摘要
背景阳明腑实证是指无形燥热弥漫在经,充斥于表里内外,劫伤津液,肠中并无燥屎阻结的热证；亦包括热邪与宿食糟粕相搏结,津液被耗,形成有形的实邪壅滞在大肠致燥屎内结的腑实证。阳明腑实证患者有明显胃肠运动障碍的表现。下法代表中药方剂大承气汤可有效地治疗阳明腑实证。近些年来的研究表明肠神经和Cajal间质细胞(interstitia cells of Cajal, ICC)以及平滑肌细胞间形成网络状连接(胃肠道神经-ICC—平滑肌网络)。胃肠道神经—ICC—平滑肌网络与胃肠运动功能密切相关。
目的通过观察阳明腑实证大鼠小肠组织中肠神经-ICC之间形态学结构的变化和小肠全厚肌层组织中ICC-平滑肌之间缝隙连接和ICC细胞超微结构的改变,探讨阳明腑实证大鼠胃肠动力功能障碍时肠神经-ICC-平滑肌网络的细胞形态学改变和细胞通讯传导障碍机制以及大承气汤的施治机制。
方法健康成年Wistar大鼠100只,随机分为：对照组(20只)、阳明腑实证模型组(40只)和大承气汤治疗组(40只)。对照组动物腹腔注射1.0ml生理盐水。阳明腑实证模型组服热性中药、自身粪便灌胃,然后腹腔注射1.0ml E.coloi.混悬液建立阳明腑实证模型。治疗组于造模成功后即给予大承气汤灌胃,2次／日,每次1.Oml／1 00g,连用5日。取各组存活大鼠上段小肠组织制作小肠全厚肌层组织标本,进行c-Kit和乙酰胆碱能神经(vesicular acetylcholine transporter,VAChT),氮能神经(neuronal nitric oxide synthase,nNOS),肽能神经(substance P, SP)和VIP神经(vasoactive intestinal peptide,VIP)免疫荧光双标记染色后,激光扫描共聚焦显微镜检测肠神经-ICC网络结构的变化；免疫组化染色分析ICC-平滑肌网络缝隙连接蛋白Cx43的表达,透射电镜观测ICC-平滑肌网络和ICC超微结构的改变。
结果对照组：胃肠形态正常,肠管颜色呈粉红色,浆膜光泽鲜亮,肠管之间无粘连,胃肠蠕动频率和形态正常。阳明腑实证模型组：胃肠极度扩张,胃内有大量气体和内容物滞留,肠腔内大量液体潴留,多为粪水样液体。大部分肠管肌层呈灰白色,肥厚,浆膜充血肿胀,偶可见黑色,散在的缺血表现。小肠系膜水肿肥厚,系膜根部淋巴结肿大。大网膜充血肥厚,有散出血斑点,腹腔内少量淡黄色清亮渗出液,部分出现血性腹水,壁层腹膜充血,肥厚,腹腔有明显感染现象。胃肠蠕动频率减少,存在明显肠道运动功能减弱征象。治疗组：胃肠道扩张不明显,胃肠腔内潴留物较少或胃内无物滞留。小肠浆膜壁红润,轻度肥厚,无充血,小肠壁和系膜水肿较轻,腹腔内未见血性腹水,腹腔无污染。肠道蠕动频率较模型组明显增加。
共聚焦显微镜检测肠神经—ICC网络的变化对照组：小肠ICC相互连接形成较完整的网络样结构。小肠胆碱能神经(VACHT)／氮能神经(NO)／肽能神经(SP)/VIP神经各自彼此相接形成网状结构,神经网络含有成串的神经节。ICC网络围绕着胆碱能神经／氮能神经／肽能(SP)神经／VIP神经形成网络,肠神经—ICC网络结构完整。
模型组较对照组小肠ICC数量明显减少(p<0.01),细胞荧光强度明显减弱,彼此之间的网络样结构不完整。与对照组比较,小肠胆碱能神经(VACHT)／氮能神经神经(NO)／肽能神经(SP)/VIP神经纤维数目明显减少(p<0.01),彼此间的连接减少,神经网络结构紊乱；神经网络的荧光强度减弱(p<0.01), ICC与肠神经纤维间的连接松散或消失,网络结构不完整,肠神经—ICC网络结构遭受明显破坏。
治疗组较模型组小肠ICC数量明显增多(p<0.01),网络样结构的完整性有所恢复。与模型组比较,ICC细胞荧光强度明显增强(p<0.01)。治疗组小肠胆碱能神经(VACHT)／氮能神经神经(NO)／肽能神经(SP)／VIP神经神经纤维数目明显增加(p<0.01)。神经网络结构好转,彼此间的网络连接的完整性恢复明显,神经网络的荧光强度增加(p<0.01)。ICC与肠神经纤维间的连接接近正常,ICC—肠神经网络结构恢复明显。
免疫组化观察分析连接蛋白Cx43对照组的小肠纵肌层大部分细胞存在呈阳性表达的Cx43蛋白,环肌层有少数细胞呈弱阳性表达,肌间神经丛处大部分细胞呈阳性表达。模型组小肠肌层Cx43蛋白阳性灰度较对照组减弱(p<0.01),尤其以纵肌层明显。治疗组与模型组比较,Cx43蛋白阳性灰度增强(p<0.01),尤其以纵肌层明显。
透射电镜观察对照组大鼠ICC特征：①胞质和突起内含丰富的线粒体、可见内质网和发育良好的高尔基体；②有膜内陷空泡；③有较多中间丝,无粗肌丝；④非常接近三级神经束；⑤ICC相互之间、与平滑肌细胞间可见缝隙连接(gap junctions)；神经纤维末梢与平滑肌细胞见紧密连接,间距约为1 00nm左右,形成完整的神经—ICC—平滑肌网络形态学基础。模型组大鼠ICC细胞突起明显减少或消失,大量神经纤维末梢肿胀,伴随神经递质小泡减少或消失；平滑肌细胞粗面内质网扩张,高尔基体肿胀,出现大量细胞膜内陷空泡,相互间的缝隙连接缺失；ICC与神经纤维间的突触样连接缺如,间距增加；ICC相互间及其与平滑肌细胞间缝隙连接消失,存在较大间隙,神经—ICC—平滑肌网络形态学基础受到明显损伤。治疗组大鼠部分ICC细胞突起损伤不明显；神经组织末梢较为丰富,少量神经纤维末梢肿胀,神经递质小泡无明显减少；平滑肌细胞损伤不明显,有少量细胞膜内陷空泡；ICC与神经纤维间保持突触样连接；ICC相互间及ICC与平滑肌细胞间存在缝隙连接,无明显间隙；组织间隙内存在少量液性物质,神经—ICC—平滑肌网络形态基本保持完整。
