Electroacupuncture stimulating Zusanli (ST36), Sanyinjiao (SP6) in mice with collagen-induced arthritis leads to adenosine A2A receptor-mediated alteration of p38α mitogen-activated protein kinase signaling and inhibition of osteoclastogenesis

doi:10.19852/j.cnki.jtcm.2023.06.001

Abstract

Abstract:

OBJECTIVE: To observe the effect of electroacupuncture (EA) stimulating Zusanli (ST36), Sanyinjiao (SP6) on inhibition of osteoclastogenesis and the role of the adenosine A2A receptor (A2AR) and the p38α Mitogen-Activated Protein Kinase (MAPK) signaling pathway in mediating this effect.
METHODS: Mice with collagen induced arthritis (CIA) received different treatments. Immunohistochemistry and western blotting were used to determine the levels of multiple signaling molecules in these joints [receptor activator of nuclear transcription factor-κB (NF-κB) ligand (RANKL), receptor activator of NF-κB (RANK), tumor necrosis factor receptor associated factor 6 (TRAF6), p38α, NF-κB, and nuclear factor of activated T cells C1 (NFATc1)]. Osteoclasts were identified using tartrate-resistant acid phosphatase (TRAP) staining.
RESULTS: The immunohistochemistry results indicated upregulation of p38α, NF-κB, and NFATc1 in the CIA-control and CIA-EA-SCH58261 groups, but reduced levels in the CIA-EA group. Western blotting indicated upregulation of RANKL, RANK, TRAF6, p38α, NF-κB, and NFATc1 in the CIA-control and CIA-EA-SCH58261 groups, but reduced expression in the CIA-EA group. Osteoclasts were more abundant in the CIA-control and CIA-EA-SCH58261 groups than in the CIA-EA group.
CONCLUSIONS: EA treatment enhanced the A2AR activity and inhibited osteoclast formation by inhibition of RANKL, RANK, TRAF6, p38α, NF-κB, and NFATc1. SCH58261 reversed the effect of EA. These results suggest that EA regulated p38α-MAPK signaling by increasing A2AR activity, which inhibited osteoclastogenesis.

Key words: electroacupuncture, arthritis, experimental, receptor, adenosine A2A, mitogen-activated protein kinases, signal transduction, osteoclasts

DU Zhongheng, CONG Wenjie, TANG Kejing, ZHENG Qiqi, SONG Zhiwei, CHEN Yong, YANG Su, ZHANG Chunwu, YE Tianshen. Electroacupuncture stimulating Zusanli (ST36), Sanyinjiao (SP6) in mice with collagen-induced arthritis leads to adenosine A2A receptor-mediated alteration of p38α mitogen-activated protein kinase signaling and inhibition of osteoclastogenesis[J]. Journal of Traditional Chinese Medicine, 2023, 43(6): 1103-1109.

Figures/Tables 3

Figure 1 Expression of p38α, NF-κB, NFATc1 in the ankle joints of mice in the different treatment groups A: expression of p38α in Blank group (× 100); B: expression of p38α in CIA-control group (× 100); C: expression of p38α in CIA-EA group (× 100); D: expression of p38α in CIA-EA-SCH58261 group (× 100); E: quantitation of p38α in all mice; F: expression of NF-κB in Blank group (× 100); G: expression of NF-κB in CIA-control group (× 100); H: expression of NF-κB in CIA-EA group (× 100); I: expression of NF-κB in CIA-EA-SCH58261 group (× 100); J: quantitation of NF-κB in all mice; K: expression of NFATc1 in Blank group (× 100); L: expression of NFATc1 in CIA-control group (× 100); M: expression of NFATc1 in CIA-EA group (× 100); N: expression of NFATc1 in CIA-EA-SCH58261 group (× 100); O: quantitation of NFATc1 in all mice. NF-κB: nuclear transcription factor-κB; NFATc1: nuclear factor of activated T cells C1; CIA: collagen induced arthritis; EA: electroacupuncture. Dyeing method of all pictures are the immunohistochemical SP method, PBS was used as negative control. Blank group: not have CIA and daily headgear fixation 30 min for 14 d (n =10); CIA-control group: become a CIA model and daily headgear fixation 30 min for 14 d (n = 10); CIA-EA group: become a CIA model, electroacupuncture stimulating Zusanli (ST36), Sanyinjiao (SP6) and daily headgear fixation 30 min at the same time for 14 d (n = 10); CIA-EA-SCH58261 group: received intraperitoneal injections of SCH58261 (adenosine A2A receptor antagonist, 5 mg/kg), and then headgear fixation, EA as in the CIA-EA group (n = 10). aP < 0.05, compared with the Blank group. bP < 0.05, compared with the CIA-control group. cP < 0.05, compared with the CIA-EA group. Data were compared using a one-way analysis of variance.

Figure 2 Western blotting of proteins in the p38α-MAPK signaling pathway in the different treatment groups 1: Blank group, not have CIA and daily headgear fixation 30 min for 14 d; 2: CIA-control group, become a CIA model and daily headgear fixation 30 min for 14 d; 3: CIA-EA group, become a CIA model, electroacupuncture stimulating Zusanli (ST36), Sanyinjiao (SP6) and daily headgear fixation 30 min at the same time for 14 d; 4: CIA-EA-SCH58261 group, received intraperitoneal injections of SCH58261 (Adenosine A2A receptor antagonist, 5 mg/kg), and then headgear fixation, EA as in the CIA-EA group. RANKL: receptor activator of nuclear transcription factor-κB ligand; RANK: receptor activator of NF-κB; TRAF6: tumor necrosis factor receptor associated factor 6; NF-κB: nuclear transcription factor-κB; NFATc1: nuclear factor of activated T cells C1; MAPK: mitogen-activated protein kinase; CIA: collagen induced arthritis; EA: electroacupuncture.

Figure 3 TRAP-staining of osteoclasts in representative mice from the different treatment groups A: expression of osteoclasts in Blank group (× 200); B: expression of osteoclasts in CIA-control group (× 200); C: expression of osteoclasts in CIA-EA group (× 200); D: expression of osteoclasts in CIA-EA-SCH58261 group (× 200). E: quantitation of osteoclasts in all mice. TRAP: tartrate-resistant acid phosphatase; CIA: collagen induced arthritis; EA: electroacupuncture. Dyeing method of all pictures are the TRAP staining method. Blank group: not have CIA and daily headgear fixation 30 min for 14 d (n = 10); CIA-control group: become a CIA model and daily headgear fixation 30 min for 14 d (n = 10); CIA-EA group: become a CIA model, electroacupuncture stimulating Zusanli (ST36), Sanyinjiao (SP6) and daily headgear fixation 30 min at the same time for 14 d (n = 10); CIA-EA-SCH58261 group: received intraperitoneal injections of SCH58261 (Adenosine A2A receptor antagonist, 5 mg/kg), and then headgear fixation, EA as in the CIA-EA group (n = 10). Data were compared using a one-way analysis of variance. aP < 0.05, compared with the Blank group; bP < 0.05, compared with the CIA-control group. cP < 0.05, compared with the CIA-EA group.

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