Acupoint Peripheral Sensitization And Analgesic Effect Mechanism Of Heat-sensitive Moxibustion For KOA Rabbits Based On TRPV1

Objective:To observe the effects of heat-sensitive moxibustion on the MAST cells,inflammatory pain-causing substances and TRPV1,and the changes of SP,TRPV1 and PKC in the dorsal root ganglia of Rabbit Dubi acupoint in KOA.To explore the mechanism of the peripheral sensitization and analgesic effect of heat-sensitive moxibustion.Methods:Research1: Using random number table method.36 New Zealand rabbits were randomly divided into blank group(n=6),sham operation group(n=6),model establishment group(n=24).The knee joint cavity was injected with papain solution for 15 days,and the sham operation group was injected with an equivalent amount of 0.9% Na Cl solution into the joint cavity.After the model was successfully built,the model establishment group was randomly divided into model group(n=6)and moxibustion group(n=18).Moxibustion group moxibustion "Dubi" point,once a day,40 min each time,a total of 7 days.Infrared thermal tomography imager and temperature recorder were used to measure the temperature of the abdomen,hip joint and ankle joint.When the temperature fluctuation of the rabbits within7 days was all > 1?,they were divided into the heat-sensitive moxibustion group(n=10).However,the temperature fluctuation of the rabbits within 7 days was all ?1?,they were divided into the non-heat-sensitive moxibustion group(n=7).The rabbits whose temperature fluctuated around 1? for 7 days were not included in the observation(n=1).Six rabbits were randomly selected from heat-sensitive moxibustion group and non-heat-sensitive moxibustion group.In the course of moxibustion process,the rabbits in the model group and the blank group were not allowed to intervene.After the intervention,(1)Observe the behavioral changes of the rabbits in each group.(2)The synovial tissue was observed by naked eye;The histopathological changes of cartilage and synovial membrane and Mankin's scores of cartilage were observed by HE staining.(3)Toluidine blue staining method was used to observe the expression of mast cells in the "Dubi" acupoint area.(4)The content of inflammatory pain-causing substances(5-HT,HA,SP,CGRP)in "Dubi" acupoint area was observed by immunofluorescence single label method.(5)Real-time PCR method was used to detect the content of TRPV1 mRNA in the skin of the "Dubi" acupoint area of rabbit calves in each group.Research2:Using random number table method.36 New Zealand rabbits were randomly divided into blank group(n=6),sham operation group(n=6),model group(n=6)and moxibustion group(n=48).The method for modeling and acupoint sensitization was the same as in Research 1.Among them,in the moxibustion group,temperature fluctuations>1 ? within 7 days of treatment(n=29),temperature fluctuations ? 1 ?(n=9),and fluctuated around 1 ?(n=10).In the heat-sensitive moxibustion group,randomly selected from the 29 animals in the CPZ(TRPV1 antagonist)group(n=6),CAP(TRPV1 agonist)group(n=6),the vehicle group(n=6),and heat-sensitive moxibustion control group(n=6),non-heat-sensitive moxibustion group,6 out of 9 were randomly selected for observation.Before treatment in the CPZ group,CAP group,and vehicle group,apply 0.1% CPZ,0.5% CAP,and the same amount of solvent on the Dubi acupoint area(0.5cm×0.5cm)before treatment.Apply once every 10 minutes for a total of 4 times,then continue moxibustion for 40 minutes.The non-heat-sensitive moxibustion group and the heat-sensitive moxibustion group were also moxibustion for 40 min.No intervention was given to the blank group,sham operation group and model group.After the intervention,(1)Real-time PCR method was used to detect the content of PKC mRNA in the nasal acupoint area of rabbits in each group.(2)Western blot method was used to detect the expression of SP and p-TRPV1 protein in the dorsal root ganglia of rabbits in each group.Result:Research1:1.After modeling,Compare with blank group,the sham operation group,model group,non-heat-sensitive moxibustion group,and heat-sensitive moxibustion group increased(P<0.01).After intervention,compared with the model group,the Lequesne MG score of the non-heat-sensitive moxibustion group and the heat-sensitive moxibustion group decreased(P<0.05,P<0.01),but the difference between the two groups was not statistically significant(P>0.05).2.Histopathological observation and scoring of articular cartilage under light microscope in each group of rabbits after intervention:compared with blank group,the cartilage pathological damage of the model group was severe,and the Markin's score of cartilage increased(P<0.01).Compared with the model group,pathological damage of cartilage in the non-heat-sensitive moxibustion and heat-sensitive moxibustion group was reduced,and the Markin's score of