慢性胆道感染对Oddi括约肌功能影响的初步研究
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摘要
研究背景与目的
临床上,Oddi括约肌(sphincter of Oddi,SO)松弛在肝胆管结石患者比较常见,由于SO松弛可能诱发返流性胆管炎,从而使肝胆管结石复发增加,因此,SO松弛与否是术中是否废弃SO行胆肠吻合的主要参考因素。但随着胆肠吻合术后吻合口狭窄、癌变以及结石复发等远期并发症逐渐被认识,越来越多的研究者提出尽可能地保留SO的结构和功能并恢复胆道的正常生理结构,这一观点的提出使得一些研究者致力于弄清肝胆管结石患者SO松弛的发生原因。但迄今为止SO松弛的病因不明,学术界对肝胆管结石患者SO松弛的原因存在较大争议,一部分研究者认为排石过程对SO组织的机械性损伤是SO松弛的主要原因,另一部分研究者却发现SO松弛与否和可能存在的排石过程并无密切关系。
炎性肠道病变和非结石性胆囊炎引起的平滑肌功能变化是近年来消化系统功能学研究的热点之一。炎症状态下平滑肌运动功能减弱与活性氧簇和亚硝酸盐等炎症代谢产物对平滑肌细胞膜的损害有密切关系,表现为钙离子稳态失衡,肌球蛋白轻链(myosin light chain, MLC)磷酸化障碍甚至纤丝状肌动蛋白(filamentous actin, F-actin)发生重塑。这些研究结果强烈的提示我们反复发作的胆道感染可能与肝胆管结石患者SO松弛有着密切的关系。但关于慢性胆道感染对SO功能有何影响目前尚未见相关报道。
基于以上背景,我们推测:反复发作的胆道感染可能是SO平滑肌收缩功能减弱的重要原因。为验证我们的假说,本研究旨在通过临床研究,观察SO松弛患者的SO压力和肌电活动特征,分析肝胆管结石患者SO松弛的主要危险因素。随后通过建立兔胆道感染的动物模型,研究慢性胆道感染对兔在体和离体SO肌电活动的影响;最后从细胞水平阐明慢性胆道感染后SO平滑肌细胞收缩相关的钙离子动员、MLC磷酸化、F-actin重塑的发生情况。
材料与方法
一、采用环状电极记录肌电和三通道测压导管测压的方法,我们记录了肝胆管结石患者SO松弛时的SO压力和肌电特征,分析其SO基础压(SO basal pressure,SOBP)、峰压(SO amplitude,SOAP)和收缩持续时间(duration of SO contraction)、收缩频率(frequency of SO phasic contraction, SOF)以及SO肌电幅值(amplitude of SO spike burst, SOSB)的变化情况。进一步,采用多因素分析回顾性研究293例肝胆管结石患者SO松弛的发生情况,分析年龄、性别、病程、结石分布、结石大小、结石性状、既往手术次数、手术方式以及既往胆管炎发作次数等因素与SO松弛的相关性。
二、用改良的Thomas法建立清醒状态下兔SO功能检测的动物模型,在清醒状态下记录兔SO的肌电活动,分析消化间期、静脉给予不同剂量的CCK和进食后SO肌电的变化情况。
三、用胆总管内置丝线和经胆囊管连续3周(每周1次)向胆总管内注入大肠杆菌的方法建立慢性胆道感染的动物模型,以对照组和非感染组为对照,采用一般情况(饮食量、肛温、体重变化)、SO病理检测评分和肝功能检测的方法对模型进行判断。在模型终点进行在体和离体SO肌电记录,对慢性胆道感染组、对照组和非感染组的肌电活动进行比较。在体SO肌电记录分别比较在消化间期、静脉给予不同剂量的CCK后各组SO的肌电变化情况,分析慢性胆道感染对SO肌电活动的影响。离体实验采用完整切取SO组织、在离体器官浴槽中记录SO肌电活动的方法进行,比较各组SO在基础状态、CCK诱发的SO肌电频率、幅值变化情况,进一步,我们研究了在激动剂[60 mM KCl,50 nM ionomycin,10 nM cholecystokinin (CCK)]作用下和容量依赖的钙离子内流(capacitative calcium entry, CCE)诱导下胞外钙离子内流和胞浆内钙池钙离子释放引发SO肌电活动的变化。
四、为研究慢性胆道感染对平滑肌细胞钙离子稳态、MLC磷酸化及F-actin表达的影响,我们对对照组,非感染组和慢性胆道感染组SO平滑肌细胞进行了原代分离和培养,首先用Fluo-3 AM负载后在激光共聚焦显微镜下观察激动剂(60 mM KCl,50 nM ionomycin,10 nM CCK)和CCE对胞浆内[Ca2+]i的影响,然后用Western-blot方法检测SO平滑肌组织中MLC磷酸化水平的改变,最后检测了FITC- phalloidin标记的平滑肌细胞骨架F-actin的表达情况。
