Journal of Traditional Chinese Medicine ›› 2025, Vol. 45 ›› Issue (2): 335-347.DOI: 10.19852/j.cnki.jtcm.20220419.001
• Original articles • Previous Articles Next Articles
Efficacy of electro-acupuncture at “Weizhong” (BL40) on macrophage polarization in rats with injured lumbar multifidus
TIAN Yuan1,2, BU He2, WANG Tieshan3, YANG Dongliang4, ZHANG Wei7, LIU Tong5,6, ZHANG Li1(
), HUO Zejun8()
- 1 Department of Acupuncture-Moxibustion and Tuina, Beijing University of Chinese Medicine, Beijing 100029, China
2 Department of Acupuncture-Moxibustion and Tuina, the Second Affiliated Hospital of Baotou Medical College, Inner Mongolia Autonomous Region, Baotou 014030, China
3 Beijing Research Institute of Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029, China
4 Cangzhou Medical College, Cangzhou 061000, China
5 the Fifth College of Clinical Medicine, Guangzhou University of Traditional Chinese Medicine, Guangzhou 510006, China
6 Department of Acupuncture and Rehabilitation, Guangdong Second Hospital of Traditional Chinese Medicine, Guangzhou 510095, China
7 Department of Pathology, the First Affiliated Hospital of Baotou Medical College, Inner Mongolia University of Science and Technology, Inner Mongolia 014010, China
8 Department of Chinese Medicine, Peking University 3rd Hospital, Beijing 100191, China
-
Received:2022-01-22Accepted:2022-03-17Online:2025-04-15Published:2022-04-19 -
Contact:Prof. ZHANG Li, Department of Acupuncture-Moxibustion and Tuina, Beijing University of Chinese Medicine, Beijing 100029, China. zhangli1572@sina.com; HUO Zejun, Department of Chinese Medicine, Peking University 3rd Hospital, Beijing 100191, China. huozejun@163.com, Telephone: +86-10-64287525 -
Supported by:Natural Science Foundation-funded Project: Regulation of Autophagy by Electroacupuncture at Weizhong Point on the Proliferation of Multifidus Muscle Satellite Cells(81574052);Exosomes Arounding Acupoints Deliver miR-206 to Mediate Insulin - like Growth Factor 1/Phosphatidylinositol 3 - Kinase/Protein Kinase B Pathway and Regulate Muscle Satellite Cell Differentiation: Mechanism of Electroacupuncture in Promoting Rehabilization of Lumbar Multifidus Muscles Injury in Model Rats(82174482);Major Program of Foundation of Inner Mongolia Autonomous Region: Study on the Eeffect of Pediatric Tuina Combined with Acupuncture on the Intestinal Microflora Structure in Children with Provisional Tic Disorder and its Therapeutic Mechanism(201802127)
Cite this article
TIAN Yuan, BU He, WANG Tieshan, YANG Dongliang, ZHANG Wei, LIU Tong, ZHANG Li, HUO Zejun. Efficacy of electro-acupuncture at “Weizhong” (BL40) on macrophage polarization in rats with injured lumbar multifidus[J]. Journal of Traditional Chinese Medicine, 2025, 45(2): 335-347.
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Figure 1 LM muscular H&E staining at different time points (1-, 2-, 3-, and 5-days) in each group BPVC injection induced the infiltration of local macrophages at the L4-L5 segment of lumbar multifidus. Original magnification: A × 100, B × 400, scale bar = 50 μm. A1-A2: control group, B1-B2: 1d Model group, B3-B4: 1d EA group, C1-C2: 2d Model group, C3-C4: 2 d EA group, D1-D2: 3 d Model group, D3-D4: 3 d EA group; E1-E2: 5 d Model group, E3-E4: 5 d EA group. The rats in the EA group received EA treatment [bilateral Weizhong (BL 40) acupoints, frequency of 2/10 Hz and an intensity of 2 mA for a total of 30 min] 24 h post-anesthesia recovery and model establishment, followed by daily sessions until euthanasia was performed. In the control group and model group, only grabbing and fixing were administered. LM: Lumbar multifidus; BPVC: Bupivacaine.
