小檗碱（Berberine，BBR）是一种植物来源的苄基异喹啉季铵生物碱，在临床上主要用于胃肠炎、细菌性痢疾等肠道感染性疾病的治疗。小檗碱具有降脂、抗炎、降糖和神经保护等广泛的药理活性。但小檗碱的口服生物利用度不到1%，大部分富集在肠道，这严重限制了其临床应用。小檗碱能够通过“标本兼治”发挥多重生物学作用，“治标”为通过分子靶点或通路来改善临床症状，“治本”则是针对疾病起因, 如炎症、肠道菌群失调等生物学过程进行的干预。麻黄碱、槲皮素等天然产物具有肠道菌群、免疫功能调节的作用（治本），但无法减轻疾病症状（不治标），而小檗碱则是一种“标本兼治”的药物，具有其独特性。因此，在论文中，以提高小檗碱生物利用度及药效为目的，结合纳米技术和化学技术两种方案，开展了关于动脉粥样硬化（Atherosclerosis，AS）、非酒精性脂肪肝（Nonalcoholic fatty liver disease，NAFLD）和抑郁中的药效和机制研究。

第一部分 纳米技术

第一章 功能性纳米载体增强小檗碱对Apoe（-/-）小鼠的抗动脉粥样硬化作用

动脉粥样硬化（Atherosclerosis，AS）起始于内皮损伤，随着细胞的粘附和迁移、脂质沉积、单核细胞入侵并分化为巨噬细胞、泡沫细胞的形成等，逐渐促进了斑块的产生，使动脉内部由于斑块堆积而变窄。在疾病进程中，低密度脂蛋白和慢性炎症会诱发或加重内皮功能障碍。

D-α-生育酚聚乙二醇琥珀酸酯（TPGS）是天然维生素E（VE，D-生育酚）的水溶性衍生物，由VE琥珀酸酯与聚乙二醇（PEG）酯化形成，其作为药物递送载体的同时，也是P糖蛋白（P-GlycoProtein，P-gp）的抑制剂。

本研究合成了三个TPGS类似物（PEG400，1500，3000）对BBR包埋的纳米体系。经过对三者进行比较，我们发现，BT1500M能够在低浓度的水性载体中形成稳定的胶束，同时，在促进BBR吸收及抑制外排上的表现更好，且较BT400M和BT3000M相比能更好地促进HepG2和3T3-L1细胞对BBR的摄取，因此，我们选择了BT1500M开展药效学和机制研究。

体内实验中，利用高脂饲料喂养Apoe（-/-）小鼠5个月以构建AS动物模型，给药组分别灌胃单纯小檗碱（BP，100 mg/kg/day BBR）、空胶束（EV）和BT1500M（BM，以100 mg/kg/day BBR给药）。药效学结果显示：①BM组小鼠主动脉中内皮细胞中细胞间黏附分子（Intercellular cell adhesion molecule，ICAM）、血管细胞黏附分子（Vascular Cell Adhesion Molecule，VCAM）、肿瘤坏死因子α（Tumor necrosis factor α，TNFα）等与炎症和斑块形成过程中成正相关的细胞因子含量显著降低，内皮损伤和炎症反应减轻。②主动脉油红染色发现BM能减少斑块区域且降低主动脉组织中各类胆固醇酯含量，有效抑制了斑块的形成。

已有研究证明，肝脏和脂肪是BBR治疗代谢性疾病的靶器官，组织分布结果中BT1500M也促进了BBR在肝脏和脂肪中的富集。因此，我们以肝脏和脂肪为靶组织，进一步探讨了BT1500M治疗AS的分子机制。我们发现：BT1500M可以通过调节肝脏和脂肪组织中AMP依赖的蛋白激酶（Adenosine 5‘-monophosphate (AMP)-activated protein kinase，AMPK）和核因子κB（Nuclear factor-k-gene binding，NF-kB）来减轻高脂饮食喂养所引起的Apoe（-/-）小鼠高脂血症和代谢紊乱。在肝脏中，BT1500M能够抑制肝脏中脂质堆积和活性氧（Reactive oxygen species，ROS）生成。在脂肪组织方面，由于肥大的脂肪细胞倾向于释放脂肪细胞因子，启动巨噬细胞的浸润和极化。而极化的1型巨噬细胞（M1）则释放更多的炎性细胞因子，进而激活了脂肪细胞和巨噬细胞的炎症反应。二者信号传导不断循环，促进AS的发生。实验结果显示，BT1500M成功地抑制了巨噬细胞的活化，并中断了脂肪细胞和巨噬细胞之间的信息反馈过程，改善了Apoe（-/-）小鼠的炎症状态。此外，小鼠口服BM 5个月后的生物安全性结果较好。需要注意的是，在上述的研究过程中，BP、EV对某些指标也有改善作用，但效果小于BM。

