恭喜尤沛栋在Materials & Design ( IF 8.4 )上发表论文！！！
动脉粥样硬化是一种慢性炎症性疾病，与斑块中胞葬作用受损，凋亡细胞（apoptotic
cells, ACs）无法及时清除密切相关。应用阿托伐他汀（atorvastatin, AT）和二甲双胍
（metformin, Met）激活胞葬作用的策略在动脉粥样硬化治疗中受到越来越多的关注。
虽然这些药物治疗可以促进ACs的吞噬清除，但药物清除速度快、生物利用度差限制了
其疗效。在本研究中，我们通过将AT和Met共负载到PLGA NPs中，开发了一种用于靶
向动脉粥样硬化治疗的仿生纳米复合物。巨噬细胞膜包覆纳米复合物有效地避免了免疫
系统对NPs的清除，实现了对动脉粥样硬化斑块的靶向作用。此外，修饰透明质酸
（modified hyaluronic acid, HA）可将NPs靶向到斑块部位功能失调的胞葬细胞，实现
斑块—巨噬细胞靶向治疗。体外实验结果表明，仿生纳米复合物通过增强
ERK5/MerTK通路促进巨噬细胞从M1向M2表型极化，和通过抑制p53-p16/pRB通路抑
制细胞衰老，从而激活胞葬作用。体内研究表明，此仿生纳米复合物在动脉粥样硬化斑
块中积累，从而减少坏死核心区域，并通过重新激活胞葬作用来防止斑块破裂。这些研
究表明，通过AT和Met联合递送的纳米复合物激活胞葬作用，为靶向动脉粥样硬化治疗
提供了一种新的有希望的策略。
本文第一作者为宁夏医科大学尤沛栋博士，湖南大学刘斌教授、宁夏医科大学姜怡邓和
张慧萍教授为本文共同通讯
Fig. 1. The effect of AT/Met combination on the efferocytosis reactivation. (A&B) Immunofluorescence staining and semi-quantitative analysis of iNOS (green) and nuclei (blue) on activated macrophages treated by AT (5 μM and 7.5 μM). (C&D) Immunofluorescence staining and semi-quantitative analysis of CD206 (green) and nuclei (blue) on activated macrophages treated by AT (5 μM and 7.5 μM). (E&F) The levels of intracellular SA-β-gal and corresponding quantitative data in ox-LDL stimulated macrophages treated by Met (2.5 μM and 5 μM) for 48 h, scale bar = 50 μm. (G) In vitro cell viability of ox-LDL stimulated macrophages after incubation with Met (2.5 μM and 5 μM) for 48 h. (H&I) Representative images of CLSM and semi-quantification analysis the effect of AT and Met on reactivate efferocytosis of macrophages, scale bar = 60 μm. (J&K) Confocal fluorescence images and semi-quantification analysis of cellular uptake of DiI-oxLDL in RAW 264.7 cells, scale bar = 60 μm. (L&M) Representative images and semi-quantification analysis of ox-LDL internalization in RAW 264.7 cells, scale bar = 50 μm. 1: Control, 2: Model, 3: AT = 5 μM, 4: AT = 7.5 μM, 5: Met = 2.5 μM, 6: AT + Met = 5 μM + 2.5 μM, 7: AT + Met = 7.5 μM + 2.5 μM. n = 3 per treatment, *P < 0.05, **P < 0.01, ***P < 0.001 vs. the model. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. Design and Characterization of HA-M@P@(AT+Met) NPs. (A) TEM image of PLGA NPs. (B) TEM image of HA-M@P@(AT+Met) NPs. (C) FT-IR spectra analysis of AT and P@AT. (D) FT-IR spectra analysis of Met and P@Met. (E) SDS-PAGE analysis of retention protein bands of Møm and HA-M@P@(AT+Met) NPs. (F) Western blot of Møm and HA-M@P@(AT+Met) NPs for characteristic Møm marker CD11b. (G) ζ-potential measurement of different NPs. (H) Hydrodynamic size distribution of HA-M@P@(AT+Met) NPs. (I&J) Cumulative release of AT (I) and Met (J) from nanocomplexes.
