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研究生：	湛羽柔 CHAN, Yu-Jou
論文名稱：	內質網靶向奈米載體包覆薑黃素調控巨噬細胞極化並改善NOX2突變小鼠模型之動脈粥狀硬化斑塊 ER-targeting nanovehicle–encapsulated curcumin modulates macrophage polarization and ameliorate atherosclerotic plaques in a NOX2-mutant mouse model
指導教授：	謝奇璋 Shieh, Chi-Chang
學位類別：	碩士 Master
系所名稱：	醫學院 - 微生物及免疫學研究所 Department of Microbiology & Immunology
論文出版年：	2026
畢業學年度：	114
語文別：	英文
論文頁數：	100
中文關鍵詞：	動脈粥狀硬化、薑黃素、巨噬細胞極化、內質網標靶奈米載體、胞葬作用、CybbC1024T 、NOX2
外文關鍵詞：	atherosclerosis, curcumin, ER-targeted nanovehicle, efferocytosis, macrophage polarization, CybbC1024T, NOX2
相關次數：	點閱：20 下載：2
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動脈粥狀硬化是一種慢性發炎性血管疾病，其特徵為血管壁內脂質累積與斑塊形成。巨噬細胞在動脈粥狀硬化進展中扮演關鍵角色，其極化相關表型與功能會影響發炎活性及斑塊穩定性。NADPH oxidase 2（NOX2）由Cybb基因編碼，是參與巨噬細胞功能調控的重要活性氧來源，既有研究指出，CybbC1024T 突變會造成突變型 H338Y-gp91ᵖʰᵒˣ蛋白處理異常並滯留於內質網。因此，本研究探討 NOX2 功能狀態是否與巨噬細胞極化相關標記表現及效應功能有關，以及 ER-targeting nanovehicle–encapsulated curcumin 是否能調節巨噬細胞功能異常並影響動脈粥狀硬化斑塊形成。本研究自 wild-type、Cybb⁻/⁻、CybbC1024T 及 ApoE⁻/⁻CybbC1024T 小鼠取得骨髓來源巨噬細胞，以流式細胞儀分析 CD40、CD11c、NLRP3 及 CD206 表現以評估巨噬細胞極化相關標記表現，以 ELISA 測定 IL-6 產量，並以螢光標記凋亡細胞評估胞葬能力，進一步於高脂飲食誘導之ApoE缺失動脈粥狀硬化易感小鼠中，透過血清脂質分析與 Oil Red O 染色評估 ER-curcumin-NP 在體內的效果。結果顯示，NOX2 突變及 NOX2 完全缺失巨噬細胞皆呈現與基因型相關之促發炎標記型態，包括部分促發炎相關標記表現上升並伴隨 CD206 表現下降，功能性分析進一步顯示 NOX2 功能受損之巨噬細胞背景下IL-6分泌型態改變、胞葬能力下降，其中ApoE⁻/⁻CybbC1024T 巨噬細胞在 M1 極化條件下的 IL-6 上升尤為明顯，且此複合基因型整體呈現發炎與功能異常，其細胞表型與體內呈現的動脈粥狀硬化易感狀態大致一致。在 WT、CybbC1024T 及 ApoE⁻/⁻CybbC1024T 巨噬細胞中，ER-curcumin-NP 處理於特定條件下與促發炎標記表現降低、IL-6分泌降低、CD206表現上升或呈上升趨勢、以及胞葬能力提升有關，相對地，Cybb⁻/⁻ 巨噬細胞在多項標記與胞葬相關指標上並未觀察到具統計意義的反應。體內實驗方面，ER-curcumin-NP 治療可降低高脂飲食餵養之ApoE缺失小鼠之血清總膽固醇、三酸甘油酯濃度，以及主動脈Oil Red O陽性病灶面積。綜合而言，本研究顯示 NOX2 功能狀態與巨噬細胞發炎表型、細胞激素分泌、胞葬能力及對 ER-curcumin-NP 之反應性具有關聯性，ER-targeting nanovehicle–encapsulated curcumin 於部分巨噬細胞表型與功能指標上呈現改善並降低動脈粥狀硬化病灶負荷，此效果在NOX2功能完整或NOX2突變背景中較為明顯，於完全缺失NOX2的背景則較不明顯，此結果支持 ER-curcumin-NP 作為 NOX2 突變相關動脈粥狀硬化之巨噬細胞標的治療策略的潛力。

Atherosclerosis is a chronic inflammatory vascular disease characterized by lipid accumulation and plaque formation within the arterial wall. Macrophages play a central role in atherosclerotic progression, as their polarization-associated phenotype and effector functions influence inflammatory activity and plaque stability. NADPH oxidase 2 (NOX2), encoded by the Cybb gene, is an important enzymatic source of reactive oxygen species involved in the regulation of macrophage function. Previous studies have shown that the CybbC1024T mutation causes defective processing and endoplasmic reticulum (ER) retention of mutant H338Y-gp91ᵖʰᵒˣ protein. Therefore, this study investigated whether NOX2 functional status is associated with macrophage polarization-related marker profiles and effector functions, and