本工作由四川省科技厅重点研发项目（2020YFS0539）资助。

动物的护理和使用程序得到了绵阳师范大学伦理委员会的批准，并严格遵守了所有有关动物伦理使用的制度和政府规定。在本研究中，使用了SPF大鼠（昆明物种，雄性和雌性，体重180-220 g）。这些动物是从商业来源获得的（见 材料表）。所有动物都被饲养在无病原体的环境中，并可以 随意 获取食物。
1.试剂的制备
<ol><li>用电子秤称取2.5g水杨酸钠粉末（见 材料表）制备5%水杨酸钠溶液。将粉末溶解在少量蒸馏水中，并将其稀释至总体积为50mL。</li>
	<li>制备 D. caudatum （12.8g/kg）的汤剂。称取 128 g D. caudatum （见 材料表），将其放入装有 1000 mL 实验室蒸馏水的圆底烧瓶中，并将其浓缩至最终体积为 100 mL。</li>
</ol>2. 分组和管理
<ol><li>将大鼠分成不同的组，并在胃内施用所需的溶液。 注：本研究将18只大鼠（9只雄性，9只雌性）分为三组：对照组（C组）、模型组（M组）和治疗组（T组），每组6只大鼠。对照组接受生理盐水溶液，模型组接受 5% 水杨酸钠溶液，剂量为 10 mL/kg/天15 周。治疗组用 5% 水杨酸钠溶液以 10 mL/kg/天的剂量预处理 5 周，然后以 1 mL/100 g 的剂量给予 D. caudatum 汤剂，持续10 天 15,16。</li>
	<li>在实验的最后一天，使用携带尾巴的方法17 在干燥和无菌的微量离心管中收集大鼠的粪便，并将它们冷冻在液氮超低温冰箱中。</li>
	<li>采样结束后，通过宫颈脱位处死大鼠（遵循机构批准的方案）。用剪刀打开腹腔，露出腹主动脉。<ol><li>将针头（23-25G）几乎平行地插入腹主动脉，收集血清，并将其保存在超低温冰箱中。从腹腔16 中取出胃，并浸泡在10%福尔马林溶液中保存。 注意：在M组中，如果胃粘膜扩张并充血并具有规则的腺体形态15，同时观察到固有层中的少量炎症细胞浸润，则慢性胃炎模型被认为是成功的。</li>
	</ol></li>
</ol>3.胃壁病理切片
<ol><li>从每组大鼠身上取胃壁样本进行病理切片。随后，进行常规苏木精-伊红染色以观察和分析病理变化18。</li>
</ol>4.血清胃肠道激素的测定
<ol><li>在血清样品中，按照制造商的说明使用ELISA检测胃泌素（GAS）和丙二醛（MDA）19 的含量（参见 材料表）。使用酶标仪进行分析。</li>
</ol>5. 粪便总DNA提取及PCR扩增纯化
<ol><li>使用市售的DNA分离试剂盒（参见 材料表）进行微生物DNA提取，并使用紫外-可见分光光度计16测定浓度和纯度。随后，通过 1% 琼脂糖凝胶电泳18 评估 DNA 完整性。</li>
	<li>使用热循环仪PCR系统17，用引物338F（5′-ACT、CCT、ACG、GGA、GGC、AGC、AG-3′）和806R（5′-GAC、TAC、HVG、GGG、GTW、TCT、AT-3′）扩增细菌16S rRNA基因。引物是从商业来源获得的（见 材料表）。 注意：遵循PCR程序：变性（3分钟，95°C），然后是27个循环（30秒，95°C;30秒，55°C;45秒，72°C），最后延长（10分钟，72°C）。使用以下组分进行PCR反应：4μL 5× DNA 聚合酶缓冲液、2 μL 2.5 mM dNTP、0.8 μL 每种引物 （5 μM）、0.4 μL DNA 聚合酶和 10 ng 模板 DNA（参见 材料表）。</li>
	<li>分别使用 2% 琼脂糖凝胶、市售 DNA 凝胶提取试剂盒和荧光计依次提取、纯化和定量PCR 产物 17。</li>
</ol>6. 测序
注意：步骤6和步骤7按照先前发布的方法20,21执行。
<ol><li>利用Illumina平台进行测序。 注意：DNA片段的一端与嵌入芯片的连接器的底座相辅相成，将其固定在芯片上。另一端随机补充另一个相邻埋地接头的底座，形成一个“桥”。</li>
	<li>进行PCR扩增以生成DNA簇。将 DNA 扩增线性化为单链。</li>
	<li>将改变的 DNA 聚合酶与具有四种不同荧光标记物的脱氧核糖核苷酸三磷酸 （dNTP） 一起引入。每个周期仅合成一个碱基。</li>
	<li>利用激光扫描反应板的表面，并确定在每个模板序列的初始反应期间聚合的核苷酸类型。</li>
	<li>化学裂解“荧光基团”和“终止基团”以重新建立 3' 粘性末端。随后，推进到聚合第二核苷酸。</li>
	<li>通过分析每轮收集的荧光信号结果，获得模板DNA片段的序列。</li>
</ol>7. 测序数据的处理
<ol><li>最初，修剪任何分数< Q20 的原始 fastq 数据的读取，并消除基数不确定的读取。此外，允许在精确匹配的引物中出现两次错配。随后，合并重叠>10 bp的序列。</li>
	<li>使用 UPARSE 对相似度为 97% 的操作分类单元 （OTU） 进行聚类，并使用 UCHIME20,21 去除嵌合序列。</li>
	<li>采用 RDP 分类器算法根据 Silva （SSU123） 16S rRNA 数据库分析每个 16S rRNA 基因序列的分类，置信阈值为 70%。</li>
	<li>分别使用 Mothur 和 Qiime 进行 Alpha 多样性分析和 Beta 多样性分析（参见 材料表）。</li>
</ol>8. 统计分析
<ol><li>使用统计分析软件分析本研究中的数据（见 材料表）。采用单因素方差分析 （ANOVA） 进行多组之间的比较，并利用最小显着差异 （LSD） 检验进行两组之间的比较。</li>
	<li>在所有比较中， 将 P < 0.05 视为具有统计学意义。 P < 0.01 被认为是极其显著的。</li>
</ol>

