摘要
目的：
1.利用甘草制作胃动力低下大鼠模型，观察其胃残留率及小肠推进率的影响；
2.观察槟榔对胃动力低下大鼠胃残留率、小肠推进率的影响，并利用SPECT测定胃半排空时间，观察槟榔对胃动力作用的影响；
3.透射电镜观察槟榔对Cajal间质细胞超微结构的改变及其与周围肠神经系统的关系，免疫组化方法探索槟榔对c-kit在模型大鼠胃组织中表达的影响。
4.采用HPLC对槟榔成分进行分析，观察其对离体胃平滑肌细胞收缩效应的影响，并使用LC-MS分析有效组分。
方法：
1.胃动力低下大鼠模型的建立
制备生甘草溶液，分为2.5、5、7.5、10、15g/kg5个剂量组，予大鼠每日灌胃，持续5d。以苯酚红测定法，紫外分光光度计测胃残留率，并测量计算小肠推进率。使用SPECT检测99mTc-MIBI溶液在各组大鼠胃内的半排空时间。
2.槟榔对胃动力的影响
采用方法1建立胃动力低下大鼠模型，以莫沙必利作为对照药物，分为模型组（10g/kg甘草）、正常对照组（0.9％NaCl）、西药组（0.4mg/kg莫沙必利）以及中药槟榔组（10g/kg槟榔），灌胃7d后，测量各组大鼠的胃残留率、小肠推进率、胃半排空时间。
3.槟榔对胃内ICC超微结构及c-kit表达的影响
造模方法同1，分组方案同2，摘取全胃，并取胃底、胃体、胃窦组织各两块。其中一块制作超薄切片，透射电子显微镜观察记录；另一块制作免疫组化标本，观察c-kit抗体表达，计算阳性细胞面积。
4.槟榔有效组分的分析提取及其对离体平滑肌的作用制备槟榔冻干粉，制作豚鼠平滑肌肌条，观察不同批次提取物对肌条的效应，选取有效批次槟榔提取物，HPLC记录其紫外吸收图谱并提取有效成分，观察其对豚鼠离体平滑肌的收缩效应，LC-MS测定其分子量。
结果：
1.不同剂量（2.5、5、7.5、10、15g/kg）甘草溶液灌胃后发现，10g/kg剂量组的胃残留率比其它组别要高，明显高于正常对照组（P＜0.01），且胃半排空时间延长（P＜0.05）。而5g/kg剂量组的小肠推进率与正常对照组相比，具有统计学意义（P＜0.05）。
2.与正常对照组相比，中药槟榔组及莫沙必利组的胃残留率降低（P＜0.05），胃半排空时间缩短，而小肠推进率增大；同时，模型组胃残留率高于其他三组（P＜0.05），胃半排空时间延长。槟榔组与莫沙必利组无统计学差异。
3.电镜观察发现模型组大鼠胃内ICC形状较扁，甚至不规则，向外伸展的突起变短小；其余三组大鼠ICC呈纺锤形,有巨大的卵圆形核及向外伸展的长突起，25000倍放大观察发现，胞质内有丰富的线粒体，大量滑面内质网和粗面内质网。槟榔组ICC与周围的肠神经元轴突间突触样连接增多。四组中，胃内C-kit阳性表达最高部位均为胃窦部，高于胃体部、胃底部。槟榔组的c-kit阳性表达面积最高，尤其在胃窦部（7.97±0.39万μm2）。
4.分取槟榔不同组分进行豚鼠离体平滑肌实验，选取有效组分进行HPLC成分分析，紫外吸收光谱显示69个峰值，其中34、42最高，提取该组分，发现10μmol/L剂量BL42对平滑肌胃环形肌和胃纵行肌的收缩效应最强，其曲线下面积分别为5.96±0.58,5.64±0.52g/s，接近于Ach效应。LC-MS检测发现，该有效成分分子量不同于槟榔碱。
结论：
1.在剂量为10g/kg时，中药甘草可以增加大鼠的胃残留率，延长胃半排空时间，因此可以用来建立胃动力低下大鼠模型。
2.槟榔能够降低胃动力低下模型大鼠的胃残留率，缩短胃半排空时间，并增高小肠推进率，推测槟榔具有促进胃动力作用。
3.槟榔能够增加胃动力低下大鼠胃内ICC与周围肠神经系统的联系，并使c-kit在胃窦、胃体、胃底的阳性表达面积加大，提示槟榔的促胃动力作用与ICC具有一定联系。
4.HPLC制备的槟榔内组分BL34、BL42对豚鼠离体平滑肌具有较强收缩效应，且该作用类似于Ach，该物质成分的具体分子结构尚需进一步实验研究。
Objectives:
1. To observe the effects of Chinese medicinal glycyrrhiza on the gastric residual rateand the intestinal propulsive rate of rat models of low gastric motility, which areestablished by utilizing glycyrrhiza;
2. To observe the effects of areca on the gastric residual rate and the intestinalpropulsive rate of rats with low gastric motility, to determine the gastrichalf-emptying time (GET1/2) by using SPECT (Single Photon Emission ComputedTomography), and to observe the effects of the areca on the gastric motility;
3. To observe the effects of the areca on changing the ultrastructure of interstitial cellsof Cajal (ICC) and the relationship between the ICC with the peripheral entericnervous system by using a transmission electron microscope (TEM), and to explorethe effects of the areca on the expression of c-kit in gastric tissues of model rats byusing an immunohistochemistrical method.
