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| Investigation on the effect of electroacupuncture on pulmonary alveolar macrophage polarization in sepsis mice based on mitochondrial transfer |
| Hits 395 Download times 165 Received:May 10, 2025 |
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| DOI
10.11656/j.issn.1672-1519.2025.09.14 |
| Key Words
electroacupuncture;sepsis;extracellular vesicles;mitochondrial transfer;macrophage polarization |
| Author Name | Affiliation | E-mail | | ZHENG Yaman | School of Acupuncture-Moxibustion and Tuina, Tianjin University of Traditional Chinese Medicine, Tianjin 301617, China | | | ZHANG Jingyu | School of Acupuncture-Moxibustion and Tuina, Tianjin University of Traditional Chinese Medicine, Tianjin 301617, China | | | CHEN Bo | School of Acupuncture-Moxibustion and Tuina, Tianjin University of Traditional Chinese Medicine, Tianjin 301617, China
National Clinical Research Center for Chinese Medicine Acupuncture and Moxibustion, Tianjin 300381, China | tjutcmchenbo@163.com | | LI Ningceng | School of Acupuncture-Moxibustion and Tuina, Tianjin University of Traditional Chinese Medicine, Tianjin 301617, China | | | LIAN Jinyu | School of Acupuncture-Moxibustion and Tuina, Tianjin University of Traditional Chinese Medicine, Tianjin 301617, China | | | LI Changqi | School of Acupuncture-Moxibustion and Tuina, Tianjin University of Traditional Chinese Medicine, Tianjin 301617, China | | | LI Xinyang | School of Acupuncture-Moxibustion and Tuina, Tianjin University of Traditional Chinese Medicine, Tianjin 301617, China | | | MA Jiani | School of Medical Technology, Tianjin University of Traditional Chinese Medicine, Tianjin 301617, China | | | WANG Jingwen | School of Acupuncture-Moxibustion and Tuina, Tianjin University of Traditional Chinese Medicine, Tianjin 301617, China | | | KUANG Xiaobing | School of Medical Technology, Tianjin University of Traditional Chinese Medicine, Tianjin 301617, China | |
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| Abstract
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| [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. |
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