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罗雯鹏,王真权,周佳敏,肖俐敏,王适,陆文洪,王军文.基于网络药理学和实验验证探究生血通便颗粒治疗慢传输型便秘的作用机制[J].湖南中医药大学学报,2024,44(3):408-418[点击复制] |
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| 基于网络药理学和实验验证探究生血通便颗粒治疗慢传输型便秘的作用机制 |
| 罗雯鹏,王真权,周佳敏,肖俐敏,王适,陆文洪,王军文 |
| (湖南中医药大学第二附属医院, 湖南 长沙 410005;湖南中医药大学, 湖南 长沙 410208) |
| 摘要: |
| 目的 采用网络药理学预测生血通便颗粒治疗慢传输型便秘（slow transit constipation，STC）的潜在作用机制，并进行动物实验验证。方法 利用TCMSP数据库和BATMAN-TCM数据库筛选生血通便颗粒的活性成分及作用靶点，通过GeneCards数据库和OMIM数据库筛选STC疾病相关靶点，构建“活性成分-疾病靶点”网络，使用R语言对交集基因进行GO及KEGG富集分析。大鼠采用盐酸洛哌丁胺混悬液[3 mg/（kg·d）]灌胃建立STC模型。将大鼠分为正常组（等体积蒸馏水）、模型组（等体积蒸馏水）、莫沙比利组[1.35 mg/（kg·d）]、生血通便颗粒低剂量组[1.44 g/（kg·d）]、生血通便颗粒高剂量组[2.88 g/（kg·d）]，每组8只，每日灌胃1次，连续治疗14 d。记录治疗后肠道推进率；HE染色观察结肠组织病理学改变；免疫组织化学法检测结肠组织酪氨酸激酶受体（tyrosine kinase receptor， c-Kit）表达；TUNEL染色检测结肠组织Cajal间质细胞（interstitial cells of Cajal， ICC）凋亡情况；Western blot检测结肠组织葡萄糖调节蛋白78（glucose regulated protein 78， GRP78）、C/EBP同源蛋白（C/EBP homologous protein， CHOP）、B细胞淋巴瘤-2（B-cell lymphoma-2， Bcl-2）、Bcl-2相关X蛋白（Bcl-2 associated X protein， Bax）、活化型胱天蛋白酶-3（cleaved cysteine aspartic acid specific protease-3， cleaved Caspase-3）蛋白表达水平。结果 筛选出生血通便颗粒活性成分74个，对应的作用靶点为492个，CASP3（Caspase-3）、Bcl-2、Bax凋亡基因是STC的核心靶点之一，其相关的作用通路为内质网应激信号通路。动物实验结果表明，与正常组比较，模型组大鼠肠道推进率降低（P<0.01）；结肠黏膜萎缩、变薄，黏膜上皮层有缺失，固有层腺体缺损破坏明显，可见炎性改变；结肠组织中c-Kit表达降低（P<0.01）；凋亡细胞绿色荧光增加、c-Kit红色荧光减少，ICC凋亡指数升高（P<0.01）；结肠组织GRP78、CHOP、Bax、cleaved Caspase-3蛋白表达水平升高（P<0.01），Bcl-2蛋白表达水平降低（P<0.01）。与模型组相比，莫沙必利组及生血通便颗粒低、高剂量组大鼠肠道推进率提高（P<0.01）；结肠黏膜结构较完整，炎性情况有所恢复，腺体排列较整齐；结肠组织中c-Kit表达升高（P<0.01）；凋亡细胞绿色荧光减少、c-Kit红色荧光增加，ICC凋亡指数降低（P<0.01）；结肠组织GRP78、CHOP、Bax、cleaved Caspase-3蛋白表达水平降低（P<0.01），Bcl-2蛋白表达水平升高（P<0.01）。与莫沙必利组相比，生血通便颗粒高剂量组肠道推进率提高（P<0.05）、c-Kit表达升高（P<0.05）、ICC凋亡指数降低（P<0.05）；生血通便颗粒低、高剂量组结肠组织中GRP78、CHOP、Bax、cleaved Caspase-3蛋白表达水平降低（P<0.01），生血通便颗粒高剂量组Bcl-2蛋白表达水平升高（P<0.01）。结论 生血通便颗粒可通过抑制内质网应激GRP78/CHOP信号通路，调控Bax、Bcl-2、cleaved Caspase-3凋亡蛋白表达，从而抑制ICC凋亡以达到治疗STC的作用。 |
| 关键词: 生血通便颗粒 慢传输型便秘 Cajal间质细胞 内质网应激 细胞凋亡 网络药理学 |
| DOI：10.3969/j.issn.1674-070X.2024.03.009 |
| 投稿时间：2023-08-14 |
| 基金项目:湖南省自然科学基金青年基金项目（2023JJ40496）；湖南省中医药管理局中医防治肛肠疾病临床研究中心（湘中医药函〔2022〕93号）；湖南省中医药管理局一般项目（2021063）；湖南中医药大学校级科研项目（2020XJJJ059）；湖南省中医药管理局中医药防治肛肠疾病重点研究室建设项目（湘中医药函〔2020〕51号）。 |
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| Mechanism of Shengxue Tongbian Granules in treating slow transit constipation based on network pharmacology and experimental validation |
| LUO Wenpeng,WANG Zhenquan,ZHOU Jiamin,XIAO Limin,WANG Shi,LU Wenhong,WANG Junwen |
| (The Second Hospital of Hunan University of Chinese Medicine, Changsha, Hunan 410005, China;Hunan University of Chinese Medicine, Changsha, Hunan 410208, China) |
| Abstract: |
| Objective To predict the potential mechanism of action of Shengxue Tongbian Granules (SXTBG) in treating slow transit constipation (STC) by network pharmacology and to verify it by animal experiments. Methods TCMSP and BATMAN-TCM databases were used to screen out the active ingredients and targets of SXTBG, and GeneCards and OMIM databases were used to screen out STC disease-related targets, then an "active ingredient-disease target" network was established. GO and KEGG enrichment analyses were performed on intersection genes through R language. A STC rat model was established by gavage with loperamide hydrochloride suspension [3 mg/(kg·d)]. Rats were randomized into normal