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艾坤, 谢万健, 佘艳, 宋泉, 瞿启睿, 陈彦灵, 江浩清, 张蕾, 邓奕辉.左归降糖通脉方治疗糖尿病神经源性膀胱的效应及分子靶点预测与机制验证研究[J].湖南中医药大学学报,2026,46(7):1323-1335[点击复制] |
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| 左归降糖通脉方治疗糖尿病神经源性膀胱的效应及分子靶点预测与机制验证研究 |
| 艾坤,谢万健,佘艳,宋泉,瞿启睿,陈彦灵,江浩清,张蕾,邓奕辉 |
| (湖南中医药大学, 湖南 长沙 410208;福建省漳州市医院, 福建 漳州 363000) |
| 摘要: |
| 目的 基于网络药理学及分子对接技术，筛选左归降糖通脉方治疗糖尿病神经源性膀胱（DNB）的主要活性成分及潜在作用靶点，并通过建立DNB动物模型验证左归降糖通脉方对该疾病的作用机制。方法 应用中药系统药理学数据库与分析平台和本草组鉴数据库筛选左归降糖通脉方的活性成分及作用靶点，通过通用蛋白质资源数据库规范靶点对应的基因名称。依托人类基因综合数据库和人类孟德尔遗传数据库检索DNB的相关靶点基因，利用Venny软件获取复方与DNB的交集靶点韦恩图，借助STRING在线平台与Cytoscape软件进行可视化处理，构建蛋白质-蛋白质相互作用网络，通过相关数据库进行基因本体（GO）功能和京都基因与基因组百科全书（KEGG）通路富集分析，在微生信云平台呈现结果。运用CB-Dock2对复方相应活性成分与核心靶点蛋白受体进行分子对接验证。在筛选出左归降糖通脉方治疗DNB的潜在作用靶点后，通过动物实验进行效应验证。将36只雌性SD大鼠随机分为空白组6只与造模组30只，采用单次腹腔注射链脲佐菌素（STZ）溶液制备模型。成模后将造模组随机分为模型组、低剂量组、中剂量组、高剂量组及阳性药物组，每组6只。低、中、高剂量组分别予以6.5、13、26 g/kg左归降糖通脉方灌胃，阳性药物组予以α-硫辛酸100 mg/kg灌胃，每日1次，持续4周。实验过程中各组各剔除1只不合格样本，最终每组有效样本量为5。给药1个月后取材，采用尿流动力学检测膀胱功能，Western blot检测相关蛋白[磷酸化蛋白激酶B（p-AKT）、磷酸化信号转导与转录激活因子3（p-STAT3）、磷酸化原癌基因酪氨酸蛋白激酶Src（p-SRC）、肿瘤蛋白p53、90 kDa热休克蛋白（HSP90）]表达。结果 共获得左归降糖通脉方治疗DNB的交集靶点357个，主要活性成分包括槲皮素、黄芪异黄烷苷、二氢辣椒素、2-去甲基质体醌-3和木犀草素，核心靶点包括TP53、AKT1、SRC、HSP90AA1、STAT3。GO功能富集获得生物过程1 077条、细胞组分120条和分子功能260条，KEGG通路富集获得186条通路，其中AGE-RAGE信号通路可能是重要作用通路。分子对接结果显示，主要活性成分与核心靶点均具有较好的结合活性，其中黄芪异黄烷苷与AKT1的结合活性最强。与模型组比较，左归降糖通脉方低、中、高剂量组大鼠排尿时长均缩短、排尿速度均增加（P＜0.05），中剂量组漏尿点压升高（P＜0.05），各剂量组最大膀胱容量和膀胱顺应性差异均无统计学意义（P＞0.05）；蛋白质印迹结果显示，中剂量组p-AKT、p-STAT3、p53蛋白表达降低（P＜0.05或P＜0.01），高剂量组p-AKT蛋白表达降低（P＜0.05），中、高剂量组AGE、RAGE蛋白表达降低（P＜0.05或P＜0.01）。结论 左归降糖通脉方可显著改善DNB模型大鼠的排尿功能，其作用机制可能与调控AKT、STAT3、SRC、p53、HSP90等核心靶点及抑制AGE-RAGE信号通路有关。 |
| 关键词: 左归降糖通脉方|糖尿病神经源性膀胱|网络药理学|分子对接|尿流动力学|AGE-RAGE信号通路|AKT|STAT3 |
| DOI：10.3969/j.issn.1674-070X.2026.07.003 |
| 投稿时间：2026-03-26 |
| 基金项目:国家自然科学基金面上项目（81874510）。 |
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| Efficacy, molecular target prediction, and mechanism validation of Zuogui Jiangtang Tongmai Formula in treating diabetic neurogenic bladder |
| AI Kun, XIE Wanjian, SHE Yan, SONG Quan, QU Qirui, CHEN Yanling, JIANG Haoqing, ZHANG Lei, DENG Yihui |
| (Hunan University of Chinese Medicine, Changsha, Hunan 410208, China;Zhangzhou Municipal Hospital of Fujian Province, Zhangzhou, Fujian 363000, China) |
| Abstract: |
| Objective To screen the main active components and potential action targets of Zuogui Jiangtang Tongmai Formula (ZGJTTMF) in treating diabetic neurogenic bladder (DNB) based on network pharmacology and molecular docking technology, and to verify its mechanism of action by establishing a DNB animal model. Methods The active components and corresponding action targets of ZGJTTMF were screened using the Chinese Medicine Systems Pharmacology Database and Analysis Platform and the HERB database, and gene names corresponding to the action targets were standardized using the Universal Protein Resource (UniProt) database. DNB-related target genes were retrieved from the GeneCards and Online Mendelian Inheritance in Man (OMIM) databases. Venny software was used to construct a Venn diagram of the intersecting action targets between the formula and DNB. A protein-protein interaction (PPI) network was constructed and visualized using the STRING online platform and Cytoscape software. Gene Ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using relevant databases, with the