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.