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Objectives: To observe the effect of heat-reinforcing needling (HRN) on synovial inflammation, hypoxia-inducible factor-1α (HIF-1α) and glycolytic activity in serum and synovial tissue in rabbits with cold syndrome of rheumatoid arthritis (RA), so as to explore its mechanisms underlying improvement of RA., Methods: A total of 32 rabbits were randomly divided into normal, model, inhibitor and HRN groups, with 8 rabbits in each group. The RA with cold syndrome model was induced by injecting ovalbumin dry powder and Freund's complete adjuvant combined with cold freezing. Rabbits in the inhibitor group were intraperitoneally injected with 2-methoxyestradiol (2.5 mg/kg), rabbits in the HRN group were received HRN at bilateral "Zusanli" (ST36) for 30 min. The treatments were conducted once daily for 14 consecutive days. After the interventions, the knee circumference and pain threshold were measured. The contents of nicotinamide adenine dinucleotide phosphoric (NADPH), Hexokinase II (HK2) and 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3 (PFKFB3) in serum of rabbits were detected by ELISA. The pathological morphology of synovial tissue of the knee joints were observed by HE staining. The positive expressions of tumor necrosis factor (TNF-α), interleukin (IL)-1β, IL-6 and IL-17 in synovial tissue of knee joint were detected by immunohistochemistry. The content of lactic acid in synovial tissue of rabbit knee joint was detected by spectrophotometry. The expression levels of HIF-1α, pyruvate kinase 2 (PKM2) and lactate dehydrogenase (LDHA) in synovial tissue of knee joint were detected by Western blot., Results: After intervention, compared with the normal group, the knee circumference was significantly enlarged ( P <0.05), the pain threshold was significantly decreased ( P <0.05)；the synovial tissue of knee joints showed significant cell proliferation and inflammatory infiltration, the pathological score was significantly increased ( P <0.05)；positive expressions of TNF-α, IL-1β, IL-6 and IL-17, the content of lactic acid in synovial tissue, the contents of NADPH, HK2 and PFKFB3 in serum, and the protein expression levels of HIF-1α, PKM2 and LDHA in synovial tissue were increased (all P <0.05) in the model group. Compared with model group, the circumference of knee joint was significantly decreased ( P <0.05), the pain threshold was significantly increased ( P <0.05)；in synovial tissue, the pathological score was decreased ( P <0.05)；the positive expressions of TNF-α, IL-1β, IL-6 and IL-17 in synovial tissue were decreased ( P <0.05), the lactic acid content in synovial tissue was decreased ( P <0.05)；the contents of NADPH, HK2 and PFKFB3 in serum and the protein expression levels of HIF-1α, PKM2 and LDHA in synovial tissue were decreased ( P <0.05) in inhibitor group and HRN group. Compared with the inhibitor group, the synovial pathological score was significantly increased ( P <0.05), positive expressions of TNF-α, IL-1β, IL-6 and IL-17, the content of lactic acid in synovial tissue, the contents of NADPH, HK2 and PFKFB3 in serum, and the protein expression levels of HIF-1α, PKM2 and LDHA in synovial tissue were increased (all P <0.05) in HRN group., Conclusions: HRN can increase the pain threshold, reduce the knee circumference and inhibit the inflammatory response in rabbits with cold syndrome of RA. The possible mechanism is related to the down-regulation of HIF-1α and glycolysis activity.