Desmodium caudatum （金银 草）煎剂对大鼠肠道菌群的影响

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作者没有什么可透露的。

羌族12人常用的民间药材，在治疗胃肠道疾病方面显示出显著的疗效。随着现代药理学研究的进步，胃肠道微生态失衡导致的菌群失衡被确定为急性和慢性胃肠道疾病的关键因素22,23。肠道中的某些微生物在维持体内的动态平衡方面起着至关重要的作用，血清中的激素反映了身体的生理和病理状态。因此，该研究的重点是肠道菌?...

慢性胃炎（CG）是最常见的临床疾病之一，其特征是胃黏膜上皮的慢性和持续的炎症性变化，经常受到各种致病因素的反复攻击1。CG的发病率在所有胃病中排名第一，占胃肠科门诊服务率的40%-60%2。此外，发病率通常随着年龄的增长而增加，尤其是在中老年人中 3.毫无疑问，CG大大降低了人们的生活质量，强调了发现新治疗药物的迫切需要。
大量研究报告称，CG的发生和发展与胃肠道激素（如胃泌素（GAS））的分泌有关4,5。GAS是消化道中常见的胃肠道肽激素，通过促进嗜酸性粒细胞释放组胺来刺激细胞分泌胃酸。此外，它还能改善胃粘膜的营养和血液供应，促进胃粘膜和壁细胞的增殖6。因此，胃泌素可以作为评价CG发展水平的指标。此外，由活性氧化物触发的脂质过氧化产物可以激活炎症细胞，导致CG。丙二醛（MDA）是一种脂质过氧化标志物，是测量氧化应激程度的常用指标。它在一定程度上反映了胃粘膜中自由基的水平。MDA水平可以表明由自由基引起的局部胃粘膜中不饱和脂肪酸的攻击7,8。
肠道微生态学由数百万种微生物组成，它们存在于宿主肠道中，在维持宿主健康和调节宿主免疫力方面发挥着至关重要的作用。健康的肠道菌群具有高度丰富、多样性和稳定的微生物群功能，通过参与新陈代谢作为防止病原微生物入侵的保护屏障。肠道菌群的紊乱使个体更容易患上急性和慢性胃肠道疾病9,10。近年来，胃肠道疾病的微生物群治疗进展迅速，并显示出显著的疗效11。总之，肠道菌群的考虑对于理解和解决胃肠道疾病至关重要。
民间药材作为中医药宝库中不可或缺的组成部分，对临床实践和民族医药现代化具有重要意义。Desmodium caudatum （Thunb.） 的根部和整个部分在绵阳市北川羌地区长期被广泛用于缓解胃部不适。一些文章指出了用尾蛾治疗胃肠道疾病的科学依据，引用了其止血、抗氧化和胃肠道保护等作用12,13,14。然而，由于缺乏深入研究，尚未建立临床药效学机制。本文旨在基于胃肠道激素和肠道菌群研究尾曲霉治疗胃炎的效果，为其合理的临床应用提供依据。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alpha diversity analysis</td>
			<td>Mothur 1.30.2</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AxyPrep deoxyribonucleic acid (DNA) gel extraction kit&#160;</td>
			<td>Axygen Biosciences&#160;</td>
			<td>AP-GX-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Beta diversity analysis</td>
			<td>Qiime 1.9.1</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cryogenic refrigerator</td>
			<td>Forma-86C ULT freezer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Desmodium caudatum</td>
			<td>The Traditional Chinese Medicine Hospital of Beichuan Qiang Autonomous County</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E.Z.N.A. soil kit</td>
			<td>Omega Bio-tek</td>
			<td>D5625-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Illumina MiSeq Platform</td>
			<td>Illumina Miseq PE300/NovaSeq PE250</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multiskan Spectrum</td>
			<td>spectraMax i3</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>OTU clustering</td>
			<td>Uparse 7.0.1090</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>OTU statistics</td>
			<td>Usearch 7.0</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PCR instrument</td>
			<td>TransGen AP221-02</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PCR instrument</td>
			<td>ABI GeneAmp 9700</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>QuantiFluor-ST double-stranded DNA (dsDNA) system</td>
			<td>Promega Corp.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sequence classification annotation</td>
			<td>RDP Classifier 2.11</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium salicylate</td>
			<td>Sichuan Xilong Chemical Co., Ltd</td>
			<td>54-21-7</td>
			<td></td>
		</tr>
		<tr>
			<td>SPF rats</td>
			<td>Chengdu Dashuo Experimental Animal Co., Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SPSS 18.0</td>
			<td>IBM</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