结论阳明腑实证大鼠胃肠扩张,胃肠功能紊乱征象明显；大承气汤能够显著改善胃肠运动功能功能。
阳明腑实证时,小肠胆碱能神经(VACHT)／氮能神经(NO)／肽能神经(SP)/VIP神经数目减少；神经网络结构遭到破坏,ICC数量减少,ICC网络结构破坏,肠神经—ICC之间网络结构受到破坏。由于小肠上行兴奋性神经和下行的抑制性神经形态学改变以及他们之间的网络破坏,导致神经之间的传导功能改变。这可能是阳明腑实证大鼠小肠胃肠动力功能障碍的细胞形态学基础。大承气汤可能通过抑制炎症,抗菌、抑制内毒素吸收,以及稳定细胞膜等功能增加阳明腑实证大鼠小肠肠神经和ICC的细胞数量,维持肠神经—ICC网络结构的完整。进而恢复小肠运动功能。
阳明腑实证时,大鼠小肠肌层ICC—平滑肌网络缝隙连接蛋白Cx43阳性表达减少,细胞通讯功能蛋白减少。是阳明腑实证大鼠胃肠动力功能改变的细胞学病理基础。ICC—平滑肌细胞之间缝隙连接消失或间隙增加,导致肠神经—ICC—平滑肌细胞网络的信号传导通路破坏。大承气汤治疗后,大鼠小肠肌层ICC—平滑肌网络缝隙连接蛋白Cx43阳性表达增多,ICC—平滑肌细胞之间缝隙连接间隙接近正常,小肠肌层ICC—平滑肌网络细胞间通讯传导通路恢复。
以上结果提示,阳明腑实证大鼠小肠肠神经—ICC—平滑肌细胞网络结构改变是胃肠道动力障碍的细胞学基础,大承气汤能够减轻肠神经—ICC—平滑肌三者间结构的损伤,维持肠神经—ICC—平滑肌细胞网络形态和信号传导通路完整性,改善阳明腑实证大鼠胃肠道运动功能。
Objective To observe the morphological changes in the network of enteric nerve-interstitia cells of Cajal (ICC)-smooth muscle in rats with Yang Ming Fu Shi Syndrome, and to investigate the therapeutic effect of purgative therapy on the function of gastrointestinal motility.
Methods One handred Wistar rats were randomly divided into three groups:control group (n=20), Yang Ming Fu Shi Sydrom model group (n=40) and Da Cheng Qi decoction treated group (n=40). The rats of control group were injected 1.0ml of normal saline into the peritoneal cavity. The rats of Yang Ming Fu Shi Syndrome model group were perfused chinese herb of the hot and itself dung, then 1.0ml suspension of Escherichia coli was injected into the peritoneal cavity. The rats of Da Cheng Qi decoction treated group(DCQT group) were administrated by gavage with Da Cheng Qi decoction (twice a day,each one 1.0ml/100g) after the Yang Ming Fu Shi Syndrome was established. After five days, the proximal 10cm segment of jejunum was taken and the whole-mount preparation from each group were studied used c-Kit, vesicular acetylcholine transporter (VAChT), neuronal nitric oxide synthase (nNOS), substance P(SP) and vasoactive intestinal peptide (VIP) immunofluorescence double-staining with confocal laser scanning microscopy. The expression of protein Cx43 in gap junction between ICC and smooth muscle cell with immunohistochemical analysis was observed, and the morphologic changes in the network of enteric nerve-ICC-smooth muscle was studied with transmission election micros-cope.
