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.