whether ER-targeting nanovehicle-encapsulated curcumin modulates macrophage dysfunction and atherosclerotic lesion formation. Bone marrow-derived macrophages were generated from wild-type, Cybb⁻/⁻, CybbC1024T, and ApoE⁻/⁻CybbC1024T mice. Macrophage polarization-associated marker profiles were evaluated by flow cytometric analysis of CD40, CD11c, NLRP3, and CD206 expression. IL-6 production was measured by ELISA, and efferocytotic capacity was assessed using fluorescently labeled apoptotic cells. The in vivo effects of ER-curcumin-NP were further evaluated in high-fat diet-fed ApoE-deficient atherosclerosis-prone mice by serum lipid analysis and Oil Red O staining. NOX2-mutant and NOX2-deficient macrophages showed genotype-associated pro-inflammatory marker patterns, including increased expression of selected pro-inflammatory-associated markers and reduced CD206 expression. Functional analyses further showed altered IL-6 secretion and reduced efferocytotic capacity in NOX2-impaired macrophage backgrounds, with particularly evident IL-6 elevation in ApoE⁻/⁻CybbC1024T macrophages under M1-polarizing conditions. ApoE⁻/⁻CybbC1024T macrophages displayed an inflammatory and functional profile that was broadly consistent with the atherosclerosis-prone phenotype observed in the compound genotype in vivo. In WT, CybbC1024T, and ApoE⁻/⁻CybbC1024T macrophages, which represent NOX2-intact or NOX2-mutant backgrounds rather than complete NOX2-deficient backgrounds, ER-curcumin-NP treatment was associated with reduced pro-inflammatory marker expression, reduced IL-6 secretion, increased or upward-trending CD206 expression, and increased efferocytotic capacity under selected conditions. In contrast, Cybb⁻/⁻ macrophages showed no statistically detectable response in several marker-based and efferocytosis-related readouts. In vivo, ER-curcumin-NP treatment reduced serum total cholesterol, serum triglyceride, and aortic Oil Red O-positive lesion burden in high-fat diet-fed ApoE-deficient mice, both with and without the CybbC1024T mutation. These findings suggest that NOX2 functional status is associated with macrophage inflammatory phenotype, cytokine output, efferocytotic capacity, and responsiveness to ER-curcumin-NP. ER-targeting nanovehicle-encapsulated curcumin was associated with partial improvement in selected macrophage phenotypic and functional readouts and reduced atherosclerotic lesion burden, with more evident responses in NOX2-intact or NOX2-mutant backgrounds than in the complete NOX2-deficient background. These results are consistent with the potential of ER-curcumin-NP as a macrophage-targeted therapeutic strategy for NOX2 mutation-associated atherosclerosis.