Figure 1 LM muscular H&E staining at different time points (1-, 2-, 3-, and 5-days) in each group BPVC injection induced the infiltration of local macrophages at the L4-L5 segment of lumbar multifidus. Original magnification: A × 100, B × 400, scale bar = 50 μm. A1-A2: control group, B1-B2: 1d Model group, B3-B4: 1d EA group, C1-C2: 2d Model group, C3-C4: 2 d EA group, D1-D2: 3 d Model group, D3-D4: 3 d EA group; E1-E2: 5 d Model group, E3-E4: 5 d EA group. The rats in the EA group received EA treatment [bilateral Weizhong (BL 40) acupoints, frequency of 2/10 Hz and an intensity of 2 mA for a total of 30 min] 24 h post-anesthesia recovery and model establishment, followed by daily sessions until euthanasia was performed. In the control group and model group, only grabbing and fixing were administered. LM: Lumbar multifidus; BPVC: Bupivacaine.
Figure 2 IHC showed electro-acupuncture reduced the positive expression of macrophages at the L4-L5 segment of lumbar multifidus A1-A2: control group; B1-B2: 2 d Model group; B3-B4: 2 d EA group; C1-C2: 2 d Model group; C3-C4: 2 d EA group; D1-D2: 5 d Model group; D3-D4: 5d EA group. Yellow brown-colored cells represent CD68+ macrophages. Black scale bar: 50 μm. Original magnification: A1, B1, C1, D1, B3, C3, D3 × 100, A2, B2, C2, D2, B4, C4, D4 × 400. E-F: The sum area and mean integrated optical density represents the expression level of CD68+ macrophages (n = 6). “a” indicates a significant difference (P < 0.001) when compared with the Control group; “b” indicates a difference (P < 0.001) when compared with the Model group; “c” indicates a difference (P < 0.05) when compared with the Control group; “d” indicates a difference (P < 0.05) when compared with the Model group. IHC: immunohistochemical staining.
Figure 2 IHC showed electro-acupuncture reduced the positive expression of macrophages at the L4-L5 segment of lumbar multifidus A1-A2: control group; B1-B2: 2 d Model group; B3-B4: 2 d EA group; C1-C2: 2 d Model group; C3-C4: 2 d EA group; D1-D2: 5 d Model group; D3-D4: 5d EA group. Yellow brown-colored cells represent CD68+ macrophages. Black scale bar: 50 μm. Original magnification: A1, B1, C1, D1, B3, C3, D3 × 100, A2, B2, C2, D2, B4, C4, D4 × 400. E-F: The sum area and mean integrated optical density represents the expression level of CD68+ macrophages (n = 6). “a” indicates a significant difference (P < 0.001) when compared with the Control group; “b” indicates a difference (P < 0.001) when compared with the Model group; “c” indicates a difference (P < 0.05) when compared with the Control group; “d” indicates a difference (P < 0.05) when compared with the Model group. IHC: immunohistochemical staining.
Figure 3 IFA showed EA regulated M1-macrophage polarization in injured LM A1-A4: control group; B1-B4: model 2 d group; C1-C4: EA 2 d group; D1-D4: model 3 d group; E1-E4: EA 3 d group; F: Counting and analysis were carried out in the same visual field (315.87 μm × 315.87 μm), and the cell counts of iNOs+ CD68+ M1-macrophagess at different time points (2- and 3 d) in each group (n = 6) were recorded. “a” indicates a significant difference (P < 0.001) when compared with the Control group; “b” indicates a difference (P < 0.001) when compared with the Model group; “c” indicates a difference (P < 0.05) when compared with the control group. Double immunofluorescence images of CD68 (green) and iNOS (red). Asterisks indicate co-expression. Arrowheads indicate myofibers. Arrows represents cells only expressing CD68 or iNOs, respectively. DAPI (blue) was used for nuclear staining. Scale bar: 50 μm. Original magnification: × 400. LM: Lumbar multifidus; IFA: Immunofluorescence Assay; EA: Electro-acupuncture.
Figure 3 IFA showed EA regulated M1-macrophage polarization in injured LM A1-A4: control group; B1-B4: model 2 d group; C1-C4: EA 2 d group; D1-D4: model 3 d group; E1-E4: EA 3 d group; F: Counting and analysis were carried out in the same visual field (315.87 μm × 315.87 μm), and the cell counts of iNOs+ CD68+ M1-macrophagess at different time points (2- and 3 d) in each group (n = 6) were recorded. “a” indicates a significant difference (P < 0.001) when compared with the Control group; “b” indicates a difference (P < 0.001) when compared with the Model group; “c” indicates a difference (P < 0.05) when compared with the control group. Double immunofluorescence images of CD68 (green) and iNOS (red). Asterisks indicate co-expression. Arrowheads indicate myofibers. Arrows represents cells only expressing CD68 or iNOs, respectively. DAPI (blue) was used for nuclear staining. Scale bar: 50 μm. Original magnification: × 400. LM: Lumbar multifidus; IFA: Immunofluorescence Assay; EA: Electro-acupuncture.
