Chuan-Ying Zhang1, Ling Hu2, Rong-Lin Cai2, Chuan-Yu Peng2, Juan Yuan3. 1. School of Nursing, Anhui University of Chinese Medicine, Hefei 230012, China. 2. Institute of Acupuncture-moxibustion and Meridian, Anhui University of Chinese Medicine, Hefei 230012, China. 3. School of Nursing, 2Institute of Acupuncture-moxibustion and Meridian, Anhui University of Chinese Medicine, Hefei 230012, China.
Abstract
OBJECTIVE: To observe the effect of moxibustion on Toll-like receptor 4/nuclear factor-κB (TLR 4/NF-κB) signaling in the synovial tissue of the ankle joint in rats with rheumatoid arthritis (RA), so as to analyze its biological mechanism underlying improvement of RA. METHODS: Fifty male SD rats were randomly divided into normal control, model, moxibustion, moxibustion ＋ TLR 4 agonist, and moxibustion ＋ TLR 4 antagonist groups (n＝10 rats in each). The RA model was established by subcutaneous injection of Freund's complete adjuvant (FCA, 0.1 mL/rat) at the right hind-paw and by being raised in a wind (air fan blowing), cold (about 10 ℃) and wet (purling) environment for 20 days. After 3 days of modeling, mild moxibustion was applied to bilateral "Shenshu" (BL 23) and "Zusanli" (ST 36) for 20 minutes, once daily for successive 10 days. The TLR 4 agonist (lipopolysaccharide) or TLR 4 antagonist (TAK-242) (1 mg/mL) was separately administered via the tail vein 30 min before performing moxibustion every time in the agonist group and the antagonist group. The expression of NF-κB inhibitory factor ɑ (IκBɑ), IκB kinase complex β(IκKβ), myeloid differentiation factor 88 (MyD 88), TLR 4, and NF-κB p 65 proteins in the synovial tissue of the ankle joint was detected by using Western blot. RESULTS: Following modeling, the girth of the swollen ankle joint was obviously bigger (P<0.01), and the expression levels of IκBɑ， IκKβ， TLR 4, MyD 88, and NF-κB p 65 proteins in the synovial tissue were considerably increased in the model group relevant to the normal control group (P<0.01). After moxibustion intervention, the girth of the swollen ankle joint and the expression levels of IκBɑ， IκKβ， TLR 4, MyD 88, and NF-κB p 65 proteins were significantly down-regulated in the moxibustion, moxibustion ＋TLR 4 agonist, moxibustion＋TLR 4 antagonist groups compared with the model group (P<0.01, P<0.05). Comparison among the 3 moxibustion groups showed that the lowered levels of ankle-joint girth, and IκBɑ， IκKβ， TLR 4, MyD 88, and NF-κB p 65 expression were significantly smaller in the moxibustion＋TLR 4 agonist group than in the simple moxibustion and moxibustion＋TLR 4 antagonist groups (P<0.05，P<0.01). No significant differences were found between the moxibustion and moxibustion＋TLR 4 antagonist groups in the decreased ankle joint girth and IκBɑ， IκKβ， TLR 4, MyD 88, and NF-κB p 65 expression levels (P>0.05), suggesting that activation of TLR 4 reduced the anti-inflammatory effect of moxibustion intervention. CONCLUSION: Moxibustion can reduce the ankle joint swelling in RA rats, which may be closely associated with its effect in down-regulating the expression of IκBɑ， IκKβ， TLR 4, MyD 88, and NF-κB p 65 proteins and in inhibiting TLR 4/NF-κB signaling in the synovial tissue of the ankle joint.
OBJECTIVE: To observe the effect of moxibustion on Toll-like receptor 4/nuclear factor-κB (TLR 4/NF-κB) signaling in the synovial tissue of the ankle joint in rats with rheumatoid arthritis (RA), so as to analyze its biological mechanism underlying improvement of RA. METHODS: Fifty male SD rats were randomly divided into normal control, model, moxibustion, moxibustion ＋ TLR 4 agonist, and moxibustion ＋ TLR 4 antagonist groups (n＝10 rats in each). The RA model was established by subcutaneous injection of Freund's complete adjuvant (FCA, 0.1 mL/rat) at the right hind-paw and by being raised in a wind (air fan blowing), cold (about 10 ℃) and wet (purling) environment for 20 days. After 3 days of modeling, mild moxibustion was applied to bilateral "Shenshu" (BL 23) and "Zusanli" (ST 36) for 20 minutes, once daily for successive 10 days. The TLR 4 agonist (lipopolysaccharide) or TLR 4 antagonist (TAK-242) (1 mg/mL) was separately administered via the tail vein 30 min before performing moxibustion every time in the agonist group and the antagonist group. The expression of NF-κB inhibitory factor ɑ (IκBɑ), IκB kinase complex β(IκKβ), myeloid differentiation factor 88 (MyD 88), TLR 4, and NF-κB p 65 proteins in the synovial tissue of the ankle joint was detected by using Western blot. RESULTS: Following modeling, the girth of the swollen ankle joint was obviously bigger (P<0.01), and the expression levels of IκBɑ， IκKβ， TLR 4, MyD 88, and NF-κB p 65 proteins in the synovial tissue were considerably increased in the model group relevant to the normal control group (P<0.01). After moxibustion intervention, the girth of the swollen ankle joint and the expression levels of IκBɑ， IκKβ， TLR 4, MyD 88, and NF-κB p 65 proteins were significantly down-regulated in the moxibustion, moxibustion ＋TLR 4 agonist, moxibustion＋TLR 4 antagonist groups compared with the model group (P<0.01, P<0.05). Comparison among the 3 moxibustion groups showed that the lowered levels of ankle-joint girth, and IκBɑ， IκKβ， TLR 4, MyD 88, and NF-κB p 65 expression were significantly smaller in the moxibustion＋TLR 4 agonist group than in the simple moxibustion and moxibustion＋TLR 4 antagonist groups (P<0.05，P<0.01). No significant differences were found between the moxibustion and moxibustion＋TLR 4 antagonist groups in the decreased ankle joint girth and IκBɑ， IκKβ， TLR 4, MyD 88, and NF-κB p 65 expression levels (P>0.05), suggesting that activation of TLR 4 reduced the anti-inflammatory effect of moxibustion intervention. CONCLUSION: Moxibustion can reduce the ankle joint swelling in RArats, which may be closely associated with its effect in down-regulating the expression of IκBɑ， IκKβ， TLR 4, MyD 88, and NF-κB p 65 proteins and in inhibiting TLR 4/NF-κB signaling in the synovial tissue of the ankle joint.