cartilage decreased(P<0.01).The heat-sensitive moxibustion group was better than the non-heat-sensitive moxibustion group(P<0.05).3.Histopathological observation of synovium under naked eye and light microscope after intervention: Naked eye observation: there was a large amount of effusion in the joint cavity of the model group,part of which showed purulent changes,synovium hyperplasia and hypertrophy,obvious congestion and edema could be seen.In the non-heat-sensitive moxibustion group,there was a small amount of fluid in the joint cavity,synovium thickened and capillary congestion.The synovium in the heat-sensitive moxibustion was thickened,but there was no obvious congestion and edema.Under light microscope,there were moderate hyperplasia of synovial cells and connective tissue in the synovial tissue of the model group,accompanied by vascular hyperplasia and aggregation of a large number of inflammatory cells.Slight hyperplasia of synovial tissue and infiltration of inflammatory cells were observed in the non-heat-sensitive moxibustion group.No obvious synovial tissue hyperplasia was observed in the heat-sensitive moxibustion group,and a small amount of inflammatory cells gathered around the blood vessels.4.Mast cell counts in the Dubi acupoint area after rabbit intervention in each group: Compared with the blank group,the number of mast cells in the model group increased(P<0.01);compared with the model group,the number of mast cells in non-heat-sensitive moxibustion group and heat-sensitive moxibustion group decreased(P<0.01);compared with the non-heat-sensitive moxibustion group,the number of mast cells in the heat-sensitive moxibustion group decreased(P<0.05).5.Inflammatory sensitizers in the Dubi acupoint area after the intervention of rabbits in each group: Compared with the blank group,the average optical density of 5-HT,HA,SP,CGRP in the model group increased significantly(P<0.01);and Compared with the model group,the average optical density of 5-HT,HA,CGRP and SP decreased in the non-heat-sensitive moxibustion group and the heat-sensitive moxibustion group(P<0.01,P<0.05);compared with non-heat-sensitive moxibustion group,the average optical density of 5-HT,HA,SP and CGRP in heat-sensitive moxibustion group decreased(P<0.01,P<0.05).6.The expression of TRPV1 mRNA in the Tubi points area after the intervention of rabbits in each group: compared with the blank group,the expression of TRPV1 mRNA in the model group was increased(P <0.01);Compared with model group,TRPV1 mRNA expression in non-heat-sensitive moxibustion group and heat-sensitive moxibustion group decreased(both P < 0.01).Compared with non-heat-sensitive moxibustion group,TRPV1 mRNA expression in heat-sensitive moxibustion group was decreased(P<0.05).Research2:1.The expression of PKC mRNA in the dorsal root ganglia of rabbits in each group after intervention: Compared with the blank group,the expression of PKC mRNA in the model group increased(P<0.01);compared with the model group,The expression of PKC mRNA in the CPZ group,the CAP group,the vehicle group,non-heat-sensitive moxibustion group and the heat-sensitive moxibustion group decreased(all P<0.01);compared with non-heat-sensitive moxibustion group,the CPZ group and heat-sensitive moxibustion group PKC mRNA the expression decreased(P<0.01,P<0.05);compared with the CPZ group,the expression of PKC mRNA in the CAP group,the vehicle group,and the non-heat-sensitive moxibustion group increased(P<0.01,P<0.05);Compared with the CAP group,the expression of PKC mRNA in the vehicle group and heat-sensitive moxibustion group decreased(P<0.05,P<0.01).2.Compared with blank group,SP and p-TRPV1 protein expression in model group increased(all P<0.01).Compared with model group,SP and p-TRPV1 protein expressions in non-heat-sensitive moxibustion group,CPZ group,CAP group,solvent group and heat-sensitive moxibustion group were decreased(all P<0.01).Compared with non-heat-sensitive moxibustion group,SP and p-TRPV1 protein expressions in CPZ group,solvent group and heat-sensitive moxibustion group were decreased(all P<0.01).Compared with CPZ group,SP and p-TRPV1 protein expressions were increased in CAP group,solvent group and heat-sensitive moxibustion group(all P <0.01).Compared with the CAP group,SP and p-TRPV1 protein expressions in the solvent group and heat-sensitive moxibustion group were decreased(P<0.01).Conclusion:1.Mast cells and inflammatory sensitization substances 5-HT,HA,SP,CGRP,TRPV1 may be the material basis of causing heat sensitization in Dubi point.2.TRPV1 is the key target of heat-sensitive moxibustion analgesia.Heat-sensitive moxibustion can play its role in the treatment of KOA by reducing SP,PKC and p-TRPV1 content,inhibiting the activation of SP/PKC/TRPV1 pathway,and alleviating the pain sensitive response of inflammatory local areas.