results presented on the Bioinformatics Cloud platform. Molecular docking between the active components and core target protein receptors was validated using CB-Dock2. After screening the potential action targets of ZGJTTMF against DNB, animal experiments were conducted for in vivo validation. Thirty-six female SD rats were randomly divided into a blank group (n=6) and a modeling group (n=30). The DNB model was established by a single intraperitoneal injection of streptozotocin (STZ) solution. After successful modeling, the rats in the modeling group were randomly divided into model group, low-dose group, medium-dose group, high-dose group, and positive drug group, with 6 rats in each group. The low-, medium-, and high-dose groups were administered 6.5, 13, and 26 g/kg of ZGJTTMF by gavage, respectively, while the positive drug group received α-lipoic acid at 100 mg/kg by gavage, once daily for 4 weeks. During the experiment, one unqualified sample was excluded from each group, leaving a final effective sample size of 5 per group. After one month of administration, bladder function was assessed by urodynamic testing. Protein expression levels of phosphorylated protein kinase B (p-AKT), phosphorylated signal transducer and activator of transcription 3 (p-STAT3), phosphorylated proto-oncogene tyrosine-protein kinase Src (p-SRC), tumor protein p53 (p53), and heat shock protein 90 kDa (HSP90) were determined by Western blot. Results A total of 357 intersecting targets of ZGJTTMF against DNB were identified. The main active components included quercetin, isomucronulatol 7-O-β-D-glucoside, dihydrocapsaicin, 2-demethylplastoquinone-3, and luteolin. The core targets included TP53, AKT1, SRC, HSP90AA1, and STAT3. GO functional enrichment analysis yielded 1,077 biological processes, 120 cellular components, and 260 molecular functions, while KEGG pathway enrichment identified 186 pathways, among which the AGE-RAGE signaling pathway might be a key pathway. Molecular docking results showed good binding affinity between the main active components and the core targets, with isomucronulatol 7-O-β-D-glucoside exhibiting the strongest binding activity to AKT1. Compared with the model group, the rats in the low-, medium-, and high-dose groups of ZGJTTMF all showed shortened urination duration and increased urination flow rate (P<0.05), while the medium-dose group showed an increased leak point pressure (P<0.05). There were no statistically significant differences in maximum bladder capacity or bladder compliance among the dose groups (P>0.05). Western blot results revealed that p-AKT, p-STAT3, and p53 protein expression decreased in the medium-dose group (P<0.05 or P<0.01), p-AKT expression decreased in the high-dose group (P<0.05), and AGE and RAGE protein expression decreased in the medium- and high-dose groups (P<0.05 or P<0.01). Conclusion ZGJTTMF can significantly improve urinary function in DNB model rats, and its mechanism of action may be related to the regulation of core targets such as AKT, STAT3, SRC, p53, and HSP90, as well as the inhibition of the AGE-RAGE signaling pathway. |
| Key words: Zuogui Jiangtang Tongmai Formula|diabetic neurogenic bladder|network pharmacology|molecular docking|urodynamics|AGE-RAGE signaling pathway|AKT|STAT3 |
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