group (equal volume of distilled water), model group (equal volume of distilled water), mosapride group [1.35 mg/(kg·d)], low-dose SXTBG group [1.44 g/(kg·d)], and high-dose SXTBG group [2.88 g/(kg·d)], with eight rats in each group. All the groups were treated by gavage once a day for 14 consecutive days. Intestinal propulsion rate of the rats after treatment was recorded. In the rat colon tissue, HE staining was used to observe its pathological changes, immunohistochemical staining to check the expression level of tyrosine kinase receptor (c-Kit), TUNEL staining to examine the apoptosis of interstitial cells of Cajal (ICC), and Western blot to test expressions of glucose regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), Bcl-2 associated X protein (Bax), B-cell lymphoma-2 (Bcl-2), and cleaved cysteine aspartic acid specific protease-3 (cleaved Caspase-3). Results A total of 74 active ingredients of SXTBG were screened out, with 492 corresponding targets. CASP3 (Caspase-3), Bcl-2, and Bax apoptosis genes were one of the core targets of STC, and their related pathways of action were endoplasmic reticulum stress signaling pathways. Animal experiment results showed that compared with normal group, the intestinal propulsion rate of model group decreased (P<0.01); the colonic mucosa atrophied and became thinner, with loss of mucosal epithelial layer, severe damage to the glandular tissue in the lamina propria, and visible inflammatory changes; the expression of c-Kit in the colon tissue decreased (P<0.01); the green fluorescence of apoptotic cells increased, the red fluorescence of c-Kit decreased, and the ICC apoptosis index increased (P<0.01); the protein expression levels of GRP78, CHOP, Bax, and cleaved Caspase-3 in the colon tissue increased (P<0.01), while that of Bcl-2 decreased (P<0.01). Compared with model group, the intestinal propulsion rates of mosapride group and low- and high-dose SXTBG groups increased (P<0.01); the colonic mucosal structure was relatively intact, and the inflammation was alleviated, and the glandular arrangement was relatively neat; the expression of c-Kit in the colon tissue increased (P<0.01); the green fluorescence of apoptotic cells decreased, the red fluorescence of c-Kit increased, and the ICC apoptosis index decreased (P<0.01); the protein expression levels of GRP78, CHOP, Bax, and cleaved Caspase-3 in the colon tissue decreased (P<0.01), while that of Bcl-2 increased (P<0.01). Compared with mosapride group, high-dose SXTBG group showed an increase in the intestinal propulsion rate (P<0.05) and c-Kit expression (P<0.05), and a decrease in the ICC apoptosis index (P<0.05); the protein expression levels of GRP78, CHOP, Bax, and cleaved Caspase-3 in the colon tissue of low- and high-dose SXTBG groups decreased (P<0.01), while that of Bcl-2 increased in high-dose SXTBG group (P<0.01). Conclusion SXTBG can inhibit ICC apoptosis and exert therapeutic effects on STC by inhibiting the endoplasmic reticulum stress GRP78/CHOP signaling pathway and regulating the expressions of Bax, Bcl-2, and cleaved Caspase-3 apoptotic proteins. |
| Key words: Shengxue Tongbian Granules slow transit constipation interstitial cells of Cajal endoplasmic reticulum stress apoptosis network pharmacology |
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