负载白细胞介素-4的水凝胶微球调控卒中后免疫微环境对神经功能修复的影响

doi:10.3969/j.issn.1674-8115.2025.09.008

上海交通大学学报（医学版） ›› 2025, Vol. 45 ›› Issue (9): 1161-1170.doi: 10.3969/j.issn.1674-8115.2025.09.008

负载白细胞介素-4的水凝胶微球调控卒中后免疫微环境对神经功能修复的影响

徐彤彤¹^,², 阮慧瞳¹^,³()

^1.上海交通大学医学院附属瑞金医院骨科，上海市伤骨科研究所，上海 200025
^2.上海交通大学生物医学工程学院，上海 200030
^3.上海交通大学医学院医学技术学院，上海 200025

收稿日期:2025-05-05 接受日期:2025-08-16 出版日期:2025-09-28 发布日期:2025-09-30
通讯作者: 阮慧瞳，副研究员，博士；电子信箱：ruanhuitong@163.com/ruanhuitong@sjtu.edu.cn。
基金资助:
国家自然科学基金(82372120);上海交通大学医学院“双百人”项目(2024-0701)

Effects of hydrogel microspheres loaded with interleukin-4 on neural functional recovery by modulating the immune microenvironment after stroke

XU Tongtong¹^,², RUAN Huitong¹^,³()

^1.Shanghai Institute of Traumatology and Orthopaedics, Department of Orthopaedics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
^2.School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai 200030, China
^3.College of Health Science and Technology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China

Received:2025-05-05 Accepted:2025-08-16 Online:2025-09-28 Published:2025-09-30
Contact: RUAN Huitong, E-mail: ruanhuitong@163.com/ruanhuitong@sjtu.edu.cn.
Supported by:
National Natural Science Foundation of China(82372120);“Two-hundred Talents” Program of Shanghai Jiao Tong University School of Medicine(2024-0701)

摘要/Abstract

摘要：

目的·探讨负载免疫调控因子白细胞介素-4（interleukin-4，IL-4）的水凝胶微球（microsphere，MP）对卒中损伤后再生与修复的影响。方法·通过乳化法生成负载IL-4的纳米颗粒，并联合气流控法构建的聚乙烯醇（polyvinyl alcohol，PVA）水凝胶微球制得负载IL-4的PVA水凝胶微球（MP@IL-4）。使用光学显微镜对MP@IL-4的形态进行表征。体外培养BV2小胶质细胞，经MP@IL-4处理后，分别采用CCK-8法、活/死细胞染色法对其细胞活力、细胞存活进行检测。经脂多糖继续处理BV2小胶质细胞后，采用免疫荧光染色对其促炎标志物诱导型一氧化氮合酶（inducible nitric oxide synthase，iNOS）和抑炎标志物精氨酸酶1（arginase 1，Arg-1）的表达水平进行分析。同时，采用大脑中动脉栓塞法构建小鼠卒中模型，随机分为假手术组（Sham组）、缺血性卒中（ischemic stroke，IS）组和微球治疗组（IS-MP@IL-4组）。通过神经行为学评分、悬空旋转实验和悬丝实验评估3组小鼠的神经行为学功能，焦油紫染色检测3组小鼠的脑萎缩体积，免疫荧光染色表征3组小鼠小胶质细胞促炎因子CD86和抑炎因子CD206的表达水平。结果·光学显微镜的观察结果显示成功制备了粒径为200 μm的MP@IL-4，且不影响BV2小胶质细胞的细胞活力和存活。体外和体内实验均证实，MP@IL-4可下调小胶质细胞的促炎标志物的表达，上调抑炎标志物的表达。此外，与IS组相比，IS-MP@IL-4组小鼠的神经行为学功能有较大改善（P<0.05），脑萎缩体积也有所减小（P<0.001）。结论·MP@IL-4可通过改善免疫微环境发挥对卒中后神经功能损伤的治疗作用。

关键词: 水凝胶微球, 药物递送, 缺血性卒中, 抑炎作用, 神经修复

Abstract:

Objective ·To explore the effects of hydrogel microsphere (MP) loaded with the immunoregulatory factor interleukin-4 (IL-4) on the regeneration and repair after stroke injury. Methods ·IL-4-loaded nanoparticles were prepared using an emulsification method and subsequently incorporated into polyvinyl alcohol (PVA) hydrogel microspheres via an airflow technique to obtain IL-4-loaded PVA hydrogel microspheres (MP@IL-4). The morphology of MP@IL-4 was characterized by optical microscopy. BV2 microglia were cultured in vitro and treated with MP@IL-4. Cell viability and survival were detected by the CCK-8 assay and live/dead cell staining, respectively. Subsequently, BV2 microglia were further treated with lipopolysaccharide (LPS), and the expression levels of the pro-inflammatory marker inducible nitric oxide synthase (iNOS) and the anti-inflammatory marker arginase 1 (Arg-1) were detected by immunofluorescence staining. Meanwhile, a mouse stroke model was constructed using the middle cerebral artery occlusion method, and the mice were randomly divided into the sham operation group (Sham group), the ischemic stroke (IS) group and the microsphere treatment group (IS-MP@IL-4 group). The neurobehavioral functions of mice in the three groups were detected by the modified Neurological Severity Score, elevated body swing test, and hanging wire test. The brain atrophy volume was detected by cresyl violet staining. Immunofluorescence staining was used to assess the expression levels of the pro-inflammatory factor CD86 and the anti-inflammatory factor CD206 in microglia. Results ·Optical microscopy confirmed the successful fabrication of MP@IL-4 with a particle size of 200 μm, which did not affect the cell viability and survival of BV2 microglia. Both in vitro and in vivo experiments demonstrated that MP@IL-4 downregulated the expression of pro-inflammatory markers and upregulated the expression of anti-inflammatory markers in microglia. Moreover, compared with the IS group, the neurobehavioral function of mice in the IS-MP@IL-4 group was significantly improved (P<0.05), and the brain atrophy volume was reduced (P<0.001). Conclusion ·MP@IL-4 can exert a therapeutic effect on post-stroke neurofunctional injury by improving the immune microenvironment.

Key words: hydrogel microsphere, drug delivery, ischemic stroke (IS), anti-inflammatory effect, neural repair

中图分类号:

R743

徐彤彤, 阮慧瞳. 负载白细胞介素-4的水凝胶微球调控卒中后免疫微环境对神经功能修复的影响[J]. 上海交通大学学报（医学版）, 2025, 45(9): 1161-1170.

XU Tongtong, RUAN Huitong. Effects of hydrogel microspheres loaded with interleukin-4 on neural functional recovery by modulating the immune microenvironment after stroke[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2025, 45(9): 1161-1170.

图/表 5

图 1 MP@IL-4的表征及释放曲线Note： A. Characterization of IL4-NP size distribution by dynamic light scattering. B. Observation of the morphology and diameter size of MP@IL-4 by microscopy (×100). C. Drug release profile of MP@IL-4 (n=3).

Fig 1 Characterization and release profile of MP@IL-4

图 2 BV2小胶质细胞的增殖与存活分析Note： A/C. Cell viability of PBS and MP@IL-4 co-cultured with BV2 microglia for 24 h (A) or 48 h (C), respectively (n=3). B/D. Representative Calcein-AM/PI staining images of PBS and MP@IL-4 co-cultured with BV2 microglia for 24 h (B) or 48 h (D), respectively (×100).

Fig 2 Analysis of proliferation and viability of BV2 microglia

图3 MP@IL-4在体外对BV2小胶质细胞极化水平的影响（×100）Note：A. Representative immunostaining images of iNOS with IBA-1. B. Representative immunostaining images of Agr-1 with IBA-1.

Fig 3 Effect of MP@IL-4 on the polarization level of BV2 microglia in vitro (×100)

图4 MP@IL-4对IS小鼠神经功能损伤的影响Note： A. Statistical analysis of body weight in the three groups. B‒D. Statistical analysis of mNSS (B), hanging wire test (C), and EBST (D) of the three groups after tMCAO model establishment (n=7). E/F. Quantification of brain atrophy volume by cresyl violet staining at 14 d after tMCAO model establishment (n=3).

Fig 4 Effects of MP@IL-4 on the neural functional recovery of IS mice

图5 MP@IL-4对小鼠小胶质细胞极化水平的影响（×200）Note： A. Representative immunostaining images of CD86 and IBA-1 at day 14 post-IS. B. Representative immunostaining images of CD206 and CD11b at day 14 post-IS.