综上所述，本研究开发的纳米递送系统BT1500M提高了BBR的抗动脉粥样硬化功效。通过对其表征、组织分布、药效和分子机制进行深入的研究，阐明了BT1500M的生物活性。结果表明，BT1500M对高脂饮食诱导的动脉粥样硬化的治疗效果优于单纯小檗碱及胶束载体，该效果是基于纳米技术增加了药物吸收以及药物与载体的协同作用来实现的。

第二部分 化学技术

小檗碱-水飞蓟宾盐通过调节脂质代谢提高抗非酒精性脂肪肝的效果

非酒精性脂肪肝（Nonalcoholic fatty liver disease，NAFLD）是一个不断进展的疾病，能够向肝炎、肝硬化、肝癌发展。该疾病的特点是脂质代谢异常、炎症和肝损伤，肝脏中脂肪的积累和氧化应激不断增多。

小檗碱具有潜在的肝脏保护作用，许多研究已经证明它在治疗2型糖尿病、脂质代谢紊乱和肿瘤方面的具有很好的疗效，且副作用较小。水飞蓟宾（Silybin，SIY）目前用于治疗由化学或环境毒素引起的肝炎及脂肪肝患者，能够降低CHO、TG、LDL-c、谷丙转氨酶（Alanine Aminotransferase，ALT）和谷草转氨酶（Aspartate aminotransferase，AST）水平，并改善胰岛素抵抗。考虑到BBR和SIY可通过不同的机制保护肝脏功能，拟将这两种化合物联用以增强其在肝病中的药理作用。

在该部分研究中，将碱性药物BBR和微酸性药物SIY等摩尔剂量下形成了盐（BSS），利用核磁共振氢谱、傅里叶红外光谱等技术对盐和物理混合物（BSP）进行了表征比较，确认了盐的合成。体外溶出度实验表明两药成盐后，能够有效促进水飞蓟宾成分在人工胃液及肠液中的溶出；在MDCK-MDR1单细胞模型中，BSS较BSP能够增加BBR和SIY的吸收，减少药物外排；同时在体内组织分布实验中，BSS也表现出了增加BBR和SIY在小鼠血液和肝脏内含量的效果，这为BSS增强对NAFLD的治疗作用提供了物质基础。

    在药效学实验中，我们使用高脂饲料构建了小鼠NAFLD模型（MC），给药组分别为物理混合物组（BSP）和盐组（BSS），对照组（NC）给予正常饮食，持续给药4周。给药结束后，BSS显著减少了小鼠体重、肝脏指数和附睾脂肪指数的增加，减轻了高脂饮食导致的附睾脂肪组织中脂肪细胞的肥大及肝脏内脂肪空泡的增多。同时，BSS组小鼠血浆中CHO、TG、LDL-C、ALT和AST生化标记物的含量显著减少。转录组学结果显示BSS对NAFLD的影响与对脂肪酸代谢的调节有关。我们进一步采用了免疫组化、PCR和WB等方法验证了BSS对关键抗NAFLD分子靶标（脂肪酸转运蛋白CD36、脂肪酸结合蛋白 4（Fatty acid binding protein 4，FABP4）和硬脂酰辅酶 A 去饱和酶（Stearoyl Coenzyme A desaturase 1，SCD1））表达的影响，发现BSS能够更好地抑制上述基因和蛋白的表达，从而减少脂肪酸的合成和转运。