结果
一、293例肝胆管结石患者中伴发SO松弛60例(20.5%),与SO不松驰的患者相比较,其SO基础压、峰压、收缩频率和收缩持续时间降低,SO肌电幅值和频率也相应降低。肝胆管结石的病程和多次发作的急性胆管炎与SO松弛的发生密切相关。
二、建立了清醒状态下SO肌电检测的动物模型。禁食期间,SO的肌电活动呈周期性变化,肌电活动由较弱的1期经逐渐升高的2期过渡至肌电活动最强的第3期,4期为3期向1期的短暂过渡期。20 ng·kg-1和100 ng·kg-1的CCK以及进食可以明显兴奋SO的肌电活动。
三、建立了慢性胆道感染的动物模型,慢性胆道感染后γ-谷氨酰转肽酶(γ-glutamyltransferase, GGT)和碱性磷酸酶(alkaline phosphatase, ALP)的明显升高,SO组织在病理上表现为黏膜层、肌层炎细胞浸润和肌层胶原纤维沉积增多,炎症评分明显增高。慢性胆道感染后SO肌电频率、运动指数在2期、3期明显减低,20 ng·kg-1和100 ng·kg-1的CCK兴奋SO肌电活动的作用减弱。同样,慢性胆道感染后静息状态及10 nM CCK诱导下离体SO肌电活动明显减低,促进胞外钙离子内流和胞内钙离子释放的激动剂诱发的肌电活动也明显减弱。
四、利用胶原酶消化法,我们对兔SO平滑肌细胞进行了分离和培养。慢性胆道感染后,兔SO平滑肌细胞钙离子稳态失衡,包括KCl、CCE诱发的胞外钙离子内流和CCK、Caffeine诱发的胞内钙离子释放减少。此外,MLC磷酸化障碍、F-actin骨架重塑在慢性胆道感染后SO平滑肌细胞也有发生。
结论
一、肝胆管结石患者SO松弛的发生率较高,其运动特征为SO肌电活动减弱和压力降低,病程和多次发作的胆管炎是SO松弛的主要危险因素。
二、慢性胆道感染后兔在体及离体SO肌电频率、幅值和运动指数均明显降低,其对CCK等激动剂的反应减弱。
三、钙离子稳态失衡、MLC磷酸化障碍、F-actin表达下降可能是慢性胆道感染后SO平滑肌运动功能减弱的重要原因。
BACKGROUND AND OBJECTS
It is very common that a loose sphincter of Oddi (SO) is encountered during exploration of common bile duct in patients with hepatolithiasis or common bile duct stones. Because of its potential risk to induce reflux cholangitis and result in stones recurrence, loose SO is one of the decisive reasons to adopt cholangiojejunostomy. However, with the long-term complications such as the stenosis and the carcinomatous change of the stoma as well as the high incidence of stones recurrence be recognized, more and more researchers proposed to preserve the structure and the function of SO. This point of view encouraged many researchers to reveal the underlying cause of loose SO accompanied with hepatolithiasis, but completely different results were draw and effects of cholelithiasis on the motility of SO were quiet controversy, it is conceivable that passage of stones through the sphincter can result in loose SO, however, other researchers argued that the passage of stones through SO was not in close relationship to the occurrence of loose SO.
