胃饥饿素介导幽门螺杆菌相关消化不良的动物实验研究

doi:10.3877/cma.j.issn.1674-1358.2022.05.005

目的

建立慢性幽门螺杆菌感染的动物模型，在此模型中通过加强胃饥饿素的表达，探讨胃饥饿素在幽门螺杆菌相关性消化不良中的影响。

方法

选取4周龄雌性C57BL/6小鼠24只，随机分为对照组（蒸馏水灌胃+生理盐水腹腔注射）、实验组A（幽门螺杆菌菌液灌胃+生理盐水腹腔注射）和实验组B（幽门螺杆菌菌液灌胃+雷帕霉素腹腔注射）。实验结束后处死小鼠，分别检测3组小鼠胃饥饿素表达水平、胃内容物残留率及小肠推进率及HE染色观察小鼠胃炎的病理变化。采用独立样本t检验分析小鼠的胃排空率、小肠推进速度、胃饥饿素分泌水平；多组间数据的整体比较采用ANOVA检验，再应用LSD-t进行组间两两比较。

结果

实验组A小鼠胃残留率[（43.6 ± 7.2）%]较对照组小鼠[（61.6 ± 3.8）%]显著下降，差异有统计学意义（t = 4.579、P = 0.013）；实验组B小鼠胃残留率[（61.6 ± 3.8）%]较实验组A[（43.6 ± 7.2）%]升高，差异均有统计学意义（t = 5.232、P = 0.012）。对照组、实验组A、实验组B小鼠的小肠推进率经两两比较差异均无统计学意义（P均> 0.05）。与对照组小鼠的胃饥饿素[（39.6 ± 1.3）%]分泌相比，实验组A小鼠胃饥饿素[（25.4 ± 5.0）%]分泌显著减少（t = 6.104、P < 0.001），且病理染色发现伴有胃炎发生；实验组B小鼠胃饥饿素[（31.8 ± 4.5）%]较实验组A小鼠分泌显著增多，差异有统计学意义（t = 3.196、P = 0.041），同时病理染色提示胃炎得到不同程度改善。

结论

提高胃饥饿素水平可促进幽门螺杆菌相关消化不良症状的改善。

关键词: 幽门螺杆菌, 消化不良, 胃排空, 胃饥饿素, 雷帕霉素

Objective

To establish an animal model of chronic Helicobacter pylori (H. pylori) infection, and to investigate the effect of ghrelin on H. pylori related dyspepsia by increasing ghrelin expression.

Methods

Twenty-four 4-week-old female C57BL/6 mice were randomly divided into control group (water gavage + saline injection), group A (H. pylori gavage + saline injection) and group B (H. pylori gavage + rapamycin injection). The mice were sacrificed at the end of the experiment, and the expression level of ghrelin, residual rate of gastric contents and small intestine propelling rate of the three groups were detected, and the pathological changes of gastritis in mice were observed by HE staining. Independent sample t test was used to analyze the gastric emptying mice, intestinal propulsion speed and ghrelin secretion of mice. Overall comparison of data between multiple groups was performed by ANOVA test and pairwise comparisons between the groups were performed by LSD-t test.

Results

The gastric residual rate of group A mice [(43.6 ± 7.2)%] was significantly lower than that of control mice [(61.6 ± 3.8)%], with significant difference (t = 4.579, P = 0.013). The gastric residual rate of group B mice [(61.6 ± 3.8)%] was higher than that of group A mice (43.6 ± 7.2%), with significant difference (t = 5.232, P = 0.012). There was no significant difference in small intestine propulsion mice between two groups (all P > 0.05). Compared with ghrelin secretion of control mice [(39.6 ± 1.3)%], ghrelin secretion of group A mice significantly reduced [(25.4 ± 5.0)%], (t = 6.104, P < 0.001), accompanied by gastritis. Compared with ghrelin secretion of group A mice, group B mice secretion ghrelin [(31.8 ± 4.5)%] was significantly increased (t = 3.196, P = 0.041), and the levels of gastritis were improved.

Conclusions

Increased ghrelin levels could alleviate symptoms of H. pylori-related dyspepsia.

Key words: Helicobacter pylori, Dyspepsia, Gastric emptying, Ghrelin, Rapamycin

图1 使用产气袋培养幽门螺杆菌模式图

图2 幽门螺杆菌固体培养图注：幽门螺杆菌菌落颜色透明色泽很浅为半透明色，在强光下观察结果不明显。因此使用侧位观察，可见透明大量透明针尖样菌落铺满平板

图3 幽门螺杆菌液体培养显微镜下图注：幽门螺杆菌液体培养后未染色直接于电子显微镜下图片，可见图中存在大量形态各异的棒状细菌，呈海鸥形，弯曲状，直棒状等多种形态

表1 三组小鼠胃残留率和小肠推进率（± s，%）

组别	只数	胃残留率	小肠推进率
对照组	6	61.6 ± 3.8	55.9 ± 3.9
实验组A	6	43.6 ± 7.2	53.4 ± 6.8
实验组B	6	61.2 ± 6.6	62.4 ± 4.4
F值		14.658	2.394
P值		< 0.001	0.128
^at值		4.579	0.646
^aP值		0.013	0.539
^bt值		5.232	0.754
^bP值		0.012	0.468
^ct值		0.114	2.411
^cP值		0.912	0.037

表1 三组小鼠胃残留率和小肠推进率（± s，%）

组别	只数	胃残留率	小肠推进率
对照组	6	61.6 ± 3.8	55.9 ± 3.9
实验组A	6	43.6 ± 7.2	53.4 ± 6.8
实验组B	6	61.2 ± 6.6	62.4 ± 4.4
F值		14.658	2.394
P值		< 0.001	0.128
^at值		4.579	0.646
^aP值		0.013	0.539
^bt值		5.232	0.754
^bP值		0.012	0.468
^ct值		0.114	2.411
^cP值		0.912	0.037

图4 小鼠胃肠蠕动功能模式解剖图注：A、B、C分别为3组小鼠灌胃结扎后胃组织的图像，可见A、C中的胃内仍留存有较多的半固体营养液，B中的胃内半固体营养液含量明显减少；D为小鼠灌胃后游离小肠的图像

图5 幽门螺杆菌鉴定与小鼠胃组织染色图注：A：对照组（左）与实验组（右）的小鼠胃组织尿素酶实验结果对比；B和C中所指为Hp在胃黏膜中定植；E、F、G分别为对照组、实验组A和实验组B胃组织

表2 ELISA法检测3组小鼠血清胃饥饿素水平（± s，pg/ml）

组别	数量	血清胃饥饿素
对照组	6	39.6 ± 1.3
实验组A	6	25.4 ± 5.0
实验组B	6	31.8 ± 4.5
F值		19.222
P值		< 0.001
^at值		6.104
^aP值		< 0.001
^bt值		3.196
^bP值		0.041
^ct值		4.018
^cP值		0.002

表2 ELISA法检测3组小鼠血清胃饥饿素水平（± s，pg/ml）

组别	数量	血清胃饥饿素
对照组	6	39.6 ± 1.3
实验组A	6	25.4 ± 5.0
实验组B	6	31.8 ± 4.5
F值		19.222
P值		< 0.001
^at值		6.104
^aP值		< 0.001
^bt值		3.196
^bP值		0.041
^ct值		4.018
^cP值		0.002

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Animal experimental study of Helicobacter pylori associated dyspepsia mediated by ghrelin

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Animal experimental study of Helicobacter pylori associated dyspepsia mediated by ghrelin