Fig 5 Effects of MP@IL-4 on the polarization level of mouse microglia (×200)

参考文献 36

[1]	XU T T, CHEN P Q, WANG J X, et al. Introduction to biomedical engineering in stroke diagnosis and treatment[J]. Stroke, 2022, 53(11): e487-e489.
[2]	JIANG Y X, WANG R Q, WANG C, et al. Brain microenvironment responsive and pro-angiogenic extracellular vesicle-hydrogel for promoting neurobehavioral recovery in type 2 diabetic mice after stroke[J]. Adv Healthc Mater, 2022, 11(22): e2201150.
[3]	HILKENS N A, CASOLLA B, LEUNG T W, et al. Stroke[J]. Lancet, 2024, 403(10446): 2820-2836.
[4]	DELONG J H, OHASHI S N, O'CONNOR K C, et al. Inflammatory responses after ischemic stroke[J]. Semin Immunopathol, 2022, 44(5): 625-648.
[5]	MENDELSON S J, PRABHAKARAN S. Diagnosis and management of transient ischemic attack and acute ischemic stroke: a review[J]. JAMA, 2021, 325(11): 1088-1098.
[6]	WASSÉLIUS J, ARNBERG F, VON EULER M, et al. Endovascular thrombectomy for acute ischemic stroke[J]. J Intern Med, 2022, 291(3): 303-316.
[7]	JOLUGBO P, ARIËNS R A S. Thrombus composition and efficacy of thrombolysis and thrombectomy in acute ischemic stroke[J]. Stroke, 2021, 52(3): 1131-1142.
[8]	DORDOE C, HUANG W T, BWALYA C, et al. The role of microglial activation on ischemic stroke: modulation by fibroblast growth factors[J]. Cytokine Growth Factor Rev, 2023, 74: 122-133.
[9]	MO Y, XU W L, FU K J, et al. The dual function of microglial polarization and its treatment targets in ischemic stroke[J]. Front Neurol, 2022, 13: 921705.
[10]	QIN C, ZHOU L Q, MA X T, et al. Dual functions of microglia in ischemic stroke[J]. Neurosci Bull, 2019, 35(5): 921-933.
[11]	MA Y Y, WANG J X, WANG Y T, et al. The biphasic function of microglia in ischemic stroke[J]. Prog Neurobiol, 2017, 157: 247-272.
[12]	LIU X, LIU J, ZHAO S, et al. Interleukin-4 is essential for microglia/macrophage M2 polarization and long-term recovery after cerebral ischemia[J]. Stroke, 2016, 47(2): 498-504.
[13]	JIANG X, YI S, LIU Q, et al. The secretome of microglia induced by IL-4 of IFN-γ differently regulate proliferation, differentiation and survival of adult neural stem/progenitor cell by targeting the PI3K-Akt pathway[J]. Cytotechnology, 2022, 74(3): 407-420.
[14]	XU J, CHEN Z Q, YU F, et al. IL-4/STAT6 signaling facilitates innate hematoma resolution and neurological recovery after hemorrhagic stroke in mice[J]. Proc Natl Acad Sci U S A, 2020, 117(51): 32679-32690.
[15]	SONG G N, ZHAO M, CHEN H M, et al. The role of nanomaterials in stroke treatment: targeting oxidative stress[J]. Oxid Med Cell Longev, 2021, 2021: 8857486.
[16]	GHUMAN H, GERWIG M, NICHOLLS F J, et al. Long-term retention of ECM hydrogel after implantation into a sub-acute stroke cavity reduces lesion volume[J]. Acta Biomater, 2017, 63: 50-63.
[17]	ROTARU-ZĂVĂLEANU A D, DINESCU V C, ALDEA M, et al. Hydrogel-based therapies for ischemic and hemorrhagic stroke: a comprehensive review[J]. Gels, 2024, 10(7): 476.
[18]	LI X, HAN Z H, WANG T Y, et al. Cerium oxide nanoparticles with antioxidative neurorestoration for ischemic stroke[J]. Biomaterials, 2022, 291: 121904.
[19]	BAI Y, HAN B, ZHANG Y, et al. Advancements in hydrogel application for ischemic stroke therapy[J]. Gels, 2022, 8(12): 777.
