组目的蛋白的灰度值标准化为100%,痢疾杆菌处理组目的蛋白的灰度值与对照组目的蛋白的灰度值之比为统计指标。
所有数据均采用均数±标准误(mean±SEM)表示,采用单因素方差分析和t检验进行统计学处理,以P＜0.05为显著性差异界值。
结果
1.大鼠在痢疾杆菌(1×108 CFU/ml,1 ml)灌胃后第3-4天陆续开始出现腹泻、稀便,在7-11天最严重,第18天后消失。HE染色显示在第4天-11天粘膜水肿,粘膜下中性粒细胞浸润。与对照组相比,感染组大鼠体重增长只在第11天明显低于对照组；结肠组织MPO活性从第7天开始增加,到第11天达到最高,第18天恢复正常。
2.PAR-2-AP(1-10μM)抑制大鼠远端结肠纵行肌条和环形肌条自发性收缩且有一定的剂量-反应关系,该抑制效应在加入PAR-2-AP 2 min内最明显。同样剂量的PAR-2-RP不影响各肌条的自发性收缩活动。
加入三种浓度(终浓度分别为1,5,10μM)的PAR-2-AP后2 min,远端结肠纵行肌条的张力分别下降8.52±1.37%(P< 0.05 vs. RP)、13.77±2.91%(P< 0.05 vs. RP)和15.27±4.04%(P<0.05 vs. RP),远端结肠环形肌条的张力分别下降10.29±2.71%(P<0.05 vs. RP)、12.95±3.04%(P< 0.05 vs. RP)和16.11±3.93%(P<0.05 vs. RP),加入TTX(10μM)预处理30 min不影响PAR-2-AP对结肠肌条自发性收缩活动的抑制效应。
3.大鼠痢疾杆菌感染后结肠远端环形肌条对PAR-2-AP舒张作用的敏感性降低,远端纵行肌条敏感性没有明显改变。
4.免疫组织化学证实PAR-2表达于结肠的环形肌、纵行肌以及肌间神经丛,痢疾杆菌灌胃后第18天,环形肌上PAR-2表达下降。
5. Western blot检测结果表明痢疾杆菌感染大鼠后第11-35天远端结肠PAR-2的表达量降低。
结论
1.痢疾杆菌灌胃可以引起远端结肠的感染和炎症反应,远端结肠局部炎症第11天最明显,第18天消失
2. PAR-2-AP引起结肠平滑肌发生舒张,该效应是由平滑肌细胞上的PAR-2介导的。
3.痢疾杆菌感染大鼠后结肠远端环形肌对PAR-2-AP的敏感性降低,这种改变可能与痢疾杆菌感染下调环形肌上PAR-2表达有关。
第二部分痢疾杆菌感染后空肠肠系膜传入神经敏感性的改变
目的
肠易激综合征(Irritable Bowel Syndorme, IBS)是最常见的功能性肠道疾病,内脏高敏感性是IBS的重要临床表现。部分IBS病人发病前有肠道感染的病史,这类IBS被称为感染后IBS (post infections IBS, PI-IBS)。目前常用的PI-IBS动物模型是用化学物质刺激和寄生虫感染造成肠道炎症,本研究拟采用痢疾杆菌灌胃造成大肠炎模型,研究痢疾杆菌感染后空肠肠系膜传入神经对机械和化学刺激敏感性的改变。
方法
1.肠炎模型建立同第一部分,取空肠组织做HE染色和髓过氧化物酶活性测定,方法同第一部分
2.肠系膜传入神经放电记录技术
2%戊巴比妥钠麻醉大鼠后小心取出带有肠系膜血管的空肠肠管2-3 cm,置于预冷的Krebs液中。冲洗干净肠腔内容物后固定于灌流浴槽。蠕动泵与外灌流系统相连,灌流浴槽的内槽,灌流速度为10 ml min-1;微量注射泵与内灌流系统相连,灌流肠腔,灌流速度为10 ml h-1可,外灌流的Krebs液持续供给95%O2和5%CO2,保持内槽恒温33℃。40倍镜下分离出肠系膜神经并从中枢端剪断,游离一段与神经同样粗细的结缔组织,肠系膜神经中枢端和结缔组织分别悬挂于两根直径为0.1 m的铂金电极上。电信号通过生物电放大器(1902,CED,Cambridge,英国)放大10000倍后经过模／数转换器(Micro1401,CED,Cambridge,英国)后输入计算机,通过Spike 2(Version 5.20,CED,Cambridge,英国)记录并分析神经纤维动作电位。
4.实验步骤
4.1化学刺激时,待肠系膜传入神经自发性放电稳定15 min后,关闭外循环泵,内浴槽内加入药物作用5 min后,打开外循环泵冲洗。
4.2机械刺激时,待自发性放电稳定15 min后,保持肠腔内灌流速度30ml h-1并关闭内灌流出口,使肠腔内压力升高60 mmHg.记录肠神经放电活动的变化。
统计分析
每3 s对神经放电频率进行一次累计,在某一特定时间段内放电次数最多的3 s期间的放电频率称为peak放电。
采取加药前120s内的神经peak放电为baseline,在化学性刺激时,加药后0-30 s、30-60 s、60-90 s及90-120 s的peak放电减去baseline为统计指标。在机械扩张刺激时,每升高10 mmHg后肠神经peak放电减去basel ine为统计指标。
所有数据采用均数±标准误(mean±SEM)表示,采用单因素方差分析和t检验进行统计学处理,以P＜0.05位显著性差异界值。
结果