Fig. 3. Cellular uptake of HA-M@P@(AT+Met) NPs. (A) Representative fluorescence images of different NPs (P@ce6, HA-RBCm@P@ce6, and HA-Møm@P@ce6 (red)), HUVECs (blue), and ICAM-1/P-selectin (green) in the LPS-treated group, scale bar = 50 μm. (B) Fluorescence intensity of different NPs in each group. (C&D) Phagocytosis of fluorescent P@ce6 and HA-M@P@ce6 NPs in HUVECs, activated macrophages, and senescent macrophages. (E) Quantitative statistical analysis of fluorescence signal in HUVECs, activated macrophages, and senescent macrophages. (F) Flow cytometry profiles of P@ce6 and HA-M@P@ce6 uptake in RAW264.7 cells, activated macrophages, and senescent macrophages for different times. n = 3 per treatment, **P < 0.01, ***P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. The effect and mechanism of HA-M@P@(AT+Met) NPs on polarization and senescence of macrophages. (A&B) Immunofluorescence staining and quantitative analysis of iNOS (green) in macrophages with different treatments, scale bar = 60 μm. (C&D) Immunofluorescence staining and semi-quantitative analysis of CD206 (green) in macrophages with different treatments, scale bar = 60 μm. [LPS] = 100 ng/mL, [AT] = 5 μM, [Met] = 2.5 μM. (E) ELISA assay of TNF-α, MCP-1, and IL-10. (F&G) Western blot and semi-quantitative analysis of p-ERK5, ERK5, and MerTK levels. (H&I) Expression of intracellular SA-β-gal and corresponding quantitative data in senescent macrophages treated with AT + Met, P@(AT+Met) NPs, and HA-M@P@(AT+Met) NPs. [AT] = 5 μM, [Met] = 2.5 μM, scale bar = 50 μm. (J&K) Western blot and semi-quantitative analysis of p53, p16, and pRB. (L) Mechanism diagram of HA-M@P@(AT+Met) NPs to simultaneously reactivate efferocytosis. 1: Control, 2: Model, 3: AT + Met, 4: P@(AT+Met) NPs, 5: HA-M@P@(AT+Met) NPs. n = 3 per treatment, *P < 0.05, **P < 0.01, and ***P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. In vitro anti-atherosclerosis effects and mechanism of HA-M@P@(AT+Met) NPs (A&B) CLSM images and semi-quantitative analysis the effect of HA-M@P@ (AT+Met) NPs on reactivate efferocytosis of macrophages. [AT] = 5 μM, [Met] = 2.5 μM, scale bar = 60 μm. (C&D) Intracellular ACs levels (by flow cytometry analysis) in macrophages treated with LPS (100 ng/mL), w or w/o AT+Met, P@(AT+Met) NPs, and HA-M@P@(AT+Met) NPs, respectively. (E&F) Flow cytometry analysis of intracellular DiI-ox-LDL levels in macrophages after incubation for 4 h. (G&H) Confocal fluorescence images and semi-quantitative analysis of DiI-ox-LDL internalization in RAW264.7 cells. (I&J) Optical microscopy images and semi-quantitative analysis of ORO staining, scale bar = 50 μm. 1: Control, 2: Model, 3: AT + Met, 4: P@(AT+Met) NPs, 5: HA-M@P@(AT+Met) NPs. n = 3 per treatment, *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig. 6. HA-M@P@(AT+Met) NPs targeting and bio-distributive assay. (A) Relative fluorescence intensity of PBS, P@ce6 NPs, and HA-M@P@ce6 NPs in the blood. (B&C) Ex vivo fluorescence images and semi-quantitative analysis of HA-M@P@(AT+Met) NPs in the aorta. (D&E) Distribution of HA-M@P@(AT+Met) NPs in various organs of ApoE−/− mice after i.v. administration. n = 3 per treatment, *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig. 7. Therapeutic efficacy of HA-M@P@(AT+Met) NPs in vivo. (A) Schematic diagram of prophylactic therapeutic regimens (a) and remedial therapeutic regimens (b). (B&C) Representative photographs and Semi-quantitative analysis of ORO stained en face aortas. n = 3 per treatment. (D) The representative images of ORO-stained cross-cryosections, scale bar = 200 μm. (E-F) Semi-quantitative analysis of ORO-stained cross-cryosections. (G) The number of WBC in serum. n = 6 per treatment. *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig. 8. Efferocytosis reactivated in ApoE−/− mice caused by HA-M@P@(AT+Met) NPs treatment. (A) The representative immunohistochemistry staining images with Tunel (red), scale bar = 200 μm. (B) Semi-quantitative analysis of the percentage of Tunel+ ACs. (C-E) ELISA analysis of TNF-α, MCP-1, and IL-10 levels in serum after different treatments. (F) Representative immunohistochemistry staining images using antibodies against ERK5, iNOS, CD206, and pRB (red). (G-J) Semi- quantitative analysis of plaque ERK5 expression (G), iNOS expression (H), CD206 expression (I), and pRB expression (J). n = 6 per treatment, *P < 0.05, **P < 0.01, and ***P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 9. Investigation of plaque stability in ApoE−/− mice by HA-M@P@(AT+Met) NPs treatment. (A) Representative immunohistochemistry staining photographs with H&E, Masson’s trichrome, antibody against α-SMA, and antibody against MMP-9. (B-E) Semi-quantitative analysis of the necrotic core area (B), collagen contents (C), VSMCs number (D), and MMP-9 expression (E) after different treatments. n = 6 per treatment, *P < 0.05, **P < 0.01, and ***P < 0.001.