[20]	CAI L, GONG Q, QI L, et al. ACT001 attenuates microglia-mediated neuroinflammation after traumatic brain injury via inhibiting AKT/NF-κB/NLRP3 pathway[J]. Cell Commun Signal, 2022, 20(1): 56.
[21]	LI G Q, ZHANG R L, CHEN K Y, et al. Zinc sulfide nanoparticles serve as gas slow-release bioreactors for H₂S therapy of ischemic stroke[J]. Biomaterials, 2025, 315: 12291.
[22]	LALANI J, PATIL S, KOLATE A, et al. Protein-functionalized PLGA nanoparticles of lamotrigine for neuropathic pain management[J]. AAPS PharmSciTech, 2015, 16(2): 413-427.
[23]	SU Y, ZHANG B L, SUN R W, et al. PLGA-based biodegradable microspheres in drug delivery: recent advances in research and application[J]. Drug Deliv, 2021, 28(1): 1397-1418.
[24]	STOJILJKOVIĆ A, KUEHNI-BOGHENBOR K, GASCHEN V, et al. High-content analysis of factors affecting gold nanoparticle uptake by neuronal and microglial cells in culture[J]. Nanoscale, 2016, 8(37): 16650-16661.
[25]	PEVIANI M, CAPASSO PALMIERO U, CECERE F, et al. Biodegradable polymeric nanoparticles administered in the cerebrospinal fluid: brain biodistribution, preferential internalization in microglia and implications for cell-selective drug release[J]. Biomaterials, 2019, 209: 25-40.
[26]	LUO L X, YANG J, OH Y, et al. Controlled release of corticosteroid with biodegradable nanoparticles for treating experimental autoimmune uveitis[J]. J Control Release, 2019, 296: 68-80.
[27]	BENHABBOUR S R, SHEARDOWN H, ADRONOV A. Cell adhesion and proliferation on hydrophilic dendritically modified surfaces[J]. Biomaterials, 2008, 29(31): 4177-4186.
[28]	FERNÁNDEZ-ALBARRAL J A, RAMÍREZ A I, DE HOZ R, et al. Neuroprotective and anti-inflammatory effects of a hydrophilic saffron extract in a model of glaucoma[J]. Int J Mol Sci, 2019, 20(17): 4110.
[29]	GORIELY A, GEERS M G, HOLZAPFEL G A, et al. Mechanics of the brain: perspectives, challenges, and opportunities[J]. Biomech Model Mechanobiol, 2015, 14(5): 931-965.
[30]	BLASCHKE S J, DEMIR S, KÖNIG A, et al. Substrate elasticity exerts functional effects on primary microglia[J]. Front Cell Neurosci, 2020, 14: 590500.
[31]	MOSHAYEDI P, NG G, KWOK J C, et al. The relationship between glial cell mechanosensitivity and foreign body reactions in the central nervous system[J]. Biomaterials, 2014, 35(13): 3919-3925.
[32]	KHODOUN M, LEWIS C C, YANG J Q, et al. Differences in expression, affinity, and function of soluble (s)IL-4Rα and sIL-13Rα2 suggest opposite effects on allergic responses[J]. J Immunol, 2007, 179(10): 6429-6438.
[33]	LIU H, HE Y, LU C, et al. Efficacy of pulmonary transplantation of engineered macrophages secreting IL-4 on acute lung injury in C57BL/6J mice[J]. Cell Death Dis, 2019, 10(9): 664.
[34]	WANG J, WANG L L, WU Q J, et al. Interleukin-4 modulates neuroinflammation by inducing phenotypic transformation of microglia following subarachnoid hemorrhage[J]. Inflammation, 2024, 47(1): 390-403.
[35]	HE Y, GAO Y, ZHANG Q, et al. IL-4 switches microglia/macrophage M1/M2 polarization and alleviates neurological damage by modulating the JAK1/STAT6 pathway following ICH[J]. Neuroscience, 2020, 437: 161-171.
[36]	SMALL D L, BUCHAN A M. Animal models[J]. Br Med Bull, 2000, 56(2): 307-317.

负载白细胞介素-4的水凝胶微球调控卒中后免疫微环境对神经功能修复的影响

Effects of hydrogel microspheres loaded with interleukin-4 on neural functional recovery by modulating the immune microenvironment after stroke

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