Journal of Traditional Chinese Medicine ›› 2024, Vol. 44 ›› Issue (5): 981-990.DOI: 10.19852/j.cnki.jtcm.2024.05.005
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Regulatory role of electroacupuncture on satellite glial cell activity in the colon and dorsal root ganglion of rats with irritable bowel syndrome
ZHANG Fang1,2, YAN Cuina3, WENG Zhijun1,2, WU Luyi3,4, QI Li3, ZHAO Min3, XIN Yuhu5, WU Huangan1,2, LIU Huirong1,2(
)
- 1 Key Laboratory of Acupuncture and Immunological Effects, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 200437, China
2 Shanghai Research Institute of Acupuncture and Meridian, Shanghai 200030, China
3 Key Laboratory of Acupuncture and Immunological Effects, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 200437, China
4 Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
5 Cancer Hospital, Fudan University, Shanghai 200032, China
-
Received:2023-06-11Accepted:2023-11-27Online:2024-10-15Published:2024-09-11 -
Contact:Prof. LIU Huirong, Shanghai Research Institute of Acupuncture and Meridian, Shanghai 200030, China; lhr_tcm@139.com Telephone: +86-21-64644238 -
Supported by:Research on the Initiation Mechanism of Moxibustion Effect and Its Endogenous Regulation Mechanism (973 program, No. 2015CB554501);Interaction Mechanism of the Information between Electroacupuncture Stimulation to Zusanli and Visceral Pain in Dorsal Root Ganglion of Rats with Irritable Bowel Syndrome(81873367);Study on the Mechanism of Periaqueductal gray Purinergic Ion Channel Receptor 3 Mediated in Electro-acupuncture Relieving Visceral Hypersensitivity in Mice with Irritable Bowel Syndrome(81904301);based on Transient Receptor Potential Vanilloid 1 Mediated Calcium/Calmodulin-dependent Protein Kinase Ⅱ Signaling Pathway Involved in Electroacupuncture to Relieve Irritable Bowel Syndrome Mice Visceral Pain Mechanism Study(22ZR1458600);Shanghai Clinical Research Center for Acupuncture and Moxibustion(20MC1920500)
Cite this article
ZHANG Fang, YAN Cuina, WENG Zhijun, WU Luyi, QI Li, ZHAO Min, XIN Yuhu, WU Huangan, LIU Huirong. Regulatory role of electroacupuncture on satellite glial cell activity in the colon and dorsal root ganglion of rats with irritable bowel syndrome[J]. Journal of Traditional Chinese Medicine, 2024, 44(5): 981-990.
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Table 1 AWR scores before EA and drug intervention [M (Q25, Q75)]
| Group | n | 20 mm Hg | 40 mm Hg | 60 mm Hg | 80 mm Hg |
|---|---|---|---|---|---|
| NG | 8 | 0.33 (0, 0.33) | 1.33 (1.33, 1.33) | 2.67 (2.67, 2.67) | 3.84 (3.33, 4) |
| MG | 24 | 0.67 (0.33, 1.00)a | 2.33 (2, 2.67)a | 3.33 (3, 3.33)a | 4 (3.67, 4) |
Table 1 AWR scores before EA and drug intervention [M (Q25, Q75)]
| Group | n | 20 mm Hg | 40 mm Hg | 60 mm Hg | 80 mm Hg |
|---|---|---|---|---|---|
| NG | 8 | 0.33 (0, 0.33) | 1.33 (1.33, 1.33) | 2.67 (2.67, 2.67) | 3.84 (3.33, 4) |
| MG | 24 | 0.67 (0.33, 1.00)a | 2.33 (2, 2.67)a | 3.33 (3, 3.33)a | 4 (3.67, 4) |
Table 2 AWR scores after EA and drug intervention [M (Q25, Q75)]
| Group | n | 20 mm Hg | 40 mm Hg | 60 mm Hg | 80 mm Hg |
|---|---|---|---|---|---|
| NG | 8 | 0.33 (0, 0.33) | 1.5 (1.33, 1.67) | 2.33 (2.08, 2.59) | 3.33 (3.08, 3.67) |
| MG | 8 | 0.67 (0.67, 0.92)a | 2 (2, 2.33)a | 3 (2.75, 3.25)a | 4 (3.67, 4)a |
| EA | 8 | 0.33 (0.08, 0.67)b | 2 (1.67, 2)b | 2.67 (2.67, 2.92)b | 3.5 (3.08, 3.92)b |
| FCA | 8 | 0.17 (0, 0.33)c | 2 (1.67, 2)b | 2.67 (2.42, 2.92)b | 3.33 (3.33, 3.59)b |
Table 2 AWR scores after EA and drug intervention [M (Q25, Q75)]
| Group | n | 20 mm Hg | 40 mm Hg | 60 mm Hg | 80 mm Hg |
|---|---|---|---|---|---|
| NG | 8 | 0.33 (0, 0.33) | 1.5 (1.33, 1.67) | 2.33 (2.08, 2.59) | 3.33 (3.08, 3.67) |
| MG | 8 | 0.67 (0.67, 0.92)a | 2 (2, 2.33)a | 3 (2.75, 3.25)a | 4 (3.67, 4)a |
| EA | 8 | 0.33 (0.08, 0.67)b | 2 (1.67, 2)b | 2.67 (2.67, 2.92)b | 3.5 (3.08, 3.92)b |
| FCA | 8 | 0.17 (0, 0.33)c | 2 (1.67, 2)b | 2.67 (2.42, 2.92)b | 3.33 (3.33, 3.59)b |
Figure 1 HE staining of colon tissues A-D: HE staining of colon tissues, scale bar: 100 μm. A: NG; B: MG; C: EA; D: FCA. NG: normal group, the same fixation as the EA group; MG: model group, the same fixation as the EA group; EA: electroacupuncture group, EA at bilateral Tianshu (ST25) and Shangjuxu (ST37) (2/100 Hz, 1 mA, 30 min, 7 d); FCA: DL-fluorocitric acid barium salt group, intrathecal injection of FCA (10 μL, 1 nmol/μL) every three days. HE: hematoxylin and eosin.
