[Effect of moxibustion on JAK2/STAT3 signal pathway in synovial tissues of rabbits with adjuvant arthritis under the overexpression of NLRP3 inflammatory bodies].

OBJECTIVE: To observe the effect of moxibustion on Janus protein tyrosine kinase 2/signal transducer and activator of transcription 3 (JAK2-STAT3) signal pathway and interleukin (IL)-1ß and IL-18 in synovial fluid of adjuvant arthritis (AA) rabbits, so as to explore the mechanism of moxibustion in the treatment of rheumatoid arthritis. METHODS: Twenty-eight rabbits were randomized into control, model, moxibustion, and NLRP3 overexpression groups, with 7 rabbits in each group. AA rabbit model was established by injection of Freund's complete adjuvant (FCA, 0.5 mL/kg) into the rabbits' bilateral hind-knee joint cavities. On the third day after modeling, the NLRP3 lentiviral vector (40 µL) were injected into the bilateral hind-knee joint cavities of rabbits in NLRP3 overexpression group. Moxibustion was used to bilateral "Shenshu" (BL23) and "Zusanli" (ST36), 5 cones every time, once daily, 6 days a week for 3 weeks in the moxibustion and NLRP3 overexpression groups. The perimeters of rabbits' hind legs were measured after modeling and after the intervention. The contents of IL-1ß, IL-18 in synovial fluid were detected by ELISA and the expression levels of NLRP3, JAK2 and STAT3 mRNAs in synovial tissue were detected by real-time PCR. RESULTS: In comparison with the control group, the perimeters of bilateral knee joints were significantly increased at each time point (P<0.05),and the contents of IL-1ß, IL-18 in synovial fluid and the expression levels of NLRP3, JAK2, STAT3 mRNA in synovial tissue were significantly increased (P<0.05) in the model group. Compared with the model group, the perimeters of bilateral knee joints were significantly decreased (P<0.05), and the contents of IL-1ß, IL-18 in synovial fluid and the expression levels of NLRP3, JAK2, STAT3 mRNAs in synovial tissue were significantly decreased (P<0.05) in the moxibusion group. Compared with the moxibustion group, the above indexes were higher in the NLRP3 overexpression group (P<0.05). CONCLUSION: Moxibustion may play an anti-inflammatory and detumescent role in AA rabbits by regulating JAK2-STAT3 signal pathway, and its therapeutic effect may be closely related to the expression of NLRP3.

[Mechanism on moxibustion for rheumatoid arthritis based on PD-1/PD-L1 signaling pathway].

OBJECTIVE: To explore the mechanism of moxibustion on the treatment of rheumatoid arthritis (RA) in the perspective of programmed cell death-1 and its ligand 1 (PD-1/PD-L1). METHODS: A total of 30 Japanese big ear white rabbits were randomly divided into a control group, a model group and a moxibustion group, 10 rabbits in each one. In the model group and the moxibustion group, RA model was prepared by the injection of Freund's complete adjuvant (FCA) into the hind knee joint cavities of each rabbit. In the control group, 0.9% sodium chloride solution of the same dose was injected. On the 8th day of experiment, in the moxibustion group, moxibustion was applied to "Shenshu" (BL 23) and "Zusanli" (ST 36), 5 cones at each acupoint, on the bilateral sides alternatively, once a day, 6 treatments as one course, with an interval of 1 days between the treatment courses. Totally, 3 courses of treatment were required. On the 1st, 7th, 14th, 21st and 28th days of experiment, successively, the circumference of the bilateral knee joints was measured with the tape. On the 28th day of experiment, H.E. staining was adopted to observe the histopathological morphology and to evaluate the score of knee synovial tissue. ELISA was used to determined the concentrations of soluble PD-1 (sPD-1) and its ligand 1 (sPD-L1), the interleukin 2 (IL-2) and IL-17 in knee synovial fluid and the concentrations of sPD-1 and sPD-L1 in serum. The histochemistry method was used to determine the expressions of membrane PD-1 (mPD-1) and its ligand 1 (mPD-L1) in spleen tissue. RESULTS: On the 14th, 21st and 28th days of experiment, the circumference of both knee joints was increased in each of the rabbits in the model group as compared with the control group (P<0.01), and it was reduced significantly in the moxibustion group as compared with the model group (P<0.01). Compared with the control group, the hyperplasia of synovial tissue and fibrous tissue, as well as inflammatory cell infiltration were increased obviously in the model group (P<0.01), and they were reduced significantly in the moxibustion group as compared with the model group (P<0.01, P<0.05). Compared with the control group, the concentrations of IL-2 and IL-17 in knee synovial fluid were increased in the rabbits of the model group (P<0.01). Compared with the model group, after the intervention with moxibustion, the concentrations of IL-2 and IL-17 in knee synovial fluid were reduced in the rabbits of the moxibustion group (P<0.05). Compared with the control group, the concentrations of sPD-1 and sPD-L1 in knee synovial fluid and serum in the rabbits of the model group were all increased (P<0.05). Compared with the model group, the concentration of sPD-1 in the knee synovial fluid and serum were reduced in the rabbits of moxibustion group (P<0.05). Compared with the control group, the expressions of mPD-1 and mPD-L1 in spleen tissue were increased obviously in the rabbits of the model group (P<0.05). Compared with the model group, the expression of mPD-L1 in spleen tissue was up-regulated in the rabbits of the moxibustion group (P<0.05). CONCLUSION: Moxibustion could inhibit the over-activation of T cells by enhancing the negative regulation of PD-1/PD-L1 signaling pathway so as to play its effect in treatment of RA.