这项研究的结果表明，成盐会改变BBR和SIY的物理性质，与常见的物理混合物相比，成盐可以通过其更佳的调节脂质代谢作用来提高这些化合物的抗NAFLD功效。

第二章 小檗碱-水飞蓟宾盐对糖皮质激素不良反应的改善作用及抗抑郁作用

糖皮质激素（Glucocorticoid）是临床一线用药，用于自身免疫性疾病等的治疗及危重症抢救等。当长期或大剂量使用激素类药物时，会引起患者出现高血糖、高血脂等代谢紊乱的不良反应，进一步表现为向心性肥胖，部分患者会出现精神异常，使患者具有较大的身体及心理负担，因此，寻找一种具有改善长期使用糖皮质激素所致副反应作用、且安全性高的药物，是临床用药中的一大需求。

在本部分研究中，将雄性C57小鼠随机分为5组，通过泼尼松（Prednisone）灌胃给药（5 mg/kg/day）的方法，构建长期口服激素类药物的动物模型（GC），给药组分别同时给予低（100 mg/kg，LB）、中（150 mg/kg，MB）、高（200 mg/kg，HB）剂量小檗碱处理，持续8周。给药结束后，利用小动物核磁成像技术及双能X射线仪检测小鼠体脂成分、肌肉含量及骨密度。实验结果表明，C57小鼠在连续服用人类临床等剂量下的泼尼松8周后：①模型组小鼠内脏脂肪含量增加，小檗碱对其有降低趋势；②模型组小鼠肌肉出现明显萎缩，小檗碱在在中、高剂量下，能够通过调节肌细胞生成素（Myogenin，MyoG）和肌细胞特异性增强因子2D（Myocyte enhancer factor 2D，MEF2D）来提高肌纤维直径及肌纤维平均面积，促进肌肉的生长；③在骨密度方面，各组之间没有明显变化。     

小鼠粪便菌群16SrRNA测序分析显示，长期口服激素类药物影响了小鼠肠鼠杆菌（Muribaculum）、瘤胃球菌（norank-f-Ruminococcaceae、unclassified-f-Ruminococcaceae）等菌群丰度，而小檗碱具有逆转作用。同时，GC组和NCD组小鼠差异菌群与粪便代谢组学中丰度排名前30的差异代谢物有显著相关性，小檗碱在回调激素对菌群改变的同时也能够影响代谢物的变化。通过对GC组与NCD组差异代谢物进行通路富集，发现血清素（Serotonin）代谢途径排在首位。由于血清素与情绪尤其是抑郁密切相关，于是我们猜想，泼尼松可能对小鼠也具有精神系统的影响，随后我们开展了小檗碱对激素所引起的抑郁是否具有改善作用的实验。

体内实验中，将C57小鼠随机分为空白对照组（NCD），模型组（MC），小檗碱组（BBR）。模型组以5 mg/kg/day剂量泼尼松灌胃，小檗碱组在泼尼松造模的同时以200 mg/kg/day 等剂量药饲饲养，持续8周。饲养结束后，先后开展糖水实验、旷场实验、悬尾实验、强迫游泳实验等用于评价动物抑郁样指标的公认行为学实验，实验结果表明模型组小鼠出现明显的快感缺失、紧张、求生欲降低、绝望等抑郁样行为，小檗碱改善了小鼠的抑郁症状。

考虑到水飞蓟宾（Sylibin，SLN）可通过调节脑源性神经营养因子（Brain-derived neurotrophic factor，BDNF）, 酪氨酸激酶受体B（Tyrosine Kinase receptor B，TRKB）的表达发挥出神经保护作用，且与小檗碱一样具有生物利用度差的缺点，我们设想将前述制备的小檗碱-水飞蓟宾盐应用在该模型中，期望能在相关指标中发挥出更好的改善效果。为检验这一想法，我们设置了小檗碱组（BBR），水飞蓟宾组（SLN），BBR与SLN物理混合物组（BBR+SLN）及BBR与SLN盐组（BBR-SLN），各组以小檗碱100 mg/kg/day等剂量灌胃给药，持续8周。我们发现，BBR＋SLN盐较单纯BBR可以降低泼尼松灌胃所引起的脂肪堆积和肌肉萎缩；在中枢神经系统方面，BBR＋SLN盐较单药组或物理混合物组，在改善抑郁样行为指标方面作用更好。

