基于线粒体转移探讨电针调控脓毒症小鼠肺泡巨噬细胞极化的作用研究

Investigation on the effect of electroacupuncture on pulmonary alveolar macrophage polarization in sepsis mice based on mitochondrial transfer

关键词:  电针  脓毒症  细胞外囊泡  线粒体转移  巨噬细胞极化

Key words:  electroacupuncture  sepsis  extracellular vesicles  mitochondrial transfer  macrophage polarization

摘要:

[目的] 重点聚焦于骨髓细胞外囊泡（EVs）介导线粒体转移，探究电针是否通过小鼠胫骨骨髓EVs介导的线粒体转移来改善脓毒症小鼠肺泡巨噬细胞的线粒体功能，调节肺泡巨噬细胞极化。[方法] 1）将55只C57BL/6J雄性小鼠随机分为空白组、模型组和模型+电针1 d 0.5 mA组、模型+电针1 d 1.5 mA组、模型+电针1 d 2.5 mA组、模型+电针3 d 0.5 mA组、模型+电针3 d 1.5 mA组、模型+电针3 d 2.5 mA组、模型+电针7 d 0.5 mA组、模型+电针7 d 1.5 mA组、模型+电针7 d 2.5 mA组，每组5只，空白组腹腔注射磷酸缓冲盐溶液（PBS），模型组腹腔注射脂多糖造模，电针组在造模前对双侧足三里进行电针预处理。造模后24 h，采用流式细胞术检测小鼠肺脏的线粒体膜电位和活性氧。2）将15只C57BL/6J雄性小鼠随机分为空白组、模型组和电针组，每组5只，空白组腹腔注射PBS溶液，模型组腹腔注射脂多糖造模，电针组在造模前进行电针预处理（双侧足三里，3 d，连续波，10 Hz，1.5 mA）。造模后24 h，采用流式细胞术检测肺泡巨噬细胞的M₂、M₁的数量、线粒体膜电位和活性氧。3）使用EXODUS全自动外泌体提取系统提取未针刺正常小鼠骨髓EVs（NC-EVs）和电针3 d 正常小鼠骨髓EVs（ACU-EVs），并用线粒体红色荧光染料标记。选取15只C57BL/6J雄性健康小鼠为研究对象，随机分为空白组、NC-EVs组和ACU-EVs组，每组5只。空白组尾静脉回输PBS溶液作为对照，NC-EVs组回输NC-EVs，ACU-EVs组回输ACU-EVs，回输剂量均为200 μL，EVs浓度均为50 μg/200 μL。回输30 min后，空白组、模型+NC-EVs组和模型+ACU-EVs组进行造模。造模后8 h 用小动物活体成像仪观测肺脏的荧光富集情况；采用HIS-SIM超分辨显微镜观察骨髓EVs中是否含有线粒体或线粒体片段；将ACU-EVs与小鼠肺泡巨噬细胞系（MH-S）共培养，观察细胞摄取情况。[结果] 1）与空白组相比，模型组小鼠肺脏的线粒体膜电位显著降低（P＜0.01），活性氧显著升高（P＜0.01）；与模型组相比，电针1 d 2.5 mA组、3 d 1.5 mA组和7 d 0.5 mA组的小鼠肺脏线粒体膜电位显著升高（P＜0.05），电针1 d 0.5 mA组、1 d 2.5 mA组、3 d 1.5 mA组、3 d 2.5 mA组、7 d 0.5 mA组和7 d 2.5 mA组活性氧显著降低（P＜0.05），肺脏线粒体功能得到恢复，因此后续实验选择电针参数为3 d 1.5 mA。2）与空白组相比，模型组肺泡巨噬细胞M₂/M₁的比例显著降低（P＜0.01），线粒体膜电位显著降低（P＜0.01），活性氧显著升高（P＜0.01）；与模型组相比，电针组肺泡巨噬细胞M₂/M₁的比例显著升高（P＜0.05），线粒体的膜电位明显升高（P＜0.01），活性氧明显降低（P＜0.01）。3）HIS-SIM超分辨显微镜下观察到ACU-EVs中存在线粒体或线粒体片段；与回输NC-EVs组相比，回输ACU-EVs组小鼠肺脏荧光强度显著升高（P＜0.05）；激光共聚焦显微镜下观察到ACU-EVs被MH-S细胞所摄取。[结论] 电针可以改善脓毒症小鼠肺泡巨噬细胞线粒体功能，调节肺泡巨噬细胞极化，其作用机制可能与电针驱动骨髓EVs介导线粒体转移有关。

Abstract:

[Objective] This study focused on the process of mitochondrial transfer mediated by bone marrow-derived extracellular vesicles（EVs），aiming to investigate whether electroacupuncture（EA） treatment improves mitochondrial function in alveolar macrophages of septic mice and modulates their polarization through EV-mediated mitochondrial transfer from tibial bone marrow cells. The experimental models and multi-omics data were collected to validate this regulatory mechanism and elucidate the potential therapeutic pathway for sepsis. [Methods] 1）A total of 55 male C57BL/6J mice were randomly assigned to 11 groups（n=5 per group）：a blank control group，a model group，and nine experimental groups that received electroacupuncture at different durations and intensities. The experimental groups included：model + electroacupuncture for 1 day at 0.5 mA，1.5 mA，and 2.5 mA；model + electroacupuncture for 3 days at 0.5 mA，1.5 mA，and 2.5 mA；and model + electroacupuncture for 7 days at 0.5 mA，1.5 mA，and 2.5 mA. Mice in the blank control group were intraperitoneally injected with PBS solution，while those in the model group were intraperitoneally injected with LPS to induce the model. Prior to LPS injection，bilateral Zusanli acupoints were pre-treated with electroacupuncture according to the respective group conditions. Mitochondrial membrane potential and reactive oxygen species levels were assessed by flow cytometry 24 hours post-modeling. 2）The 15 male C57BL/6J mice were randomly assigned to three groups（n=5 per group）：control，sepsis model，and EA pre-treatment. The control group received intraperitoneal injections of phosphate-buffered saline（PBS），while the sepsis model group was injected with lipopolysaccharide（LPS） to induce sepsis. The EA pre-treatment group underwent electroacupuncture at bilateral Zusanli acupoints（3 days，continuous wave，10 Hz，1.5 mA） prior to LPS injection. After 24 hours post-LPS administration，flow cytometry was employed to assess the M2/M1 ratio of alveolar macrophages，mitochondrial membrane potential，and reactive oxygen species（ROS） levels. 3）Extracellular vesicles（EVs） were isolated from the bone marrow of non-acupuncture-treated normal mice（NC-EVs） and normal mice subjected to 3 days of acupuncture treatment（Acupuncture-derived EVs，ACU-EVs） using the EXODUS automated exosome extraction system. The EVs were labeled with a mitochondrial red fluorescent dye. A total of 15 healthy male C57BL/6J mice were selected as experimental subjects and randomly divided into three groups：a control group，an NC-EVs group，and an ACU-EVs group，with five mice in each group. In the control group，phosphate-buffered saline（PBS） solution was intravenously administered via the tail vein as a control. The NC-EVs group received NC-EVs，and the ACU-EVs group received ACU-EVs，with a dosage of 200 μL per injection and an EV concentration of 50 μg/200 μL. Thirty minutes after administration，all groups underwent modeling. Eight hours after modeling，fluorescence accumulation in the lungs was observed using a small animal in vivo imaging system. Subsequently，the presence of mitochondria or mitochondrial fragments within the bone marrow-derived EVs was examined using HIS-SIM super-resolution microscopy. Additionally，ACU-EVs were co-cultured with mouse alveolar macrophage cells（MH-S） to observe cellular uptake. [Results] 1） Compared with the control group，the mitochondrial membrane potential in the lungs of the model group was significantly reduced（P<0.01），and reactive oxygen species（ROS） levels were markedly elevated（P<0.01）. These findings indicate impaired mitochondrial function in the lungs. In comparison to the model group，the mitochondrial membrane potential in the lungs of mice subjected to electroacupuncture at 2.5 mA for 1 day，1.5 mA for 3 days，and 0.5 mA for 7 days showed significant increases（P<0.05）. Additionally，ROS levels were significantly decreased by electroacupuncture in groups treated with 0.5 mA for 1 day，2.5 mA for 1 day，1.5 mA for 3 days，2.5 mA for 3 days，0.5 mA for 7 days，and 2.5 mA for 7 days（P<0.05）. These results suggest that electroacupuncture restored lung mitochondrial function. Based on these findings，the optimal electroacupuncture parameter for subsequent experiments was determined to be 1.5 mA for 3 days. 2）Compared to the control group，the sepsis model group exhibited a significant decrease in the M2/M1 ratio of alveolar macrophages（P<0.01），reduced mitochondrial membrane potential（P<0.01），and elevated ROS levels（P<0.01）. Conversely，the EA pre-treatment group showed a significant increase in the M2/M1 ratio（P<0.05），enhanced mitochondrial membrane potential（P<0.01），and decreased ROS levels（P<0.01）. 3）The presence of mitochondria or mitochondrial fragments in ACU-EVs was observed under HIS-SIM superresolution microscope. Compared with NC-EVs group，the pulmonary fluorescence intensity in ACU-EVs group was significantly increased（P<0.05）. ACU-EVs uptake by MH-S cells was observed under confocal laser microscopy. [Conclusion] Electroacupuncture improves mitochondrial function and regulates alveolar macrophage polarization in septic mice，potentially through the transfer of mitochondria mediated by bone marrow EVs.