Objective: To observe the effect of heat-tonifying needling on Keap1-Nrf2/ARE/HO-1 signal transduction pathway in knee synovium in rabbits with cold syndrome type rheumatoid arthritis (RA), so as to explore its mechanisms underl-ying improvement of RA., Methods: New Zealand rabbits were randomly divided into normal control, RA model, uniform reinforcing-reducing acupuncture, twisting reinforcing acupuncture and heat-tonifying acupuncture groups, with 6 rabbits in each group. The cold syndrome type RA model was established by subcutaneous injection of mixture fluid of ovalbumin and Freund's complete adjuvant at the shoulder-back as well as injection of mixture of ovalbumin and normal saline into knee-joint cavity combined with ice-compress freezing. Acupuncture stimulation (uniform reinforcing-reducing, or twisting reinforcing or heat-tonifying) was applied to bilateral "Zusanli"(ST36) for 1 min with the needle retained for 30 min, once a day for 7 consecutive days. The general conditions of rabbits in each group were recorded, the thermal pain threshold (TPT) and perimeter of knee joints was measured. Conditions of the synovium in the knee cavity, hydrops, blood flow signal, articular surface, and related muscles were observed by using a color Doppler ultrasonic diagnostic apparatus, and the blood flow signals inside the synovium (image scores) were divided into 0 (no signals), I (1 or 2 dot-like signal), II (less than half) ad III (more than half). After H.E. staining, the pathological changes (0-3 points) were assessed according to the state of inflammatory cell infiltration, and hyperplasia of synovial matrix and coating cells. The expression levels of Keap1, Nrf2, HO-1 and GSH-PX1 mRNAs in the knee synovium were detected by quantitative real-time PCR, and the expression of knee synovial HO-1 protein was measured by Western blot., Results: In comparison with the normal control group, the model group had a significant increase in the perimeter, pathological score, expression of Nrf2, HO-1 mRNAs and HO-1 protein ( P <0.05), and an obvious decrease in the TPT, expression levels of Keap1 and GSH-PX1 mRNAs ( P <0.05). Relevant to the model group, all the three acupuncture maneuvers reversed modeling-induced increase of perimeter and pathological score ( P <0.05), decrease of TPT and expression of GSH-PX1 mRNA( P <0.05), further down-regulated expression of Keap1 mRNA ( P <0.05), further up-regulated the expression of Nrf2, HO-1 mRNAs and HO-1 protein ( P <0.05). The heat-reinforcing manipulation was significantly superior to uniform reinforcing-reducing and twirling reinforcing manipulations in up-regulating TPT, and expression of Nrf2 mRNA, GSH-PX1 mRNA, HO-1 mRNA and protein ( P <0.05), and in down-regulating pathological score and Keap1 mRNA expression ( P <0.05)., Conclusion: Heat-tonifying, uniform reinforcing-reducing and twirling reinforcing needling manipulations may relieve pain and improve pathological state in RA rabbits, which may be associated with their functions in raising the ability of anti-oxidative stress by regulating Keap1-Nrf2/ARE/ HO-1 signaling pathway, the therapeutic effect of heat-tonifying needling is superior to that of uniform reinforcing-reducing and twirling reinforcing needling.

Objective: To observe the effect of heat-reinforcing needling (HRN) on inflammatory factors and necrotizing apoptosis of synovial cells in synovial tissues of knee joint in rabbits with cold syndrome rheumatoid arthritis (RA), so as to explore its underlying mechanisms in treating RA., Methods: By using the random number table method, 40 New Zealand rabbits were randomly divided into normal, model, antagonist(AG), twist-reinforceing needling (TRN) and HRN groups, with 8 rabbits in each group. The model of cold syndrome RA was established by ovalbumin induction combined with Freund's complete adjuvant injection and cryogenic freezing method. In the AG group, the antagonist TAK-632 (25 mg/kg) was administered intragastrically, once every 2 days, for a total of 7 times. Rabbits of TRN and HRN groups were treated with corresponding acupuncture techniques on bilateral "Zusanli" (ST36) for 30 min, once a day for 14 days. After intervention, the changes of knee skin temperature and circumference were measured. Color Doppler ultrasound was used to observe the joint cavity effusion, synovial thickness and internal blood flow signal. The histomorphological changes of synovial tissues were observed after HE staining. ELISA was used to detect the contents of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 in serum. Transmission electron microscope was used to observe the ultrastructure, necrosis and apoptosis of synovial cells. Western blot was used to detect the protein expressions of receptor-interacting protein kinase1 (RIPK1), RIPK3, mixed lineage kinase domain-like protein (MLKL), and phosphorylation （p）-MLKL in synovial tissues., Results: Compared with the normal group, the synovial was diffusely hyperplasia, joint cavity effusion and abnormal blood flow signal were obvious, inflammatory cells were clustered, arranged closely and disordered in the model group. The findings of transmission electron microscopy showed disruption of cell membrane