Results In control group, morphological of gastrointestinal tract was normal, and the colour was pink, there was no adhesion each other, motility funtion was normal.. In model group, gastrointestinal tract was significantly distended, there were much gas and contents in gastrointestinal tract, serosa was congestive and edema; there were bleeding point on great omentum and exudate in peritoneal cavity, inflammation in peritoneal cavity was exited. Many intestinal were gray, mesangial hypertrophy; many liquit content like dung exited in gastrointestinal tract, motility funtion of gastrointestinal tract was significantly decreased. In DCQT group, there were less or no content in gastrointestinal tract. Intestinal was pink, no edema, and motility function recoveried.
Observion with confocal laser scanning microscopy In control group, ICCs constituted network in circular muscle layer, longitudinal muscle layer and the myenteric plexus. Enteric nerve of VAChT, nNOS, SP and VIP constructed network each other, and there were many ganglions. ICCs distributed arround the nerve of VAChT, nNOS, SP and VIP, and formed network. The long synapse of ICC distributed with smooth muscle parellely, and short synapse connected each other and formed tight junction with nerve and muscle.
In the model group, the number of ICC were significantly decreased compare with control group(p<0.01), the network was disrupted, fluorescence intensity of ICC decreased. The number of nerve fiber of VAChT, nNOS, SP and VIP were significantly decreased compare with control group(p<0.01), nerves network disordered, each connection decrea-sed, fluorescence intensity of enteric nerve decreased(p<0.01). The conne-ction of ICCs and enteric nerves were loosen or disappeared. ICC-enteric nerve network was significantly disrupted.
In DCQT group, the number of ICC were significantly increased (p<0.01) compare with control group, the network was recovered, fluore-scence intensity of ICC increased. The number of nerve fiber of VAChT, nNOS, SP and VIP were significantly increased (p<0.01), the network was recovered, fluorescence intensity of nerves increased (p<0.01). The conne-ction of ICCs and enteric nerves were approaching normal, ICC-enteric nerve network was significantly recovered.
Expression of protein Cx43 by immunohistochemistry In control group, positive expression of protein Cx43 was distributed in most of cells of longitudinal muscle layer, less of cells distributed circular muscle layer expressed weak postively, and the cells which were distributed in myenteric plexus expressed postively. In model group, postive expression in muscle layer of small intestine was significantly decreased compared with control group (p<0.01), especially in longitudinal muscle layer. In DCQT group, postive expression in muscle layer of small intestine was significantly increased compared with model group (p<0.01), especially in longitudinal muscle layer.
Obsercation with transmission electron microscopy The main characteristic of ICCs in control group were abundant mitochondrion, endoplasmic reticulum and mature Golgi body in cytoplasm and synapse; vacuole in falled membrane; many intermediate filaments, no thick filaments; approximate three class tractus; many gap junctions were formed between ICCs and neuron cells, between ICC and myocyte,and among ICCs. The distance of tight junction between nerves and smooth muscle was100nm, and form morphological basic unit:nerves-ICC-smooth muscle.
In model group, the synapse of ICC were decreased or disappeared, many nerves swelled; accompanied neurotransmitter vacuole to decreased or disapperaed; granular reticulum of smooth muscle distended, Golgi body swelled, appeared many vacuole because of membrane was in falls; the gap junction between them disappeared; synapse-like junction between ICCs and nerves disappeared, and distance between them was larger; the gap junction between ICCs and smooth muscle disappeared, and distance between them was larger, morphological basic unit of nerves-ICC-smooth muscle was significantly disrupted.
In DCQT group, the synapse of ICC was sligherly damaged, nerve ending were abundent, there were less amiunt of swelling nerves, The number of neurotransmitter vacuole was not obviously decreased. The damage in smooth muscle cells was not obvious, synapse-like junction between ICC and nerve was maintenance; the gap junction between ICC and smooth muscle cell was maintained, the distance of gap junction was smaller; There was a little of ligaite substance in tissue intertice, The basic morphology of the unit of nerve-ICC-smooth muscle was maintenanced.
Conclusion Gastrointestinal tract was distended in Yang Ming Fu Shi Syndrome rats. Function of gastrointestinal motility was significantly damaged; and the function was recoveried after Da Cheng Qi decoction treatment.
In Yang Ming Fu Shi Syndrome rats, number of nerve fiber of VAChT, nNOS, SP and VIP were significantly decreased; and number of ICC were decreased too, enteric nerve-ICC network was disrupted; after treatment with Da Cheng Qi Tang, the numbers of nerves and ICC were increased, and network was recoveried.
In rats with Yang Ming Fu Shi Syndrome, the postive expression of protein Cx43 of gap junction in network of enteric nerve-ICC-smooth muscle was decreased, the distance between gap junction was larger. Signal pathway in network of enteric nerve-ICC-smooth muscle was damaged; after treatment with Da Cheng Qi Tang, gap junction and signal pathway was recoveried.
引文
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