中文摘要	I
Abstract	III
誌謝	V
Abbreviations	XI
Chapter 1 Introduction	1
1.1 Atherosclerosis is a chronic inflammatory disease	2
1.2 Pathological progression of atherosclerotic plaques	2
1.3 Macrophage function in plaque progression and resolution	5
1.4 Mechanisms and phenotypic classification of macrophage polarization	6
1.5 NOX2-dependent regulation of macrophage phenotype and function	8
1.6 ER-associated gp91ᵖʰᵒˣ maturation and NOX2 dysfunction	10
1.7 Therapeutic rationale of ER-targeting nanovehicle–encapsulated curcumin	11
1.8 Hypothesis and Study Objectives	13
Chapter 2 Materials and Methods	15
2.1 Experimental Animals and Disease Models	16
2.2 Isolation and cultivation of bone marrow-derived macrophages	16
2.3 Macrophage polarization and ER-curcumin-NP treatment	17
2.4 Flow cytometry analysis	18
2.5 Heatmap Visualization of Macrophage Polarization-Associated Marker Expression	19
2.6 Quantification of IL-6 secretion by enzyme-linked immunosorbent assay	20
2.7 Assessment of Macrophage Efferocytotic Capacity	20
2.8 In Vivo Atherosclerosis Mouse Model and ER-curcumin-NP Treatment	21
2.9 Body Weight Monitoring and Serum Lipid Analysis	22
2.10 Histological Assessment of Atherosclerotic Lesions	23
2.11 Statistical Analysis	23
Chapter 3 Results	25
3.1 NOX2 Status Is Associated with Macrophage Polarization Phenotype and the Therapeutic Response to ER-Targeting Nanovehicle–Encapsulated Curcumin	26
3.1.1 NOX2 Deficiency and Mutation Are Associated with a Proinflammatory Macrophage Phenotype	26
3.1.2 ER-targeting nanovehicle–encapsulated curcumin attenuates NOX2-associated proinflammatory macrophage phenotypes in a Genotype-Dependent Manner	27
3.1.3 Additional polarization-associated markers provide supportive phenotypic information.	28
3.1.4 Heatmap Visualization Summarizes Genotype- and Treatment-Associated Macrophage Phenotypic Patterns	30
3.2 NOX2 Functional Status Is Associated with Macrophage Atherogenic Functions and Responses to ER-Curcumin-NP	31
3.2.1 Genotype-Dependent IL-6 Secretion and Its Response to ER-Curcumin-NP in M1 Macrophages	32
3.2.2 NOX2 Status Affects Macrophage Efferocytotic Capacity	33
3.2.3 ER-curcumin-NP Enhances Efferocytosis in WT and NOX2-Mutant Macrophages with Limited Efficacy in NOX2-Deficient Macrophages	34
3.3 ER-Curcumin-NP Reduces Serum Lipids and Atherosclerotic Lesion Burden in HFD-Fed ApoE-Deficient Mice	36
3.3.1 Experimental Design of In Vivo ER-curcumin-NP Treatment in Atherosclerotic Mouse Models	36
3.3.2 ER-Curcumin-NP Reduces Serum Total Cholesterol and Triglyceride in HFD-Fed ApoE-Deficient Mice Without Significantly Altering Body Weight Gain	37
3.3.3 ER-Curcumin-NP Treatment Is Associated with Reduced Atherosclerotic Lesion Burden in ApoE-Deficient Mice	38
Chapter 4 Discussion	41
4.1 NOX2 Functional Status and Macrophage Polarization-Associated Phenotypes	42
4.2 Differential Therapeutic Responsiveness to ER-Curcumin-NP Across NOX2 Genotypes	43
4.3 Biological Interpretation of Macrophage Dysfunction in the ApoE⁻/⁻CybbC1024T Compound Genotype	45
4.4 In Vivo Relevance and Therapeutic Implications	47
4.5 Limitations and Concluding Remarks	48
Chapter 5 References	51
Chapter 6 Figure, Legends, and Table	57