Researches on the functional changes of smooth muscle motility in the inflammatory bowel disease (IBD) and acalculous cholecystitis were prevalent in the past few years. Many studies have reported that there is a correlation between the impaired smooth muscle motility and the increased reactive oxygen species (ROS) and /or peroxynitrite, which induce cellular injury via several mechanisms including peroxidation of membrane lipids, and result in impaired calcium homeostasis, abnormal phosphorylation of myosin light chain (MLC) as well as distortion of filamentous actin (F-actin). It is imperative to assume that recurrent cholangitis may be involved in the progress of loose SO, However, few researches focused on the cause and effect of cholangitis and loose SO.
Then we assumed that recurrent cholangitis be the most important factor of impaired smooth muscle motility of SO. To identify this hypothesis, the aim of our study was, first of all, to collect clinical data, observe the motility characteristics of loose SO and analyze its related risk factors in hepatolithiasis patients. Then we devised a novel cholangtis model in rabbits and observed the effects of chronic cholangitis on the myoelectric motility of SO in-vivo and in-vitro. Finally, we investigated the effects of chronic cholangitis on the calcium mobilization, phosphorylation of myosin light chain (MLC) as well as the remodeling of filamentous actin (F-actin) in SO smooth muscle cells.
MATERIALS AND METHODS
1. With the methods of myoelectric and manometry recording via circular electrode (CE) or perfused triplelumen system, we recorded the myoelectric motility and manometry change of SO including the SO basal pressure (SOBP), SO amplitude (SOAP), frequency of SO phasic contraction(SOF), duration of SO contraction, as well as the frequency and amplitude of SO spike burst (SOSB). Then, we analyzed the correlation between loose SO and independent variables involved in the progress of hepatolithiasis including age, gender, course of disease, distribution of stones, size of stones, characteristic of stones, operation frequency, occurrence frequency of cholangitis, and diameter of common bile duct in 293 cases.
2. With modified Thomas process, we devised a novel model of myoelectric recording of SO in fully conscious state in rabbits. We recorded the cyclic change of myoelectric motility during fasting, and the myoelectric response to different doses (20 ng·kg-1 or 100ng·kg-1) of cholecystokin (CCK) or feeding.
3. To establish the model of chronic cholangitis in rabbits, we introduced three 2-0 silk and sequentially injected E Coli into the bile duct, and then observed the changes of weight, rectal temperature, liver function [alanine aminotransferase (ALT); alkaline phosphatase (ALP);γ-glutamyltransferase (GGT) and direct bilirubin (DB)], as well as inflammation score assessed by pathological changes of SO including inflammatory infiltration and collagen deposition. In in-vivo SO myoelectric motility experiment, the SO myoelectric activity was recorded by cyclic electrode (CE) through the jejunum stump in conscious rabbits or application of CCK. In in-vitro experiment, the SO was completely isolated under operating microscope and the myoelectric activity was recorded by CE in a 10-ml organ bath filled with Krebs solution with or without addition of CCK, KCl, ionomycin or induction of capacitative calcium entry (CCE). All the results from chronic cholangitis rabbits were in comparison with that in control and non-infective subjects.
4. To investigate the effects of chronic cholangitis on the calcium mobilization, phosphorylation of MLC as well as the remodeling of F-actin in SO smooth muscle. We isolated the smooth muscle cells from the SO with the modified enzymatic digestion method, then we observed the changes of [Ca2+]i induced by agonists in Fluo-3 AM loaded cells, or the expression of F-actin by FITC-phalloidin loaded smooth muscle cells under laser scanning confocal microscope (LSCM). The phosphorylated MLC was also detected by Western-blot.
RESULTS
1. 60 cases (20.5%) of loose SO was found in 293 hepatolithiasis patients, in comparison with the intact SO, the SOBP, SOAP, SOF and duration of SOAP recorded by SO manometry were significantly decreased, the frequency and amplitude of SOSBs were also decreased. The recurrent cholangitis and course of disease were in close relationship with loose SO.
2. The myoelectric motility was recorded in fully conscious rabbits with this model; sphincter motility exerted a cyclic change because of the cyclical variation of fast waves and almost invariant slow waves. Feeding caused excitation of the sphincter activity as well as the effect of CCK in the dosage of 20 ng·kg-1 or 100 ng·kg-1.
