| Objective: To observe the effect of heat-reinforcing acupuncture on the ubiquitination level of TRAF6-K63 and inflammatory factors such as Interleukin-1β(IL-1β),Interleukin-6(IL-6)and tumor necrosis factor α(TNF-α)in the synovial tissue of knee joint of rabbit model with cold syndrome of rheumatoid arthritis,From the perspective of inhibiting NF-κB inflammatory pathway,the mechanism of heat-reinforcing acupuncture in RA was investigated,which provides the experimental theoretical basis for the treatment of RA in clinical.Methods: From Thirty-two New Zealand White rabbits(domestic rabbits),Eight rabbits were randomly selected as the normal group.In addition to the above 8,the other rabbits used RA cold model combined with Fredrin’s complete adjuvant induction and cryofreezing.Two rabbits from the normal group and six rabbits after mold making were randomly selected for model validation.After successful replication of the model,they were randomly divided into model group,inhibitor group and heat-reinforcing acupuncture group according to the knee circumference diameter and given corresponding intervention.In the heat-reinforcing acupuncture group,the heat-reinforcing acupuncture method was used to acupuncture both sides of "Zusanli",once a day,leaving the needle for 30 min.Inhibitor group was given intraperitoneal injection of C25-140 2.5mg/kg,twice a day;The same grasping fixation was adopted simultaneously in the normal group and the model group.Four groups of continuous intervention for 14 days.The same grasping fixation was adopted simultaneously in the normal group and the model group.Four groups of continuous intervention for 14 days.After 14 days,we measured the pain threshold and knee circumference value and observed the general condition of rabbits.Beside,we used the color ultrasoundand to detect the joint cavity effusion,synovial thickness and internal blood flow signal.The synovial tissue of the knee joint was taken for pathomorphological observation.The protein expressions of UBC13 and NF-κBp65 in synovium tissues were detected by WB.Co IP-WB was used to detect the ubiquitination level of TRAF6-K63 in synovium.The expressions of TNF-α,IL-1β and IL-6 inflammatory factors in synovium were observed by immunohistochemistry.Results:1.Behavioral research(1)General situation: rabbits in the normal group had good mental state and free movement;Rabbits in the model group had poor mental condition,disordered and lustrous body hair,unhealed ulcers on the back of some rabbits,swelling of the knee joint,low skin temperature,Like curled up,and occasional loose stool.Compared with the model group,the mental state of rabbits in the heat-reinforcing acupuncture group and the inhibitor group was polish up,the body hair was Clean and shiny,the knee swelling was light,the back ulcer scab healed,like to move,and no loose stool.(2)Pain threshold: The pain threshold of rabbits in model group was significantly lower than that in normal group(P<0.05);Compared with model group,The rabbits,spain threshold in heat-reinforcing acupuncture group and inhibitor group was increased(P<0.05).Moreover,the pain threshold of rabbits in the heat-reinforcing acupuncture group was higher than that in the inhibitor group(P<0.05).(3)Knee circumference: compared with normal group,the knee circumference of model group was distinctly increased(P<0.05).The knee circumference of rabbits in the inhibitor group and the heat-reinforcing acupuncture group decreased by comparison with the model group(P<0.05),and there was no statistical difference in the knee circumference between the two groups(P>0.05).2.Morphological study(1)Color Doppler ultrasound: normal group of rabbits knee synovium no abnormal hyperplasia,no joint effusion and abnormal blood flow signal;Color ultrasound showed diffuse hyperplasia and hypertrophy of synovium in the model group,forming substantial mass echo of varying size and irregular shape,and a large number of linear or punctate blood flow signals and obvious joint effusion in the synovium.Compared with the model group,the synovial hyperplasia and hypertrophy of the inhibitor group and the heat-reinforcing acupuncture group were improved,the blood flow signal was reduced,and the joint effusion was significantly reduced.(2)Pathological changes of synovial tissue: the normal group had uniform staining,complete cell morphology and structure,no obvious pannus formation and inflammatory cell infiltration;In the model group,the synovium coated cells were enlarged obviously,and the structure and morphology were seriously damaged.The endostromal cells were hyperplasia and enlargement,and extensive inflammatory cells and lymphocyte infiltration could be seen.The proliferation,swelling and inflammatory infiltration of synovial cells in the inhibitor group and the heat-reinforcing acupuncture group were improved compared with the model group.(3)Pathological score of synovial tissue: Pathological score of synovial tissue in model group was clearly higher than that in normal group(P<0.05).The scores of the inhibitor group and the heat-reinforcing acupuncture group were both reduced by comparison with the model group(P<0.05),and the scores of the inhibitor group were more substantially decreased than the heat-reinforcing acupuncture group(P<0.05).3.Ubiquitination of TRAF6-K63 and expression of TNF-α,IL-1β and IL-6inflammatory factors in NF-κB signaling pathway(1)Expression levels of TNF-α,IL-1β and IL-6 :Compared with normal group,the average optical density of TNF-α,IL-1β and IL-6 in synovial tissue of knee joint of rabbits in model group was increased(P<0.05).Compared with the model group,the average optical density in the inhibitor group and the heat-reinforcing acupuncture group was decreased(P<0.05),and the decrease in the inhibitor group was more obvious than the heat-reinforcing acupuncture group(P<0.05).(2)Ubiquitination levels of TRAF6-K63 : Ubiquitination levels of TRAF6 and K63 in model group were increased in comparison to the normal group(P<0.05);Compared to the model group,Ubiquitin expressions of TRAF6 and K63 were lessened in inhibitor group and heat-reinforcing acupuncture group(P<0.05).The ubiquitin expression of TRAF6-K63 in the inhibitor group was more significantly decreased than that in the heat-reinforcing acupuncture group(P<0.05).(3)Expressions of UBC13 and NF-κBp65 protein : compared with normal group,the expression level of NF-κBp65 protein in model group was increased(P<0.05),while the expression level of UBC13 protein was decreased(P<0.05).Compared with model group,the expression of NF-κBp65 protein in inhibitor group and heat-reinforcing acupuncture group was decreased(P<0.05),the decrease of UBC13 protein in inhibitor group and heat-reinforcing acupuncture group was more obvious than that in heat-reinforcing acupuncture group(P<0.05),and the expression level of UBC13 protein in inhibitor group and heat-reinforcing acupuncture group was increased(P<0.05).The increase in the inhibitor group was more obvious than that in the heat-reinforcing acupuncture group(P<0.05).Conclusion:heat-reinforcing acupuncture can restrain the activity of NF-κB pathway by inhibiting the polyubiquitination level of TRAF6-K63,inhibit the secretion of downstream inflammatory factors,and play an anti-inflammatory role.Therefore,inhibition of polyubiquitination of TRAF6 is one of the anti-inflammatory mechanisms in the treatment of RA by heat-reinforcing acupuncture. |
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