Figure 1.	58
Figure 1. Experimental workflow for BMDM differentiation, macrophage polarization, ER-curcumin-NP treatment, and downstream functional analyses.	58
Figure 2.	59
Figure 2. Flow cytometry gating strategy	59
Figure 3.	60
Figure 3. Macrophage polarization-associated marker expression in M0 BMDMs across different genotypes.	60
Figure 4.	61
Figure 4. Expression of macrophage polarization-associated markers in M1 BMDMs from different NOX2 genetic backgrounds following ER-curcumin-NP treatment.	61
Figure 5.	62
Figure 5. Quantitative analysis of CD40 and CD206 expression in polarized BMDMs following ER-curcumin-NP treatment.	63
Figure 6.	64
Figure 6. Quantitative analysis of CD11c and NLRP3 expression in polarized BMDMs following ER-curcumin-NP treatment.	65
Figure 7.	66
Figure 7. Quantitative analysis of TNF-α and TGF-β expression in polarized BMDMs following ER-curcumin-NP treatment.	67
Figure 8.	68
Figure 8. Quantitative analysis of Arg1 expression in polarized BMDMs following ER-curcumin-NP treatment.	68
Figure 9.	69
Figure 9. Heatmap analysis of macrophage polarization-associated marker expression following ER-curcumin-NP treatment.	70
Figure 10.	71
Figure 10. Heatmap analysis of core M1 and M2-associated marker expression in WT, CybbC1024T, and Cybb⁻/⁻ macrophages under M1-polarizing conditions.	71
Figure 11.	72
Figure 11. ER-curcumin-NP suppresses IL-6 secretion in M1 macrophages.	72
Figure 12.	73
Figure 12. Schematic overview of the macrophage efferocytosis assay protocol.	73
Figure 13.	74
Figure 13. Representative immunofluorescence images of efferocytosis in polarized WT BMDMs following ER-curcumin-NP treatment.	74
Figure 14.	75
Figure 14. Representative immunofluorescence images of efferocytosis in polarized CybbC1024T BMDMs following ER-curcumin-NP treatment.	75
Figure 15.	76
Figure 15. Representative immunofluorescence images of efferocytosis in polarized ApoE-/-CybbC1024T BMDMs following ER-curcumin-NP treatment.	76
Figure 16.	77
Figure 16. Representative immunofluorescence images of efferocytosis in polarized Cybb-/- BMDMs following ER-curcumin-NP treatment.	77
Figure 17.	78
Figure 17. ER-curcumin-NP enhances M1 efferocytotic capacity in WT and NOX2-mutant macrophages with limited efficacy in NOX2-deficient macrophages.	78
Figure 18.	79
Figure 18. Experimental design for in vivo ER-curcumin-NP treatment in diet-induced atherosclerosis models.	79
Figure 19.	80
Figure 19. ER-curcumin-NP reduces HFD-induced dyslipidemia without altering body weight gain in ApoE-deficient atherosclerotic mice.	80
Figure 20.	81
Figure 20. HFD-Induced Aortic Lesion Burden in ApoE-Deficient Mice Is Attenuated by ER-Curcumin-NP Treatment.	81
Figure 21.	82
Figure 21. ER-curcumin-NP attenuates HFD-induced lipid-rich lesion accumulation in aortic root sections of ApoE⁻/⁻ and ApoE⁻/⁻CybbC1024T mice.	83
Figure 22.	84
Figure 22. NOX2 dysfunction promotes pro-inflammatory polarization and impairs efferocytosis. ER-curcumin-NP shows genotype-dependent efficacy and reduces atherosclerosis.	84
Table 1. Antibodies to identify macrophage phenotypes by flow cytometry analyses	85
                                    

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