[Effect of heat-reinforcing needling on expression profile of LncRNAs in synovial tissues of rabbits with cold-syndrome type rheumatoid arthritis].

Abstract

Objectives: To observe the effect of heat-reinforcing needling on the expression profile of long non-coding RNA (LncRNA) in the synovial tissue of rabbits with cold-syndrome type rheumatoid arthritis (RA), so as to explore its mechanisms underlying improvement of RA.

Methods: A total of 18 New Zealand rabbits were randomly divided into normal, model and heat-reinforcing needling (acupuncture) groups, with 6 rabbits in each group. The RA model was established by injection of mixed solution (emulsifier) of ovalbumin powder(80 mg) + NaCl (20 mL) +Freund's complete adjuvant (20 mL), combined with cold compress (ice cube +crystal calcium chloride) of the knee-joint. In addition, rabbits of the normal and model groups received regular grasping and fixation in the same way as those of the acupuncture group. For rabbits of the acupuncture group, the heat-reinforcing needling stimulation was applied to bilateral "Zusanli" (ST36) for about 1 min, followed by retaining the needle for 30 min, once a day, for a total of 7 d. The rabbits' general condition was observed, and the knee joint circumference and pain threshold after intervention were measured. The knee-joint cavity effusion, synovial thickness, and internal blood flow signals were observed using a color Doppler ultrasound apparatus. Histopathological changes of synovium were observed using H.E. staining. The contents of serum tumor necrosis factor α (TNF-α), interleukin (IL)-1β and IL-6 were detected using enzyme linked immunosorbent assay (ELISA), and the differential LncRNAs and biological functions in the synovial tissues were detected and analyzed using high-throughput sequencing technology. The expression levels of 5 significantly differentially expressed LncRNAs were verified using qPCR.

Results: Compared with the normal group, the model group of rabbits showed a significant increase in the knee joint circumference (P<0.05) and the content of serum TNF-α, IL-1β, IL-6 (P<0.05), and a significant decrease in the pain threshold (P<0.05), and diffuse synovial hyperplasia of the synovial membrane, joint effusion and abnormal blood flow signals, proliferated and thickened synovial lining, with visible capillary formation beneath the lining and a large number of inflammatory cell clustering. In comparison with the model group, the knee joint circumference, and the serum contents of TNF-α, IL-1β and IL-6 were obviously decreased (P<0.05), while the pain threshold was notably increased (P<0.05), and H.E. staining showed a reduction in the hyperplasia of synovial lining, with a milder inflammatory cell infiltration. High-throughput sequencing and bioinformatics analysis displayed that a total of 142 LncRNAs were found to have opposite expression trends between the acupuncture group and the model group, of which 83 LncRNAs were up-regulated in the model group vs the normal group and down-regulated in the acupuncture group vs the model group, mainly involving the core functional clusters of cell cycle, mitogen-activated protein kinase (MAPK) signaling and cell senescence, and 59 LncRNAs were down-regulated in the model group vs the normal group, and up-regulated in the acupuncture group vs the model group, mainly involving the metabolic pathway, peroxisome proliferator-activated receptor (PPAR) signaling and AMP-dependent protein kinase (AMPK) signaling. The results of qPCR showed that compared with the normal group, the expression levels of MSTRG.15448.1 and MSTRG.17571.1 in the synovial tissue of knee joint were significantly decreased (P<0.05), and those of MSTRG.5134.1, MSTRG.13294.1 and MSTRG.15972.1 were significantly increased (P<0.05) in the model group. Compared with the model group, the expression levels of MSTRG.15448.1 and MSTRG.17571.1 were significantly increased (P<0.05), and those of MSTRG.5134.1, MSTRG.13294.1 and MSTRG.15972.1 were significantly decreased (P<0.05) in the acupuncture group.

Conclusions: The heat-reinforcing needling can regulate the expression profile of LncRNAs in the RA synovial tissue, which may be one of the mechanisms underlying inhibition of synovial inflammatory response and improvement of symptoms.