Figure 1 HE staining of colon tissues A-D: HE staining of colon tissues, scale bar: 100 μm. A: NG; B: MG; C: EA; D: FCA. NG: normal group, the same fixation as the EA group; MG: model group, the same fixation as the EA group; EA: electroacupuncture group, EA at bilateral Tianshu (ST25) and Shangjuxu (ST37) (2/100 Hz, 1 mA, 30 min, 7 d); FCA: DL-fluorocitric acid barium salt group, intrathecal injection of FCA (10 μL, 1 nmol/μL) every three days. HE: hematoxylin and eosin.
Figure 2 GFAP expression in the colonic myenteric plexus and colon A: immunofluorescence was used to detect the distribution of GFAP in the colonic myenteric plexus, scale bar: 20 μm; A1: NG; A2: MG; A3: EA; A4: FCA. B: representative gel images show the protein level of GFAP in the colon, β-Actin was used as a loading control; Western blotting was used to detect the expression of GFAP protein in the colon. C: quantitative analysis of GFAP protein expression in the colon. D: relative expression of GFAP mRNA in the colon, real-time polymerase chain reaction was used to detect the expression of GFAP mRNA in the colon. NG (n = 8): normal group, the same fixation as the EA group; MG (n = 8): model group, the same fixation as the EA group; EA (n = 8): electroacupuncture group, EA stimulation at bilateral Tianshu (ST25) and Shangjuxu (ST37) (2/100 Hz, 1 mA, 30 min, 7 d); FCA (n = 8): DL-fluorocitric acid barium salt group, intrathecal injection of FCA (10 μL, 1 nmol/μL) every three days. GFAP: glial fibrillary acidic protein; mRNA: messenger ribonucleic acid. All data was easured by one-way analysis, and least significance difference test was performed for inter-group comparisons. All data was presented as mean ± standard deviation. Compared with the normal group, aP<0.01; compared with the model group, bP<0.05, cP<0.01.
Figure 2 GFAP expression in the colonic myenteric plexus and colon A: immunofluorescence was used to detect the distribution of GFAP in the colonic myenteric plexus, scale bar: 20 μm; A1: NG; A2: MG; A3: EA; A4: FCA. B: representative gel images show the protein level of GFAP in the colon, β-Actin was used as a loading control; Western blotting was used to detect the expression of GFAP protein in the colon. C: quantitative analysis of GFAP protein expression in the colon. D: relative expression of GFAP mRNA in the colon, real-time polymerase chain reaction was used to detect the expression of GFAP mRNA in the colon. NG (n = 8): normal group, the same fixation as the EA group; MG (n = 8): model group, the same fixation as the EA group; EA (n = 8): electroacupuncture group, EA stimulation at bilateral Tianshu (ST25) and Shangjuxu (ST37) (2/100 Hz, 1 mA, 30 min, 7 d); FCA (n = 8): DL-fluorocitric acid barium salt group, intrathecal injection of FCA (10 μL, 1 nmol/μL) every three days. GFAP: glial fibrillary acidic protein; mRNA: messenger ribonucleic acid. All data was easured by one-way analysis, and least significance difference test was performed for inter-group comparisons. All data was presented as mean ± standard deviation. Compared with the normal group, aP<0.01; compared with the model group, bP<0.05, cP<0.01.