integrity, swollen or ruptured mitochondria, obviously ruptured nucleus, condensed and pyknotic chromatin and nucleolus in the model group. Also, the skin temperature of the knee joint was significantly decreased ( P <0.01), while the circumference of the knee joint, the contents of TNF-α, IL-1β and IL-6 in serum, the protein expressions of RIPK1, RIPK3, p-MLKL and MLKL in synovial tissues were significantly increased ( P <0.01) in the model group. Compared with the model group, synovial tissue hyperplasia, joint cavity effusion, abnormal blood flow signals, synovial cell proliferation, inflammatory cell infiltration, disruption of cell membrane integrity, cell swelling, cell rupture, and nuclear pyknosis were reduced to different degrees in the AG, TRN and HRN groups. Additionally, the skin temperature of the knee joint was increased ( P <0.01, P <0.05), while the circumference of the knee joint, the contents of TNF-α, IL-1β and IL-6 in serum, the expressions of RIPK1, RIPK3, p-MLKL and MLKL in synovial tissues were decreased ( P <0.01, P <0.05) in the AG, TRN and HRN groups. The effects of HRN and AG were notably superior to that of TRN in up-regulating skin temperature of the knee joint, and down-regulating the circumference of the knee joint, the contents of TNF-α, IL-1β and IL-6 in serum, the expressions of RIPK1, RIPK3, p-MLKL and MLKL in synovial tissues ( P <0.01, P <0.05)., Conclusion: HRN can reduce synovial inflammation of knee joint in rabbits with cold syndrome RA, which may be related to its function in inhibiting the necrotizing apoptosis of synovial cells.

Objective: To observe the effects of heat-reinforcing needling on synovial inflammation and microRNA-155 (miR-155)/Toll-like receptor 4 (TLR4)/nuclear transcription factor-κB (NF-κB) signaling axis, so as to investigate its anti-inflammatory mechanism in rabbits with cold syndrome of rheumatoid arthritis (RA)., Methods: A total of 36 rabbits were randomly divided into normal, model, agonist, inhibitor, heat-reinforcing needling (HRN) and agonist+heat-reinforcing needling (A+HRN) groups, with 6 rabbits in each group. The RA with cold syndrome model was induced by injecting ovalbumin dry powder and Freund's complete adjuvant combined with cold freezing. Rabbits in agonist group were intraperitoneally injected with miR-155 agomir 4.5 OD; rabbits in the inhibitor group were intraperitoneally injected with miR-155 antagomir 6.1 OD; rabbits in HRN group received heat-reinforcing needling at bilateral "Zusanli" (ST36) for 30 min；rabbits in A+HRN group received the same treatment as agonist group, and 30 min later, received the same treatment as the HRN group; rabbits in the normal and model groups were grasped and fixed in the same way, all groups received continuous treatment once a day for 7 d. After modeling, the knee joints of rabbits were examined by ultrasound, the pain threshold and the circumference were determined. After the interventions, the pain threshold and knee circumference were measured; the pathological morphology of synovial tissue of the knee joints were observed by HE staining; the mRNA levels of miR-155 and suppressor of cytokine signaling protein 1 (SOCS1), the expression levels of SOCS1, TLR4, NF-κB p65, interleukin (IL)-1β and IL-17A proteins in synovial tissue of knee joints were detected by real-time PCR and Western blot respectively., Results: Compared with the normal group, the pain threshold was significantly decreased ( P <0.05), and the knee circumference was significantly increased ( P <0.05); the synovial tissue of knee joints showed significant hyperplasia, abundant blood flow signal, joint cavity effusion and obvious inflammatory invasion, the pathological score was significantly increased ( P <0.05), the expressions of miR-155 mRNA and IL-1β, IL-17A, TLR4, NF-κB p65 proteins were significantly increased ( P <0.05), the expressions of SOCS1 mRNA and protein were significantly decreased ( P <0.05) in the model group. Compared with model group, the pain threshold was significantly increased ( P <0.05), the circumference of knee joint was significantly decreased ( P <0.05); in synovial tissue, the pathological score was decreased ( P <0.05), the expression levels of miR-155 mRNA and IL-1β, IL-17A, TLR4, NF-κB p65 proteins were significantly decreased ( P <0.05), and the expressions of SOCS1 mRNA and protein were significantly increased ( P <0.05) in inhibitor group and HRN group, while the above changes in agonist group were reversed ( P <0.05). Compared with the agonist group, the pain threshold was significantly increased ( P <0.05), the knee circumference was significantly decreased ( P <0.05), the synovial pathological score was significantly decreased ( P <0.05), the expressions of miR-155 mRNA and IL-1β, IL-17A, TLR4, NF-κB proteins in synovial tissue were significantly decreased ( P <0.05), and the expression levels of SOCS1 mRNA and protein were significantly increased ( P <0.05) in A+HRN group., Conclusion: The heat-reinforcing needling can increase the pain threshold, reduce the knee circumference and inhibit the inflammatory response in rabbits with RA cold syndrome. The possible mechanism is related to the regulation of miR-155/TLR4/NF-κB signaling axis.