4. To analyze components of the areca by adopting HPLC (High Performance LiquidChromatography) and LC-MS (Liquid Chromatography-Mass Spectrometry) andextract effective components of the areca in order to observe the effects of theeffective components on contraction of in-vitro gastric smooth muscle cells.
Methods:
1. Establishment of rat models of low gastric motilityRaw glycyrrhiza solutions are prepared and divided into five dose groups, namely,
2.5g/kg,5g/kg,7.5g/kg,10g/kg and15g/kg. Rats are continuously fed with the rawglycyrrhiza solutions by gavage for5days. The gastric residual rate is measured byusing an ultraviolet spectrophotometer through pH-sensitive assay using phenol red,and the intestinal propulsive rate is measured and calculated. The GET1/2of99mTc-MIBI (Technetium-Methoxy Isobutyl Isonitrile) solution in rat stomachs in allgroups is detected by using SPECT.
2. Effects of the areca on the gastric motilityBy taking mosapride as a control medicine, the rat models of low gastric motility,established by adopting the method1, are divided into a model group (10g/kgglycyrrhiza), a normal control group (0.9%NaCl), a western medicine group(0.4mg/kg mosapride) and an areca group (10k/kg areca), and are fed by gavage for7days. The gastric residual rate, the intestinal propulsive rate and the GET1/2of therats in each group are measured.
3. Effects of the areca on the ultrastructure of intragastric ICC and the expression ofc-kitModels and groups are established according to the methods1and2. A wholestomach is removed, and two pieces of tissues are taken from each of the gastric
fundus, the gastric body and the gastric antrum. One piece of each kind of tissue isused for making an ultrathin section to be observed and recorded by the TEM, and theother piece of each kind of tissue is used for making an immunohistochemistricalspecimen for observation of c-kit antibody expression and calculation of positive cellareas.
4. Extraction of areca effective components and effects of the areca effectivecomponents on in-vitro smooth muscleLyophilized areca powder is prepared, smooth muscle strips of guinea pigs are made,and the effects of extractives of different batches on the muscle strips are observed; the areca extractives of effective batches are selected, the ultraviolet absorptionspectra of the selected extractives are recorded by means of HPLC, and the effectivecomponents of the selected extractives are extracted; and the effects of the effectivecomponents on the contraction of the in-vitro smooth muscle of the guinea pigs areobserved, and a mechanism is analyzed.
Results:1. According to the gavage using the glycyrrhiza solutions with different doses
(2.5g/kg,5g/kg,7.5g/kg,10g/kg and15g/kg), the gastric residual rate in the group of10g/kg dose is higher than that in other groups and is significantly higher than that inthe normal control group (P<0.01), and the GET1/2is prolonged (P<0.05). Whilecomparing the intestinal propulsive rate in the group of5k/kg dose with that in thenormal control group, a statistical significance can be achieved (P<0.05).2. Compared with the normal control group, in the areca group and the western
medicine group, the gastric residual rate is decreased (P<0.05), the GET1/2isshortened, and the intestinal propulsive rate is increased; meanwhile, the gastricresidual rate in the model group is significantly higher (P<0.05), and the GET1/2isprolonged. No statistical differences are presented between the areca group and themosapride group.
3. According to TEM-based observations, the shapes of the ICC in the rat stomachs ofthe model group are more flat, or even irregular, and the protrusions of the ICCbecome shot and small; and the ICC in the rats of the other three groups take theshape of a spindle, with huge oval nuclear cores and long protrusions extendingoutwards, and the cytoplasm of the ICC are rich in mitochondria, smoothendoplasmic reticulum and rough endoplasmic reticulum based on observations of25,000×magnification. The connection between the ICC and the surrounding intestinal neurons axoaxonic synapse is increased in the areca group. In the fourgroups, the intragastric positive expression of the c-kit in the gastric antrum is highestand is higher than that in the gastric body and the gastric fundus. The positiveexpression area of the c-kit in the areca group is highest, particularly in the gastricantrum (79,700±3,900μm2).
4. The lyophilized areca powder of ten batches is respectively used for performingguinea pig in-vitro smooth muscle experiments. The lyophilized areca powder of thebatches1and2are selected to undergo HPLC component analysis, and69peakvalues are shown on the ultraviolet absorption spectrum, wherein the34th and42thpeak value are higher. The component corresponding to the34th and42th peak valueis extracted, and10μmol/L of dose BL42is found to have the strongest effect on thecontraction of the gastric circular muscle and the gastric longitudinal muscle of thesmooth muscle, and the areas under a curve are respectively5.96±0.585g/s and5.64±0.52g/s, which approaches anAch (Acetylcholine) effect.
Conclusion
1. When the dose is10g/kg, the glycyrrhiza has the effects of increasing the gastricresidual rate and prolonging the GET1/2for the rats, thus, the glycyrrhiza can beused for establishing the rat models of low gastric motility.
2. The areca has the effects of lowering the gastric residual rate, shortening the GET1/2and increasing the intestinal propulsive rate for the rat models of low gastricmotility, thus, the areca is proved to have the effect on promoting the gastric motility.
3. The areca has the effects of enhancing the relationship between intragastric ICC ofrats of low gastric motility and the peripheral enteric nervous system, and enlargingthe positive expression area of c-kit in the gastric antrum, the gastric body and thegastric fundus, thus, a certain relationship is presented between the gastric motility promotion effect of the areca and ICC.
4. BL34and BL42prepared by means of HPLC has a stronger effect on thecontraction of the in-vitro smooth muscle of the guinea pig, and is similar to Ach, andthe components of BL34and BL42need further experimental studies.
引文
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