3. 总结与展望
本研究构建了一种共递送阿托伐他汀和二甲双胍到活化巨噬细胞和衰老巨噬细胞的仿生纳米药物（HA-M@P@(AT+Met) NPs），靶向治疗动脉粥样硬化。HA-M@P@(AT+Met) NPs通过调节ERK5/MerTK通路促进巨噬细胞从M1型转化到M2型和通过调节p53-p16/pRB抑制巨噬细胞衰老，最终重新激活巨噬细胞胞葬作用。更重要的是，HA-M@P@(AT+Met) NPs可以促进药物在动脉粥样硬化斑块中的积累，并通过双重靶向策略提高药物的生物利用度。同时，HA-M@P@(AT+Met) NPs对动脉粥样硬化的预防和治疗均有显著的疗效，为动脉粥样硬化治疗提供了一种新的策略。此外。此纳米药物具有炎症环境趋化作用和巨噬细胞靶向能力，使其可以应用于其他炎症类疾病，如类风湿性关节炎。
4. 文献链接
https://www.sciencedirect.com/science/article/pii/S0264127523007311
5. 作者简介
第一作者：尤沛栋，医学博士，山东大学第二医院助理研究员。研究方向为动脉粥样硬化发病机制和靶向治疗，以第一作者在Applied Materials Today和Materials&Design学术期刊发表论文2篇。
通讯作者：姜怡邓，医学博士、二级教授、博士研究生导师，享受国务院特殊津贴专家。现任宁夏医科大学党委副书记、校长，国家卫健委代谢性心血管疾病研究重点实验室主任、宁夏血管损伤与修复研究重点实验室主任。荣获国家百千万人才工程“有突出贡献中青年专家”、教育部新世纪优秀人才、宁夏313人才、宁夏科技创新领军人才、宁夏创新争先奖、宁夏青年科技奖、宁夏青年五四奖章、宁夏科研贡献奖等。教育部高等学校基础医学类教学指导委员会委员、国际动脉粥样硬化学会中国分会副主席、中国病理生理学会第九届动脉粥样硬化专业委员会常务委员、中国动脉硬化杂志副主编、ISHR中国转化医学工作委员会委员、ISHR中国分会委员、中国生理学会循环专业委员会委员、宁夏医学会副会长等。先后主持国家自然科学基金项目7项，宁夏重点研发计划重点项目等16项；已在J Control Release、Aging Cell等国内外30余种国际权威杂志发表论文200余篇，其中SCI收录70余篇；出版学专著及教材14部；申请/获批国家专利34项；获宁夏科技进步一等奖1项、二等奖4项、三等奖2项；中国生理学报最佳研究论文、宁夏自然科学优秀学术论文等各类奖励共12项；培养博士研究生17名。
通讯作者：刘斌，湖南大学教授，博士生导师，中国民族医药协会委员，湖南中医药大学中药民族药创新发展实验室学术委员会委员。1994年毕业于湖南师范大学生物系，2001年于中南大学肿瘤研究所获硕士学位，研究方向为鼻咽癌发病分子机制；2002年至2007年在湖南大学攻读博士学位，研究方向为纳米荧光探针与肿瘤生化分析，2007年9月赴美国Texas Tech University 医学院开展博士后研究，研究内容为肾类疾病分子机制。目前研究方向主要集中在药用植物活性成分群的靶向筛选、抗肿瘤、心血管疾病的天然药物靶向递送和抗感染复合纳米材料研究。先后主持和参与各类科研项目20余项，以第一和通讯作者身份在ACS Nano,Biomaterials, Journal of Controlled Release, Applied materials today, Acta Biomaterialia, Analchem,Biosensor and Bioelectrics等国际知名学术期刊发表论文100余篇，总影响因子达到240，论文他引2000余次。
通讯作者：张慧萍，主任医师，副教授，硕士生导师。中华预防医学会出生缺陷预防与控制专业委员会委员，中华医学会围产医学分会第七届委员会青年委员会委员，宁夏妇幼保健协会理事，宁夏回族自治区产前诊断（筛查）技术组组长。2011年主持筹建宁夏医科大学总医院产前诊断中心。主要研究方向为产前诊断、异常妊娠的早期诊断和发病机制，从事染色体病、基因病诊断、产前诊断、遗传咨询和临床遗传学等科研教学工作，先后主持和参与国家自然科学基金、教育部春晖计划项目、宁夏自然科学基金、宁夏科技攻关项目等10余项课题，曾获宁夏回族自治区优秀学术论文一等奖、宁夏回族自治区科技进步二等奖、宁夏自然科学学术论文二等奖等。目前在国内外学术刊物上发表学术论文20余篇，其中SCI收录10余篇。入选2022年湖南省卫生健康高层次人才。