Objective: To observe the effects of heat-reinforcing needling on the expression of purinergic ligand-gated ion channel 7 receptor(P2X7R)，Nodlike receptor protein 3(NLRP3) and Caspase-1 in synovium tissues of knee joint of rabbits with cold syndrome rheumatoid arthritis(RA)，so as to explore the anti-inflammatory mechanism of heat reinforcing needling in the treatment of RA., Methods: Thirty male New Zealand white rabbits were randomly divided into normal group, model group, reinforcing-reducing needling group(RRG), heat-reinforcing needling group(HRG), and antagonist group(AG), with 6 rabbits in each group.The model of cold syndrome RA was established by ovalbumin combined with Freund's adjuvant and cryogenic freezing. Rabbits of RRG and HRG were treated with corresponding acupuncture techniques on both sides of "Zusanli"(ST36) for 30 min, once a day for 7 days; Rabbits of the AG was intraperitoneally injected with A438079(2.5 mg/kg), once a day for 7 days. After intervention, color Doppler ultrasound was used to observe the joint cavity effusion, synovial thickness and internal blood flow signal.The histomorphological changes of synovial tissues were observed by HE staining. Quantitative real-time PCR method was used to detect the mRNA expression levels of P2X7R, NLRP3, and Caspase-1 in synovial tissues. Western blot was used to detect the protein expression of interleukin(IL)-1β, IL-6 and tumor necrosis factor(TNF)-α in synovial tissues., Results: Compared with the normal group, the synovial tissues in the model group was thickened, linear and punctate blood flow signals were increased, joint effusion was obvious, synovial coating cells were enlarged, the synovial matrix was severely hyperplasia, inflammatory cells infiltration was obvious.The mRNAs expression levels of P2X7R, NLRP3, Caspase-1 and IL-1β, IL-6, TNF-α proteins in synovial tissues were significantly increased( P <0.01). Compared with model group, abnormal blood flow signals, synovial thickness, joint effusion, proliferation of synovial matrix, inflammatory cell infiltration and synovial coating cells in the RRG, HRG and AG were improved. The mRNAs expression levels of P2X7R, NLRP3, Caspase-1 and IL-1β, IL-6, TNF-α proteins in synovial tissues were decreased in the RRG, HRG and AG ( P <0.05, P <0.01). Compared with the RRG, the above indexes were lower in the HRG and AG ( P <0.05, P <0.01).There was no significant difference in all indexes above between the HRG and AG ( P <0.01, P <0.05)., Conclusion: Heat reinforcing needling can improve synovial inflammation of RA, which may be related to regulating the expressions of P2X7R/NLRP3/Caspase-1 signaling pathway.

