目的 探究巨噬細(xì)胞膜包被的黃芩苷脂質(zhì)體（macrophage membrane-coated baicalin liposomes，MM-BA-LP）的血清穩(wěn)定性、免疫逃逸及腦靶向性，以及其對(duì)小鼠腦全缺血再灌注損傷（global cerebral ischemia-reperfusion injury，GCIRI）的保護(hù)作用。方法 通過(guò)吸光度分析MM-BA-LP血清穩(wěn)定性；利用熒光顯微鏡和流式細(xì)胞儀評(píng)估MM-BA-LP的免疫逃逸能力；使用活體成像系統(tǒng)研究MM-BA-LP在腦組織中的分布；通過(guò)CCK-8法確定安全給藥范圍；構(gòu)建細(xì)胞氧糖剝奪/再灌注（oxygen glucose deprivation/reperfusion，OGD/R）模型和小鼠GCIRI模型，進(jìn)行神經(jīng)功能評(píng)分、曠場(chǎng)實(shí)驗(yàn)、蘇木素-伊紅（hematoxylin-eosin）染色以評(píng)價(jià)藥效，并通過(guò)ELISA和Western blotting實(shí)驗(yàn)進(jìn)行抗炎機(jī)制研究。結(jié)果 MM-BA-LP具有良好的血清穩(wěn)定性；活體成像結(jié)果顯示，MM-BA-LP組腦部熒光強(qiáng)度高于BA-LP組；CCK-8實(shí)驗(yàn)結(jié)果顯示，MM-BA-LP對(duì)BV2細(xì)胞的毒性低于BA-LP；藥效結(jié)果顯示，MM-BA-LP顯著改善GCIRI小鼠的運(yùn)動(dòng)和神經(jīng)功能（P＜0.001），且能顯著改善腦組織炎性病變；機(jī)制研究結(jié)果顯示，MM-BA-LP能顯著降低OGD/R細(xì)胞與GCIRI小鼠腦組織中高遷移率族蛋白B1（high mobility group box 1 protein，HMGB1）、Toll樣受體4（Toll-like receptor 4，TLR4）和磷酸化核因子-κB（phosphorylated nuclear factor-κB，p-NF-κB）/NF-κB的表達(dá)（P＜0.05、0.01、0.001），且能顯著下調(diào)炎癥因子白細(xì)胞介素-6（interleukin-6，IL-6）、白細(xì)胞介素-1β、腫瘤壞死因子-α（tumor necrosis factor-α，TNF-α）的水平（P＜0.05、0.01、0.001）；且HMGB1特異性抑制劑甘草酸與MM-BA-LP聯(lián)用能顯著改善GCIRI小鼠腦組織炎性病變。結(jié)論 MM-BA-LP具有良好的穩(wěn)定性、免疫逃逸能力和腦靶向性，能顯著抑制GCIRI后的炎癥反應(yīng)和神經(jīng)功能損傷，其機(jī)制可能涉及HMGB1/TLR4/NF-κB通路。

Objective To investigate the serum stability, immune evasion and brain targeting of macrophage membrane-coated baicalin liposomes (MM-BA-LP), and study its protective effect on global cerebral ischemia-reperfusion injury (GCIRI) in mice. Methods The serum stability of MM-BA-LP was analyzed by absorbance; Fluorescence microscopy and flow cytometry were used to assess immune evasion of MM-BA-LP; The distribution of MM-BA-LP in brain tissue was studied using a in vivo bioluminescence imaging; The safe dosing range was determined by CCK-8 method; Oxygen glucose deprivation/reperfusion (OGD/R) model and mice GCIRI model were constructed, neurological severity score, open field experiments and hematoxylin-eosin (HE) staining were performed to evaluate drug efficacy, and the anti-inflammatory mechanism was studied by ELISA and Western blotting. Results MM-BA-LP had good serum stability. The results of in vivo imaging showed that the fluorescence intensity of MM-BA-LP group was higher than that of BA-LP group. The results of CCK-8 experiments showed that the toxicity of MM-BA-LP to BV2 cells was lower than that of BA-LP. The efficacy results showed that MM-BA-LP significantly improved the motor and nerve function of GCIRI mice (P < 0.001), and significantly improved the inflammatory lesions of brain tissue. Mechanistic studies showed that MM-BA-LP significantly reduced the expressions of high mobility group box 1 protein (HMGB1), Toll-like receptor 4 (TLR4) and phosphorylated nuclear factor-κB (p-NF-κB)/NF-κB in OGD/R cells and brain tissues of GCIRI mice (P < 0.05, 0.01, 0.001), and significantly down-regulated the levels of inflammatory cytokines such as interleukin-6 (IL-6), IL-1β and tumor necrosis factor-α (TNF-α) (P < 0.05, 0.01, 0.001). In addition, the combination of HMGB1 specifically inhibitor glycyrrhizic acid and MM-BA-LP significantly improved the inflammatory lesions of brain tissue in GCIRI mice. Conclusion MM-BA-LP has good serum stability, immune escape ability and brain targeting, and could significantly inhibit the inflammatory response and neurological impairment after GCIRI, and the mechanism may involve HMGB1/TLR4/NF-κB pathway.

R285.5

國(guó)家自然科學(xué)基金資助項(xiàng)目（82474220）