Figure 4 IFA showed EA facilitated M2-macrophage polarization in injured LM A1-A4: control group; B1-B4: model 3 d group; C1-C4: EA 3 d group; D1-D4: model 5-days group; E1-E4: EA 5-d group; F: Counting and analysis were carried out in the same visual field (315.87 μm × 315.87 μm), and the positive percent of CD163+CD206+ M2-macrophages at different time points (3- and 5- d) in each group (n = 6) was recorded. “a” indicates a significant difference (P < 0.001) when compared with the Model group. Double immunofluorescence images of CD206 (green) and CD163 (red). Asterisks indicate co-expression. Arrowheads indicate myofibers. Arrows represents cells only expressing CD206 or CD163, respectively. DAPI (blue) was used for nuclear staining. Scale bar: 50 μm. Original magnification: × 400. LM: Lumbar multifidus; IFA: immunofluorescence assay; EA: Electro-acupuncture.
Figure 4 IFA showed EA facilitated M2-macrophage polarization in injured LM A1-A4: control group; B1-B4: model 3 d group; C1-C4: EA 3 d group; D1-D4: model 5-days group; E1-E4: EA 5-d group; F: Counting and analysis were carried out in the same visual field (315.87 μm × 315.87 μm), and the positive percent of CD163+CD206+ M2-macrophages at different time points (3- and 5- d) in each group (n = 6) was recorded. “a” indicates a significant difference (P < 0.001) when compared with the Model group. Double immunofluorescence images of CD206 (green) and CD163 (red). Asterisks indicate co-expression. Arrowheads indicate myofibers. Arrows represents cells only expressing CD206 or CD163, respectively. DAPI (blue) was used for nuclear staining. Scale bar: 50 μm. Original magnification: × 400. LM: Lumbar multifidus; IFA: immunofluorescence assay; EA: Electro-acupuncture.
Figure 5 Exosomal miRNAs are involved in EA's regulation after LM muscular injury A: Venn diagram: N_M represents DEmiRs between the control group and model group; M_D represents DEmiRs between model group and electroacupuncture group. B: List of DEmiRs in each group. C: Volcano plots: DEmiRs between control and model groups. D: Volcano plots: DEmiRs between model and electroacupuncture groups. Each point in the volcano plot represents a miRNA. Blue dots represent non-differentially expressed miRNAs. Red dots represent the upregulated miRNAs while green dots represent the downregulated miRNAs. E: Clustering analysis of DEmiRs in the control, model, and EA groups. Blue indicates lower gene expression while red color indicates higher gene expression. LM: Lumbar multifidus; EA: Electro-acupuncture; DEmiRs: differentially expressed miRNAs.
Figure 5 Exosomal miRNAs are involved in EA's regulation after LM muscular injury A: Venn diagram: N_M represents DEmiRs between the control group and model group; M_D represents DEmiRs between model group and electroacupuncture group. B: List of DEmiRs in each group. C: Volcano plots: DEmiRs between control and model groups. D: Volcano plots: DEmiRs between model and electroacupuncture groups. Each point in the volcano plot represents a miRNA. Blue dots represent non-differentially expressed miRNAs. Red dots represent the upregulated miRNAs while green dots represent the downregulated miRNAs. E: Clustering analysis of DEmiRs in the control, model, and EA groups. Blue indicates lower gene expression while red color indicates higher gene expression. LM: Lumbar multifidus; EA: Electro-acupuncture; DEmiRs: differentially expressed miRNAs.
Figure 6 GO-BP and KEGG enrichment analyses of target genes corresponding to 24 DEmiRs A: the bar chart of the KEGG pathway enrichment comparing the control group and the model group; B: the bar chart of the KEGG pathway enrichment comparing the model group and the electro-acupuncture group; C: the bubble plot of biological processes comparing the control group and the model group; D: the bubble plot of biological processes comparing the model group and the electro-acupuncture group. GO-BP: Gene Ontology - Biological Process; KEGG: Kyoto Encyclopedia of Genes and Genomes.
Figure 6 GO-BP and KEGG enrichment analyses of target genes corresponding to 24 DEmiRs A: the bar chart of the KEGG pathway enrichment comparing the control group and the model group; B: the bar chart of the KEGG pathway enrichment comparing the model group and the electro-acupuncture group; C: the bubble plot of biological processes comparing the control group and the model group; D: the bubble plot of biological processes comparing the model group and the electro-acupuncture group. GO-BP: Gene Ontology - Biological Process; KEGG: Kyoto Encyclopedia of Genes and Genomes.
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