Figure 3 GFAP protein and mRNA expression in the colon-related DRG A: immunohistochemical assay was used to detect the expression of GFAP protein in the colon-related DRG, scale bar: 50 μm; A1: NG; A2: MG; A3: EA; A4: FCA; dyeing method of all pictures are the immunohistochemical DAB method. B: semiquantitative analysis of GFAP protein expression in the colon-related DRG. C: representative gel images show the protein level of GFAP in the colon-related DRG, β-Actin was used as a loading control; Western blotting was used to detect the expression of GFAP protein in the colon-related DRG. D: quantitative analysis of GFAP protein expression in the colon-related DRG. E: relative expression of GFAP mRNA in the colon-related DRG, Real-time Polymerase Chain Reaction was used to detect the expression of GFAP mRNA in the colon. NG (n = 8): normal group, the same fixation as the EA group; MG (n = 8): model group, the same fixation as the EA group; EA (n = 8): electroacupuncture group, EA stimulation at bilateral Tianshu (ST25) and Shangjuxu (ST37) (2/100 Hz, 1 mA, 30 min, 7 d); FCA (n = 8): DL-fluorocitric acid barium salt group, intrathecal injection of FCA (10 μL, 1 nmol/μL) every three days. GFAP: glial fibrillary acidic protein; DAB: diaminobenzidine; IOD: immunohistochemical optical density. mRNA: messenger ribonucleic acid; DRG: dorsal root ganglion. All data was measured by one-way analysis, and least significance difference test was performed for inter-group comparisons. All data was presented as mean ± standard deviation. Compared with the normal group, aP<0.01; compared with the model group, bP<0.01.
Figure 3 GFAP protein and mRNA expression in the colon-related DRG A: immunohistochemical assay was used to detect the expression of GFAP protein in the colon-related DRG, scale bar: 50 μm; A1: NG; A2: MG; A3: EA; A4: FCA; dyeing method of all pictures are the immunohistochemical DAB method. B: semiquantitative analysis of GFAP protein expression in the colon-related DRG. C: representative gel images show the protein level of GFAP in the colon-related DRG, β-Actin was used as a loading control; Western blotting was used to detect the expression of GFAP protein in the colon-related DRG. D: quantitative analysis of GFAP protein expression in the colon-related DRG. E: relative expression of GFAP mRNA in the colon-related DRG, Real-time Polymerase Chain Reaction was used to detect the expression of GFAP mRNA in the colon. NG (n = 8): normal group, the same fixation as the EA group; MG (n = 8): model group, the same fixation as the EA group; EA (n = 8): electroacupuncture group, EA stimulation at bilateral Tianshu (ST25) and Shangjuxu (ST37) (2/100 Hz, 1 mA, 30 min, 7 d); FCA (n = 8): DL-fluorocitric acid barium salt group, intrathecal injection of FCA (10 μL, 1 nmol/μL) every three days. GFAP: glial fibrillary acidic protein; DAB: diaminobenzidine; IOD: immunohistochemical optical density. mRNA: messenger ribonucleic acid; DRG: dorsal root ganglion. All data was measured by one-way analysis, and least significance difference test was performed for inter-group comparisons. All data was presented as mean ± standard deviation. Compared with the normal group, aP<0.01; compared with the model group, bP<0.01.
Figure 4 Changes in the electrical properties of membrane of the colon-related DRG neurons A: representative traces of APs after a 200 pA depolarization current injection into colon-related DRG neurons of the NG, MG, and the EA, and FCA groups; single cell patch clamp recording was used to detect the RMP, rheobase and AP frequency of the colon-related DRG neurons. B: changes in the cell RMP in colon-related DRG neurons; C: number of APs of neuronal cells in colon-related DRG; D: changes in the rheobase in colon-related DRG neurons. NG (n = 8): normal group, the same fixation as the EA group; MG (n = 8): model group, the same fixation as the EA group; EA (n = 8): electroacupuncture group, EA stimulation at bilateral Tianshu (ST25) and Shangjuxu (ST37) (2/100 Hz, 1 mA, 30 min, 7 d); FCA (n = 8): DL-fluorocitric acid barium salt group, intrathecal injection of FCA (10 μL, 1 nmol/μL) every three days. RMP: resting membrane potential; No: number; AP: action potential; DRG: dorsal root ganglion. All data was measured by one-way analysis, and least significance difference test was performed for inter-group comparisons. All data was presented as mean ± standard deviation. Compared with the normal group, aP<0.01; Compared with the model group, bP<0.05, cP<0.01.
Figure 4 Changes in the electrical properties of membrane of the colon-related DRG neurons A: representative traces of APs after a 200 pA depolarization current injection into colon-related DRG neurons of the NG, MG, and the EA, and FCA groups; single cell patch clamp recording was used to detect the RMP, rheobase and AP frequency of the colon-related DRG neurons. B: changes in the cell RMP in colon-related DRG neurons; C: number of APs of neuronal cells in colon-related DRG; D: changes in the rheobase in colon-related DRG neurons. NG (n = 8): normal group, the same fixation as the EA group; MG (n = 8): model group, the same fixation as the EA group; EA (n = 8): electroacupuncture group, EA stimulation at bilateral Tianshu (ST25) and Shangjuxu (ST37) (2/100 Hz, 1 mA, 30 min, 7 d); FCA (n = 8): DL-fluorocitric acid barium salt group, intrathecal injection of FCA (10 μL, 1 nmol/μL) every three days. RMP: resting membrane potential; No: number; AP: action potential; DRG: dorsal root ganglion. All data was measured by one-way analysis, and least significance difference test was performed for inter-group comparisons. All data was presented as mean ± standard deviation. Compared with the normal group, aP<0.01; Compared with the model group, bP<0.05, cP<0.01.
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