3. Chronic cholangitis was successfully established in rabbits. In control (CN) and non-infective (NI) groups, no changes were found in weight, rectal temperature as well as liver function. However, in chronic cholangitis (CC) group, rectal temperature, ALT, ALP, GGT, and DB were significantly elevated at the first 3~4 days. Three months latter, ALP, GGT and inflammation score were significantly higher than that in NI and CN groups, but the ALT and DB was not changed. The cyclic change of myoelectric motility was recorded in 25 rabbits. In comparison with the CN and NI group, the duration of phase 3 was significantly decreased. The frequency and amplitude of SOSBs were lower in CC than that in the CN and NI group in phase 2 and 3, and the myoelectric motility index (MI) was especially lower in phase 3 in CC group. The excitative response of myoelectric motility was decreased in CC group with shortened effective duration, lower amplitude of SOSBs and MI.
In-vitro study also showed decreased frequency and amplitude of SOSBs as well as MI in resting state and response to 10 nM CCK in CC group in comparison with NI and CN group. CC group also showed significantly lower MI of elevated myoelectric motility in the presence of 60 mM KCl and induction of CCE. In CC group, agonists (10 nM CCK and 50 nM ionomycin) activated calcium release in calcium-free medium was also fail to induce the significant increase of myoelectric MI as that in CN and NI group.
4. With the modified enzymatic digestion method, we successfully isolated and cultivated the smooth muscle cells. Smooth muscle cells of SO isolated from the chronic cholangitis rabbits showed impaired calcium homeostasis including the decrease of 60 mM KCl or CCE-induced calcium influx from extracellular and 10 nM CCK or 10 mM Caffeine-induced calcium release from the calcium pools. In addition, abnormal phosphorylation of MLC and distortion or fragmentation of F-actin was also found in the SO smooth muscle cell in chronic cholangitis rabbits.
CONCLUSIONS
1. The occurrence rate of loose SO is relatively high in hepatolithiasis patients with decreased SOBP, SOAP, SOF, as well as the frequency and amplitude of SOSB. Course of disease recurrent cholangitis were the independent variable correlated with loose SO.
2. The frequency, amplitude and motility index of SO myoelectric activity and of its response to CCK was decreased either in-vivo or in-vitro in chronic cholangitis rabbits.
3. Impaired calcium homeostasis and deregulation of MLC phosphorylation as well as remodeling of F-actin might be the key events of impaired motility of SO in chronic cholangitis rabbits.
临床上,Oddi括约肌(sphincter of Oddi,SO)松弛在肝胆管结石患者比较常见,由于SO松弛可能诱发返流性胆管炎,从而使肝胆管结石复发增加,因此,SO松弛与否是术中是否废弃SO行胆肠吻合的主要参考因素。但随着胆肠吻合术后吻合口狭窄、癌变以及结石复发等远期并发症逐渐被认识,越来越多的研究者提出尽可能地保留SO的结构和功能并恢复胆道的正常生理结构,这一观点的提出使得一些研究者致力于弄清肝胆管结石患者SO松弛的发生原因。但迄今为止SO松弛的病因不明,学术界对肝胆管结石患者SO松弛的原因存在较大争议,一部分研究者认为排石过程对SO组织的机械性损伤是SO松弛的主要原因,另一部分研究者却发现SO松弛与否和可能存在的排石过程并无密切关系。
