桑枝总生物碱（Morus alba L. (Sangzhi) alkaloids，SZ-A）是本课题组参与研发的有效组分降糖中药新药，其主要活性成分是从中药材桑枝中分离提取纯化得到的总生物碱。2020年3月，桑枝总生物碱片经中国国家药品监督管理局批准上市，作用靶点为肠道糖苷酶^[1]。SZ-A作为有效组分天然药物，其成分中含有多个小分子生物碱，具有多成分的特点；且药物代谢动力学研究验证，SZ-A在体内广泛分布。这也决定了其可能具有多靶点、能够发挥全身范围的多重药理作用的特点。现阶段，该品种正处于临床IV期，即上市后再评价阶段。因此，针对SZ-A挖掘其更多靶点及探讨作用机制，拓展其更多适应症及推广临床应用，具有重要意义。

基于以上背景，本研究论文主要分为以下两部分内容。

第一部分，基于前期研究基础，运用多种生物信息学方法挖掘SZ-A改善胰岛β细胞的潜在靶点和作用机制。首先，运用网络药理学方法，对比SZ-A中所包含活性成分与已上市药物的结构相似性，进一步探索和广泛挖掘SZ-A中各组分改善胰岛β细胞功能的可能作用靶点。经分析统计，共计获得24个潜在靶点。其次，运用基因表达综合数据库（The Gene Expression Omnibus, GEO），检索上述24个基因的表达水平在正常人及2型糖尿病患者胰腺组织中是否存在显著差异。结果提示，PLA2G1B基因表达水平在糖尿病患者胰腺组织中显著升高。接下来，在高脂喂养的C57小鼠及自发性db/db小鼠中，验证PLA2G1B与代谢应激及2型糖尿病状态的相关性。q-PCR结果显示，PLA2G1B在高脂饲料喂养的C57小鼠和糖尿病db/db小鼠胰腺组织中的基因表达升高。综合网络药理学、GEO数据库及转录组学结果，我们推测，PLA2G1B可能在SZ-A改善糖尿病状态下胰岛β细胞功能的过程中发挥重要作用。最后，采用长期给予SZ-A后的KKAy小鼠胰腺组织样本，使用q-PCR、WB、免疫荧光的方法，进一步考察SZ-A给药对于糖尿病KKAy小鼠胰腺组织PLA2G1B蛋白的影响。实验结果均表明：SZ-A可显著降低KKAy小鼠胰腺组织中PLA2G1B的基因及蛋白表达水平，提示PLA2G1B可能是SZ-A参与机体糖代谢稳态调节及β细胞功能改善的靶点之一。此外，将SZ-A的活性成分分别与PLA2G1B蛋白进行分子对接，结果初步提示，DAB、DNJ、FAG、N-M-DNJ均与PLA2G1B有较好的结合。

第二部分，考察SZ-A与临床一线用药二甲双胍（Metformin，Met）联合应用改善2型糖尿病KKAy小鼠糖脂代谢的作用特点及机制。本部分研究主要采用自发性2型糖尿病动物模型KKAy小鼠，在此模型中探讨SZ-A与Met两种药物联用后的抗糖尿病作用。为实现联合用药减量和增效的目的，本研究在剂量设置上，SZ-A（100 mg/kg）与Met（100 mg/kg）均采用了较低有效剂量。研究结果表明，在整体动物水平，与单药治疗相比，SZ-A和Met在100 mg/kg剂量下联用对KKAy小鼠的摄食饮水、空腹血糖、随机血糖、糖化血红蛋白、血TG及FFA水平等指标的改善作用，均显著优于单药组。在改善口服葡萄糖耐量异常作用上，SZ-A和Met联用要优于Met单药，与SZ-A作用接近；抑制糖异生环节上，SZ-A和Met联用要优于SZ-A单药，与Met作用接近。SZ-A和Met联用可显著增加KKAy小鼠的胰岛素敏感性，作用显著优于SZ-A及Met单药组，该结果提示：两药联用可共同改善外周组织的胰岛素抵抗状态，这是明显优于两种单药的作用之一。因此，本研究后续将重点聚焦于探索两药联用改善外周组织胰岛素敏感性的潜在机制。首先，采用生化检测及HE染色，考察SZ-A与Met联合给药对于KKAy小鼠肝脏及脂肪组织的脂质堆积及氧化应激的影响。结果表明，SZ-A及Met联用可改善肝脏组织脂质堆积，缩小脂肪组织细胞面积，降低机体氧化应激水平，提示联合用药提高外周组织胰岛素敏感性的作用可能部分来源于对脂质堆积和氧化应激的改善。其次，采用Western blot技术检测了KKAy小鼠肝脏、肌肉及脂肪组织的胰岛素信号通路各关键蛋白的表达。结果显示，SZ-A及Met联用可明显激活KKAy小鼠外周组织PI3K/PDK1/Akt/GLUT2信号通路，上调糖利用关键组织中葡萄糖转运体GLUT2和GLUT4的蛋白表达。这可能是联合用药改善外周组织胰岛素敏感性的主要机制。