effects of desmodium caudatum on gastrointestinal hormones and intestinal flora in rats with gastritis

为初步探究 尾鲷对 大鼠胃炎及肠道菌群的影响，采用经典水杨酸钠法建立慢性胃炎大鼠模型。将18只SPF大鼠分为对照组（C组）、模型组（M组）和治疗组（T组）3组。从每组大鼠中取出胃壁的病理切片。此外，通过ELISA测定各组大鼠血清中胃泌素和丙二醛的浓度。此外，通过对三组肠道细菌群落进行详细比较，采用基于Illumina的16S rRNA基因测序，探索了D . caudatum 对胃炎大鼠肠道菌群的影响。结果表明， 尾醛 汤可降低丙二醛含量，增加胃泌素含量。此外，还发现 尾苔 汤可增强肠道菌群的多样性和丰度，通过调节和恢复肠道菌群对胃炎的治疗产生积极影响。

Chronic gastritis (CG), one of the most common clinical diseases, is characterized by chronic and persistent inflammatory changes in the gastric mucosa epithelium, which is frequently and repeatedly attacked by various pathogenic factors1. The incidence rate of CG ranks first among all types of stomach diseases, accounting for 40% to 60% of the outpatient service rate in the Department of Gastroenterology2. Moreover, the incidence rate generally increases with age, especially in individuals who are middle-aged and older3. Undoubtedly, CG significantly reduces people&#39;s quality of life, emphasizing the critical need to discover new therapeutic agents.
Numerous studies have reported that the occurrence and development of CG are linked to the secretion of gastrointestinal hormones, such as gastrin (GAS)4,5. GAS, a common gastrointestinal peptide hormone in the digestive tract, stimulates cells to secrete gastric acid by promoting the release of histamine from eosinophils. Additionally, it improves the nutrition and blood supply of the gastric mucosa, promoting the proliferation of gastric mucosa and parietal cells6. Therefore, gastrin can be utilized as an indicator to evaluate the development level of CG. Furthermore, lipid peroxidation products triggered by reactive oxides can activate inflammatory cells, leading to CG. Malondialdehyde (MDA), a lipid peroxidation marker, is a commonly used indicator to measure the degree of oxidative stress. It reflects the level of free radicals in the gastric mucosa to a certain extent. The MDA level can indicate the attack of unsaturated fatty acids in the local gastric mucosa caused by free radicals7,8.
Intestinal microecology consists of millions of microorganisms residing in the host gut, playing a vital role in maintaining host health and regulating host immunity. A healthy intestinal flora, characterized by high richness, diversity, and stable microbiota function, acts as a protective barrier against the invasion of pathogenic microorganisms by participating in metabolism. Disturbances in the intestinal flora make individuals more susceptible to acute and chronic gastrointestinal diseases9,10. In recent years, microbiota therapy for gastrointestinal diseases has progressed rapidly and demonstrated significant efficacy11. In summary, the consideration of intestinal flora is crucial in understanding and addressing gastrointestinal diseases.
As an indispensable component of the treasure trove of traditional Chinese medicine, folk medicinal materials hold great significance for clinical practice and the modernization of national medicine. The roots and the entire parts of Desmodium caudatum (Thunb.) DC. has been widely used for relieving stomach discomfort in the Beichuan Qiang area of Mianyang City for an extended period. Some articles indicate the scientific basis for treating gastrointestinal diseases with D. caudatum, citing its effects such as hemostasis, anti-oxidation, and gastrointestinal protection12,13,14. However, due to the lack of in-depth research, no clinical pharmacodynamics mechanism has been established. This article aims to study the effects of D. caudatum in treating gastritis based on gastrointestinal hormones and intestinal flora, providing a basis for its rational clinical application.