炎性肠道病变和非结石性胆囊炎引起的平滑肌功能变化是近年来消化系统功能学研究的热点之一。炎症状态下平滑肌运动功能减弱与活性氧簇和亚硝酸盐等炎症代谢产物对平滑肌细胞膜的损害有密切关系,表现为钙离子稳态失衡,肌球蛋白轻链(myosin light chain, MLC)磷酸化障碍甚至纤丝状肌动蛋白(filamentous actin, F-actin)发生重塑。这些研究结果强烈的提示我们反复发作的胆道感染可能与肝胆管结石患者SO松弛有着密切的关系。但关于慢性胆道感染对SO功能有何影响目前尚未见相关报道。
基于以上背景,我们推测:反复发作的胆道感染可能是SO平滑肌收缩功能减弱的重要原因。为验证我们的假说,本研究旨在通过临床研究,观察SO松弛患者的SO压力和肌电活动特征,分析肝胆管结石患者SO松弛的主要危险因素。随后通过建立兔胆道感染的动物模型,研究慢性胆道感染对兔在体和离体SO肌电活动的影响;最后从细胞水平阐明慢性胆道感染后SO平滑肌细胞收缩相关的钙离子动员、MLC磷酸化、F-actin重塑的发生情况。
材料与方法
一、采用环状电极记录肌电和三通道测压导管测压的方法,我们记录了肝胆管结石患者SO松弛时的SO压力和肌电特征,分析其SO基础压(SO basal pressure,SOBP)、峰压(SO amplitude,SOAP)和收缩持续时间(duration of SO contraction)、收缩频率(frequency of SO phasic contraction, SOF)以及SO肌电幅值(amplitude of SO spike burst, SOSB)的变化情况。进一步,采用多因素分析回顾性研究293例肝胆管结石患者SO松弛的发生情况,分析年龄、性别、病程、结石分布、结石大小、结石性状、既往手术次数、手术方式以及既往胆管炎发作次数等因素与SO松弛的相关性。
二、用改良的Thomas法建立清醒状态下兔SO功能检测的动物模型,在清醒状态下记录兔SO的肌电活动,分析消化间期、静脉给予不同剂量的CCK和进食后SO肌电的变化情况。
三、用胆总管内置丝线和经胆囊管连续3周(每周1次)向胆总管内注入大肠杆菌的方法建立慢性胆道感染的动物模型,以对照组和非感染组为对照,采用一般情况(饮食量、肛温、体重变化)、SO病理检测评分和肝功能检测的方法对模型进行判断。在模型终点进行在体和离体SO肌电记录,对慢性胆道感染组、对照组和非感染组的肌电活动进行比较。在体SO肌电记录分别比较在消化间期、静脉给予不同剂量的CCK后各组SO的肌电变化情况,分析慢性胆道感染对SO肌电活动的影响。离体实验采用完整切取SO组织、在离体器官浴槽中记录SO肌电活动的方法进行,比较各组SO在基础状态、CCK诱发的SO肌电频率、幅值变化情况,进一步,我们研究了在激动剂[60 mM KCl,50 nM ionomycin,10 nM cholecystokinin (CCK)]作用下和容量依赖的钙离子内流(capacitative calcium entry, CCE)诱导下胞外钙离子内流和胞浆内钙池钙离子释放引发SO肌电活动的变化。
四、为研究慢性胆道感染对平滑肌细胞钙离子稳态、MLC磷酸化及F-actin表达的影响,我们对对照组,非感染组和慢性胆道感染组SO平滑肌细胞进行了原代分离和培养,首先用Fluo-3 AM负载后在激光共聚焦显微镜下观察激动剂(60 mM KCl,50 nM ionomycin,10 nM CCK)和CCE对胞浆内[Ca2+]i的影响,然后用Western-blot方法检测SO平滑肌组织中MLC磷酸化水平的改变,最后检测了FITC- phalloidin标记的平滑肌细胞骨架F-actin的表达情况。
结果
一、293例肝胆管结石患者中伴发SO松弛60例(20.5%),与SO不松驰的患者相比较,其SO基础压、峰压、收缩频率和收缩持续时间降低,SO肌电幅值和频率也相应降低。肝胆管结石的病程和多次发作的急性胆管炎与SO松弛的发生密切相关。
二、建立了清醒状态下SO肌电检测的动物模型。禁食期间,SO的肌电活动呈周期性变化,肌电活动由较弱的1期经逐渐升高的2期过渡至肌电活动最强的第3期,4期为3期向1期的短暂过渡期。20 ng·kg-1和100 ng·kg-1的CCK以及进食可以明显兴奋SO的肌电活动。
三、建立了慢性胆道感染的动物模型,慢性胆道感染后γ-谷氨酰转肽酶(γ-glutamyltransferase, GGT)和碱性磷酸酶(alkaline phosphatase, ALP)的明显升高,SO组织在病理上表现为黏膜层、肌层炎细胞浸润和肌层胶原纤维沉积增多,炎症评分明显增高。慢性胆道感染后SO肌电频率、运动指数在2期、3期明显减低,20 ng·kg-1和100 ng·kg-1的CCK兴奋SO肌电活动的作用减弱。同样,慢性胆道感染后静息状态及10 nM CCK诱导下离体SO肌电活动明显减低,促进胞外钙离子内流和胞内钙离子释放的激动剂诱发的肌电活动也明显减弱。
四、利用胶原酶消化法,我们对兔SO平滑肌细胞进行了分离和培养。慢性胆道感染后,兔SO平滑肌细胞钙离子稳态失衡,包括KCl、CCE诱发的胞外钙离子内流和CCK、Caffeine诱发的胞内钙离子释放减少。此外,MLC磷酸化障碍、F-actin骨架重塑在慢性胆道感染后SO平滑肌细胞也有发生。
结论
一、肝胆管结石患者SO松弛的发生率较高,其运动特征为SO肌电活动减弱和压力降低,病程和多次发作的胆管炎是SO松弛的主要危险因素。
二、慢性胆道感染后兔在体及离体SO肌电频率、幅值和运动指数均明显降低,其对CCK等激动剂的反应减弱。
三、钙离子稳态失衡、MLC磷酸化障碍、F-actin表达下降可能是慢性胆道感染后SO平滑肌运动功能减弱的重要原因。
BACKGROUND AND OBJECTS
It is very common that a loose sphincter of Oddi (SO) is encountered during exploration of common bile duct in patients with hepatolithiasis or common bile duct stones. Because of its potential risk to induce reflux cholangitis and result in stones recurrence, loose SO is one of the decisive reasons to adopt cholangiojejunostomy. However, with the long-term complications such as the stenosis and the carcinomatous change of the stoma as well as the high incidence of stones recurrence be recognized, more and more researchers proposed to preserve the structure