Berberine (BBR) is a benzylisoquinoline quaternary ammonium alkaloid belonging to the protoberberine family. It is currently mainly used to treat intestinal infectious diseases such as gastroenteritis and bacillary dysentery. It has a wide range of pharmacological activities, such as lipid-lowering, hypoglycemic, anti-inflammatory, neuroprotective, etc. However, the oral bioavailability of berberine is less than 1%, which severely limits its clinical application. Berberine can exert multiple biological effects by addressing both the symptoms and root causes of disease. The "symptom relief" is achieved through molecular targets or pathways to improve clinical symptoms, while the "root cause" is targeted at biological processes such as inflammation and gut flora imbalance. In addition to berberine, other natural products such as quercetin and puerarin can also address the root causes of disease, but berberine has its own unique characteristics in terms of "symptom relief". Therefore, during the entire research period, with the aim of improving the efficacy of berberine, combined with pharmacy (nanotechnology) or silybin (chemical technology), I carried out research on atherosclerosis, non-alcoholic fatty liver disease and depression models.

Part 1 Nanotechnology

Chapter 1  Functional nano-vector boost anti-atherosclerosis efficacy of berberine in Apoe(-/-) mice

Atherosclerosis (AS) begins with endothelial damage. With cell adhesion and migration, lipid deposition, monocyte invasion and differentiation into macrophages, and the formation of foam cells, plaques are gradually promoted. The production of plaque causes the inside of the arteries to narrow due to plaque accumulation. Increasing evidence confirms that endothelial dysfunction is associated with low-density lipoprotein particles and chronic inflammation.

D-α-Tocopherol polyethylene glycol succinate (TPGS）is a water-soluble derivative of natural vitamin E (VE, D-tocopherol), which is esterified with VE succinate and polyethylene glycol (PEG). It serves as a drug delivery carrier and is also an inhibitor of P-glycoprotein (P-Glyco Protein, P-gp).

In this study, three TPGS analogs (PEG400, 1500, 3000) were synthesized to encapsulate BBR in a nanosystem. After comparing the three, we found that BT1500M can form stable micelles in low-concentration aqueous carriers, and at the same time, it has better performance in promoting BBR absorption and inhibiting efflux. Compared with BT400M and BT3000M, it can better promote the uptake of BBR by HepG2 and 3T3-L1 cells. Therefore, we chose BT1500M for pharmacodynamic and mechanism studies.

In vivo experiments, Apoe(-/-) mice were fed with high-fat diet for 5 months to construct an AS animal model. The drug administration groups were given intragastric administration of simple berberine (BP, 100 mg/kg/day BBR), empty micelles (EV), and BT1500M (BM, administered with 100 mg/kg/day BBR). The pharmacodynamic results showed that: ①The contents of cytokines positively correlated with inflammation and plaque formation, such as Intercellular cell adhesion molecule (ICAM), Vascular Cell Adhesion Molecule (VCAM), and Tumor necrosis factor α (TNFα), in endothelial cells of the aorta in the BM group were significantly reduced, and endothelial injury and inflammatory response were alleviated. ②Oil red staining of the aorta showed that BM could reduce the plaque area and reduce the content of various cholesterol esters in aortic tissue, effectively inhibiting the formation of plaques.