Objective: To observe the effect of heat-reinforcing needling on the expression of serum inflammatory factors and autophagy of knee synovial tissue in rheumatoid arthritis (RA) rabbits with cold syndrome, so as to explore its mechanism of anti-inflammatory in the treatment of RA., Methods: Fifty rabbits were randomly divided into normal, model, heat-reinforcing needling, inhibitor and agonist groups ( n =10 rabbits in each group). The model of RA with cold syndrome was established by Freund's adjuvant and ovalbumin mixed solution injection combined with freezing and wind-cold dampness method. Heat-reinforcing needling was applied at "Zusanli" (ST36) for 30 min, once a day for 14 days. Rabbits of the inhibitor and agonist groups were given intraperitoneally injected with autophagy inhibitor 3-methyladenine (3-MA) or autophagy agonist rapamycin, once every 2 days for 7 days. The knee circumference and skin temperature of the rabbits in each group were measured. Color doppler ultrasonography was applied to examine the synovial membrane, joint effusion and blood flow signals in the knee joints of the rabbits in each group. Serum tumor necrosis factor (TNF) -α, interleukin (IL)-1β, IL-6 and C-creactive protein (CRP) were detected by ELISA. Transmission electron microscopy was applied to observe the ultrastructure and autophagosomes of synovial cells. The protein expressions of autophagy-related protein Atg5, serine/threonine protein kinase-dysregulated 51-like kinase 1 (ULK1), microtubule-associated protein light chain 3B (LC3B), and Beclin-1 were detected by Western blot. Fluorescence quantitative PCR was used to detect the mRNA expressions of NOD-like receptor 3 (NLRP3) and nuclear factor-κB (NF-κB)., Results: Compared with the normal group, the circumference of the knee joint was increased ( P <0.01), the skin temperature was decreased ( P <0.01), the knee joint synovium was thickened and the blood flow signal was abundant, the contents of serum TNF-α, IL-1β, IL-6, and CRP were increased ( P <0.01), the protein expressions of Atg5, ULK1, Beclin-1 and LC3BⅡ/LC3BⅠof synovial tissue were significantly decreased ( P <0.01), the mRNA expressions of NLRP3 and NF-κB were increased ( P <0.01) in the model group. In comparison with the model and inhibitor groups, the circumference of the knee joint was decreased ( P <0.01), whlie the skin temperature was increased ( P <0.01), the synovial membrane became thinner and the blood flow signal was wea-kened, the contents of TNF-α, IL-1β, IL-6 and CRP were decreased ( P <0.01), the protein expressions of Atg5, ULK1, Beclin-1 and LC3B Ⅱ/LC3B Ⅰ were increased ( P <0.01), and the mRNA expressions of NLRP3 and NF-κB were decreased ( P <0.01) in the heat-reinforcing needling and agonist groups., Conclusion: Heat-reinforcing needling can alleviate the inflammatory response of the knee joint synovium in RA rabbits with cold syndrome, which may be related to its function in enhancing the autophagy activity of synovial cells and inhibiting the synthesis and release of inflammatory factors TNF-α, IL-1β, IL-6 and CRP.

Objective: To explore the specific mechanism of heat-reinforcing needling (HRN) for rheumatoid arthritis (RA) rabbits with cold syndrome based on metabolomics., Methods: A total of 40 healthy rabbits were randomly divided into a normal group, a model group, a reinforcing-reducing needling(RRN) group, a twirling-reinforcing needling (TRN) group and a HRN group ( n =8 rabbits/group). The RA model with cold syndrome was established with ovalbumin and freezing. Except the normal and model groups, RRN, TRN and HRN were given at "Zusanli" (ST 36) in the corresponding acupuncture groups for 30 min, once a day for 7 days. After the interventions, the pain threshold and the local skin temperature of each group were observed. The endogenous metabolites in the serum were analyzed by the gas chromatography mass spectrometry (GC-MS) technology combined with the pattern-recognition method., Results: The pain threshold and the local skin temperature in the model group were lower than those in the normal group ( P <0.05). The pain thresholds and the local skin temperatures in the three acupuncture groups were higher than those in the model group after interventions ( P <0.05), and those in the HRN group were obviously higher than those in the RRN and TRN groups ( P <0.05). The serum metabolites of leucine, valine, isoleucine, and arachidonic acid in the model group were increased compared with those in the normal group ( P <0.05), and the metabolites of α-ketoglutaric acid, citric acid, succinic acid, glucose, inositol, palmitic acid, stearic acid, lactose, d-ribose, and D-mannose were decreased ( P <0.05). The above-mentioned metabolites in the three acupuncture groups were significantly reversed relevant to the model group ( P <0.05), and the effect of HRN group was significantly superior to those of the RRN and TRN groups in regulating α-ketoglutaric acid, citric acid, succinic acid, glucose, inositol, d-ribose, and D-mannose ( P <0.05)., Conclusions: HRN for RA with cold syndrome is effective, which may be related to the specific regulation for the krebs cycle and glycometabolism.