1.空肠段肠系膜传入神经呈现规律的自发放电,平均放电频率为8±2 spikes／sec。
2.痢疾杆菌灌胃处理后不同时间点空肠段没有观察到炎症反应,与对照组大鼠相比,SF-treated组大鼠空肠髓过氧化物酶活性没有明显改变。
3.灌流液中加入5-HT后,正常和痢疾杆菌感染大鼠空肠段肠系膜传入神经放电都明显增加；与对照组相比,灌胃处理后第25天,大鼠空肠段肠系膜传入神经电活动对5-HT的反应性没有明显改变。
4.机械扩张刺激引起正常和痢疾杆菌灌胃大鼠空肠段肠系膜传入神经放电明显增加；与对照组相比,在痢疾杆菌灌胃处理后第25天,空肠段肠系膜传入神经对机械扩张刺激的敏感性增加。
5.缓激肽兴奋正常和痢疾杆菌灌胃大鼠空肠段肠系膜传入神经自发放电活动,与对照组相比,在痢疾杆菌灌胃处理后第25天,大鼠空肠段肠系膜传入神经对缓激肽刺激的敏感性明显增加。
结论
痢疾杆菌灌胃处理后第25天,空肠肠系膜传入神经对Bradykinin和机械刺激的敏感性增高,对5-HT的刺激没有明显改变,说明肠道痢疾杆菌感染导致了空肠交感传入神经的高敏感性。
PartⅠ:Change of the PAR-2-AP Induced Relaxation of the Colonic Muscle Strips of Rats following Bacillary Dysentery and the Underlying Mechanism
Objective
Although 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.
Methods
Bacteria preparation
Clinical 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 preparation
Healthy 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 analysis
The 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 protocol
After 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.
Immunohistochemistry
Frozen 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 analysis
Tissue 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 analyses
1. 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.
Results
1. 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.
Conclusions
PAR-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 Dysentery
Objective
IBS (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.
Methods
The rat model of Shigella flexneri infection was the same with that of PartⅠ
The 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 preparation
At 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 discharge
The 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 protocols
For 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 analysis
The 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.
Results
1. 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.
Conclusions
The sensitivity of the jejunal mesenteric afferent nerve to Bradykinin and mechanical stimuli were increased significantly at 25 days after the treatment of SF.
引文
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