Previous studies have shown that the liver and fat are the target organs of BBR in the treatment of metabolic diseases. The tissue distribution results also showed that BT1500M promoted the enrichment of BBR in the liver and fat. Therefore, we further explored the molecular mechanism of BT1500M in the treatment of AS by targeting the liver and fat. We found that BT1500M can alleviate the hyperlipidemia and metabolic disorders caused by high-fat diet feeding in Apoe (-/-) mice by regulating the adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) and nuclear factor-kappa B (NF-kB) in the liver and fat tissues. In the liver, BT1500M can inhibit lipid accumulation and reactive oxygen species (ROS) production in the liver. In terms of fat tissue, due to the tendency of hypertrophic adipocytes to release adipocytokines, macrophage infiltration and polarization are initiated. Polarized type 1 macrophages (M1) release more inflammatory cytokines, which in turn activate the inflammatory response of adipocytes and macrophages. The continuous cycle of signal transduction between the two promotes the occurrence of AS. The experimental results showed that BT1500M successfully inhibited the activation of macrophages and interrupted the information feedback process between adipocytes and macrophages, improving the inflammatory state of Apoe (-/-) mice. In addition, the biological safety results of mice after oral administration of BM for 5 months were good. It should be noted that in the above research process, BP and EV also had an improving effect on certain indicators, but the effect was less than that of BM.

In summary, this study developed the nano-delivery system BT1500M to improve the anti-atherosclerotic efficacy of BBR. Through in-depth research on its characterization, tissue distribution, efficacy and molecular mechanism, the biological activity of BT1500M was elucidated. The results show that the therapeutic effect of BT1500M on high-fat diet-induced atherosclerosis is better than that of simple berberine and micellar carriers. This effect is achieved based on nanotechnology to increase drug absorption and the synergistic effect of drugs and carriers.

Part 2 Chemical Technology

Chapter 1 Berberine-silybin salt achieves improved anti-nonalcoholic fatty liver disease effect through regulating lipid metabolism

Nonalcoholic fatty liver disease (NAFLD) is a progressive disease that can develop into hepatitis, cirrhosis, and liver cancer. The disease is characterized by abnormal lipid metabolism, inflammation, and liver damage, together with increasing fat accumulation and oxidative stress in the liver.

Berberine has potential hepatoprotective effects, and many studies have proven its efficacy in the treatment of type 2 diabetes, lipid metabolism disorders, and tumors with minimal side effects. Silybin (SIY) is currently used to treat patients with hepatitis and fatty liver caused by chemical or environmental toxins. It can reduce CHO, TG, LDL-c, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, and improve insulin resistance. Considering that BBR and SIY can protect liver function through different mechanisms, it is proposed to combine these two compounds to enhance their pharmacological effects in liver diseases.

In this part of the research, the alkaline drug BBR and the slightly acidic drug SIY were combined into equimolar doses to form a salt (BSS). The salt and physical mixture (BSP) were characterized and compared using other techniques to confirm the synthesis of the salt. In vitro dissolution experiments show that after the two drugs are salted, they can effectively promote the dissolution of silybin ingredients in simulated gastric fluid and intestinal fluid; in the MDCK-MDR1 cell model, BSS can increase the absorption of BBR and SIY and reduce the efflux of drugs compared with BSP; at the same time, in vivo tissue distribution experiments, BSS also showed the effect of increasing the contents of BBR and SIY in the blood and liver of mice, which provides a material basis for BSS to enhance the treatment of NAFLD.

In the pharmacodynamic experiment, we used high-fat diet to construct a mouse model of NAFLD (MC), and the drug groups were physical mixture group (BSP) and salt group (BSS). The control group (NC) was given normal diet, and the drug was administered for 4 weeks. After the end of the administration, BSS significantly reduced the increase in body weight, liver index, and epididymal fat index in mice, alleviating the hypertrophy of adipocytes in epididymal adipose tissue and the increase in fat vacuoles in the liver caused by high-fat diet. At the same time, the levels of biochemical markers such as CHO, TG, LDL-C, ALT, and AST in the plasma of BSS group mice significantly decreased. The transcriptomic results showed that the effect of BSS on NAFLD was related to the regulation of fatty acid metabolism. We further used immunohistochemistry, PCR, and WB methods to verify the effect of BSS on the expression of key anti-NAFLD molecular targets (fatty acid transporter CD36, fatty acid binding protein 4 (FABP4), and stearoyl Coenzyme A desaturase 1 (SCD1)). We found that BSS could better inhibit the expression of these genes and proteins, thereby reducing the synthesis and transport of fatty acids.