[Effects of Chronological Moxibustion on Circadian Rhythm Activities of Hypothalamus-Pituitary-Axis in Rheumatoid Arthritis Rats].

OBJECTIVE: To observe the effect of chronological moxibustion on plasma corticosterone (CORT), corticotrophin releasing factor (CRF) and adrenocorticotropic hormone (ACTH) levels in rheumatoid arthritis (RA) rats, so as to explore its mechanisms underlying improvement of RA. METHODS: A total of 144 SD rats were randomly divided into normal control, model, moxibustion, sham-adrenalectomy (ADX, sham-ADX), ADX and ADX+moxibustion groups which were further separately divided into 4 subgroups (4 time-points: 12 pm, 6 am, 12 am, 6 pm, n = 6/subgroup). The RA model was established by subcutaneous injection of Freund's complete adjuvant (FCA, 0. 1 mL) into the right footpad (for rats of the latter 5 groups). In rats of the ADX and ADX+ moxibustion groups, bilateral adrenal glands were removed under anesthesia. Moxibustion was applied to unilateral "Shenshu" (BL 23) and "Zusanli" (ST 36) for 10 min, once daily for 6 days in a week, and continuously for 3 weeks. The paw swelling volume was measured and plasma CORT, ACTH and CRF contents were assayed by ELISA and the circadian rhythm was analyzed by cosine curve fitting (cosinor) method. RESULTS: The paw swelling volume was significantly increased in the model group than in the normal control group (P<0.05), and decreased remarkably in the moxibustion group (P<0.05). The paw volume was markedly increased in the ADX +moxibustion group in comparison with the moxibustion group (P<0.05). In intact rats, plasma CRF and ACTH contents were significantly increased (P<0.05), and plasma CORT was obviously decreased in the model group (P<0.05). After moxibustion, the increased plasma CRF and ACTH contents and the decreased plasma CORT level Nere considerably reversed (P<0.05). In ADX rats, plasma ORE, ACTH and CORT contents of ADX and ADX + moxibustion groups had no significant.changes compared with the sham-ADX group (P>0.05). Results of the cosine curve fitting analysis showed that the peak phases of plasma CORT contents were -355.78 degrees at about 23:43 in the normal control group, -309.05 degrees at about 20:36 in the model group, -326.5 degrees at about 21:46 in the moxibustion group, -291.65 degrees at about 19:27 in the ADX group and -300.87 degrees at about 20:31 in the ADX + moxibustion group. The peak phases of plasma ACTH contents were -324.08 degrees at about 21:37 in the normal control group, -295.39 degrees at about 19:41 in the model group, -310.81 degrees at about 20:43 in the moxibustion group, -146.51 degrees at about 9:46 in the ADX group and -267.64 degrees at about 17:50 in the ADX+ moxibustion group. The peak phases of plasma CRF contents were -257.47 degrees at about 17:10 in the normal control group, -184.74 degrees at about 12:19 in the model group, -263.00 degrees at about 17:32 in the moxibustion group, -202.46 degrees at about 13:30 in the ADX group and -232.84 degrees at about 15:31 in the ADX+ moxibustion group. It suggests that in the intact RA rats, moxibustion intervention may inhibit modeling-induced decrease of the circadian rhythm of plasma CORT, and hyperactivity of ACTH and CRF circadian rhythm. CONCLUSION: VAoxibustion intervention can relieve paw swelling in RA rats and modulate the circadian rhythm activities of plasma CORT, ACTH and ORE levels (HPA axis activities) in intact animals.