作者聚焦：探索四川中草药在中医研究中的奥秘

Desmodium caudatum对胃炎大鼠胃肠道激素和肠道菌群的影响

胃壁病理切片的结果如 图1所示。与C组相比，M组表现出轻度胃壁萎缩和轻度炎症。然而，与M组相比，T组没有明显的炎症、肠化生或萎缩。这表明 D.caudatum 汤可以有效改善胃炎。
血清胃肠道激素测定结果如 图2所示。M组丙二醛（MDA）含量显著高于C组。用 D.caudatum 汤处理后，它显着减少。胃泌素含量（GAS）明显低于C组...

Watch this Scientific Journal Video about Author Spotlight: Exploring the Mysteries of Sichuan's Herbal Medicine in Chinese Medicine Research at JoVE.com

本方案描述了使用基于Illumina的16S rRNA基因测序分析肠道菌群的方法，并为评估草药汤剂的有效性提供了框架。

In order to preliminarily explore the effects of Desmodium caudatum on gastritis and intestinal flora in rats, a chronic gastritis rat model was ...

民间医药资料作为中医药宝库中不可或缺的组成部分，对临床实践和民族医药现代化具有重要意义。本文旨在基于胃肠道激素和肠道菌群研究D.caudatum治疗胃炎的效果，为其在临床上的应用提供依据。许多研究报告说，慢性胃的发生和发展与胃肠道激素（如胃泌素）的分泌有关。它可以优化为评估慢性胃发育水平的指标。此外，由活性氧化物触发的重复过氧化产物可以激活炎症细胞，导致慢性胃病。在16S rRNA高通量基因测序中，低分辨率可能使区分序列高度相似的菌株和属具有挑战性，导致在更高分类水平（如属、科和门）进行分类困难。对于ELISA试剂盒方法，通常需要稀释和洗涤等样品预处理步骤，从而引入潜在的误差和变异性。

effects-desmodium-caudatum-on-gastrointestinal-hormones-intestinal

Research

JoVE Journal

Medicine

Effects of Desmodium caudatum on Gastrointestinal Hormones and Intestinal Flora in Rats with Gastritis

372 次观看.  Mianyang Normal University. 民间医药资料作为中医药宝库中不可或缺的组成部分，对临床实践和民族医药现代化具有重要意义。本文旨在基于胃肠道激素和肠道菌群研究D.caudatum治疗胃炎的效果，为其在临床上的应用提供依据。许多研究报告说，慢性胃的发生和发展与胃肠道激素（如胃泌素）的分泌有关。它可以优化为评估慢性胃发育水平的指标。此外，由活性氧化物触发的重复过氧化产物可以激...

视频: 作者聚焦：探索四川中草药在中医研究中的奥秘

In order to preliminarily explore the effects of Desmodium caudatum on gastritis and intestinal flora in rats, a chronic gastritis rat model was established using the classic sodium salicylate method. Eighteen SPF rats were divided into three groups: the control group (Group C), the model group (Group M), and the treatment group (Group T). Pathological sections of the gastric wall were taken from rats in each group. Furthermore, the concentrations of gastrin and malondialdehyde in the serum of rats in each group were determined by ELISA. Additionally, the effects of D. caudatum on the intestinal flora of rats with gastritis were explored through a detailed comparison of gut bacterial communities in the three groups, employing Illumina-based 16S rRNA gene sequencing. The results indicated that D. caudatum decoction could reduce the malondialdehyde content and increase the gastrin content. Moreover, D. caudatum decoction was found to enhance the diversity and abundance of intestinal flora, exerting a positive impact on the treatment of gastritis by regulating and restoring the intestinal flora.

The present protocol describes the method for analyzing intestinal flora using Illumina-based 16S rRNA gene sequencing and provides a framework for evaluating the effectiveness of herbal decoctions.