and the function of SO. This point of view encouraged many researchers to reveal the underlying cause of loose SO accompanied with hepatolithiasis, but completely different results were draw and effects of cholelithiasis on the motility of SO were quiet controversy, it is conceivable that passage of stones through the sphincter can result in loose SO, however, other researchers argued that the passage of stones through SO was not in close relationship to the occurrence of loose SO.
Researches on the functional changes of smooth muscle motility in the inflammatory bowel disease (IBD) and acalculous cholecystitis were prevalent in the past few years. Many studies have reported that there is a correlation between the impaired smooth muscle motility and the increased reactive oxygen species (ROS) and /or peroxynitrite, which induce cellular injury via several mechanisms including peroxidation of membrane lipids, and result in impaired calcium homeostasis, abnormal phosphorylation of myosin light chain (MLC) as well as distortion of filamentous actin (F-actin). It is imperative to assume that recurrent cholangitis may be involved in the progress of loose SO, However, few researches focused on the cause and effect of cholangitis and loose SO.
Then we assumed that recurrent cholangitis be the most important factor of impaired smooth muscle motility of SO. To identify this hypothesis, the aim of our study was, first of all, to collect clinical data, observe the motility characteristics of loose SO and analyze its related risk factors in hepatolithiasis patients. Then we devised a novel cholangtis model in rabbits and observed the effects of chronic cholangitis on the myoelectric motility of SO in-vivo and in-vitro. Finally, we investigated the effects of chronic cholangitis on the calcium mobilization, phosphorylation of myosin light chain (MLC) as well as the remodeling of filamentous actin (F-actin) in SO smooth muscle cells.
MATERIALS AND METHODS
1. With the methods of myoelectric and manometry recording via circular electrode (CE) or perfused triplelumen system, we recorded the myoelectric motility and manometry change of SO including the SO basal pressure (SOBP), SO amplitude (SOAP), frequency of SO phasic contraction(SOF), duration of SO contraction, as well as the frequency and amplitude of SO spike burst (SOSB). Then, we analyzed the correlation between loose SO and independent variables involved in the progress of hepatolithiasis including age, gender, course of disease, distribution of stones, size of stones, characteristic of stones, operation frequency, occurrence frequency of cholangitis, and diameter of common bile duct in 293 cases.
2. With modified Thomas process, we devised a novel model of myoelectric recording of SO in fully conscious state in rabbits. We recorded the cyclic change of myoelectric motility during fasting, and the myoelectric response to different doses (20 ng·kg-1 or 100ng·kg-1) of cholecystokin (CCK) or feeding.