综上，本论文围绕中药桑枝总生物碱抗糖尿病的作用靶点及调节机制展开研究。通过现代药理学研究技术手段，采用网络药理学、GEO数据库生信分析并结合组学研究，初步确定PLA2G1B可能是SZ-A改善胰岛β细胞功能的潜在靶点，为后续深入挖掘SZ-A的作用机制提供依据。在2型糖尿病实验动物模型中，初步明确了SZ-A与Met联合用药的抗糖尿病作用特点，并初步探讨了其改善胰岛素抵抗的作用机制，为临床应用中二者联合用药及复方制剂的开发提供了研究数据支撑。

Morus alba L. (Sangzhi) alkaloids (SZ-A) is isolated and purified from traditional Chinese medicine Sangzhi. In March 2020, SZ-A was approved for marketing by China National Medical Products Administration with the action target on intestinal glycosidase^[1]. As a natural drug, SZ-A contains various small molecular alkaloids. What’s more, Pharmacokinetic studies have revealed that SZ-A is widely distributed throughout the body, suggesting its potential for systemic pharmacological effects. At present, SZ-A is in clinical stage IV. Therefore, it is of great significance to explore more targets and mechanisms of SZ-A, expand more indications as well as promote clinical application.

Based on the aforementioned background, this study is divided into two parts.

In the first part, based on previous studies, various bioinformatics methods were employed to investigate the protective mechanism of SZ-A on β cells. Firstly, the network pharmacology method was employed to elucidate the potential therapeutic targets of SZ-A in enhancing β cell function by comparing the structural similarity between the active ingredients in SZ-A and the marketed drugs. A total of 24 potential targets were identified. Secondly, Gene Expression Omnibus (GEO) database was utilized to retrieve expression levels of the aforementioned 24 genes in the pancreas tissue of both normal individuals and those with type 2 diabetes. The findings indicated that PLA2G1B gene expression level are significantly higher in pancreatic tissues of type 2 diabetic patients than those in normal individuals. Furthermore, q-PCR results demonstrated that the PLA2G1B gene expression was upregulated in the pancreatic tissues of mice fed with high-fat as well as in db/db mice. Findings of network pharmacology analysis, GEO database and transcriptome analysis collectively suggested an important role of PLA2G1B for SZ-A in improving β-cell function. Additionally, q-PCR, Western blotting and immunofluorescence were employed to further investigate the effect of SZ-A on the expression of PLA2G1B in pancreatic tissue of diabetic KKAy mice. The results demonstrated that SZ-A could significantly reduce the level of PLA2G1B in pancreatic tissue of KKAy mice, suggesting that PLA2G1B may be one of the targets through which SZ-A regulates glucose metabolism homeostasis and improves β cell function. In addition, results of molecular docking indicated a favorable combination between DAB, DNJ, FAG, N-M-DNJ and PLA2G1B.

The second part of this study aims to evaluate the effects and characteristics of the combination of SZ-A with Metformin (Met) on glucose and lipid metabolism in diabetic KKAy mice, and explores the synergistic pathways of this combination. Lower effective doses of SZ-Aand Met, both at 100 mg/kg, were used to achieve dose reduction and to enhance the combination's efficacy. In KKAy mice, the combination of SZ-A and Met at a dose of 100 mg/kg showed significantly better effects than individual drug treatments regarding food and water intake, fasting and non-fasting blood glucose levels, glycosylated hemoglobin level, blood triglyceride levels, and free fatty acid levels.

Furthermore, the performance of combination of SZ-A and Met showed better improvement in oral glucose tolerance compared to Met alone and better inhibition of gluconeogenesis in KKAy mice than SZ-A alone. Additionally, the combined administration enhanced insulin sensitivity more effectively than either SZ-A or Met monotherapy, suggesting a synergistic effect between SZ-A and Met in improving insulin resistance of peripheral tissues. As a result, our research has been directed towards investigating peripheral tissues. Firstly, biochemical detection methods and Hematoxylin-eosin staining (HE) were used to investigate the effects of the combined administration of SZ-A and Met on lipid accumulation in the liver tissue and adipose tissue and oxidative stress of KKAy mice. The results showed that the combination effectively reduced lipid accumulation of the liver tissue, decreased adipose cell size, and mitigated systemic oxidative stress levels, suggesting that the beneficial impact of combined administration on peripheral insulin sensitivity may be, at least in part, due to its ability to alleviate lipid accumulation and oxidative stress. To further explore the molecular mechanism by which combined administration improves insulin resistance, Western blot analysis was conducted to assess the expression levels of key proteins in the insulin signaling pathway in liver, muscle, and adipose tissues of KKAy mice. It was observed that the combined administration significantly activated PI3K/PDK1/Akt/GLUT2 signaling pathway in peripheral tissue of KKAy mice, particularly upregulated protein expression levels of crucial glucose transporters involved in tissue glucose uptake.

In summary, our research focus on identifying the target and mechanism of SZ-A for diabetes. By using modern pharmacological techniques, including network pharmacology, GEO database analysis, and RNA-seq, PLA2G1B has been identified as a potential target for SZ-A in improving β-cell function, laying a foundation for further mechanistic studies. Additionally, this research has provided initial insights into the pharmacological effects and synergistic mechanisms of the combination therapy involving SZ-A and Met, providing foundational data to support clinical applications as well as the development of compound preparations.