Fibroblast-like synoviocytes (FLS) with aberrant expression of microRNA (miRNA) are critical pathogenic regulators in rheumatoid arthritis (RA). Previous studies have found that overexpression or silencing of miRNA can contribute to the development of miRNA-based therapeutics in arthritis models. In this study, we explored the effects of miR-27a on cell migration and invasion in cultured FLS from RA patients. We found that miR-27a was markedly downregulated in the serum, synovial tissue, and FLS of RA patients. Meanwhile, the expression of follistatin-like protein 1 (FSTL1) was upregulated, which suggests that FSTL1 plays a key role in RA development. The results of a Transwell assay showed that miR-27a inhibited FLS migration and invasion. However, miR-27a inhibition promoted the migration and invasion of FLS. In addition, the down-regulated expression of matrix metalloproteinases (MMP2, MMP9, and MMP13) and Rho family proteins (Rac1, Cdc42, and RhoA) was detected after treatment with miR-27a in RA-FLS by quantitative reverse transcription-PCR and western blot analysis. Then, a luciferase reporter assay validated that miR-27a targeted the 3-untranslated region (3'-UTR) of FSTL1. Moreover, miR-27a caused a significant decrease of FSTL1. In addition, the expression of TLR4 and NFκB was inhibited by miR-27a but increased by FSTL1 overexpression. In conclusion, we found that miR-27a inhibited cell migration and invasion of RA-FLS by targeting FSTL1 and restraining the TLR4/NFκB pathway.

Objective: To observe influences of heat-reinforcing needling (HRN) on scores of traditional Chinese medicine (TCM) symptoms and expression of plasma ATP synthase subunit O ( Atp 5 O ) mRNA and lysosomal V 1 subunit B 2 ( Atp 6 V 1 B 2 ) mRNA in patients with wind-cold-damp retention type rheumatoid arthritis (RA), so as to investigate its biological mechanisms in "heat production"., Methods: Sixty wind-cold-damp retention type RA patients were randomly allocated to HRN group ( n =30) and control group ( n =30). Guanyuan (CV 4), Qihai (CV 6), bilateral Zusanli (ST 36), and local acupoints near the knee-joint were selected for needling stimulation. Patients of the HRN group were treated by manipulating the acupuncture needle with HRN, and those of the control group treated by manipulating the needle with uniform reinforcing-reducing method. The treatments were performed once daily, 5 days a week, and two weeks altogether. The other 30 healthy volunteers were recruited as the normal control group. The TCM symptom scoring system (0-31 points, 11 items as the severities of pain, swelling and tenderness of the knee-joint) was used to evaluate the status of RA, and quantitative real-time PCR (RT-PCR) was used to detect the expression of plasma Atp 5 O mRNA and Atp 6 V 1 B 2 mRNA following removal of red blood cells., Results: After the treatment, the TCM scores of both the HRN and control groups were significantly decreased ( P <0.05), and that of the HRN group was significantly lower than that of the control group ( P <0.05). Before the treatment, the expression levels of plasma Atp 5 O mRNA and Atp 6 V 1 B 2 mRNA in RA patients were significantly lower than those of the normal group ( P <0.05), and after the treatment, the expression levels of plasma Atp 5 O mRNA and Atp 6 V 1 B 2 mRNA were significantly increased in both HRN and control groups compared to pre-treatment in the same one group ( P <0.05), and the up-regulated Atp 5 O mRNA and Atp 6 V 1 B 2 mRNA levels were remarkably higher in the control group than in the HRN group ( P <0.05)., Conclusions: Both heat-producing needling and uniform reinforcing-reducing needling can improve RA patients' clinical symptoms, which may be associated with their actions in up-regulating expressions of plasma Atp 5 O mRNA and Atp 6 V 1 B 2 mRNA.