3. To establish the model of chronic cholangitis in rabbits, we introduced three 2-0 silk and sequentially injected E Coli into the bile duct, and then observed the changes of weight, rectal temperature, liver function [alanine aminotransferase (ALT); alkaline phosphatase (ALP);γ-glutamyltransferase (GGT) and direct bilirubin (DB)], as well as inflammation score assessed by pathological changes of SO including inflammatory infiltration and collagen deposition. In in-vivo SO myoelectric motility experiment, the SO myoelectric activity was recorded by cyclic electrode (CE) through the jejunum stump in conscious rabbits or application of CCK. In in-vitro experiment, the SO was completely isolated under operating microscope and the myoelectric activity was recorded by CE in a 10-ml organ bath filled with Krebs solution with or without addition of CCK, KCl, ionomycin or induction of capacitative calcium entry (CCE). All the results from chronic cholangitis rabbits were in comparison with that in control and non-infective subjects.
4. To investigate the effects of chronic cholangitis on the calcium mobilization, phosphorylation of MLC as well as the remodeling of F-actin in SO smooth muscle. We isolated the smooth muscle cells from the SO with the modified enzymatic digestion method, then we observed the changes of [Ca2+]i induced by agonists in Fluo-3 AM loaded cells, or the expression of F-actin by FITC-phalloidin loaded smooth muscle cells under laser scanning confocal microscope (LSCM). The phosphorylated MLC was also detected by Western-blot.
RESULTS
1. 60 cases (20.5%) of loose SO was found in 293 hepatolithiasis patients, in comparison with the intact SO, the SOBP, SOAP, SOF and duration of SOAP recorded by SO manometry were significantly decreased, the frequency and amplitude of SOSBs were also decreased. The recurrent cholangitis and course of disease were in close relationship with loose SO.
2. The myoelectric motility was recorded in fully conscious rabbits with this model; sphincter motility exerted a cyclic change because of the cyclical variation of fast waves and almost invariant slow waves. Feeding caused excitation of the sphincter activity as well as the effect of CCK in the dosage of 20 ng·kg-1 or 100 ng·kg-1.
3. Chronic cholangitis was successfully established in rabbits. In control (CN) and non-infective (NI) groups, no changes were found in weight, rectal temperature as well as liver function. However, in chronic cholangitis (CC) group, rectal temperature, ALT, ALP, GGT, and DB were significantly elevated at the first 3~4 days. Three months latter, ALP, GGT and inflammation score were significantly higher than that in NI and CN groups, but the ALT and DB was not changed. The cyclic change of myoelectric motility was recorded in 25 rabbits. In comparison with the CN and NI group, the duration of phase 3 was significantly decreased. The frequency and amplitude of SOSBs were lower in CC than that in the CN and NI group in phase 2 and 3, and the myoelectric motility index (MI) was especially lower in phase 3 in CC group. The excitative response of myoelectric motility was decreased in CC group with shortened effective duration, lower amplitude of SOSBs and MI.
In-vitro study also showed decreased frequency and amplitude of SOSBs as well as MI in resting state and response to 10 nM CCK in CC group in comparison with NI and CN group. CC group also showed significantly lower MI of elevated myoelectric motility in the presence of 60 mM KCl and induction of CCE. In CC group, agonists (10 nM CCK and 50 nM ionomycin) activated calcium release in calcium-free medium was also fail to induce the significant increase of myoelectric MI as that in CN and NI group.
4. With the modified enzymatic digestion method, we successfully isolated and cultivated the smooth muscle cells. Smooth muscle cells of SO isolated from the chronic cholangitis rabbits showed impaired calcium homeostasis including the decrease of 60 mM KCl or CCE-induced calcium influx from extracellular and 10 nM CCK or 10 mM Caffeine-induced calcium release from the calcium pools. In addition, abnormal phosphorylation of MLC and distortion or fragmentation of F-actin was also found in the SO smooth muscle cell in chronic cholangitis rabbits.
CONCLUSIONS
1. The occurrence rate of loose SO is relatively high in hepatolithiasis patients with decreased SOBP, SOAP, SOF, as well as the frequency and amplitude of SOSB. Course of disease recurrent cholangitis were the independent variable correlated with loose SO.
2. The frequency, amplitude and motility index of SO myoelectric activity and of its response to CCK was decreased either in-vivo or in-vitro in chronic cholangitis rabbits.
3. Impaired calcium homeostasis and deregulation of MLC phosphorylation as well as remodeling of F-actin might be the key events of impaired motility of SO in chronic cholangitis rabbits.
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