The results of this study indicate that salt formation changes the physical properties of BBR and SIY. Furthermore, animal experiments have shown that salt formation can enhance the anti-NAFLD efficacy of these compounds through their better regulation of lipid metabolism compared with common physical mixtures.

Chapter 2 Improvement of adverse effects of glucocorticoids and antidepressant effects of berberine-silybin salt

Glucocorticoid is a first-line drug used clinically to treat autoimmune diseases and rescue critical illness. When used for a long time or in large doses, it can cause patients to develop adverse reactions such as central obesity, hyperglycemia, hyperlipidemia and other metabolic disorders, causing patients to have a greater physical and psychological burden. Therefore, finding a drug that is safe and can effectively improve the side effects caused by long-term use of glucocorticoids is a major clinical need.

In this part of the study, male C57 mice were randomly divided into five groups and treated with prednisone (5 mg/kg/day) by gavage to establish a long-term oral hormone drug animal model (GC). The treatment groups were simultaneously treated with low (100 mg/kg, LB), medium (150 mg/kg, MB), and high (200 mg/kg, HB) doses of berberine for 8 weeks. After the end of the treatment, the body fat composition, muscle content, and bone density of the mice were detected using small animal MRI technology and dual-energy X-ray absorptiometry. The experimental results showed that after continuous administration of human clinical equivalent doses of prednisone for 8 weeks in C57 mice: ①The visceral fat content of the model group mice increased, and berberine had a decreasing trend; ②The muscles of the model group mice showed significant atrophy, and berberine at medium and high doses could increase the diameter and average area of muscle fibers by regulating myogenin (MyoG) and Myocyte enhancer factor 2D (MEF2D), promoting muscle growth; ③There was no significant change in bone density among the groups.

The 16S rRNA sequencing analysis of the fecal microbiota in mice showed that long-term oral administration of hormone drugs affected the abundance of bacterial groups such as Muribaculum and norank-f-Ruminococcaceae, unclassified-f-Ruminococcaceae in the intestines of mice, while berberine had a reverse effect. At the same time, there was a significant correlation between the differential microbial groups in the GC group and the NCD group and the top 30 differential metabolites in fecal metabolomics. Berberine can also affect the changes in metabolites while reversing the changes in microbial groups caused by hormones. Through pathway enrichment analysis of the differential metabolites between the GC group and the NCD group, it was found that the serotonin metabolic pathway ranked first. Since serotonin is closely related to mood, especially depression, we speculate that prednisone may also have an impact on the mental system of mice. Subsequently, we conducted an experiment to investigate whether berberine can improve depression caused by hormones.

In the in vivo experiment, C57 mice were randomly divided into a blank control group (NCD), a model group (MC), and a berberine group (BBR). The model group was given prednisone at a dose of 5 mg/kg/day by gavage, while the berberine group was fed with the same dose of prednisone at 200 mg/kg/day for 8 weeks. After the feeding period, recognized behavioral experiments such as sugar water test, open field test, tail suspension test, forced swimming test were conducted to evaluate the depression-like indicators of the animals. The experimental results showed that the mice in the model group showed significant depression-like behaviors such as anhedonia, tension, reduced survival desire, and despair. Berberine improved the depression symptoms of the mice.

Considering that silybin (SLN) can exert neuroprotective effects by regulating the expression of brain-derived neurotrophic factor (BDNF) and tyrosine kinase receptor B (TRKB), and has the same disadvantage of poor bioavailability as berberine, we envisioned applying the previously prepared berberine- silybin salt in this model, expecting to achieve better improvement in relevant indicators. Therefore, in the third batch of animal experiments, we set up a berberine group (BBR), a silybin group (SLN), a physical mixture of BBR and SLN group (BBR+SLN), and a BBR and SLN salt group (BBR-SLN), each group was administered with berberine 100 mg/kg/day by gavage for 8 weeks. We found that BBR+SLN salt can reduce fat accumulation and muscle atrophy caused by prednisone gavage compared to pure BBR; in terms of the central nervous system, BBR+SLN salt has a better effect on improving depression-like behavior indicators than the single drug group or physical mixture group